Automated methods and systems for production of microspore-derived doubled haploids

Abstract

This disclosure provides automated, high-throughput methods and systems for generating microspore-derived doubled haploid structures, plantlets, and crop plants, and progeny thereof. Methods of integrating computer vision and artificial intelligence into a variety of steps including microspore selection, culture, sorting, and genotyping are disclosed. Multiplexed genome-editing of microspores through cargo delivery, non-destructive sampling, and genotyping are also described.

Description

Docket No.: 214105-WO-SEC-lAUTOMATED METHODS AND SYSTEMSFOR PRODUCTION OF MICROSPORE-DERIVED DOUBLED HAPLOIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US provisional application number 63 / 724,648, filed November 25, 2024, US provisional application number 63 / 810,407, filed May 22, 2025, US provisional application number 63 / 921,768, filed November 20, 2025, US provisional application number 63 / 923,996, filed November 24, 2025, US provisional application number 63 / 921,866, filed November 20, 2025, US provisional application number 63 / 921,881, filed November 20, 2025, US provisional application number 63 / 923,835, filed November 24, 2025, and US provisional application number 63 / 923,789, filed November 24, 2025, all of which are incorporated by reference herein in their entireties.FIELD OF THE DISCLOSURE

[0002] The disclosure relates to techniques used in plant breeding.BACKGROUND OF THE DISCLOSURE

[0003] Doubled haploid (DH) technology is a cornerstone of modern plant breeding, enabling the rapid development of homozygous lines that accelerate genetic gain and trait fixation. Traditional DH production methods are labor-intensive, variable, and often limited in throughput, which restricts their scalability for commercial breeding programs. Manual handling of reproductive plant parts, microspore isolation, and subsequent culture steps introduce inconsistencies and bottlenecks, making it challenging to meet the demands of high-volume crop improvement and the development of elite germplasm. As breeding programs increasingly require large populations of DH plants for selection and trait stacking, there is a critical need for methods that deliver reproducibility, efficiency, and scalability.

[0004] The present disclosure addresses these challenges by providing automated, high- throughput methods and systems for the production of microspore-derived doubled haploid structures, plantlets, and crop plants.SUMMARY OF THE INVENTIONDocket No.: 214105-WO-SEC-l

[0005] In a first aspect, the disclosure provides high-throughput, automated methods for generating microspore-derived doubled haploid structures, plantlets, and crop plants, the methods comprising:

[0006] (a) selecting tassels, tillers, anthers, immature flowers, tillers, or reproductive plant parts at an appropriate growth stage and harvesting tassels, anthers, immature flowers, or reproductive plant parts from crop plants with an automated selection and harvesting assembly, the automated selection and harvesting assembly comprising a first computer vision processing system or image processing system and a computer-vision guided robotic assembly, wherein the first computer vision processing system or image processing system images the tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts and the computer-vision guided robotic assembly removes the tassels, anthers, immature flowers, or reproductive plant parts from the crop plants, wherein the crop plants are optionally located in a field or greenhouse or have been removed from the field using agricultural harvesting equipment, and wherein selected tassels, anthers, immature flowers, or reproductive plant parts are optionally stored in an automated storage assembly;

[0007] (b) sterilizing selected tassels, anthers, immature flowers, or reproductive plant parts with an automated sterilization assembly that receives the tassels, anthers, immature flowers, or reproductive plant parts in one or more containers either singularly or in batches, dispenses and removes one or more sterilization agents from the one or more containers after a pre-set duration, and optionally provides mechanical agitation to increase surface contact of the tassels, anthers, immature flowers, or reproductive plant parts with the one or more sterilization agents to obtain sterilized tassels, anthers, immature flowers, or reproductive plant parts;

[0008] (c) extracting microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts with an automated extraction assembly that receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one or more containers or optionally in a larger batch-process container, dispenses and removes media from the containers or optionally the larger batch-process container, and separates the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by applying a mechanical force to the one or more containers;

[0009] (d) separating extracted microspores from non -microspore plant material by filtering microspore-containing media through one or more filtering assemblies to collect the extractedDocket No.: 214105-WO-SEC-l microspores in one or more containers or pooled in a large-batch processing container and optionally the one or more containers or the large-batch processing container comprising the extracted microspores are subjected to a centrifugal force to further separate remaining nonmicrospore plant material and concentrating the extracted microspores;

[0010] (e) providing separated microspores with a culture media that promotes microspore embryogenesis induction such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores are distributed in a plurality of containers, the plurality of containers capable of being handled by a robotic handler for a high-throughput, continuous or a semi-continuous workflow, optionally wherein the microspores are dispensed into the plurality of containers at a target concentration or density;

[0011] (f) optionally providing cargo to the microspores, the multicellular structures, the embryolike structures, and / or the macroscopic structures for introducing (i) one or more site-specific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing to the microspores, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex;

[0012] (g) optionally providing the culture media with a chromosome doubling agent;

[0013] (h) culturing the microspores, multicellular structures, embryo-like structures, and / or macroscopic structures in an automated incubation assembly;

[0014] (i) sorting the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures with an automated sorting assembly comprising a second computer vision processing system or image processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the second computer vision processing system or image processing system;

[0015] (j) selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures with a selection assembly, optionally wherein selecting a diploidizedDocket No.: 214105-WO-SEC-l multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model;

[0016] (k) growing the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures in a growing station adapted to work with an automated handling and sampling system that samples and tracks the multicellular structures, the embryolike structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for genetic determination, and further culturing the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to obtain microspore-derived doubled haploid plantlets; and

[0017] (1) transferring the microspore-derived doubled haploid plantlets to growth medium (i.e., in vitro culture medium or soil) for regeneration of doubled haploid crop plants.

[0018] In an example of this first aspect, the automated extraction assembly extracts microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by blending, milling, mashing, slicing, chopping, or shredding.

[0019] In an example of this first aspect, selected tassels, anthers, immature flowers, or reproductive plant parts comprise a substantial number of microspores that are in the uninucleate to binucleate stage.

[0020] In an example of this first aspect, the microspores are extracted from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by a mechanical blending process.

[0021] In an example of this first aspect, the microspores within the tassels, anthers, immature flowers, or reproductive plant parts comprise a pre-existing heterologous genetic element that promotes microspore embryogenesis.

[0022] In an example of this first aspect, the cargo comprises a site-specific genome editing agent or system.

[0023] In an example of this first aspect, the culture media comprises an embryogenesis inducing agent.Docket No.: 214105-WO-SEC-l

[0024] In an example of this first aspect, the chromosome doubling agent is a chemical agent.

[0025] In an example of this first aspect, wherein the method further comprises singulating the multicellular structures, the embryo-like structures, and / or the macroscopic structures from batch culture.

[0026] In an example of this first aspect, the method further comprises sampling the multicellular structures, the embryo-like structures, and / or the macroscopic structures at step (h), (i), (j), (k), or a combination of foregoing; and / or sampling the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures at step (k).

[0027] In an example of this first aspect, the crop plant is maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola.

[0028] In an example of this first aspect, a fluorescent dye is added to the culture media for enhanced imaging.

[0029] In an example of this first aspect, the first computer vision processing system or image processing system and / or the second image processing system obtain a series of images at various intervals to develop a time-dependent growth stage for the tassels, anthers, immature flowers, or reproductive plant parts and / or the single-cell microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures.

[0030] In an example of this first aspect, the one or more filtering assemblies comprise a reverse flow apparatus to separate the microspores from the non-microspore plant material.

[0031] In an example of this first aspect, the selected tassels, anthers, immature flowers, or reproductive plant parts are stored in a temperature-controlled chamber prior to sterilization.

[0032] In an example of this first aspect, the tassels, anthers, immature flowers, or reproductive plant parts are harvested after the crop plants are exposed to an embryogenesis inducing agent and / or to a site-specific genome editing agent or system such that the embryogenesis inducing agent and / or the site-specific genome editing agent or system acts upon the microspores within the tassels, anthers, immature flowers, or reproductive plant parts.

[0033] In an example of this first aspect, the second computer vision processing system or image processing system is under the control of an artificial intelligence model capable of classifying or determining the developmental stage, viability, ploidy, and / or predicted fertility of the single-cell microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures based on captured images and the determined developmental stage or maturation stateDocket No.: 214105-WO-SEC-l is used to facilitate further processing of the single-cell microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures selected therefrom in an automated manner.

[0034] In an example of this first aspect, containers in which the multicellular structures, embryolike structures, and / or macroscopic structures are distributed are robotically transferred to an imaging station as part of the second computer vision processing system or image processing system or alternatively, an imaging modality of the second computer vision processing system or image processing system is brought in close proximity to the containers in which the multicellular structures, embryo-like structures, and / or macroscopic structures are distributed to enable image capture of the multicellular structures, the embryo-like structures, and / or the macroscopic structures.

[0035] In an example of this first aspect, sorting the multicellular structures, the embryo-like structures, and / or the macroscopic structures comprises sorting by developmental stage, viability, ploidy, and / or predicted fertility.

[0036] In an example of this first aspect, sorting the multicellular structures and / or the embryolike structures comprises introducing a suspension of multicellular structures or embryo-like structures into a microfluidic device and applying a di electrophoretic field to separate the multicellular structures or the embryo-like structures based on developmental stage, viability, or ploidy.

[0037] In an example of this first aspect, wherein the genetic determination of the multicellular structures, the embryo-like structures, and / or the macroscopic structures comprises transferring individual multicellular structures, embryo-like structures, and / or the macroscopic structures to a container with a non-destructive medium, agitating the container to collect shed cellular material from the multicellular structures, the embryo-like structures, and / or the macroscopic structures, and genotyping DNA obtained from the shed cellular material.

[0038] In an example of this first aspect, the genetic determination of the macroscopic structures occurs 20-50 days after extracting the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts.

[0039] In an example of this first aspect, selecting the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures comprises: (a) acquiring an image of each multicellular structure, each embryo-like structure, or eachDocket No.: 214105-WO-SEC-l macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same using an imaging modality (e.g., hyperspectral or RGB); (b) segmenting or masking the image of each multicellular structure, each embryo-like structure, or each macroscopic structure; (c) extracting one or more colorimetric features or one or more wavelength-based features from each segmented or masked image; and (d) classifying each multicellular structure, each embryo-like structure, or each macroscopic structure as having a positive predicted fertility outcome or a negative predicted fertility outcome based on extracted colormetric features or extracted wavelength-based features using the artificial intelligence model.

[0040] In an example of this first aspect, selecting the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures comprises: (a) acquiring an image of each multicellular structure, each embryo-like structure, or each macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same using an imaging modality (e.g., hyperspectral or RGB) and (b) classifying each multicellular structure, each embryo-like structure, or each macroscopic structure as having a positive predicted fertility outcome or a negative predicted fertility outcome based directly on the image of each multicellular structure, each embryo-like structure, or each macroscopic structure using a deep learning transformer model.

[0041] In an example of this first aspect, the microspore-derived doubled haploid plantlets are obtained from the diploidized macroscopic structures.

[0042] In a second aspect, the disclosure provides end-to-end, high-throughput, automated systems for generating microspore-derived doubled haploid structures, plantlets, and crop plants, the systems comprising:

[0043] (a) an automated selection and harvesting assembly for selecting tassels, tillers, anthers, immature flowers, or reproductive plant parts at an appropriate growth stage and removing the tassels, anthers, immature flowers, or reproductive plant parts from crop plants, wherein the automated selection and harvesting assembly comprises a first computer vision processing system or image processing system that images the tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts and comprises a computer- vision guided robotic assembly to remove the tassels,Docket No.: 214105-WO-SEC-l anthers, immature flowers, or reproductive plant parts from the crop plants, wherein the crop plants are optionally located in the field or greenhouse or have been removed from the field using agricultural harvesting equipment;

[0044] (b) an optional automated storage assembly for storing the selected tassels, anthers, immature flowers, or reproductive plant parts;

[0045] (c) an automated sterilization assembly for sterilizing the selected tassels, anthers, immature flowers, or reproductive plant parts, wherein the automated sterilization assembly receives the tassels, anthers, immature flowers, or reproductive plant parts in one or more containers either singularly or in batches, dispenses and removes one or more sterilization agents from the one or more containers after a pre-set duration, and optionally provides mechanical agitation to increase surface contact of the tassels, anthers, immature flowers, or reproductive plant parts with the one or more sterilization agents;

[0046] (d) an automated extraction assembly for extracting microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts, wherein the automated extraction assembly receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one more containers or optionally in a larger batch-process container, dispenses and removes media from the one or more containers or optionally the larger batch-process container, and extracts the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by application of a mechanical force to the one or more containers;

[0047] (e) one or more fdtering assemblies that separate the extracted microspores from nonmicrospore plant material by filtering microspore-containing media to collect the separated microspores in one or more containers or pooled in a larger batch-processing container and optionally the one or more containers or the larger batch-processing container comprising the extracted microspores are subjected to a centrifugal force to further separate remaining nonmicrospore plant material and concentrate the extracted microspores;

[0048] (f) a robotic handler that provides the separated microspores with a culture media, wherein the robotic handler distributes the separated microspores in a plurality of containers for a high- throughput continuous or a semi-continuous workflow;

[0049] (g) an automated incubation assembly that cultures the microspores such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores,Docket No.: 214105-WO-SEC-l the multicellular structures, the embryo-like structures, and / or the macroscopic structures are optionally provided cargo for introducing (i) one or more site-specific genomic changes, (ii) one or more transgenes for integration into the genomic DNA, (iii) a morphogenic developmental polypeptide or a polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex, and wherein the automated incubation assembly optionally provides a chromosome doubling agent to the culture media;

[0050] (h) an automated sorting assembly that sorts the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly comprises a second computer vision processing system or image processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the second computer vision processing system or image processing system;

[0051] (i) an optional artificial intelligence model that selects the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures predicted to regenerate into fertile that are more likely to germinate into fertile plantlets, wherein the artificial intelligence model has been trained on a dataset comprising microspores, multicellular structures, embryo-like structures, macroscopic structures, and / or plantlets that had positive fertility outcomes (e.g., flowered or set seed);

[0052] (j) an automated selection assembly that selects diploidized multicellular structures, embryo-like structures, and / or macroscopic structures for a high-throughput continuous or a semi- continuous workflow, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model;Docket No.: 214105-WO-SEC-l

[0053] (k) a growing station that grows the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures;

[0054] (1) an automated handling and sampling system adapted to work with the growing station, wherein the automated handling and sampling system samples and tracks the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for genetic determination, and further cultures the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to obtain microspore-derived doubled haploid plantlets; and

[0055] (m) a robotic arm that transfers the microspore-derived doubled haploid plantlets to growth medium (i.e., in vitro culture medium or soil) for regeneration of doubled haploid crop plants.

[0056] In a third aspect, the disclosure provides high-throughput, automated methods for generating genome-edited microspore-derived doubled haploid structures, plantlets, and crop plants, the methods comprising:

[0057] (a) extracting microspores from sterilized tassels, tillers, anthers, immature flowers, or reproductive plant parts with an automated extraction assembly that receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one or more containers or optionally in a larger batch-process container, dispenses and removes media from the containers or optionally the larger batch-process container, and separates the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by applying a mechanical force to the one or more containers;

[0058] (b) separating extracted microspores from non -microspore plant material by filtering microspore-containing media through one or more filtering assemblies to collect the extracted microspores in one or more containers or pooled in a large-batch processing container and optionally the one or more containers or the large-batch processing container comprising the extracted microspores are subjected to a centrifugal force to further separate remaining nonmicrospore plant material and concentrating the extracted microspores;

[0059] (c) providing separated microspores with a culture media that promotes microspore embryogenesis induction such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures toDocket No.: 214105-WO-SEC-l macroscopic structures, wherein the microspores are distributed in a plurality of containers, the plurality of containers capable of being handled by a robotic handler for a high-throughput, continuous or a semi-continuous workflow, optionally wherein the microspores are dispensed into the plurality of containers at a target concentration or density;

[0060] (d) providing cargo to the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures for introducing (i) one or more site-specific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing to the microspores, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex, wherein introducing the one or more site-specific genomic modifications comprises providing to the microspores, the multicellular structures, the embryolike structures, and / or the macroscopic structures a site-specific genome editing agent or system, optionally wherein the a site-specific genome editing agent or system comprises a Cas polypeptide having DNA binding activity or a polynucleotide sequence encoding the Cas polypeptide and a guide polynucleotide that comprises a region of complementarity to a double-stranded DNA target site in the multicellular structures, the embryolike structures, and / or the macroscopic structures, wherein the Cas polypeptide and the guide polynucleotide form a complex that binds the double-stranded DNA target site and optionally induces a double-strand or single-strand break at the double-stranded DNA target site, wherein the one or more site-specific genomic modifications is insertion, deletion, single nucleotide polymorphism, inversion, or translocation;

[0061] (e) optionally providing the culture media with a chromosome doubling agent;

[0062] (f) culturing the microspores, the multicellular structures, the embryo-like structures, and or the macroscopic structures in an automated incubation assembly;

[0063] (g) sorting the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures with an automated sorting assembly comprising a second computer vision processing system or image processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-likeDocket No.: 214105-WO-SEC-l structures, and / or the macroscopic structures for culture based on images from the second computer vision processing system or image processing system;

[0064] (h) selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures with an automated selection assembly for a high-throughput continuous or a semi-continuous workflow, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model;

[0065] (i) growing the multicellular structures, the embryo-like structures, and / or the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures in a growing station adapted to work with an automated handling and sampling system that samples and tracks the multicellular structures, the embryolike structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for genetic determination, and further culturing the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to obtain genome-edited microspore- derived doubled haploid plantlets; and

[0066] (j) transferring the genome-edited microspore-derived doubled haploid plantlets to growth medium (i.e., in vitro culture medium or soil) for regeneration of doubled haploid crop plants.

[0067] In a fourth aspect, the disclosure provides high-throughput, automated methods of genotyping microspore-derived doubled haploid structures, the method comprising:

[0068] (a) extracting microspores from the sterilized tassels, tillers, anthers, immature flowers, or reproductive plant parts with an automated extraction assembly that receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one or more containers or optionally in a larger batch-process container, dispenses and removes media from the containers or optionally the larger batch-process container, and separates the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by applying a mechanical force to the one or more containers;Docket No.: 214105-WO-SEC-l

[0069] (b) providing separated microspores with a culture media that promotes microspore embryogenesis induction such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores are distributed in a plurality of containers, the plurality of containers capable of being handled by a robotic handler for a high-throughput, continuous or a semi-continuous workflow, optionally wherein the microspores are dispensed into the plurality of containers at a target concentration or density;

[0070] (c) optionally providing cargo to the microspores, the multicellular structures, the embryolike structures, and / or the macroscopic structures for introducing (i) one or more site-specific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing to the microspores, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex;

[0071] (d) optionally providing the culture media with a chromosome doubling agent;

[0072] (e) culturing the multicellular structures, the embryo-like structures, and / or the macroscopic structures in an automated incubation assembly to obtain diploidized multicellular structures, diploidized embryo-like structures, and / or diploidized multicellular structures; and

[0073] (f) genotyping the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures with an automated handling sampling system that samples and tracks the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures, wherein genotyping comprises: transferring individual multicellular structures, embryo-like structures, macroscopic structures, diploidized multicellular structures, diploidized embryo-like structures, and / or diploidized macroscopic structures to a container with a non-destructive medium, agitating the container to collect shed cellular material from the individual multicellular structure, embryo-like structure, macroscopic structure, diploidized multicellular structure, diploidized embryo-like structure, and / or diploidized macroscopic structure, and genotyping DNA obtained from the shed cellular material.Docket No.: 214105-WO-SEC-lBRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG. 1A is a flowchart illustrating a method for generating maize doubled haploids.

[0075] FIG. IB is a flowchart illustrating a method for generating doubled haploids from nonmaize crop plants.

[0076] FIG. 1C illustrates a method for generating microspore-derived doubled haploid structures, plantlets, and crop plants.

[0077] FIGS. 2A-2B are flowcharts illustrating various stages for cargo delivery to microspores and microspore-derived structures in doubled haploid production methods.

[0078] FIG. 3 is a flowchart illustrating a method for sterilization of tassels, anthers, immature flowers, or reproductive plant parts.

[0079] FIGS. 4A-4E are flowcharts illustrating stages for sampling and characterization of microspores, microspore-derived viable proliferating cell masses, and regenerated plants in doubled haploid production methods.

[0080] FIGS. 5A-5B are flowcharts illustrating stages for selecting microspores and microspore- derived viable proliferating cell masses in doubled haploid production methods.

[0081] FIG. 6 is an example of a sterilization assembly for doubled haploid production methods.

[0082] FIG. 7 is a flowchart illustrating a method for microspore extraction.

[0083] FIG. 8 is an example of a microspore extraction assembly for doubled haploid production methods.

[0084] FIG. 9 is an example of a reverse flow apparatus for separating microspores from bulk material after extraction for doubled haploid production methods.

[0085] FIG. 10 is an example of a continuous flow centrifuge apparatus for separating microspores from bulk material after extraction for doubled haploid production methods.

[0086] FIG. 11 is a robotic workstation for microspore isolation for doubled haploid production methods.

[0087] FIG. 12 is an example of a tassel sterilization and microspore extraction assembly for doubled haploid production methods.

[0088] FIGS. 13A - 13E are diagrams of a mesh culture system for haploid induction and germination of microspore-derived structures. FIG. 13A illustrates isolated microspores plated on a removable mesh insert. FIG. 13B illustrates transferring a microspore-containing mesh insert toDocket No.: 214105-WO-SEC-l a new petri dish for a media change during microspore culture. FIG. 13C illustrates microspore- derived macroscopic structures on a removable mesh insert. FIG. 13D illustrates transferring microspore-derived macroscopic structures from a mesh insert to solid media. FIG. 13E illustrates plantlet regeneration of macroscopic structures on solid media.

[0089] FIG. 14A is an image of microspore-derived macroscopic structures growing on the mesh insert in liquid media after 30 days in culture.

[0090] FIG. 14B is an image of the mesh insert of FIG. 14A inverted for transfer of macroscopic structures to solid media.

[0091] FIG. 14C is an image of the macroscopic structures of FIG. 14B after transfer to solid germination media.

[0092] FIG. 15A is an image of microspore-derived macroscopic structures growing on the mesh insert in liquid media after 30 days in culture.

[0093] FIG. 15B is an image of the mesh insert of FIG. 15A with macroscopic structures in liquid media and a chromosome doubling agent.

[0094] FIG. 15C is an image of the mesh insert of FIG. 15B inverted for transfer of macroscopic structures to solid media.

[0095] FIG. 15D is an image of the macroscopic structures of FIG. 15C after transfer to solid germination media.

[0096] FIG. 16 is a diagram of a microfluidic system for sorting microspores and microspore- derived structures by dielectrophoresis (DEP).

[0097] FIG. 17 is a graph showing percentage of multicellular structures present in 5-day old microspore cultures before and after DEP sorting.

[0098] FIGS. 18A - 18C are representative images of 5-day old microspore cultures before and after DEP sorting. FIG. 18A is an image of microspore cultures before DEP sorting. FIG. 18B is an image of flowthrough after DEP sorting. FIG. 18C is an image of microspore cultures after DEP selection.

[0099] FIG. 19 is a schematic of a two-shell model used for simulating the movement of a plant cell under an AC electric field.

[0100] FIGS. 20A - 20D are graphs showing impedance flow cytometry measurements of micropores before and after DEP sorting using flow cell chips. FIG. 20A shows impedance of the input microspore population. FIG. 20B shows impedance of the microspore output populationDocket No.: 214105-WO-SEC-l from the top channel that collected microspores experiencing positive DEP. FIG. 20C shows impedance of the microspore output population from the middle channel that collected microspores not experiencing positive DEP. FIG. 20D shows impedance of the microspore output population from the bottom channel that collected microspores experiencing positive DEP.

[0101] FIGS. 21A - 21D are violin plots showing the phase distribution of viable microspores from FIGS. 20A- 20D. FIG. 21A shows the phase distribution of the input microspore population. FIG. 21B shows the phase distribution of the microspore output population from the top channel that collected microspores experiencing positive DEP. FIG. 21C shows the phase distribution of the microspore output population from the middle channel that collected microspores not experiencing positive DEP. FIG. 21D shows the phase distribution of the microspore output population from the bottom channel that collected microspores experiencing positive DEP.

[0102] FIG. 22 is a graph of genotyping data return of DNA isolated from macroscopic structures based on isolation buffers used for DNA collection.

[0103] FIG. 23 is a graph of genotyping data return of DNA isolated from macroscopic structures based on the agitation duration used for DNA collection.

[0104] FIG. 24 is a graph of macroscopic structures having no observed further growth after solid media culture based on the agitation duration used for DNA collection.

[0105] FIG. 25 is a graph of macroscopic structures with root or shoot formation after solid media culture based on the agitation duration used for DNA collection.

[0106] FIG. 26 is a graph of genotyping data return of DNA isolated from macroscopic structures based on sampling day post microspore isolation.

[0107] FIG. 27 is a graph of macroscopic structures having no observed further growth after solid media culture based on sampling day post microspore isolation.

[0108] FIG. 28 is a graph of macroscopic structures with root or shoot formation after solid media culture based on sampling day post microspore isolation.

[0109] FIG. 29 is a representative image of macroscopic structures.

[0110] FIG. 30A is an image of diploid microspores from a tetrapioid line.

[0111] FIG. 30B is an image of haploid microspores from a diploid line.DETAILED DESCRIPTIONDocket No.: 214105-WO-SEC-l

[0112] The present disclosure relates to automated methods and systems for generating microspore-derived doubled haploid structures, plantlets, and plants for crop plants such as maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, and canola and fruits and vegetables such as peppers, potatoes, tomatoes, melons, watermelons, and berries.

[0113] Turning to FIG. 1A, maize doubled haploid production involves: (a) harvesting tassels; (b) optionally pre-treating tassels; (c) sterilizing tassels; (d) extracting microspores from the sterilized tassels and separating the extracted microspores from non-microspore plant material; (e) culturing the microspores to promote or induce embryogenesis and chromosome doubling such that singlecell microspores progress from single-cell microspores to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures to obtain microspore-derived doubled haploid structures; (f) regenerating microspore- derived doubled haploid plantlets from the microspore-derived doubled haploid structures; and (g) transferring the microspore-derived doubling haploid plantlets to growth medium (i.e., in vitro culture medium or soil).

[0114] Similarly, as shown in FIG. IB, non-maize doubled haploid production involves (a) harvesting anthers, immature flowers, or reproductive plant parts; (b) optionally pre-treating the anthers, immature flowers, or reproductive plant parts; (c) sterilizing the anthers, immature flowers, or reproductive plant parts; (d) extracting microspores from the sterilized anthers, immature flowers, or reproductive plant parts and separating the extracted microspores from nonmicrospore plant material; (e) culturing the microspores to promote or induce embryogenesis and chromosome doubling such that single-cell microspores progress from single-cell microspores to multicellular structures, from multicellular structures to embryo-like structures, and from embryolike structures to macroscopic structures to obtain microspore-derived doubled haploid structures; (f) regenerating microspore-derived doubled haploid plantlets from the microspore-derived doubled haploid structures; and (g) transferring the microspore-derived doubling haploid plantlets to growth medium (i.e., in vitro culture medium or soil)

[0115] FIG. 1C illustrates an example of a method for generating microspore-derived doubled haploid structures, plantlets, and plants from a tassel, anther, immature flower, or reproductive plant part of a crop plant such as such as maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola. Tassels, anthers, immature flowers, or reproductive plant parts are harvested from crop plants and sterilized individually or in batches with one or more sterilizationDocket No.: 214105-WO-SEC-l agents (e.g., ethanol, sodium hypochlorite (NaOCl) with a surfactant, hydrogen peroxideand other known detergents, surfactants, and disinfectants). Prior to sterilization, harvested tassels, anthers, immature flowers, or reproductive plant parts can undergo one or more pre-treatments such as a cold pre-treatment (e.g., tassels, anthers, immature flowers, or reproductive plant parts are stored at 4°C for 1-7 days to delay pollen maturation, synchronize microspore development, and improve embryogenic responses), moisture maintenance treatment (tassels, anthers, immature flowers, or reproductive plant parts are wrapped in moist paper towels or placed in sealed plastic bags during cold storage to prevent desiccation and damage of microspores), a hot or cold temperature shock treatment (e.g., tassels, anthers, immature flowers, or reproductive plant parts are incubated at 32°C for a few hours or extended cold at 4°C to boost embryogenic responses), a fungicide or antimicrobial treatment (e.g., application of fungicide or antimicrobial solutions before sterilization to reduce fungal contamination), and / or trimming damaged portions of the tassels, anthers, immature flowers, or reproductive plant parts.

[0116] Following sterilization, microspores are extracted from tassels, anthers, immature flowers, or reproductive plant parts by blending, milling, mashing, slicing, and / or shredding, and masticated bulk material, including microspores, is fdtered to separate bulk non-microspore plant material from the microspores. Microspore extraction can further comprise wash steps and centrifugation to concentrate microspores. Microspores are ultimately suspended in fresh isolation medium.

[0117] Microspores are cultured in liquid induction medium to promote or induce microspore embryogenesis such that that single-cell microspores progress from single-cell microspores to multicellular structures, from multicellular structures to embryo-like structures, and from embryolike structures to macroscopic structures. During liquid culture, developing microspores can be treated with embryogenesis inducing agents and / or chromosome doubling agents, which promote diploidization of developing microspore-derived structures (i.e., multicellular structures, embryolike structures, and / or macroscopic structures) Microspore-derived viable proliferating cell masses, typically macroscopic structures, are transferred to regeneration medium, which is usually a solid substrate (also known as a solid medium) for root and shoot formation and regeneration of microspore-derived doubled haploid plantlets. Regenerated plantlets are transferred to a suitable growth medium (i.e., in vitro culture medium or soil) for regeneration of doubled haploid crop plants.Docket No.: 214105-WO-SEC-l

[0118] As used herein, a tassel refers to the male reproductive part of a maize plant containing multiple spikelets, each spikelet containing anthers that produce pollen.

[0119] As used herein, an anther refers to a part of the stamen of a crop plant where pollen is produced.

[0120] As used herein, an immature flower refers to the staminate flower of a crop plant, which possesses only male reproductive parts.

[0121] As used herein, a reproductive plant part refers to the stamen of a crop plant, which consists of the anther and filament.

[0122] As used herein, a microspore refers to a haploid spore of a crop plant that develops into the male gametophyte (the male gametophyte of a seed plant is the pollen grain). In the methods and systems described herein, tassels, anthers, immature flowers, or reproductive plant parts are isolated from crop plants to obtain microspores of a desired developmental stage, typically between the uninucleate to binucleate stage, and more specifically around the first pollen mitosis (i.e., late uninucleate to early binucleate stage). Microspores can have a diameter of about 20 pm to about 100 pm, based on the plant species.

[0123] As used herein, “multicellular structure” and “microspore-derived multicellular structure” are used interchangeably and refer to an embryogenic, multicellular structure derived from a single-cell microspore that ranges in size from about 50 pm to about 550 pm, based on the plant species. In an example, multicellular structures range in size from about 100 pm to about 200 pm.

[0124] As used herein, “embryo-like structures” and “microspore-derived embryo-like structures” are used interchangeably and refer to an embryogenic, multicellular structure derived from a single-cell microspore that ranges in size from about 200 pm to about 1000 pm, based on the plant species. In an example, embryo-like structures range in size from about 200 pm to about 750 pm.

[0125] As used herein, “macroscopic structure” and “microspore-derived macroscopic structure” are used interchangeably and refer to an embryogenic, multicellular structure derived from a single-cell microspore that ranges in size from about 500 pm to about 10000 pm, based on the plant species. In an example, multicellular structures range in size from about 750 pm to about 3000 pm. A macroscopic structure can also be considered to be a haploid embryo, an embryoid, a somatic embryo, or an organogenic callus. A macroscopic structure is typically visible to the human eye without aid of a magnifying lens or microscope.Docket No.: 214105-WO-SEC-l

[0126] It will be understood that the size ranges for microspore-derived multicellular structures, embryo-like structures, and macroscopic structures can vary across crop plants (e.g., maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, and canola), and that such variations are known to one of skill in the art and within scope of the disclosure.

[0127] Microspore-derived structures (i.e., multicellular structures, embryo-like structures, and macroscopic structures) are each considered to be a microspore-derived viable proliferating cell mass (VPCM). A VPCM can range in size from about 50 pm to about 10000 pm. In an example, a VPCM can range in size from about 200 pm to about 3000 pm.

[0128] As used herein, “embryogenic” refers to an isolated microspore that has been artificially stimulated to bypass its normal development into a male gametophyte and instead assumes a cell fate that supports embryogenesis or organogenesis (i.e., formation of a mass of undifferentiated cells, known as a callus, that can give rise to roots and shoots).

[0129] As used herein, a doubled haploid structure is a VPCM that has undergone doubling or diploidization of its In haploid chromosome number to 2n (diploid). A doubled haploid structure can be derived from a multicellular structure, an embryo-like structure, or a macroscopic structure. Doubled haploid structures give rise to doubled haploid plantlets and doubled haploid plants.

[0130] The methods and systems disclosed herein provide a large supply of microspore-derived doubled haploid explants for producing transgenic, genome-edited, and / or doubled haploid plants. Microspore-derived doubled haploid explants, such as those generated in the first generation of doubled haploid plants obtained after chromosome doubling (DO) or any subsequent generation thereafter, include meristem explants (e.g., apical or axillary meristems that are commonly used in micropropagation), shoots, shoot tips, stems, and stem segments (e.g., nodal and internodal sections that are commonly used in clonal propagation and regeneration), leaves that are commonly used for callus induction and transformation, roots and root segments, floral explants (e.g., petals, stamens, ovules, and anthers that are commonly used for embryo rescue and (doubled) haploid production), seeds, and embryos. Microspore-derived doubled haploid explants can be, for example, produced and stored before transformation (or any other plant genetic manipulation technique for delivery of DNA, RNA, or protein) to make transgenic and / or genome-edited plants.

[0131] The generation of these microspore-derived structures, plantlets, plants, and / or explants make the process more labor-efficient and suitable for high-volume, high-throughput plant population development needs. The number of microspores processed in one day is in the range ofDocket No.: 214105-WO-SEC-l tens of thousands to tens of millions per germplasm type. The overall number of microspores processed in a working week can exceed 100 million. The number of microspore-derived multicellular structures, embryo-like structures, macroscopic structures, diploidized multicellular structures, diploidized embryo-like structures, and / or diploidized macroscopic structures processed in a day by imaging and other automation methods can readily exceed tens of thousands to several million at peak capacity per germplasm type. The methods and systems described herein can be used to generate paternal doubled haploid structures, plantlets, and plants of the same genotype. Alternatively, microspores of various genotypes can be pooled together for the automated methods described herein.

[0132] Progeny of plants regenerated from the methods described herein are also included within the scope of the disclosure.

[0133] Harvest and Storage

[0134] The methods and systems disclosed herein can utilize one or more automation steps for selecting, harvesting, and storing tassels, anthers, immature flowers, or reproductive plant parts, and subsequently transferring them from a storage facility (e.g., a cold room) for microspore isolation.

[0135] Tassels, anthers, immature flowers, or reproductive plant parts are harvested before pollen shed and stored to obtain microspores of a desired developmental stage. For the methods described, extracted microspores are between the uninucleate to binucleate stage, the mid-uninucleate to early binucleate stage, or the late uninucleate to early binucleate stage (i.e., around the first pollen mitosis) for embryogenesis induction.

[0136] Selection and harvest of tassels, anthers, immature flowers, or reproductive plant parts can be carried out by an automated selection and harvesting assembly, optionally comprising, or operably connected to, an automated storage assembly or cold room.

[0137] An automated selection and harvesting assembly can perform one or more of the following:

[0138] (a) image and select tassels, anthers, immature flowers, or reproductive plant parts to assess their developmental stage and suitability for harvest;

[0139] (b) select and remove tassels, anthers, immature flowers, or reproductive plant parts from crop plants;Docket No.: 214105-WO-SEC-l

[0140] (c) transfer and organize harvested tassels, anthers, immature flowers, or reproductive plant parts to the automated storage assembly or cold room; and

[0141] (d) provide batch or continuous processing (i.e., selection, removal, and transfer) of tassels, anthers, immature flowers, or reproductive plant parts

[0142] An automated storage assembly provides uniform storage conditions for harvested tassels, anthers, immature flowers, or reproductive plant parts with controlled environmental parameters such as temperature, moisture / humidity, light, nutrient management, and / or oxygen / gas exchange.

[0143] An automated selection and harvesting assembly can comprise or be equipped with a computer vision processing system or image processing system and a computer vision-guided robotic assembly. The computer vision processing system or image processing system images tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts. In an example, tassels, anthers, immature flowers, or reproductive plant parts are imaged to predict the developmental stage of the microspores housed within. The computer- vision guided robotic assembly removes the selected tassels, anthers, immature flowers, or reproductive plant parts from crop plants, which can be located in the field, greenhouse, or previously removed using agricultural equipment.

[0144] The automated selection and harvesting assembly can deliver harvested tassels, anthers, immature flowers, or reproductive plant parts to an automated storage assembly that provides uniform storage conditions. An automated storage assembly can be equipped with one or more robotic assemblies for organizing, selecting, and / or handling harvested tassels, anthers, immature flowers, or reproductive plant parts for subsequent processing.

[0145] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: selecting tassels, anthers, immature flowers, or reproductive plant parts at an appropriate growth stage and harvesting tassels, anthers, immature flowers, or reproductive plant parts from crop plants with an automated selection and harvesting assembly, the automated selection and harvesting assembly comprising a first computer vision processing system or image processing system and a computer-vision guided robotic assembly, wherein the first computer vision processing system or image processing system images the tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts and the computer-vision guided robotic assembly removes the tassels, anthers, immature flowers, orDocket No.: 214105-WO-SEC-l reproductive plant parts from the crop plants, wherein the crop plants are optionally located in a field or greenhouse or have been removed from the field using agricultural harvesting equipment.

[0146] In another example, a high-throughput, automated method for generating microspore- derived doubled haploid structures, plantlets, and crop plants comprises: selecting tassels, anthers, immature flowers, or reproductive plant parts at an appropriate growth stage and harvesting tassels, anthers, immature flowers, or reproductive plant parts from crop plants with an automated selection and harvesting assembly, the automated selection and harvesting assembly comprising a first computer vision processing system or image processing system and a computer-vision guided robotic assembly, wherein the first computer vision processing system or image processing system images the tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts and the computer-vision guided robotic assembly removes the tassels, anthers, immature flowers, or reproductive plant parts from the crop plants, wherein the crop plants are optionally located in a field or greenhouse or have been removed from the field using agricultural harvesting equipment; and transferring the removed tassels, anthers, immature flowers, or reproductive plant parts to a cold room or and automated storage assembly.

[0147] In another example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: an automated selection and harvesting assembly for selecting tassels, anthers, immature flowers, or reproductive plant parts at an appropriate growth stage and removing the tassels, anthers, immature flowers, or reproductive plant parts from crop plants, wherein the automated selection and harvesting assembly comprises a first computer vision processing system or image processing system that images the tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts and comprises a computer-vision guided robotic assembly to remove the tassels, anthers, immature flowers, or reproductive plant parts from the crop plants, wherein the crop plants are optionally located in the field or greenhouse or have been removed from the field using agricultural harvesting equipment.

[0148] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: an automated selection and harvesting assembly for selecting tassels, anthers, immature flowers, or reproductive plant parts at an appropriate growth stage and removing the tassels, anthers, immature flowers, orDocket No.: 214105-WO-SEC-l reproductive plant parts from crop plants, wherein the automated selection and harvesting assembly comprises a first computer vision processing system or image processing system that images the tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts and comprises a computer-vision guided robotic assembly to remove the tassels, anthers, immature flowers, or reproductive plant parts from the crop plants, wherein the crop plants are optionally located in the field or greenhouse or have been removed from the field using agricultural harvesting equipment, wherein the system further comprises an automated storage assembly that receives selected and harvested tassels, anthers, immature flowers, or reproductive plant parts from the automated selection and harvesting assembly.

[0149] Automating selection, harvest, and storage of tassels, anthers, immature flowers, or reproductive plant parts reduces variability (e.g., provides consistent imaging and robotic handling), increases throughput (e.g., multiple tassels, anthers, immature flowers, or reproductive plant parts can be processed simultaneously), and provides uniform handling and storage conditions to improve reproducibility. Further, automating selection, harvest, and storage promotes scalability for commercial production, wherein an automated selection and harvesting assembly and / or an automated storage assembly can be adapted for field or greenhouse environments and scaled for batch or continuous processing of tassels, anthers, immature flowers, or reproductive plant parts.

[0150] Sterilization

[0151] The methods and systems disclosed herein can utilize one or more automation steps for sterilization of harvested tassels, anthers, immature flowers, or reproductive plant parts. Prior to sterilization, harvested tassels, anthers, immature flowers, or reproductive plant parts can undergo one or more pre-treatments such as a cold pre-treatment (e.g., tassels, anthers, immature flowers, or reproductive plant parts are stored at 4°C for 1-7 days to delay pollen maturation, synchronize microspore development, and improve embryogenic responses), moisture maintenance treatment (tassels, anthers, immature flowers, or reproductive plant parts are wrapped in moist paper towels or placed in sealed plastic bags during cold storage to prevent desiccation and damage of microspores), a hot or cold temperature shock treatment (e.g., tassels, anthers, immature flowers, or reproductive plant parts are incubated at 32°C for a few hours or extended cold at 4°C to boost embryogenic responses), a fungicide or antimicrobial treatment (e g., application of fungicide orDocket No.: 214105-WO-SEC-l antimicrobial solutions before sterilization to reduce fungal contamination), and / or trimming damaged portions of the tassels, anthers, immature flowers, or reproductive plant parts.

[0152] Automated sterilization is designed to efficiently sterilize harvested tassels, anthers, immature flowers, or other reproductive plant parts in a scalable, high-throughput manner. This minimizes or eliminates manual handling and ensures consistent, reproducible sterilization protocols.

[0153] Sterilization of harvested of tassels, anthers, immature flowers, or reproductive plant parts can be carried out by an automated sterilization assembly. An automated sterilization assembly can perform one or more of the following:

[0154] (a) receive stored tassels, anthers, immature flowers, or reproductive plant parts in containers, either individually or in batches;

[0155] (b) dispense sterilization agents (e.g., ethanol, sodium hypochlorite (NaOCl) with a surfactant, hydrogen peroxide (H2O2), and / or other known detergents, surfactants, and disinfectants) into the containers for a predetermined period (i.e., sterilization cycle) and at a predetermined concentration and / or volume;

[0156] (c) remove sterilization agents from containers after a sterilization cycle;

[0157] (d) rinse sterilized tassels, anthers, immature flowers, or reproductive plant parts by dispensing (an subsequently removing) a wash solution to rinse to remove residual sterilization agents;

[0158] (e) provide mechanical agitation (e.g., shaking, pressure cycling, or mixing) to increase surface contact between the isolated plant material and the sterilization agent(s);

[0159] (f) monitor and control parameters of a sterilization cycle such as pressure, time, and sterilization agent concentration or volume;

[0160] (g) execute sterilization cycles optimized for different plant materials or plant species;

[0161] (h) handle both individual samples and bulk material (e.g., individual containers or large batches) to support continuous or batch workflows; and / or

[0162] (i) be operably connected to an automated selection and harvesting assembly, an automated storage assembly, and / or cold room.

[0163] An automated sterilization assembly can comprise or be equipped with one or more of the following:Docket No.: 214105-WO-SEC-l

[0164] (a) a container loading system that accepts containers with harvested and / or stored tassels, anthers, immature flowers, or reproductive plant parts from an automated selection and harvesting assembly, an automated storage assembly, or cold room;

[0165] (b) one or more dispensing and removal mechanisms such as liquid handlers, pumps, valves, and syringes for delivery and removal of sterilization agent(s) and wash solutions;

[0166] (c) an agitation module or mechanical components (e.g., shakers, mixers, pressure control systems) to agitate samples during sterilization and / or drying after sterilization;

[0167] (d) a sterilization protocol for different plant materials (e.g., a tassel, anther, immature flower, or reproductive plant part) or plant species (e.g., maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola);

[0168] (e) sensors that monitor sterilization parameters (e.g., acceleration, speed, direction, amplitude, and dwell time of agitation), sterilization agent levels, and sterilization cycle completion; and / or

[0169] (f) one or more robotic arms or assemblies (e.g., robotic arms and / or manipulative tooling) to move containers with tassels, anthers, immature flowers, or reproductive plant parts undergoing processing.

[0170] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: sterilizing selected tassels, anthers, immature flowers, or reproductive plant parts with an automated sterilization assembly that receives the tassels, anthers, immature flowers, or reproductive plant parts in one or more containers either singularly or in batches, dispenses and removes one or more sterilization agents from the one or more containers after a pre-set duration, and optionally provides mechanical agitation to increase surface contact of the tassels, anthers, immature flowers, or reproductive plant parts with the one or more sterilization agents to obtain sterilized tassels, anthers, immature flowers, or reproductive plant parts.

[0171] In another example, a high-throughput, automated method for generating microspore- derived doubled haploid structures, plantlets, and crop plants comprises: sterilizing selected tassels, anthers, immature flowers, or reproductive plant parts with an automated sterilization assembly that receives the tassels, anthers, immature flowers, or reproductive plant parts from an automated selection and harvesting assembly in one or more containers either singularly or in batches, dispenses and removes one or more sterilization agents from the one or more containersDocket No.: 214105-WO-SEC-l after a pre-set duration, and optionally provides mechanical agitation to increase surface contact of the tassels, anthers, immature flowers, or reproductive plant parts with the one or more sterilization agents to obtain sterilized tassels, anthers, immature flowers, or reproductive plant parts.

[0172] In another examples, a high-throughput, automated method for generating microspore- derived doubled haploid structures, plantlets, and crop plants comprises: sterilizing selected tassels, anthers, immature flowers, or reproductive plant parts with an automated sterilization assembly that receives the tassels, anthers, immature flowers, or reproductive plant parts from an automated storage assembly in one or more containers either singularly or in batches, dispenses and removes one or more sterilization agents from the one or more containers after a pre-set duration, and optionally provides mechanical agitation to increase surface contact of the tassels, anthers, immature flowers, or reproductive plant parts with the one or more sterilization agents to obtain sterilized tassels, anthers, immature flowers, or reproductive plant parts.

[0173] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: an automated sterilization assembly for sterilizing the selected tassels, anthers, immature flowers, or reproductive plant parts, wherein the automated sterilization assembly receives the tassels, anthers, immature flowers, or reproductive plant parts in one or more containers either singularly or in batches, dispenses and removes one or more sterilization agents from the one or more containers after a pre-set duration, and optionally provides mechanical agitation to increase surface contact of the tassels, anthers, immature flowers, or reproductive plant parts with the one or more sterilization agents. The automated sterilization assembly can be operably connected to an automated storage assembly and / or an automated selection and harvesting assembly.

[0174] Turning to FIG. 3 shows an example sterilization workflow for an automated sterilization assembly that receives harvested plant material (i.e., tassels, anthers, immature flowers, or reproductive plant parts) in containers singularly or in batches, seals the container and supplies, dispenses, and removes various sterilization agents. The tassels, anthers, immature flowers, or reproductive plant parts are soaked for a pre-determined amount of time. As plant material soaks in the container, the automated sterilization assembly mechanically agitates the container(s) to promote contact between the sterilization solution and various surfaces of the plant material. The automated sterilization assembly controls features such as acceleration, speed, direction,Docket No.: 214105-WO-SEC-l amplitude, and dwell time of agitation. Mechanical agitation includes rotation, linear movement, vibration, inversion, aeration, pressure changes, and / or vacuum. Upon completion of the sterilization cycle, the automated sterilization assembly can drain the sterilization solution and dispense sterile water or wash solution into the containers for rinsing the outer surfaces of the tassels. This wash step can be repeated one or more times until the tassels, anthers, immature flowers, or reproductive plant parts are substantially free of residue. Following the final wash, water can be drained from the container and the sterilized plant mater dries via air-drying. The automated sterilization assembly can provide additional mechanical agitation to facilitate drying. Upon completion of the drying cycle, plant material progresses to the microspore extraction step.

[0175] Additional methods and systems for sterilization of plant material such as tassels, anthers, immature flowers, and / or reproductive plant parts are disclosed in US provisional application number 63 / 923,789, which is incorporated by reference herein in its entirety.

[0176] Microspore Isolation

[0177] The methods and systems disclosed herein can utilize one or more automation steps for extraction and isolation of microspores from sterilized tassels, anthers, immature flowers, or reproductive plant parts. Microspores can be staged to an appropriate stage typically, between the uninucleate to binucleate stage, or around the first pollen mitosis (i.e., late uninucleate to early binucleate stage) for embryogenesis induction.

[0178] An automated extraction assembly can perform or more of the following:

[0179] (a) receive sterilized tassels, anthers, immature flowers, or reproductive plant parts in containers, either individually or in batches, in cups, tubes, or larger batch containers;

[0180] (b) apply mechanical force (e.g., blending, milling, mashing, juicing, slicing, or shredding) to release microspores from tassels, anthers, immature flowers, or reproductive plant parts;

[0181] (c) control speed, cycle number, duration, dwell time, number of cycles, ramp rate, tamping, and / or resting intervals of mechanical agitation to maximize microspore yield while minimizing cell damage;

[0182] (d) transfer containers between modules / processing stations of the automated extraction assembly;

[0183] (e) dispense and remove liquids and reagents for microspore isolation (e.g., isolation media, wash solutions, water);Docket No.: 214105-WO-SEC-l

[0184] (f) filter (e.g., single or multistage filtration; optionally across multiple sample tubes and / or cups) and centrifuge microspores to separate and concentrate microspores from non-microspore plant material;

[0185] (g) transfer microspores into suitable containers for microspore culture (e.g., plate microspores into liquid culture media);

[0186] (h) record microspore counts (yield) and / or provide microspore analysis (e.g., microspore developmental stage or viability);

[0187] (i) be configured for on-the-fly processing; and / or

[0188] (j) be operably connected to an automated sterilization assembly;

[0189] An automated extraction assembly can comprise or be equipped with one or more of the following:

[0190] (a) input / output modules (e.g., carousels or rotary devices for receiving and transferring containers);

[0191] (b) an agitation module, device, or mechanical components for mechanical disruption and extraction of microspores from sterilized plant material);

[0192] (c) one or more robotic arms or assemblies (e.g., robotic arms and / or manipulative tooling) to move containers with tassels, anthers, immature flowers, or reproductive plant parts undergoing processing;

[0193] (d) one or more dispensing and removal mechanisms such as liquid handlers, pumps, valves, and syringes for delivery and removal of isolation media, wash solutions, water, etc.;

[0194] (e) one or more filtration assemblies for single- or multistage filtration;

[0195] (f) one or more centrifuges for purifying and concentration extracted microspores;

[0196] (g) an extraction protocol for different plant materials (e.g., a tassel, anther, immature flower, or reproductive plant part) or plant species (e.g., maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola);

[0197] (h) sensors that monitor extraction parameters;

[0198] (i) one or more robotic arms or assemblies for transferring isolated microspores into suitable containers and dispensing liquid culture media for microspore culture; and / or

[0199] (j) resuspend concentrated microspores in liquid culture media.

[0200] A filtration assembly (or assemblies) can be part of or operably connected to an automated extraction assembly. The filtration assembly can perform one or more of the following:Docket No.: 214105-WO-SEC-l

[0201] (a) combine extracted microspores across multiple sample tubes and / or cups;

[0202] (b) concentrate microspores using tangential flow filtration in a continuous process to separate microspores based on size and / or density; and / or

[0203] (c) continuous flow centrifugation that can allow higher volumes of material to be processed in a shorter amount of time;

[0204] A filtration assembly (or assemblies) can comprise one or more of the following:

[0205] (a) a first stage course filter and a second stage fine filter;

[0206] (b) one or more automated centrifuges; and / or

[0207] (c) one or more robotic arms or assemblies (e.g., robotic arms and / or manipulative tooling) to move containers.

[0208] In an example of separating microspores non-microspore plant material after extraction, bulk material including microspores and non-microspore plant material is passed through a series of filter. A first large filter collects the bulk of the non-microspore plant material. A second filter collects the microspores and similarly sized plant particulates. Any microspores that may be retained in the bulk material can be rinsed through the filters with rinsing media. The speed of the filtering process can be improved by the application of vacuum pressure to draw the material and any rinsing media through the filters. Once the filtering is complete, the bulk material in the larger filter is discarded. The smaller filter can be manipulated to collect and transfer microspores to a test tube, along with isolation media or wash solution. The test tube can be centrifuged to pelletize the material, and the isolation media, along with residual non-microspore plant material, is removed.

[0209] A smaller quantity of isolation media or wash solution can be added, and the material is resuspended in the tube. The material can be transferred to a second tube containing densityspecific media. This tube is centrifuged again to separate the desired microspores from the denser undesired plant material. The layer of the density media containing the microspores can be transferred to new containers and the process repeated. This isolation and separation step can include aliquoting the material to multiple tubes to isolate the microspores more effectively. The number of suspension and centrifuge steps, as well as the number of aliquots, can be customized according to the plant type or plant species to achieve the desired quality and density of microspores. The microspores are finally transferred to a tube with more wash solution and concentrated via centrifugation, then transferred to the next part of the process. This process canDocket No.: 214105-WO-SEC-l be automated by means of liquid handlers to perform transfer steps, one or more transfer robot arms for loading and unloading tubes, and automated centrifuges. The process can include automation of various laboratory equipment such as SBS plates and petri dishes.

[0210] This example can further utilize ultrasonics to facilitate the separation of material in media. It is anticipated that ultrasonics will accelerate the natural flow of material in the density separation media. An automated extraction assembly for separating microspores from bulk material can include a reverse flow apparatus on the initial filter. Utilizing a centrifuge or ultrasonics, density separation media can be reverse flowed through the filter to perform an initial separation of the microspores from non-microspore plant material before being drawn off for the next step in the process.

[0211] In automating one or more steps of microspore isolation, the methods and systems of the present disclosure can increase microspore extraction and isolation efficiency and minimize microspore damage (i.e., enrich for useable microspores) as compared to manual microspore isolation.

[0212] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: extracting microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts with an automated extraction assembly that receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one or more containers or optionally in a larger batch-process container, dispenses and removes media from the containers or optionally the larger batch-process container, and separates the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by applying a mechanical force to the one or more containers; and separating extracted microspores from non-microspore plant material by fdtering microspore-containing media through one or more fdtering assemblies to collect the extracted microspores in one or more containers or pooled in a large-batch processing container and optionally the one or more containers or the large- batch processing container comprising the extracted microspores are subjected to a centrifugal force to further separate remaining non-microspore plant material and concentrating the extracted microspores;

[0213] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: an automated extraction assembly for extracting microspores from the sterilized tassels, anthers, immatureDocket No.: 214105-WO-SEC-l flowers, or reproductive plant parts, wherein the automated extraction assembly receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one more containers or optionally in a larger batch-process container, dispenses and removes media from the one or more containers or optionally the larger batch-process container, and extracts the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by application of a mechanical force to the one or more containers; and one or more filtering assemblies that separate the extracted microspores from non-microspore plant material by filtering microspore-containing media to collect the separated microspores in one or more containers or pooled in a larger batchprocessing container and optionally the one or more containers or the larger batch-processing container comprising the extracted microspores are subjected to a centrifugal force to further separate remaining non-microspore plant material and concentrate the extracted microspores.

[0214] Microspore Culture

[0215] After isolation, microspores are cultured under conditions that promote or induce embryogenesis such that single-cell microspores progress from single-cell microspores to multicellular structures, from multicellular structures to embryo-like structures, and from embryolike structures to macroscopic structures, and chromosome doubling resulting in diploidized multicellular structures, diploidized embryo-like structures, and / or diploidized macroscopic structures.

[0216] Automated microspore culture can comprise: (a) distributing microspores (e.g., separated microspores are dispensed into multiple containers at a target concentration or density); (2) providing microspores with culture media that promotes embryogenesis induction; (3) optionally providing microspores with genetic, biochemical, or chemical components (“cargo”) to introduce site-specific genomic changes, transgenes, morphogenic or embryogenesis-inducing polypeptides, and / or chromosome doubling agents; (4) culturing microspores under conditions to generate microspore-derived multicellular structures, embryo-like structures, and macroscopic structures; (5) providing controlled and / or programmable incubation conditions (e.g., temperature, light, humidity, media changes); (6) monitoring, sampling, and selection of microspores, multicellular structures, embryo-like structures, and macroscopic structures using a computer vision processing system or an image processing system for real-time assessment of culture progress; and / or (7) sorting multicellular structures, embryo-like structures, and / or macroscopic structures for further culture or regeneration.Docket No.: 214105-WO-SEC-l

[0217] Microspore culture and incubation can be carried out by an automated incubation assembly, which can perform one or more of the following:

[0218] (a) regulate microspore culture conditions including temperature, moisture / humidity, light, nutrient management, and / or oxygen / gas exchange;

[0219] (b) dispense, remove, and exchange culture media and reagents;

[0220] (c) introduce a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex to microspores, multicellular structures, embryo-like structures, and / or macroscopic structures;

[0221] (d) provide a chromosome doubling agent to the culture media;

[0222] (e) image and monitor growth and development of microspores, multicellular structures, embryo-like structures, and macroscopic structures;

[0223] (f) sample, image, and sort multicellular structures, embryo-like structures, and macroscopic structures;

[0224] (g) high-throughput processing and sampling of multicellular structures, embryo-like structures, and macroscopic structures;

[0225] (h) receive microspores from an automated extraction assembly and / or filtration assembly;

[0226] (i) provide cargo for one or more site-specific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing;

[0227] (j) high throughput bulking of one or more populations of microspore-derived multicellular structures, embryo-like structures, or macroscopic structures.

[0228] An automated incubation assembly can comprise or be equipped with one or more of the following:

[0229] (a) one or more incubation chambers;

[0230] (b) one or more robotic arms, handlers, or assemblies (e.g., robotic arms and / or manipulative tooling) to move containers;

[0231] (c) one or more dispensing and removal mechanisms such as liquid handlers, pumps, valves, and syringes for delivery and removal of culture media;

[0232] (d) one or more imaging modalities;Docket No.: 214105-WO-SEC-l

[0233] (e) a vision guided robotic arm for selecting and transferring microspores, multicellular structures, embryo-like structures, and macroscopic structures;

[0234] (f) culture protocols for different plant species (e.g., maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola); and / or

[0235] (g) sensors that monitor culture conditions such as temperature, moisture / humidity, light, nutrient management, and / or oxygen / gas exchange.

[0236] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: providing separated microspores with a culture media that promotes microspore embryogenesis induction such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores are distributed in a plurality of containers, the plurality of containers capable of being handled by a robotic handler for a high-throughput, continuous or a semi-continuous workflow, optionally wherein the microspores are dispensed into the plurality of containers at a target concentration or density, and culturing the microspores, multicellular structures, embryo-like structures, and / or macroscopic structures in an automated incubation assembly.

[0237] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: optionally providing cargo to the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures for introducing (i) one or more site-specific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing to the microspores, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex, and culturing the microspores, multicellular structures, embryo-like structures, and / or macroscopic structures in an automated incubation assembly.

[0238] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: optionally providing the culture media with a chromosome doubling agent, and culturing the microspores, multicellularDocket No.: 214105-WO-SEC-l structures, embryo-like structures, and / or macroscopic structures in an automated incubation assembly.

[0239] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: optionally providing the culture media with a chromosome doubling agent, and culturing the microspores, multicellular structures, embryo-like structures, and / or macroscopic structures in an automated incubation assembly.

[0240] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: sorting the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures with an automated sorting assembly comprising a computer vision processing system or image processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the computer vision processing system or image processing system.

[0241] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures with an automated selection assembly, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model.

[0242] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: growing the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures in a growing station adapted to work with an automated handling and sampling system that samples and tracks the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidizedDocket No.: 214105-WO-SEC-l macroscopic structures for genetic determination, and further culturing the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to obtain microspore-derived doubled haploid plantlets.

[0243] In an example, a high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: transferring the microspore-derived doubled haploid plantlets to growth medium (i.e., in vitro culture medium or soil) for regeneration of doubled haploid crop plants.

[0244] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: a robotic handler that provides the separated microspores with a culture media, wherein the robotic handler distributes the separated microspores in a plurality of containers for a high-throughput continuous or a semi-continuous workflow, and an automated incubation assembly that cultures the microspores such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures are optionally provided cargo for introducing (i) one or more sitespecific genomic changes, (ii) one or more transgenes for integration into the genomic DNA, (iii) a morphogenic developmental polypeptide or a polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex, and wherein the automated incubation assembly optionally provides a chromosome doubling agent to the culture media.

[0245] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: an automated sorting assembly that sorts the microspores, the multicellular structures, the embryolike structures, and / or the macroscopic structures, wherein the automated sorting assembly comprises a second computer vision processing system or image processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellularDocket No.: 214105-WO-SEC-l structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the second computer vision processing system or image processing system.

[0246] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: an optional artificial intelligence model that selects the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures predicted to regenerate into fertile that are more likely to germinate into fertile plantlets, wherein the artificial intelligence model has been trained on a dataset comprising microspores, multicellular structures, embryo-like structures, macroscopic structures, and / or plantlets that had positive fertility outcomes (e.g., flowered or set seed).

[0247] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: an automated selection assembly that selects diploidized multicellular structures, embryo-like structures, and / or macroscopic structures for a high-throughput continuous or a semi-continuous workflow, optionally wherein selecting a diploidized multicellular structure, a diploidized embryolike structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model.

[0248] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: a growing station that grows the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures.

[0249] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: an automated handling and sampling system adapted to work with the growing station, wherein the automated handling and sampling system samples and tracks the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for geneticDocket No.: 214105-WO-SEC-l determination, and further cultures the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to obtain microspore- derived doubled haploid plantlets.

[0250] In an example, an end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: a robotic arm that transfers the microspore-derived doubled haploid plantlets to growth medium (i.e., in vitro culture medium or soil) for regeneration of doubled haploid crop plants.

[0251] In an example of the methods described herein, isolated microspores are cultured using a mesh insert having a pore size that is smaller than the size of the microspores. Turning to FIG. 13A, microspores are plated on mesh inserts and the inserts placed in a petri dish with liquid induction media. Media can be refreshed one or more times by lifting the insert, with the microspores being retained in the mesh, from the used petri dish and reinserting the insert into a new petri dish with fresh induction media (FIG. 13B). Microspores are then cultured under conditions suitable for macroscopic structure generation (FIG. 13C). To germinate macroscopic structures, the mesh is reversed and the macroscopic structures deposited onto a solid media plate by gently shaking the mesh (FIG. 13D). Macroscopic structure-derived plantlets then regenerate on the solid media (FIG. 13E).

[0252] The methods provided herein can utilize one or more treatments to promote, improve, or increase microspore embryogenesis induction (for example, by treatment with an embryogenesis inducting agent) and / or chromosome doubling. Further, during microspore culture, cargo can be delivered for introducing one or more site-specific genomic changes and / or one or more transgenes for integration into the genomic DNA.

[0253] Microspores can be cultured in the presence of polycomb repressive complex 2 (PRC2) inhibitors and / or ethylene inhibitors. Examples of ethylene inhibitors include, but are not limited to, ethylene biosynthesis inhibitors (e.g., aminoethoxyvinylglycine) and ethylene signal perception inhibitors (e.g., silver nitrate).

[0254] Microspores can also be contacted with small molecule kinase inhibitors or other embryogenesis inducing agents that promote cellular reprogramming from an initial haploid gametic cell fate to an embryogenic cell fate. Such kinase inhibitors include N-[(2R)-2,3- dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide, anthra(l,9-cd)pyrazol- 6(2H)-one, 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)lH-imidazole, and N-Docket No.: 214105-WO-SEC-l benzyl-2-(pyrimidin-4-ylamino)-l,3-thiazole-4-carboxamide (see US11447786B2; incorporated by reference).

[0255] Sorting of Microspores and Microspore-derived Structures

[0256] The methods and systems disclosed herein can utilize one or more automation steps for microspore sorting, that is, differentiating and sorting of microspores, multicellular structures, embryo-like structures, macroscopic structures, diploidized multicellular structures, diploidized embryo-like structures, and diploidized macroscopic structures.

[0257] Sorting of microspores, multicellular structures, embryo-like structures, macroscopic structures, diploidized multicellular structures, diploidized embryo-like structures, and diploidized macroscopic structures can be carried out by an automated sorting assembly, which can perform one or more of the following:

[0258] (a) non-destructive and label -free sorting utilizing el ectrical / di electric properties, cell density, cell size, RGB imaging, hyperspectral imaging, or cytometry;

[0259] (b) sort by density using maltose cushions;

[0260] (c) sort by di electrophoretic (DEP) to determine size, viability, and ploidy

[0261] (d) sort by light scattering pattern and / or bright field images via microfluidic devices or culture vessels. Methods for microspore sorting are also disclosed in US20230191427, which is incorporated by reference in its entirety; and / or

[0262] (e) be operably connected to an automated incubation assembly to receive and transfer microspores, multicellular structures, embryo-like structures, macroscopic structures, diploidized multicellular structures, diploidized embryo-like structures, and / or diploidized macroscopic structures; and / or

[0263] (f) enrich for multicellular structures, embryo-like structures, macroscopic structures, diploidized multicellular structures, diploidized embryo-like structures, and / or diploidized macroscopic structures.

[0264] An automated sorting assembly can comprise or be equipped with one or more of the following:

[0265] (a) one or more robotic arms, handlers, or assemblies (e g., robotic arms and / or manipulative tooling) to move samples;

[0266] (b) one or more dispensing and removal mechanisms such as liquid handlers, pumps, valves, and syringes for delivery and removal of culture media;Docket No.: 214105-WO-SEC-l

[0267] (c) one or more imaging modalities;

[0268] (d) a vision guided robotic arm for a computer vision processing system or image processing system comprising guided robotic arm for selecting and transferring microspores, multicellular structures, embryo-like structures, and macroscopic structures; and / or

[0269] (e) sorting protocols for different plant species (e g., maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola).

[0270] In an example, an end-to-end, high-throughput, automated method for generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: sorting the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures with an automated sorting assembly comprising a vision processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the vision processing system and selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures with an automated selection assembly, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model

[0271] In an example, a high-throughput, automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants further comprises: (a) an automated sorting assembly that sorts the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly comprises a second vision processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the second vision processing system; (b) an optional artificial intelligence model that selects the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures predicted to regenerate into fertile that are more likelyDocket No.: 214105-WO-SEC-l to germinate into fertile plantlets, wherein the artificial intelligence model has been trained on a dataset comprising microspores, multicellular structures, embryo-like structures, macroscopic structures, and / or plantlets that had positive fertility outcomes (e.g., flowered or set seed); (c) an automated selection assembly that selects diploidized multicellular structures, embryo-like structures, and / or macroscopic structures for a high-throughput continuous or a semi -continuous workflow, optionally wherein selecting a diploidized multicellular structure, a diploidized embryolike structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model; (d) a growing station that grows the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures; (e) an automated handling and sampling system adapted to work with the growing station, wherein the automated handling and sampling system samples and tracks the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for genetic determination, and further cultures the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to obtain microspore-derived doubled haploid plantlets; and (f) a robotic arm that transfers the microspore-derived doubled haploid plantlets to growth medium (i.e., in vitro culture medium or soil) for regeneration of doubled haploid crop plants.

[0272] Chromosome Doubling of Microspore-derived Haploid Embryos

[0273] The methods and systems disclosed herein can optionally utilize one or more automation steps for application of a chromosome doubling agent to microspores, multicellular structures, embryo-like structures, and / or macroscopic structures, resulting in doubling (or diploidization) of the In haploid chromosome number to 2n (diploid). Diploidization of the haploid chromosome is necessary for producing DI seed generated from DO seedlings.

[0274] When an exogenous chemical chromosome doubling agent is used, the doubling agent can be added to induction media, culture media, or other suitable media. Chemical chromosome doubling agents are shown in Table 1.Docket No.: 214105-WO-SEC-l

[0275] In an example, a high-throughput, automated method generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises optionally providing culture media with a chromosome doubling agent and selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures with an automated selection assembly, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model.

[0276] In an example, a high-throughput, automated system generating microspore-derived doubled haploid structures, plantlets, and crop plants comprises: an automated incubation assembly that optionally provides a chromosome doubling agent to culture media and an automated selection assembly that selects diploidized multicellular structures, embryo-like structures, and / or macroscopic structures for a high-throughput continuous or a semi-continuous workflow, optionally wherein selecting a diploidized multicellular structure, a diploidized embryolike structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model.Table 1: Chemical chromosome doubling agentsDocket No.: 214105-WO-SEC-lDocket No.: 214105-WO-SEC-l

[0277] Ploidy Determination of Microspore-derived Structures

[0278] The methods and systems disclosed herein can utilize one or more automation steps for ploidy determination of microspore-derived multicellular structures, embryo-like structures, and / or macroscopic structures following chromosome doubling. For example, isolated microspores can be cultured in liquid culture media with a chromosome doubling agent such that embryogenic microspore-derived multicellular structures, microspore-derived embryo-like structures, and / or microspore-derived macroscopic structures undergo diploidization resulting in doubled haploid structures.

[0279] Alternatively or in addition to exogenous treatment with a chromosome doubling agent, microspores and microspore-derived multicellular structures, microspore-derived embryo-likeDocket No.: 214105-WO-SEC-l structures, and / or microspore-derived macroscopic structures can undergo chromosome doubling via a genetic chromosome doubling polypeptide or polynucleotide encoding the same.

[0280] In either chromosome doubling approach, multicellular structures, embryo-like structures, and / or macroscopic structures that are contacted with a chromosome doubling agent and / or provided a genetic chromosome doubling polypeptide or polynucleotide encoding the same can be sampled for ploidy determination. Non-diploidized structures can be removed to prevent nondoubled haploid plantlets from advancing to pots or soil for plant regeneration.

[0281] After microspores and microspore-derived structures are contacted with an exogenous doubling agent and / or provided a genetic chromosome doubling polypeptide or polynucleotide encoding the same, they can undergoing sampling and singulation from batch culture. Typically, once chromosome doubling-treated macroscopic structures reach a large enough size, begin to germinate, or regenerate a plantlet, they are singulated. However, smaller structures such as multicellular structures and / or embryo-like structures can also be singulated from batch culture for continued growth and plantlet regeneration.

[0282] Multicellular structures, embryo-like structures, and / or macroscopic structures that are contacted with a chromosome doubling agent in an automated incubation assembly and / or provided a genetic chromosome doubling polypeptide or polynucleotide encoding the same can be sampled for ploidy determination can be sampled by an automated selection assembly that selects diploidized multicellular structures, embryo-like structures, and / or macroscopic structures.

[0283] In an example, ploidy determination comprises non-destructive high throughput flow cytometry of microspore-derived macroscopic structures or regenerated plantlets. Macroscopic structures and / or plantlets can undergo flow cytometry analysis to determine the ploidy level of each structure or plantlet. Non-diploidized macroscopic structures or plantlets are discarded, and only confirmed doubled haploid macroscopic structures or plantlets proceed to potting and germination, decreasing the attrition rate of doubled haploid production.

[0284] Automated methods and systems for generating, sampling, and selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures comprise: (a) culturing the microspores, multicellular structures, embryo-like structures, and / or macroscopic structures in an automated incubation assembly; (b) sorting the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures with an automated sorting assembly comprising a computer vision processing system or image processing system that imagesDocket No.: 214105-WO-SEC-l the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the computer vision processing system or image processing system; (c) selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures with an automated selection assembly, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model; (d) growing the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures in a growing station adapted to work with an automated handling and sampling system that samples and tracks the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for genetic determination, and further culturing the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to generate microspore-derived doubled haploid plantlets; and (e) transferring the microspore-derived doubled haploid plantlets to growth medium for regeneration of doubled haploid crop plants.

[0285] Microspore culture, sorting, and selection can be carried out by an automated incubation assembly, an automated sorting assembly, and / or an automated selection assembly, which can perform one or more of the following:

[0286] (a) regulate culture conditions including temperature, moisture / humidity, light, nutrient management, and / or oxygen / gas exchange for microspores, multicellular structures, embryo-like structure, macroscopic structures, plantlets, diploidized multicellular structures, diploidized embryo-like structure, diploidized macroscopic structures, and / or doubled haploid plantlets;

[0287] (b) dispense, remove, and exchange culture media and reagents;

[0288] (c) introduce a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex to microspores, multicellular structures, embryo-like structures, and / or macroscopic structures;Docket No.: 214105-WO-SEC-l

[0289] (d) provide a chromosome doubling agent to the culture media;

[0290] (e) image and monitor growth and development of microspores, multicellular structures, embryo-like structure, macroscopic structures, plantlets, diploidized multicellular structures, diploidized embryo-like structure, diploidized macroscopic structures, and / or doubled haploid plantlets;

[0291] (f) sample, image, and sort multicellular structures, embryo-like structure, macroscopic structures, plantlets, diploidized multicellular structures, diploidized embryo-like structure, diploidized macroscopic structures, and / or doubled haploid plantlets;

[0292] (g) high-throughput processing and sampling of multicellular structures, embryo-like structure, macroscopic structures, plantlets, diploidized multicellular structures, diploidized embryo-like structure, diploidized macroscopic structures, and / or doubled haploid plantlets;

[0293] (h) receive microspores from an automated extraction assembly and / or fdtration assembly;

[0294] (i) provide cargo for one or more site-specific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing;

[0295] (j) perform ploidy analysis of multicellular structures, embryo-like structures, and / or macroscopic structures that were contacted with a chromosome doubling agent and / or provided a genetic chromosome doubling polypeptide or polynucleotide encoding the same.

[0296] An automated incubation assembly, an automated sorting assembly, and / or an automated selection assembly can comprise or be equipped with one or more of the following:

[0297] (a) one or more incubation chambers;

[0298] (b) one or more robotic arms, handlers, or assemblies (e.g., robotic arms and / or manipulative tooling) to move containers;

[0299] (c) one or more dispensing and removal mechanisms such as liquid handlers, pumps, valves, and syringes for delivery and removal of culture media;

[0300] (d) one or more imaging modalities;

[0301] (e) a vision guided robotic arm for selecting and transferring multicellular structures, embryo-like structure, macroscopic structures, plantlets, diploidized multicellular structures,Docket No.: 214105-WO-SEC-l diploidized embryo-like structure, diploidized macroscopic structures, and / or doubled haploid plantlets;

[0302] (f) culture protocols for different plant species (e.g., maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola);

[0303] (g) sensors that monitor culture conditions such as temperature, moisture / humidity, light, nutrient management, and / or oxygen / gas exchange; and / or

[0304] (h) a flow cytometer or other means for ploidy analysis of multicellular structures, embryolike structures, and / or macroscopic structures that were contacted with a chromosome doubling agent and / or provided a genetic chromosome doubling polypeptide or polynucleotide encoding the same.

[0305] Sampling and Selection of Microspores, Microspore-derived Structures, and Regenerated Plantlets

[0306] The methods and systems disclosed herein can utilize one or more automation steps for sampling of microspores and microspore-derived structures (i.e., microspore-derived multicellular structures (MCS), microspore-derived embryo-like structures (ELS), and microspore-derived macroscopic structures). More specifically, microspore sampling comprises non-destructive sampling of microspores and microspore-derived structures. Sampling of microspores and / or microspore-derived structures can occur at various points in the process of creating doubled haploid structures and regenerating plantlets therefrom. Automated steps for sampling include imaging characterization and enrichment prediction algorithms, selection and transfer of sampled structures from bulk culture to individual culture, plate handling for washing and sampling of microspores, liquid handling or pneumatic transfer for transfer of microspores from individual wells to solid media.

[0307] Example methods for genotyping plant components are disclosed in US20160060713 and US20170107507, each of which is incorporated by reference herein in its entirety.

[0308] Example methods for microspore automated imaging, automated cell tracking, and predictive modeling are disclosed in US20230343116, which is incorporated by reference herein in its entirety.

[0309] The methods and systems described herein can sort microspore-derived multicellular structures with an automated sorting assembly comprising a computer vision processing system or image processing system that images the microspore-derived structures, wherein the automatedDocket No.: 214105-WO-SEC-l sorting assembly selects and transfers microspore-derived multicellular structures for culture based on images from the computer vision processing system or image processing system.

[0310] In a first example, microspores, microspore-derived embryo-like structures, or microspore- derived multicellular structures are collected and sampled from bulk culture by early RNA / protein expression profiling. Single cell sequencing or profiling can also be done. Selected cells are then returned to culture. Automated steps include tissue sampling, imaging characterization, liquid handling, and selection and transfer of microspores and microspore-derived structures.

[0311] In a second example, microspore-derived macroscopic structures are singulated from bulk culture, washed, and suspended in sampling buffer and agitated to collect shed cell material. Automated steps for sampling include tissue sample, imaging characterization, selection and transfer of sampled structures from bulk culture to individual culture, plate handling for washing and sampling of microspores, liquid handling or pneumatic transfer for transfer of microspores from individual wells to solid media.

[0312] In a third example, microspore-derived macroscopic structures or seedlings are singulated into a 96-well format (rather than a petri dish) when moved to solid media. Automated steps include tissue sampling and transferring regenerated plantlets to soil.

[0313] Sampling of microspores and microspore-derived structures can involve image-based characterization. Automated sampling can occur at various stages of the doubled haploid production process. For example, automated sampling can occur during culture of microspores and / or microspore-derived structures, during sorting and / or selection of microspore-derived structures, during culture and growth of selected microspore-derived structures, and / or during transfer / regeneration of microspore-derived macroscopic structures.

[0314] In a first image-based method for sampling, sorting, and / or selecting, tassel image segmentation, morphometry, and color / spectral analysis to determine microspore quality, potential for development, and developmental stage can be used. As shown in FIG. 4A, imaging can be carried out on tassel donor plants or harvested tassels (imaging target stage indicated by dashed lines). Automated steps can include tassel image acquisition, programming image acquisition software, and processing tassel images to determine microspore health and developmental stage.

[0315] In a second image-based method for sampling, sorting, and / or selecting, microspore can be used for fluorescent label-free measurement of the count, viability, and average values of key morphological properties of microspores (FIG. 4B; imaging target stage indicated by dashedDocket No.: 214105-WO-SEC-l lines). In this method, microspores can be isolated and diluted, imaged by brightfield microscopy, and the resulting images processed to determine microspore developmental stage, count, and / or viability. Automated steps can include microspore image acquisition, programming image acquisition software, and processing microspore images to determine count and viability (e.g., binarization and segmentation, morphology and intensity analysis, viability analysis).

[0316] In a third image-based method for sampling, sorting, and / or selecting, an algorithm for label-free nuclei segmentation and morphometry can be used for label -free microspore ploidy determination (FIG. 4B; imaging target stage indicated by dashed lines). In this method, microspore are imaged by z-stack, multi-position brightfield microscopy and the resulting images processed to determine microspore ploidy and chromosome doubling. Automated steps can include microspore image acquisition, programming image acquisition software, and processing microspore images to determine ploidy (e.g., microspore segmentation, viability analysis, Z-stack edge detection and filling, identification and segmentation of nuclei, and signal processing to optimize nuclei detection). In addition to determining ploidy and chromosome doubling, this method enables deriving correlations between microspore developmental stage and potency for embryogenesis induction.

[0317] In a fourth image-based method for sampling, sorting, and / or selecting, microspore segmentation is used for cargo delivery analysis of microspores (FIG. 4B; imaging target stage indicated by dashed lines). In one example, automated measurement of the relative concentration of cargo in individual microspores and germination pore can be used to assess cargo delivery using fluorescence-based delivery assays. In another example, automated bright field / phase contrast screening of microspore, germination pore, and cell wall can be used to detect defects caused by physical or digestion-based delivery assays. In this method, microspores are imaged, simultaneously or sequentially, by brightfield and wide-field fluorescence / confocal microscopy (automated z-stack multi-position image sequence acquisition) and the resulting images processed to determine whether or not microspores contain cargo. Automated steps can include microspore image acquisition, programming image acquisition software, and processing microspore images to determine cargo presence (e.g., segmentation, z-stack / 3D edge detection, pore identification and 3D segmentation, and normalization steps and intensity analysis).

[0318] In a fifth image-based method for sampling, sorting, and / or selecting, colorimetric or morphometry of microspore structural development from microspore to microspore-derivedDocket No.: 214105-WO-SEC-l multicellular structure (MCS) to microspore-derived embryo-like structure (ELS) to microspore- derived macroscopic structures is used for sampling and selecting using z-stack brightfield microscopy (FIG. 4C; imaging target stage indicated by dashed lines). In this method, following microspore isolation, embryogenesis induction, and plating / embedding, microspores / MCS / ELS are imaged by are imaged by wide-field fluorescence / confocal microscopy (automated z-stack multi-position image sequence acquisition) at multiple time points, and the resulting microspores / MCS / ELS z-stack / 3D images processed to determine morphometry at different time points. Automated steps can include microspore / MCS / ELS image acquisition, programming image acquisition software, and processing microspore / MCS / ELS images to determine morphometry.

[0319] In a sixth image-based method for sampling, sorting, and / or selecting, analysis of microspore-derived macroscopic structures (FIG. 4D; imaging target stage indicated by dashed lines) is used for sampling, sorting, and / or selecting. More specifically, images of microspore- derived macroscopic structures are segmented and analyzed to determine count, color matrix, and morphology of macroscopic structures. In this method, following microspore isolation, embryogenesis induction, and plating / embedding, upright color images of macroscopic structures are captured with a camera and image segmentation, morphometry, and RGB or hyperspectral image analysis is carried out. Automated steps can include microspore-derived macroscopic structure image acquisition, programming image acquisition software, and processing microspore- derived macroscopic structure images to perform segmentation, morphometry, and color analysis.

[0320] In a seventh image-based method for sampling, sorting, and / or selecting, regenerated microspore-derived doubled haploid plantlets or seedlings are imaged to predict flowering and seed set (FIG. 4E). This allows for early removal of non-productive plantlets or seedlings. In this method, seedlings are imaged after treatment with a chromosome doubling agent (or after diploidization via genetic means) and negative predictive seedlings are removed from the production pipeline. Automated steps include image capture and analysis and robotic arms and / or manipulative tooling for plantlet or seedling removal.

[0321] Microspore sampling can also include automated selection methods following image analysis.

[0322] In a first selection method, robotic handling can be used to collect microspore-derived multicellular structures and microspore-derived embryo-like structures in liquid medium based onDocket No.: 214105-WO-SEC-l analyzed images (FIG. 5A; selection target stage indicated by dashed lines). Highly specific selection of desired structures early in embryogenesis can improve paternal DH-based production efficiency. In this method, microspore-derived multicellular structures and / or microspore-derived embryo-like structures are imaged, the images processed and analyzed, and the desired, selected structures collected via robotic handling. Automated steps can include image acquisition, programming image acquisition software, processing images, programming and control of robotic handling, and selection of microspore-derived structures.

[0323] In a second selection method, a robotic system can be used to collect microspore-derived macroscopic structures in solid medium based on analyzed images (FIG. 5B; selection target stage indicated by dashed lines). In this method, macroscopic structures are imaged, the images processed and analyzed, and the desired, selected structures collected via the robotic system. Automated steps can include image acquisition, programming image acquisition software, processing images, programming and control of robotic system, and selection of microspore- derived macroscopic structures.

[0324] Automated methods for generating microspore-derived doubled haploids can include sorting microspore-derived embryo-like multicellular structures with an automated sorting assembly comprising a second computer vision processing system or image processing system that images the microspore-derived structures, wherein the automated sorting assembly selects and transfers microspore-derived multicellular structures and embryo-like structures for culture based on images from the second computer vision processing system or image processing system. Optionally, the automated sorting assembly can select microspore-derived structures that are more likely to generate into fertile plants based on a microspore-regeneration artificial intelligence model that has been trained on a training dataset comprising microspore-derived plants that produced viable seeds.

[0325] Automated methods for generating microspore-derived doubled haploids can include growing selected microspore-derived structures in a growing station adapted to work with an automated handling and an automated sampling system that samples and tracks the microspore- derived structures for genetic determination, and further culturing to develop doubled haploid structures.

[0326] Automated methods for generating microspore-derived doubled haploids can include macroscopic structure selection using an automated sorting assembly comprising a computerDocket No.: 214105-WO-SEC-l vision processing system or image processing system and a robotic arm for identifying, selecting, and transferring macroscopic structures from liquid to solid media and / or from solid media to soil. The robotic arm can be equipped with a jaw gripper, vacuum tube, vacuum cup, spatula, scoop, loop, forked gripper, mesh screen (for bulk transfers), tweezers, forceps, and / or needles for macroscopic structure transfer.

[0327] Image-based characterization, selection, and / or transfer of microspores and microspore- derived structures can utilize one or more imagers / imaging systems and / or live cell incubatorimaging systems that can image microspores and microspore-derived structures in a plate, petri dish, or other bulk container.

[0328] Methods and systems for microspore imaging, analysis, culturing, and / or sorting are disclosed in US provisional application number 63 / 810,407, which is incorporated by reference herein in its entirety.

[0329] Methods and systems for selecting chromosome doubling-treated microspore-derived structures and plantlets based on predicted fertility are disclosed in US provisional application number 63 / 921,768, which is incorporated by reference herein in its entirety.

[0330] Methods and systems for selecting chromosome doubling-treated microspore-derived structures and plantlets based on predicted fertility are disclosed in US provisional application number 63 / 923,996, which is incorporated by reference herein in its entirety.

[0331] Methods and systems for selecting tassels, anthers, immature flowers, and / or reproductive plant parts based on predicted microspore and tassel / anther / immature flower / reproductive plant part properties are disclosed in US provisional application number 63 / 921,866, which is incorporated by reference herein in its entirety.

[0332] Instead of delivering cargo after microspore-isolation, the automated systems and methods described herein can deliver cargo to microspores of intact tassels, anthers, immature flowers, or reproductive plant parts. Methods and systems for anther-mediated delivery of cargo to microspores are disclosed in US provisional application number 63 / 923,835, which is incorporated by reference herein in its entirety.

[0333] Cargo Delivery

[0334] The methods and systems disclosed herein can utilize one or more automation steps for biomolecule or cargo delivery to microspores. As used herein “cargo” refers to proteins / polypeptides, DNA (e.g., expression cassettes), RNA, small molecules, and / orDocket No.: 214105-WO-SEC-l ribonucleoprotein complexes to promote microspore embryogenesis, chromosome doubling, and optionally genome-editing. Cargo can also refer to transgenes for insertion.

[0335] In the methods and systems of the present disclosure, cargo can be delivered to microspores and microspore-derived structures by bacterial-mediated transformation (e.g., Agrobacterium - mediated transformation), biolistic / particle-mediated delivery, PEG-mediated transformation, electroporation, microinjection, ribonucleoprotein delivery, cell penetrating peptides, nanotechnology-based methods including nanocarriers (e.g., carbon nanotubes, mesoporous silica nanoparticles, and lipid-based carriers), DNA nanostructures (i.e., engineered DNA frameworks that can carry and release nucleic acid molecules), magnetofection, and single-cell technologies including microfluidics, laser-assisted delivery, single-cell electroporation, and nanoneedles / nanopipettes. Cargo can be delivered by targeting the cell wall of microspores and microspore-derived structures. Cargo can be delivered by different methods and at different stages. For example, for early-stage microspore delivery (e.g., tetrad stage, uni-nucleate stage, or binucleate stage) to initiate transient expression, followed by mechanical methods like ultrasound or magnetofection for cargo delivery at later stages (e.g., embryo-like structures).

[0336] In a first example of cargo delivery, immature embryos having an Fl, or any subsequent generation thereof, genome can be transformed with one or more expression cassettes expressing morphogenic developmental polypeptide(s) for microspore embryogenesis induction, a genetic chromosome doubling polypeptide, a transgene, and genome-editing components, for example a Cas polypeptide and guide polynucleotide. Immature embryos are isolated from ears and placed in Agrobacterium infection medium to be transformed with cargo. Following transformation, embryos are cultured, grown into plants, and microspores are isolated and cultured from Fl tassel donors. Robotic arms, automated liquid handlers, and / or manipulative tooling can be used for media changes during microspore culture, transfer of microspore-derived structures to solid medium, and / or transfer of microspore-derived regenerated plantlets to a greenhouse.

[0337] In a second example of cargo delivery, microspores can be isolated and transfected with polynucleotides encoding morphogenic developmental polypeptide(s) for microspore embryogenesis induction, a genetic chromosome doubling polypeptide, a transgene, and / or genome-editing components. Isolated microspores are cultured in induction medium from a singlecell microspore to a microspore-derived multicellular structure or a microspore-derived embryolike structure. Transfection reagents and polynucleotide cargo is added to the culture medium ofDocket No.: 214105-WO-SEC-l isolated microspores. Microspores are then incubated until development of multicellular structures (MCS) and embryo-like structures (ECS). Transfection-mediated cargo delivery to microspores, MCS, and ELS is shown in FIG. 2A. Transfection reagents can be chemical (e.g., cell-penetrating peptides) or physical (e.g., nano-needle delivery). Automated liquid handlers can be used for addition and removal of transfection reagents and polynucleotide cargo. A microfluidics system can be used for automated nano-needle delivery. Robotic arms, automated liquid handlers, and / or manipulative tooling can be used for media changes during microspore culture, transfer of microspores to solid medium, and / or transfer of microspore-derived regenerated plantlets to a greenhouse.

[0338] In a third example of cargo delivery, microspore-derived embryo-like structures, macroscopic structures, and regenerated plantlets can be transformed with one or more expression cassettes encoding morphogenic developmental polypeptide(s) for microspore embryogenesis induction, a genetic chromosome doubling polypeptide, a transgene, and / or genome-editing components. Isolated microspores are cultured in an induction medium from a single-cell microspore to a microspore-derived embryo-like structure and macroscopic structure. These microspore-derived structures are subsequently placed in Agrobacterium infection medium to be transformed with plasmids encoding morphogenic developmental polypeptide(s) for microspore embryogenesis induction, a genetic chromosome doubling polypeptide, a transgene, and / or genome-editing components (FIG. 2B). Robotic arms, automated liquid handlers, and / or manipulative tooling can be used for media changes during microspore culture, transfer of microspores to solid medium, and / or transfer of microspore-derived regenerated plantlets to a greenhouse.

[0339] Methods and systems for delivering cargo to microspores, multicellular microspores, embryo-like structures, macroscopic structures, diploidized multicellular structures, diploidized embryo-like structures, and / or diploidized macroscopic structures are disclosed in US provisional application number 63 / 921,881, which is incorporated by reference herein in its entirety.

[0340] Cargo

[0341] The methods and systems disclosed herein can utilize one or more automation steps for cargo delivery to microspores, including small molecules, polynucleotides, polypeptides, and / or expression cassettes to promote microspore embryogenesis, chromosome doubling, and optionally genome editing. Cargo can also refer to transgenes for insertion. Examples of small molecules,Docket No.: 214105-WO-SEC-l polynucleotides, polypeptides, and / or expression cassettes useful for the methods and systems disclosed herein are described in US20220240467, US20220154203, US11447786, and PCT / US24 / 46207, all of which are incorporated by reference herein in their entireties.

[0342] A. Morphogenic Developmental Polypeptides

[0343] The methods and systems provided herein utilize one or more morphogenic developmental genes or polypeptides that regulate plant metabolism, organ development, stem cell development, cell growth stimulation, organogenesis, regeneration, somatic embryogenesis initiation and maturation, apical and shoot meristem initiation and development, and / or shoot formation. As used herein, “morphogenic developmental polypeptide”, “morphogenic polypeptide”, and “developmental polypeptide” are used interchangeably and refer to polypeptides that when ectopically expressed promote, stimulate, or induce embryogenesis and / or organogenesis. As used herein, “morphogenic developmental gene”, “morphogenic gene”, “developmental gene”, “morphogenic developmental polynucleotide”, “morphogenic polynucleotide”, and “developmental polynucleotide” are used interchangeably and refer to genes or polynucleotides that when ectopically expressed promote, stimulate, or induce embryogenesis and / or organogenesis to derive an embryogenic or organogenic plant structure or cell mass. This response can occur in the cell or cells in which the developmental gene is expressed or in a neighboring cell.

[0344] One class of morphogenic developmental genes includes WUSCHEL (WUS) and WUSCHEL-related homeobox (WOX) genes, including WUS1, WUS2, WUS3, W0X2A, W0X4, W0X5, W0X9 (see US Patents 7,348,468; 7,256,322; US Patent Publications 2017 / 0121722; 2007 / 0271628; Laux et al. (1996); Mayer et al. (1998); van der Graaff et al. (2009); Dolzblasz et al. (2016), all of which are incorporated by reference herein in their entireties). A WUS / WOX gene is any polynucleotide encoding a polypeptide with a homeobox DNA binding domain, a WUS box, and an EAR repressor domain (Ikeda et al., 2009).

[0345] Morphogenic developmental genes and polypeptides useful for the methods describe herein include BabyBoom (BBM) (e.g., Zea mays BBM2, Oryza sativa BBM1 / BBM2 / BBM3, Sorghum bicolor BBM2), ODP2 e.g., Zea mays ODP2, Sorghum bicolor ODP2, Setaria italica ODP2, Brachypodium distachyum ODP2, WUS / WOX (e.g., Zea mays WUS, WUS2, WUS3), MYB118, MYB115, CLAVATA (CLV), LEC1, LEC2, KN1 / STM, IPT, MONOPTEROS -DELTA, AV-6b, lAA-h / IAA-m, SERK, AGL15, FUSCA, and PICKLE. Additional methods are known in the art can use expression of ZmGRFl-ZmGIFl, ZmGRF5 -LIKE 1,2, ZmREFl, ZmWINDl,Docket No.: 214105-WO-SEC-lZmWUS2 + AtSTM, ZmWUS2 + ipt, or other known morphogenic regulators, for example, as reported by Youngstrum et al (The Plant Journal (2025) 121, el7193). A BabyBoom gene is any polynucleotide encoding a polypeptide with at least one, but typically two, AP2 DNA-binding domains. Table 2 provides some of the morphogenic developmental genes useful for the methods described herein.Table 2; Morphogenic developmental genes and polypeptides

[0346] B. Genetic Chromosome Doubling Polypeptide

[0347] The methods provided herein can utilize genetic chromosome doubling factors. As used herein, a “genetic chromosome doubling factor” refers to a polypeptide, or a polynucleotide encoding the polypeptide, that induces, stimulates, promotes, or improves the doubling of a InDocket No.: 214105-WO-SEC-l haploid chromosome number to 2n (diploid) chromosome number resulting in homozygous chromosomes. A genetic chromosome doubling factor results in diploidization of a haploid plant cell without the use of a chemical chromosome doubling agent, such as colchicine. As an alternative to using chemical chromosome doubling agents, modulating expression of genes known to impact the plant cell cycle (genetic chromosome doubling polypeptides), either through stimulation of the cell cycle (and cell division) or through stimulation of endoreduplication, can be used to double the chromosome complement in a VPCM. Increasing ploidy level in plant cells can be achieved by modulating expression of genes that stimulate key control points in the cell cycle cell.

[0348] Example of genetic chromosome doubling factors include cell cycle genes such as cyclins (e.g., A, B, C, D, E, F, G, H), Pint, E2FA / B, Cdc25, RepA, and similar plant viral polynucleotides encoding replication-associated proteins.

[0349] Genetic chromosome doubling can also involve downregulation or inhibition of genes the inhibit the cell cycle (e.g., targeting cyclin-dependent kinase inhibitor such as by microRNA or dCas-repressor fusions) or manipulation of genes affecting reduplication (e.g., overexpressing mitotic inhibitor CCS52 or downregulation or inhibition of DELI).

[0350] In a particular example, microspores or microspore-derived multicellular structures are provided a polynucleotide sequence encoding a cyclin D2 polypeptide, such as a maize cyclin D2 (Zm-CYCD2) polypeptide. Maize cyclin D2 polypeptides suitable for the methods and systems of the present disclosure can be found in US20220240467, which is incorporated by reference in its entirety.

[0351] In another example, microspores or microspore-derived multicellular structures are provided a polynucleotide sequence encoding a truncated Baby Boom polypeptide, such as the ZM-ODP (TR5) morphogenic developmental gene, which encodes a truncated maize Ovule Development Protein 2 (ZM-ODP2), also referred to herein as BBM404polypeptide. Here, BBM404polypeptide activity provided to a microspore or microspore-derived multicellular structure results in doubling of the In haploid chromosome number to 2n (diploid). Maize ZM-ODP (TR5) polypeptides suitable for the methods and systems of the present disclosure can be found in US20250101453A1, which is incorporated by reference in its entirety.

[0352] C. Genome-editing componentsDocket No.: 214105-WO-SEC-l

[0353] In the methods described herein, cargo delivered to microspores and / or microspore-derived multicellular structures can include a site-specific genome editing agent or system. A site-specific genome editing agent or system includes enzymes or polypeptides that recognize and bind to a target DNA polynucleotide and subsequently cut (i.e., induce a double-strand break) or nick (i.e., induce a single-strand break) the target DNA polynucleotide. In a first example of genome-editing cargo, a site-specific genome editing enzyme or polypeptide is an endonuclease, which cuts the phosphodiester bond within a polynucleotide chain. Endonucleases of the present disclosure include zinc finger nucleases (Urnov et al. (2010) Nat Rev Genet. 11:636-646), transcription activator-like effector nucleases (TALEN) (Joung and Sander (2013) Nat Rev Mol Cell Biol. 14:49-55), homing endonucleases (Belfort and Bonocora (2014) Methods Mol Biol. 1123: 1-26 and Stoddard (2014) Mobile DNA. 5:7), Cas endonucleases (Cong et ai. (2013) Science. 339:819-823, Zetsche et al. (2015) Cell. 163:759-771, Yan et al. (2018) Science. 363:88-91, Pausch et al. (2020) Science. 369:333-337, Karvelis et al. (2020) Nucleic Acids Res. 48:5016-5023, Yoshimi and Mashimo (2022) Gen and Genome Editing. 3-4: 100013, and Urbaitis et al. (2022) EMBO Rep. 23:e55481), transposase associated B (TnpB) nucleases (Karvelis et al. (2021) Nature. 599:692- 696 and Altae-Tran et al. (2021) Science. 374:57-65), Fanzor (Saito et al. (2023) Nature. 620:660- 668 and Jiang et al. (2023) Sci. Adv. 9:eadk0171), and hydrolytic endonucleolytic ribozymes (HYER) (Liu et al. (2024) Science. 383:eadh4859)).

[0354] Cas Polypeptides

[0355] In a second example of genome editing cargo, a site-specific genome editing agent or system is a Cas polypeptide that can be used for targeted genome-editing (via simplex and / or multiplex double-strand breaks and / or single-strand breaks) and targeted genome regulation (via tethering of epigenetic effector domains to either the Cas polypeptide or guide polynucleotide). A Cas polypeptide can also be engineered to function as a polynucleotide-guided recombinase, and via polynucleotide tethers can serve as a scaffold for the assembly of multiprotein and nucleic acid complexes (Mali et al., 2013, Nature Methods Vol. 10: 957-963).

[0356] A“Cas polypeptide” or “Cas effector protein” will be understood to mean a polynucleotide- guided CRISPR-associated protein that, when in complex with a suitable guide polynucleotide, can recognize and bind to a target DNA polynucleotide. A Cas polypeptide or Cas effector protein can have, but is not required to have, the additional functionality of: unwinding, unwinding and cutting, unwinding and nicking, cutting, or nicking a DNA polynucleotide.Docket No.: 214105-WO-SEC-l

[0357] A Cas polypeptide includes, but is not limited to, Cas9, Casl2f (Cas-alpha, Casl4), Casl21 (Cas-beta), Casl2a (Cpfl), Casl2b (a C2cl protein), Casl3 (a C2c2 protein), Casl2c (a C2c3 protein), Casl2d, Casl2e, Casl2g, Casl2h, Casl2i, Casl2j, Casl2k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, CaslO, or combinations or complexes of these.

[0358] A site-specific genome editing system can comprise a Cas endonuclease comprising one or more domains enabling it to function as a double-strand break-inducing agent. A Cas endonuclease is a type of Cas polypeptide or Cas effector protein comprising one or more nuclease domains (for example, one or more RuvC nuclease domains) that, when in complex with a suitable guide polynucleotide, recognizes, binds, and induces a double-strand break in a target DNA polynucleotide.

[0359] Alternatively, a site-specific genome editing system can comprise a Cas polypeptide having no substantial nuclease activity and is referred to as a catalytically “inactivated Cas” or a “deactivated Cas” (“dCas”). A dCas can be a Cas endonuclease comprising one or more modifications or mutations that abolish or reduce its ability to cut a double-stranded polynucleotide. The modified form of the Cas polypeptide can include an amino acid change (e.g., deletion, insertion, or substitution) that reduces or eliminates the naturally-occurring nuclease activity of a Cas endonuclease. A deactivated Cas typically lacks any functional nuclease domains. In this way, a deactivated Cas does not induce a single-strand break or a double-strand break, but can still bind to a target DNA polynucleotide. For example, a deactivated Cas-alpha endonuclease can comprise one or more amino acid substitutions in positions in the Cas-alpha endonuclease responsible for coordinating a divalent metal ion resulting in a dysfunctional RuvC domain(s).

[0360] Alternatively, a site-specific genome editing system can comprise a Cas polypeptide having nickase activity (i.e., induces a single-strand break), and is referred to herein as a “Cas nickase” (“nCas”) or a “Cas polypeptide having nickase activity”. A nCas can be a Cas endonuclease comprising one or more modifications or mutations such that DNA nicking functionality is retained. A Cas nickase typically comprises one functional endonuclease domain that allows the Cas to break only one strand (i.e., make a nick) at a target DNA sequence. For example, a Cas nickase can comprise (i) a mutant, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., wild-type HNH domain). As another example, a Cas-alpha nickase can comprise a mutant that permits the RuvC domain(s) to only or preferentially cleave a single-strand of the doubleDocket No.: 214105-WO-SEC-l stranded DNA target. Non-limiting examples of Cas nickases suitable for use herein are disclosed in US20140189896, which is incorporated by reference herein in its entirety.

[0361] Cas9 Polypeptides

[0362] A Cas polypeptide of a site-specific genome editing system can be a Cas9 polypeptide, such as a Cas9 endonuclease, and the site-specific genome editing system comprises a Cas9 polypeptide and one or more guide polynucleotides that introduce one or more site-specific modifications in a target DNA polynucleotide sequence (also referred to herein as a “guided Cas polypeptide system”). A guided Cas polypeptide system can further comprise a donor DNA or a polynucleotide modification template. Some exemplary Cas9 endonucleases are described in, for example, WO2019165168.

[0363] Cas9 (also known as Cas5, Csnl, or Csxl2) forms a complex with a crRNA and tracrRNA, or with a single guide RNA, to recognize and cut or nick target DNA. Cas9 recognizes a 3’ GC- rich PAM sequence, typically an NGG motif, on double-stranded DNA. The Cas9 polypeptide contains a RuvC nuclease domain (with subdomains I, II, III) and an adjacent HNH nuclease domain. The RuvC nuclease and HNH nuclease each can cut a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick).

[0364] Cas-alpha polypeptides

[0365] A Cas polypeptide of a site-specific genome editing system can be a Cas-alpha (e.g., Casl2f) polypeptide and the site-specific genome editing system comprises a Cas-alpha polypeptide and one or more guide polynucleotides that introduce one or more site-specific modifications in a target DNA polynucleotide sequence. The guided Cas polypeptide system can further comprise a donor DNA or a polynucleotide modification template. Some exemplary Cas- alpha polypeptides are described in, for example, US10934536, WO2022082179, WO2023244992, and WO2024196921.

[0366] A Cas-alpha endonuclease is a functional polynucleotide-guided, PAM-dependent dsDNA cleavage protein of fewer than 800 amino acids comprising: a C-terminal RuvC catalytic domain split into three subdomains, a bridge-helix, and one or more zinc finger motifs, and further comprising an N-terminal Rec subunit with a helical bundle, WED wedge-like (or “Oligonucleotide Binding Domain”, OBD) domain, and, optionally, a zinc finger motif.Docket No.: 214105-WO-SEC-l

[0367] Cas-alpha polypeptides can comprise one or more zinc finger coordination motifs that may form a zinc binding domain. Zinc finger-like motifs can aid in target and non-target strand separation and loading of the guide polynucleotide into the target DNA polynucleotide. Cas-alpha polypeptides comprising one or more zinc finger-like motifs can provide additional stability to a ribonucleoprotein complex on a target DNA polynucleotide. Cas-alpha endonucleases comprise C4 or C3H zinc binding domains.

[0368] A Cas-alpha polypeptide can function as a double- or single-strand break-inducing agent. A catalytically inactive Cas-alpha endonuclease (dCas-alpha) can be used to target or recruit to a target DNA polynucleotide sequence without inducing cleavage or nicking. A catalytically inactive Cas-alpha polypeptide can be combined with abase editing molecule, such as a cytidine deaminase or an adenine deaminase.

[0369] Guide polynucleotides

[0370] When a site-specific genome editing system comprises a Cas polypeptide, the system further comprises one or more guide polynucleotides. As used herein, a “guide polynucleotide” refers to a polynucleotide sequence that can form a complex with a Cas polypeptide, including the Cas polypeptide(s) described herein, and enables the Cas polypeptide to recognize, optionally bind to, and optionally cut or nick a target DNA polynucleotide. A guide polynucleotide is a chimeric, non-naturally occurring polynucleotide sequence (i.e., it is engineered or synthetic) comprising polynucleotide regions or domains that are not found together in nature (i.e., the regions or domains are heterologous with respect to each other), specifically, a first nucleotide sequence or domain that can hybridize to a portion of a target DNA polynucleotide and a second nucleotide sequence or domain that can recognize and interact with a Cas polypeptide. A guide polynucleotide can be a RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNA combination sequence).

[0371] A guide polynucleotide can be a duplex molecule composed of a crNucleotide (crRNA, crDNA, or crDNA-RNA) and a separate tracrNucleotide (tracrRNA, tracrDNA, or tracrDNA- RNA). Alternatively, a guide polynucleotide can be a single molecule (single guide) in which the crNucleotide and tracrNucleotide sequences are linked, forming a single guide polynucleotide (single guide RNA, single guide DNA, or single guide RNA-DNA).

[0372] Guide polynucleotide-Cas polypeptide complexDocket No.: 214105-WO-SEC-l

[0373] As used herein, a “guide polynucleotide-Cas polypeptide complex” or a “guide polynucleotide / Cas polypeptide complex” refers to at least one guide polynucleotide and at least one Cas polypeptide that form a complex capable of directing the Cas polypeptide to a target DNA polynucleotide and enabling the Cas polypeptide to recognize, optionally bind to, and optionally cut or nick a target DNA polynucleotide (i.e., introduce a double-strand break or single-strand break, respectively). A guide polynucleotide-Cas polypeptide complex can comprise a Cas polypeptide and suitable guide polynucleotide of any of the known CRISPR system (Horvath and Barrangou, 2010, Science 327: 167-170; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13: 1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13). The terms “guide polynucleotide-Cas endonuclease complex” and “guide polynucleotide / Cas endonuclease complex” refer to a guide polynucleotide-Cas polypeptide complex having a Cas endonuclease that comprises one or more domains enabling it to function as a double-strand breakinducing agent.

[0374] The terms “guide RNA-Cas polypeptide complex”, “gRNA-Cas polypeptide complex”, “guide RNA / Cas polypeptide complex”, and “gRNA / Cas polypeptide complex” are used interchangeably herein and refer to at least one guide RNA and at least one Cas polypeptide that form a complex capable of directing the Cas polypeptide to a target DNA polynucleotide and enabling the Cas polypeptide to recognize, optionally bind to, and optionally cut or nick a target DNA polynucleotide (i.e., introduce a double-strand break or single-strand break, respectively). The terms “guide RNA-Cas endonuclease complex” and “guide RNA / Cas endonuclease complex” refer to a guide RNA-Cas polypeptide complex having a Cas endonuclease that comprises one or more domains enabling it to function as a double-strand break-inducing agent.

[0375] In an example of the methods disclosed herein disclosed herein, the Cas polypeptide of a guide polynucleotide-Cas polypeptide complex is a Cas endonuclease, and genome-editing of a target DNA polynucleotide via the guide polynucleotide-Cas endonuclease complex comprises non-homologous end joining or homology-directed repair following a Cas endonuclease-mediated double-strand break. Alterations or modifications of a target DNA sequence include, for example: (i) substitution of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).

[0376] Uses for the guided Cas polypeptide systems described herein include, but are not limited to, modifying or replacing nucleotide sequences of interest (such as a regulatory elements),Docket No.: 214105-WO-SEC-l insertion of polynucleotides of interest, gene knock-out, gene-knock in, modification of splicing sites and / or introducing alternate splicing sites, modifications of nucleotide sequences encoding a protein of interest, amino acid and / or protein fusions, and gene silencing by expressing an inverted repeat into a gene of interest.

[0377] In another example of the methods disclosed herein, the Cas polypeptide of a guide polynucleotide-Cas polypeptide complex is a dCas. A guide polynucleotide-dCas polypeptide complex can be used, for example, for base editing.

[0378] In yet another example of the methods disclosed herein, the Cas polypeptide of a guide polynucleotide-Cas polypeptide complex is a nCas. A guide polynucleotide-nCas polypeptide complex can be used, for example, for base editing or prime editing.

[0379] A guide polynucleotide-Cas polypeptide complex of the methods disclosed herein can be a ribonucleoprotein (RNP) complex, wherein the Cas polypeptide is provided as a protein and the guide polynucleotide is provided as a ribonucleotide.

[0380] Target DNA Polynucleotides

[0381] As used herein, “target DNA polynucleotide”, “DNA target site”, “target DNA sequence”, “target site”, “target sequence”, “genomic target sequence”, “genomic target site”, and “target polynucleotide” are used interchangeably and refer to a polynucleotide sequence, such as but not limited to, a nucleotide sequence on a chromosome, episome, a locus, or any other DNA molecule in the genome (including chromosomal DNA, chloroplastic DNA, mitochondrial DNA, or plasmid DNA) of a cell, at which a guide polynucleotide-Cas polypeptide complex can recognize, bind to, and optionally nick or cut. The target DNA polynucleotide can be an endogenous site in the genome of a cell, or alternatively, the target DNA polynucleotide can be heterologous to the cell, and thereby not naturally occurring in the genome of the cell, or alternatively, the target DNA polynucleotide can be found in a heterologous genomic location compared to where it occurs in nature. As used herein, terms “endogenous target sequence” and “native target sequence” are used interchangeably to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell. An “artificial target site” or an “artificial target sequence” are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence can be identical in sequence to an endogenous or native target sequence in the genomeDocket No.: 214105-WO-SEC-l of a cell, but be located in a different position (i.e., a non -endogenous or non-native position) in the genome of a cell.

[0382] Methods for “modifying a target site” and “altering a target site” are used interchangeably herein and refer to methods for producing an altered target site.

[0383] As used herein, a “modified target DNA polynucleotide”, “modified DNA target site”, “modified target DNA sequence”, “modified target site”, “modified target sequence”, “modified genomic target sequence”, “modified genomic target site”, “modified target polynucleotide”, “altered target DNA polynucleotide”, “altered DNA target site”, “altered target DNA sequence”, “altered target site”, “altered target sequence”, “altered genomic target sequence”, “altered genomic target site”, and “altered target polynucleotide” are used interchangeably and refer to a DNA target sequence as that comprises at least one alteration or modification when compared to a non-altered target sequence. Such alterations or modifications include insertion, deletion, single nucleotide polymorphism, inversion, or translocation of at least one nucleotide as compared to an unmodified DNA target site.

[0384] NHEJ andHDR

[0001] In the methods of the present disclosure, cargo delivery to microspores and / or microspore- derived multicellular structures can include a guided Cas polypeptide system for genome-editing via inducing double-strand breaks at a DNA target site. For example, a genome editing system can comprise a Cas endonuclease, one or more guide polynucleotides, and optionally a donor DNA or polynucleotide modification template, and editing a target DNA polynucleotide comprises nonhom ologous end joining (NHEJ) or homology-directed repair (HDR) following a Cas endonuclease-mediated double-strand break. Once a double-strand break is induced in the DNA, the cell's DNA repair mechanism is activated to repair the break. The most common repair mechanism to bring the broken ends together is the nonhomologous end-joining pathway (Bleuyard et al., (2006) DNA Repair 5: 1-12). The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible (Siebert and Puchta, (2002) Plant Cell 14: 1121-31; Pacher et al., (2007) Genetics 175:21-9). Alternatively, the double-strand break can be repaired by homologous recombination between homologous DNA sequences. Once the sequence around the double-strand break is altered, for example, by exonuclease activities involved in the maturation of double-strand breaks, gene conversion pathways can restore the original structure if a homologous sequence is available, such as aDocket No.: 214105-WO-SEC-l homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenic DNA sequences can also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152: 1173-81).

[0385] As used herein, “homologous recombination” (HR) includes the exchange of DNA fragments between two DNA molecules at the sites of homology. The frequency of homologous recombination is influenced by a number of factors. Different organisms vary with respect to the amount of homologous recombination and the relative proportion of homologous to non- homologous recombination. Generally, the length of a region of homology affects the frequency of homologous recombination events: the longer the region of homology, the greater the frequency. The length of a homology region needed to observe homologous recombination is also speciesvariable. In many cases, at least 5 kb of homology has been utilized, but homologous recombination has been observed with as little as 25-50 bp of homology.

[0386] In another example, a genome-editing system can comprise a Cas endonuclease, one or more guide polynucleotides, and a donor DNA. As used herein, “donor DNA” is a DNA construct that comprises a polynucleotide of interest to be inserted into the DNA target site of a Cas endonuclease. The donor DNA further comprises a first and a second region of homology flanking the polynucleotide of interest. The first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the DNA target site of a cell or organism genome. Once a double-strand break is introduced in the DNA target site by the Cas endonuclease, the first and second regions of homology of the donor DNA can undergo homologous recombination with their corresponding genomic regions of homology resulting in exchange of DNA between the donor and the target genome. As such, the provided methods result in the integration of the polynucleotide of interest of the donor DNA into the double-strand break in the DNA target site in the host genome, thereby altering the original target site and producing a modified genomic target site.

[0387] In yet another example, a genome-editing system can comprise a Cas endonuclease, one or more guide polynucleotides, and a polynucleotide modification template that provides the basis for template-directed repair of a double-strand break. As used herein, a “polynucleotide modification template” includes a polynucleotide that comprises at least one nucleotide modification (i.e., substitution, addition or deletion) when compared to the nucleotide sequence toDocket No.: 214105-WO-SEC-l be edited (i.e., DNA target site). Optionally, the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

[0388] Base Editing

[0389] In the methods of the present disclosure, cargo delivery to microspores and / or microspore- derived multicellular structures can include a guided Cas polypeptide system for base editing. A base editing system comprises a base editing agent and a plurality (i.e., more than one) guide polynucleotides, and modifying a target DNA polynucleotide comprises introducing a plurality of nucleobase edits in the target polynucleotide sequence resulting in a variant nucleotide sequence. As used herein, any molecule or complex that effects a change in a nucleobase is a “base editing agent”, including glycosylase base editors.

[0390] One or more bases of a target DNA polynucleotide can be chemically altered to change the base from one type to another, for example, from a Cytosine to a Thymine, or an Adenine to a Guanine. A plurality of nucleobases, for example, 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more 90 or more, 100 or more, or even greater than 100, 200 or more, up to thousands of bases can be modified or altered, to produce a cell or an organism, such as a plant, with a plurality of modified bases.

[0391] Any base editing complex, such as a base editing agent (e.g., a deaminase or a protein having deaminase activity) fused to, linked to, or otherwise operably associated with (directly or indirectly) a guided Cas polypeptide can be used to recognize and bind to a desired locus in the genome of an organism and chemically modify one or more bases of a target DNA polynucleotide without creating a double-strand break.

[0392] A “deaminase” is an enzyme that catalyzes a deamination reaction. For example, deamination of adenine with an adenine deaminase results in the formation of inosine. Inosine selectively base pairs with cytosine instead of thymine. This results in a post-replicative transition mutation, such that the original A - T base pair transforms into a G - C base pair. In another example, cytosine deamination results in the formation of uracil, which can be repaired by cellular repair mechanisms back to a C - T base pair or to a T - A, G - C, or A - T base pair. This heterogeneity in repair can be suppressed by the introduction of a uracil glycosylase inhibitor, such that DNA repair or replication transforms the original C - T base pair into a T - A base pairDocket No.: 214105-WO-SEC-l(Burnett et al. (2022) Frontiers in Genome Editing. 4, 923718). In the case of both adenine and cytosine deaminases, the introduction of a nick promotes the respective base pair change (Burnett et al., 2022).

[0393] Site-specific base conversions can be achieved to engineer one or more nucleotide changes (i.e., single nucleotide polymorphisms) to create one or more edits in the genome. These include for example, a site-specific base edit mediated by an OG to T»A or an A»T to G*C base editing deaminase (Gaudelli et al., Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage." Nature (2017); Nishida et al. “Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.” Science 353 (6305) (2016); Komor et al. “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533 (7603) (2016): 420-4.) A catalytically “dead” or inactive Cas endonuclease (“dCas”) fused to a cytidine deaminase or an adenine deaminase becomes a specific base editor that can alter DNA bases without inducing a double-strand DNA break. Base editors convert C->T (or G- >A on the opposite strand) or an adenine base editor that would convert adenine to inosine, resulting in an A->G change within an editing window specified by the guide polynucleotide.

[0394] A base editing deaminase, such as a cytidine deaminase or an adenine deaminase, can be fused to, linked to, or otherwise operably associated with (directly or indirectly) a guided Cas polypeptide, such as an RNA-guided dCas or a partially active (i.e., having nickase activity) Cas nickase (“nCas”) so that it does not cut a target site to which it is guided. The dCas forms a functional complex with a guide polynucleotide that shares homology with a polynucleotide sequence at the target site, and is further complexed with the deaminase molecule. The guided dCas or nCas recognizes and binds to a double-stranded target sequence, opening the double-strand to expose individual bases. In the case of a cytidine deaminase, the deaminase deaminates the cytosine base and creates a uracil. Uracil glycosylase inhibitor (UGI) is provided to prevent the conversion of U back to C. DNA replication or repair mechanisms then convert the uracil to a thymine (U to T), and subsequent repair of the opposing base (formerly G in the original G-C pair) to an adenine, creating a T-Apair (Komor et al. Nature Volume 533, Pages 420-424, 19 May 2016).

[0395] Prime Editing

[0396] In the methods of the present disclosure, cargo delivery to microspores and / or microspore- derived multicellular structures can include a guided Cas polypeptide system for prime editing. A prime editing system comprises a prime editing agent and one or more guide polynucleotides, andDocket No.: 214105-WO-SEC-l modifying a target DNA polynucleotide comprises introducing one or more targeted insertions, deletions, or base-to-base conversions (also know as nucleobase swaps) without generating a double-strand break.

[0397] A prime editing agent can be, for example, a Cas polypeptide fused to a reverse transcriptase (RT), wherein the Cas polypeptide is modified to nick DNA rather than generating double-strand break. As used herein, “nick” refers to a single-strand break in a double- stranded DNA molecule. This nCas polypeptide-reverse transcriptase fusion can also be referred to as a “prime editor” or “PE”. A guide polynucleotide of a prime editing system can be a prime editing guide polynucleotide (pegRNA), which is larger than standard sgRNAs commonly used for CRISPR genome editing (e.g., >100 nucleobases). The pegRNA comprises a primer binding sequence (PBS) and a RT template containing the desired or target RNA sequence at its 3’ end.

[0398] During prime editing, the PE:pegRNA complex binds to a target DNA polynucleotide and the modified Cas polypeptide nicks one target DNA strand resulting in a flap. The PBS on the pegRNA binds to the DNA flap, and the target RNA sequence of the RT template is reverse transcribed using the reverse transcriptase. The edited strand is incorporated into the target DNA polynucleotide at the end of the nicked flap, and the target DNA strand is repaired with the new reverse transcribed DNA.

[0399] Traits

[0400] Genome modification via a Cas polypeptide may be used to effect a genotypic and / or phenotypic change in the microspore or microspore-derived multicellular structures. Such a change is preferably related to an improved trait of interest or an agronomically-important characteristic, the correction of an endogenous defect, or the expression of some type of expression marker. In some aspects, the trait of interest or agronomically-important characteristic is related to the overall health, fitness, or fertility of the plant, the yield of a plant product, the ecological fitness of the plant, or the environmental stability of the plant. In some aspects, the trait of interest or agronomically-important characteristic is selected from the group consisting of: agronomics, herbicide resistance, insecticide resistance, disease resistance, nematode resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, grain characteristics, commercial product production. In some aspects, the trait of interest or agronomically-important characteristic is selected from the group consisting of: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency,Docket No.: 214105-WO-SEC-l improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield improvement, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered starch content, altered carbohydrate content, altered sugar content, altered fiber content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of a metabolite, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, as compared to an isoline plant not comprising a modification derived from the methods or compositions herein.

[0401] Further, in any of the methods described herein, traits of interest to confer an agronomically-important characteristic, can be provided to microspores, multicellular structures, embryo-like structures, and / or macroscopic structures as transgenes.

[0402] Elite crop germplasm genotypes for selecting, isolating, and generating microspore- derived doubled-haploid structures, plantlets, and crop plants

[0403] Elite germplasm generally refers to plant materials, such as advanced breeding lines and modern cultivars, that have been genetically improved for desirable traits like high yield, disease resistance, and adaptation to specific growth environments. It represents the product of modem plant breeding and is used as the foundation for developing new commercial varieties. Unlike more primitive or earlier generations of germplasm, elite germplasm is highly adapted to a target region and breeding with elite lines has a lower risk of introducing undesirable traits. However, elite crop germplasm may not possess characteristics such as transformability, ability to produce double haploids and other cellular reprogramming techniques. Many such elite germplasm are often recalcitrant to such cellular manipulations including, cell culture, cellular reprogramming and haploid embryogenesis induction, followed by regeneration at a rate that is suitable for an advanced breeding program. Therefore, highly automated processes described herein are suitable to apply to such elite germplasm / genotypes where the efficiency may be lower compared to nonelite plant material.

[0404] Media and culture conditions for maize and wheat microspores

[0405] After isolation, maize microspores can be cultured in a vessel, e.g., in a 9% sucrose induction medium. Optionally, the cultured cells can be incubated at 28°C under dark conditions.

[0282] The methods provided herein can utilize one or more treatments to promote, improve, orDocket No.: 214105-WO-SEC-l increase microspore embryogenesis. For example, microspores can be cultured in the presence of polycomb repressive complex 2 (PRC2) inhibitors and / or ethylene inhibitors. Examples of ethylene inhibitors include, but are not limited to, ethylene biosynthesis inhibitors (e.g., aminoethoxyvinylglycine) and ethylene signal perception inhibitors (e.g., silver nitrate). Microspores can also be contacted with small molecule kinase inhibitors that promote cellular reprogramming from an initial haploid gametic cell fate to an embryogenic cell fate, including, N- [(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide, anthra(l,9- cd)pyrazol-6(2H)-one, 4-(4-Fluorophenyl)-2-(4-methylsulfmylphenyl)-5-(4- pyridyl)lH- imidazole, and N-benzyl-2-(pyrimidin-4-ylamino)-l,3-thiazole-4-carboxamide (see US11447786B2; incorporated by reference).

[0406] In some examples of the methods provided herein, the polynucleotides expressing the first morphogenic developmental polypeptide, the second morphogenic developmental polypeptide, and / or the genetic chromosome doubling factors are also used. In some examples of the methods provided herein, microspores are staged, extracted, isolated, and cultured in vitro in the presence of a sulfonylurea compound, such as ethametsulfuron, to regulate expression of the WUS, BBM, and Zm-CYCD2 expression constructs. Other known optogenetic (e.g., blue light-, green light-, and red / near-infrared-activated systems) and chemical (e.g., tetracycline-, steroid-, insecticide-, copper-, and ethanol -regulated) induction systems for regulating the expression of the first and / or second morphogenic developmental polypeptides are also suitable for the disclosed methods. Basal salts can include the N6 Formulation, designed with low ammonium (NFLf) and adequate nitrate (NOs ) levels. Carbon source can include sucrose (60-90 g / L), typically in the range of about 6% to 9% for maize induction. As sucrose hydrolyzes, the sucrose concentration is monitored during maize microspore embryo culturing. If needed, synthetic auxin such 2,4-D is also used for maize microspore division and development and the concentration can vary based on the chosen genotype (e.g., 2.0 mg / L). Cytokinins such as kinetin (0.5-1.0 mg / L) that supports cell division and is combined with 2,4-D to promote embryogenic development. Sometimes, compounds such as TIBA (Triiodobenzoic acid) that inhibit auxin transport are also used to promote the separation of embryos and prevent the fusion of callus tissue. The disclosure herein provides guidance to one of ordinary skill in the art to further optimize successful maize microspore culture. Components are chosen to address the physiological sensitivities of maize,Docket No.: 214105-WO-SEC-l particularly regarding nitrogen source, carbohydrate metabolism, hormone requirements, and the management of byproducts.

[0407] For crops such as wheat, W14 medium composition for basal salts can be used along with carbon sources such as maltose (about 9% or 90 g / L). Wheat microspore culture may also include cold pretreatment and also optionally include ovary co-culturing. Wheat tillers are harvested and cold treated for example at 4C for about 7 days, 10 days, 21 days, and 28 days. The cold-treatment period can be shorter or longer, for example for winter wheat that normally requires vernalization for flowering and / or germination. If wheat microspores exhibit a "density effect", they fail to divide if the population density is too low, due to the dilution of endogenous growth factors, ovary co-culture is employed. About 3-5 mature ovaries (from the donor plant or a responsive model like 'Igri' barley) are placed into the induction medium with the wheat microspores.

[0408] In certain embodiments, about 100,000 to 1,000,000 and up to 10,000,000 microspores are extracted from one or more tassels per maize plant. The viability percentage of such microspores can range from about 5%, 10%, 15%, 25%, 30%, 40%, 45%, 50%, 60%, 75% or higher. In certain embodiments, the isolated microspores are substantially uniform in the same stage of about 10%, 20% 30%, 40%, 50% or higher based on the tassel stage. These microspores are isolated from the anthers at the correct developmental stage and then cultured in a suspension at a density of about 10,000, 20,000, 30,000, 40,000, and 50,000-100,000 microspores per mL for embryogenesis. The microspore suspension culture can be further diluted to about 50, 100, 500, 1000, and 5000 microspores per mL.

[0409] In one aspect, this disclosure pertains to high-throughput, automated, high-volume methods for altering a maize genome through maize microspore-derived, transformation- competent haploid tissue, which includes a haploid tissue genome. A polynucleotide encoding a site-specific nuclease is delivered to this transformation-competent haploid tissue, and it is confirmed that the haploid tissue genome has been modified by the encoded site-specific nuclease. In some cases, the transformation-competent haploid tissue may be embryo or callus tissue. The polynucleotide encoding the site-specific nuclease is introduced to the transformation-competent haploid tissue using a plant transformation method. This method can include various techniques such as microparticle bombardment, Agrobacterium transformation, calcium phosphate transformation, polybrene transformation, electroporation, ultrasonic transformation, liposome transformation, microinjection, naked DNA transformation, plasmid vector transformation, viralDocket No.: 214105-WO-SEC-l vector transformation, silicon carbide-mediated transformation, aerosol beaming transformation, and PEG transformation. The site-specific nuclease polynucleotide encodes a nuclease, which can be a Zinc Finger Nuclease, TALEN nuclease, meganuclease, or CRISPR nuclease. In some embodiments, the site-specific nuclease targets and cuts a specific genomic DNA region of the haploid maize genome. This can involve cutting either both strands or a single strand of the genomic DNA. Additionally, these methods may include delivering a donor polynucleotide and stably integrating it into the modified haploid tissue genome. Each donor polynucleotide may have at least one domain that is at least 85% identical to the genomic DNA target region of the haploid tissue genome. In other cases, the donor polynucleotide may have two domains that are at least 85% identical to two different sequences in the genomic DNA target region. The microspore- derived, transformation-competent tissue can come from maize with elite performance characteristics, such as hybrid maize resulting from crossing an elite maize line with another line that has a high microspore culture response. Confirmation of genome modification in the haploid tissue can be done using assays like PCR, Southern blot, Northern blot, protein expression, Western blot, ELISA, or Next Generation Sequencing.

[0410] The transformation-competent (also genome-editing competent) haploid tissue derived from a maize microspore can be obtained by high-throughput automated harvesting of microsporecontaining tassels from maize, incubating the tassels at about 4-12°C, isolating microsporecontaining anthers from the tassels, culturing the anthers in anther culture medium to generate microspore-derived embryos, and then culturing these embryos in callus medium to produce the microspore-derived, transformation-competent haploid tissue. High-throughput automation can be integrated into these processes to enhance efficiency and scalability. This involves using automated systems for tasks such as polynucleotide delivery, tissue culture, and genomic analysis, allowing for the rapid and simultaneous processing of multiple samples, thereby accelerating the overall workflow and increasing throughput.

[0411] Automated system for generating microspore-derived doubled haploid structures, plantlets, and crop plants

[0412] An automated system for generating microspore-derived doubled haploid crop plants can further include one or more servers for storing data and one or more computing devices communicatively coupled to the one or more servers, the computing device including a memory and one or more processors to perform operations of the automated methods.Docket No.: 214105-WO-SEC-l

[0413] Computing devices of the system can be, for example, a computer, a notebook, a laptop, a mobile device, a smartphone, a tablet, wearable, smart glasses, or any other suitable computing device that is capable of communicating with a server. The computing devices can include a processor, a memory, an input / output (I / O) controller (e.g., a network transceiver), a memory unit, and a database, all of which may be interconnected via one or more address / data bus.

[0414] The system processor(s) can be any electronic device that is capable of processing data, for example a central processing unit (CPU), a graphics processing unit (GPU), a tensor processing unit (TPU), a system on a chip (SoC), or any other suitable type of processor. It should be appreciated that the various operations of example methods described herein (i.e., performed by the computing device) can be performed by one or more processors. The memory can be a randomaccess memory (RAM), read-only memory (ROM), a flash memory, or any other suitable type of memory that enables storage of data such as instruction codes that the processor needs to access in order to implement any method as disclosed herein. The computing device can be a computing device or a plurality of computing devices with distributed processing.

[0415] As used herein, the term “database” refers to a single database or other structured data storage, or to a collection of two or more different databases or structured data storage components. The database can be part of the computing device. Alternatively, the computing device can access the database via a network. The database can store data (e.g., input, output, intermediary data) used for sampling, selecting, and advancing microspores and microspore-derived structures.

[0416] The computing device(s) can further include a number of software applications stored in a memory unit, which may be called a program memory. The various software applications on the computing device(s) can include specific programs, routines, or scripts for performing processing functions associated with the methods described herein. Additionally, or alternatively, the various software applications on the computing device can include general -purpose software applications for data processing, database management, data analysis, network communication, web server operation, or other functions described herein or typically performed by a server. The various software applications can be executed on the same computer processor or on different computer processors. Additionally, or alternatively, the software applications can interact with various hardware modules installed within or connected to the computing device(s). Such modules can implement part or all of the various exemplary method functions discussed herein.Docket No.: 214105-WO-SEC-l

[0417] The server can be a single server or a plurality of servers with distributed processing, which receive data from and / or transmit data to the computing device(s). The network can be any suitable type of computer network that functionally couples at least one computing device with the server. The network can include a proprietary network, a secure public internet, a virtual private network and / or one or more other types of networks, such as dedicated access lines, plain ordinary telephone lines, satellite links, cellular data networks, or combinations thereof.

[0418] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.

[0419] While embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the instant disclosure. It should be understood that various alternatives to the embodiments described herein can be employed in practicing the disclosure.EXAMPLESExample 1: Automated method and apparatus for tassel sterilization of tassels, anthers, immature flowers, or reproductive plant parts

[0420] This example describes a method and apparatus for sterilizing the outer surface of immature plant tassels, anthers, immature flowers, or reproductive plant parts using a high throughput technique that removes potential microbial and viral contaminants.

[0421] Harvested and pre-treated tassels, anthers, immature flowers, or reproductive plant parts are placed into a container and submerged in a diluted bleach solution. The plant material soaks for a pre-determined amount of time, undergoing a series of mechanical agitations or pressure cycles. Tassels, anthers, immature flowers, or reproductive plant parts are then drained, rinsed, and undergo a drying cycle.

[0422] Alternatively or additionally, immature tassels, anthers, immature flowers, or reproductive plant parts are sterilized via exposure to a controlled atmosphere of chlorine gas or stabilized aqueous ozone (SAG) within sealed containers. SAG degrades over time, thus negating the need for a rinse cycle.Docket No.: 214105-WO-SEC-l

[0423] Harvested tassels, anthers, immature flowers, or reproductive plant parts are placed into container 12 singularly or in batches. One or more containers are then loaded on to apparatus 10 (FIG. 6) that seals the container and supplies and dispenses various liquids for sterilization, such as a disinfects (e.g., bleach), sterile water, and surfactants. The tassels, anthers, immature flowers, or reproductive plant parts are soaked for a pre-determined amount of time. As plant material soaks in the container, the apparatus mechanically agitates the container(s) to promote contact between the sterilization solution and various surfaces of the plant material. The apparatus controls features such as acceleration, speed, direction, amplitude, and dwell time of agitation. Mechanical agitation by the apparatus includes rotation, linear movement, vibration, inversion, aeration, pressure changes, and / or vacuum. Apparatus 10 dispenses liquids into the containers via inlet 14 and removes liquids via outlet 16.

[0424] Upon completion of the sterilization cycle, the apparatus drains the sterilization solution and dispenses sterile water into the containers for rinsing the outer surfaces of the tassels, anthers, immature flowers, or reproductive plant parts. This wash step is repeated one or more times until the tassels, anthers, immature flowers, or reproductive plant parts are free of residue. Following the final wash, water is drained from the container and the tassels, anthers, immature flowers, or reproductive plant parts dry via air-drying. The apparatus can provide additional mechanical agitation to facilitate drying. Upon completion of the drying cycle, tassels, anthers, immature flowers, or reproductive plant parts progress to the microspore extraction step.Example 2: Automated method and apparatus for microspore extraction

[0425] This example describes a method and apparatus for extracting microspores from sterilized immature florets or flower buds of a plant using a high throughput technique.

[0426] Following sterilization and drying, tassels, anthers, immature flowers, or reproductive plant parts are removed and gathered into a sterile container (FIG. 7). The containers are capped and loaded onto input carousel 22 of apparatus 20. Optionally, apparatus 20 dispenses isolation media from dispenser 24 into each container to promote higher extraction yields. Apparatus 20 can include various means for microspore extraction such as blending, milling, mashing, slicing, and shredding. As shown in FIG. 8, each cup is engaged onto highspeed rotary processing device 26 with programmable control. Apparatus 20 can also contain multiple drives with automated loading and unloading for high throughput. Apparatus 20 controls speed, resting, tamping, and number ofDocket No.: 214105-WO-SEC-l cycles to optimize microspore extraction without excessive cell damage. Apparatus 20 also includes transfer robot arm 28 for sample movement, one or more automated liquid handlers or syringes for media or other liquid addition / removal, and output carousel 30.

[0427] Once each sample is processed, it is prepared for filtration. A stack of filters with specific mesh sizes is used to isolate microspores as the cup of masticated tissue is poured over the top filter. Additional isolation media is poured over the tissue to further promote extraction and increase microspore yield. Microspores are collected on the fine filter, resuspended using isolation media, and pipetted into a tube for the next step.Example 3: Automated method and apparatus for separating microspores from bulk material

[0428] This example describes a method and apparatus for separating microspores from surrounding tissue after the extraction process.

[0429] After mastication (Example 2), bulk material including microspores and surrounding tissue, is passed through a series of filter. A first large filter collects the bulk of the plant matter. A second filter collects the microspores and similarly sized plant particulates. Any microspores that may be retained in the bulk material can be rinsed through the filters with rinsing media. The speed of the filtering process can be improved by the application of vacuum pressure to draw the material and any rinsing media through the filters.

[0430] Once the filtering is complete, the bulk material in the larger filter is discarded. The smaller filter is manipulated with the aid of more rinsing media to collect the material and transfer it to a test tube, along with an appropriate amount of rinsing media. The test tube is centrifuged to pelletize the material, and the isolation media, along with some undesired plant material, is removed off.

[0431] A smaller quantity of rinsing media is added, and the material is resuspended in the tube. The material is next transferred to a second tube containing density-specific media. This tube is centrifuged again to separate the desired microspores from the denser undesired plant material. The layer of the density media containing the microspores is transferred to new containers and the process is repeated. This isolation and separation step may include aliquoting the material to multiple tubes to isolate the microspores more effectively. The number of suspension and centrifuge steps, as well as the number of aliquots, can be customized according to the plant typeDocket No.: 214105-WO-SEC-l or process to achieve the desired quality and density of microspores. The microspores are finally transferred to a tube with more rinsing media and centrifuged again to concentrate them, then transferred to the next part of the process.

[0432] This process is automated by means of liquid handlers to perform transfer steps, one or more transfer robot arms for loading and unloading centrifuges, and automated centrifuges. The process can include automation of various laboratory equipment such as SBS plates and petri dishes.

[0433] This automated method further utilize ultrasonics to facilitate the separation of material in media. It is anticipated that ultrasonics will accelerate the natural flow of material in the density separation media.

[0434] The automated method and apparatus for separating microspores from bulk material can include a reverse flow apparatus (FIG. 9) on the initial filter. Utilizing a centrifuge or ultrasonics, density separation media can be reverse flowed through the filter to perform an initial separation of the microspores from undesired material before being drawn off for the next step in the process.

[0435] The automated method and apparatus for separation microspores from bulk material can utilize continuous flow centrifugation. It is anticipated that a continuous flow centrifuge setup 60 (FIG. 10) would allow higher volumes of material to be processed in a shorter amount of time.

[0436] The automated method and apparatus for separation microspores from bulk material can include a vision Al system. After the initial filtration step, the material from the smaller filter can be processed straight to the growth medium. Avision system would then be utilized to monitor the medium and subsequently isolate any microspores that may start to induce. A vision Al system would periodically image the growth plate and utilize known data to identify material that is properly inducing. The induced material would be separated from the growth plate and transferred to subsequent processes to continue the maturation.Example 4: Automated method and apparatus for microspore isolation

[0437] This example describes a method and apparatus for microspore isolation. Robotic workstation 40 includes liquid handling channels for 5 ml and 1 ml volumes, tube gripper, plate gripper, 50 ml and 15 ml tube holders, plate carriers, holder for sterile 100 micron cone filters, a centrifuge, a tube mixer / vortexer, a microplate fluorometer, SBS cell culture microplates, refrigerated reagent reservoirs, and refrigerated tube holder.Docket No.: 214105-WO-SEC-l

[0438] Robotic workstation 40 receives (FIG. 11) a sterile tassel, anther, immature flower, or reproductive plant part homogenate in a centrifuge tube capped with a septum lid which is placed on the deck in the 50ml refrigerated tube holder. Daughter centrifuge tubes and plates are prearrayed on the deck. The starting centrifuge tube is transported to the tube mixing station and agitated. A sterile cone filter is placed on to each of several 50 ml daughter centrifuge tubes prefilled with a linear gradient of 60% to 20% sucrose (w / vol in sterile ultrapure water). During agitation, the homogenate is sequentially aspirated and transferred to the cone filter and allowed to flow onto the sucrose gradient for a prearranged time. Another aliquot of induction media is dispensed to the filter cone and allowed to drain onto the substrate below. The cone and macerate are removed from the loaded gradient daughter centrifuge tubes, the centrifuge tubes are capped with a septum lid and transported to the centrifuge. After a centrifugation cycle, the centrifuge tubes are returned to the tube holders.

[0439] During centrifugation, a 24 well microplate is prepared with an aliquot of fluorescein diacetate dye. A pipetting probe and disposable tip aspirates from a predetermined depth in the sucrose gradient targeting the sucrose concentration known to be rich in intact microspores. The tip containing microspores and sucrose medium is then dispensed into a new daughter centrifuge tube containing a predetermined amount of chilled induction media and capped with a septum lid.

[0440] The centrifuge tube containing the diluted microspores is transported to the tube mixing station and agitated while a new sample aliquot is aspirated and transferred with mixing to the individual well of a microplate containing fluorescein diacetate dye. The plate is read in the calibrated fluorometer and the concentration of cells calculated for each well. Using the calculated concentrations, the daughter centrifuge tubes are diluted a second time with chilled induction media to a predetermined embryogenic target concentration. Finally, the SBS culture plates (6 well) are arrayed, the lids removed, and the diluted aliquots of induction media and microspores are distributed equally. The plates are re-lidded and the robotic application is complete. The microspore cultures are labeled and placed into the appropriate incubator for doubled haploid structure development.Example 5: Automated method and apparatus for identification of embryo-like structures

[0441] This example describes a method and apparatus for the identification and singulation of mature embryo-like structures. More specifically, this example describes a method for theDocket No.: 214105-WO-SEC-l qualitative identification of mature embryo-like structures for extraction from a sample of embryolike structures of varying maturity levels.

[0442] In a first method, a camera is used to capture images of a sample of distributed embryolike structures within growth media. The images are analyzed with computer vision and used to determine the maturity level of the embryo-like structures. This classification information is subsequently used to facilitate extraction of mature embryo-like structures.

[0443] The embryo-like structures are stored in a sterilized incubation chamber, which can hold several samples having completed extraction and the induction stage as well as samples having undergone extraction and are undergoing microspore embryogenesis. The incubation chamber performs periodic analysis of extracted microspore-derived embryo-like structures within samples plate.

[0444] Robotic arms and / or manipulative tooling are used to remove samples from the incubation chamber and deliver them to the imaging station. Once received in the imaging station, a robotic arm and / or manipulative tooling remove the lid from the sample plate. The imaging station then images the sample and sends the images to a computer for processing. A computer vision processing system or image processing system analysis the images for characteristics of mature embryo-like structures as well as characteristics indicating microspore-derived structures have surpassed their maturation window. If the computer vision processing system or image processing system determines there are mature embryo-like structures, the robotic arm and / or manipulative tooling transfer the plate to a transfer station for next stages. If the computer vision processing system or image processing system does not identify mature embryo-like structures, the robotic arm and / or manipulative tooling transfer the plate back to the incubation chamber for continued incubation.

[0445] Once a sample container with confirmed mature embryo-like structures identified is delivered to the transfer station, a robotic arm and / or manipulative tooling deposits the mature embryo-like structures into a container with other mature samples. The samples are labeled and placed in a sterile incubation chamber.Example 6: Automated method and apparatus for sterilization and microspore extraction

[0446] This example describes a method and apparatus for sample registration and sterilization of tassels, anthers, immature flowers, or reproductive plant parts, and microspore extraction.Docket No.: 214105-WO-SEC-l

[0447] Staged, harvested, and pre-treated tassels, anthers, immature flowers, or reproductive plant parts are logged with sample identifiers, the number of tassels, anthers, immature flowers, or reproductive plant parts recorded, and the sample weights collected for each subset of tissue. New batch numbers are assigned. The above data is locally logged in a software application that manages the sequence and timing of all subsequently described steps. Any user observations or meta-data is appended to the batch dataset.

[0448] Following sample registration, each sample batch is placed in a Waring lab blender homogenizer vessel (e.g., 250 ml) equipped with a variable speed control blender base unit. The speed control knob of the blender base unit has a stepper motor managed by the application software. This stepper control modulates on / off function and speed as well as timing of cycles and pulses.

[0449] The blending unit (homogenizer) has three syringe pumps nominally equipped with 50 ml syringes that are connected either to input reagents, a waste reservoir, or a sample collection stream.

[0450] The surface sterilant solution reservoir, the sterilant rinse reservoir, and the isolation media are plumbed to the first syringe pump via a 4-5 distribution valve, one port per reagent. The last port is plumbed to tubing and a cannula fitted into the lid of the blender vessel. The second pump is outfitted with a Y-valve, and connected to a waste reservoir with tubing also connected to a cannula and directed to the blender vessel. The third pump is also fitted with a Y-valve and plumbed to a cannula in a sample receiving vessel, followed by an air gap within the receiving vessel and then to another cannula also connected by tubing to final cannula in the blender reservoir. The valve positions and the aspirate / dispense modalities, the speeds and volumes and timings are maintained by the software control. The cannula positions within the blender vessel and plumbing enable their respective volumetric control functions within fractions of a milliliter.

[0451] Turning to FIG. 12, blending unit 50 includes first syringe pump port 52, overflow port 54, second syringe pump port 56, third syringe pump port 58, probe float 60, and rotary homogenizer 72. First syringe pump port 52 has probe bushing 62 with a set screw. Overflow port 54 has probe bushing 64 with a set screw. Second syringe pump port 56 has probe bushing 66 and third syringe pump port 58 has probe bushing 68. Second syringe pump port 56 and third syringe pump port 58 are joined by height stop 70 with a set screw.Docket No.: 214105-WO-SEC-l

[0452] Blending unit 50 enables process steps with precise timing and volumes: (1) addition of surface sterilant to the tassels, anthers, immature flowers, or reproductive plant parts; (2) mixing of sterilant; (3) evacuation of sterilant; (4) rinsing with rinse solution; (5) evacuation of rinse solution; (6) addition of isolation media.

[0453] Following these liquid handling events, the rotary homogenizer 72 of blending unit 50 is engaged for precise timed cycles and speeds. The process concludes when the third syringe pump transfers the homogenate to its collection vessel. The homogenate transfer is an aseptic transfer via air-displacement rather than through a valve.Example 7: Automated method and apparatus for cargo delivery

[0454] This example describes methods for delivery of cargo, that is small molecules, polynucleotides, polypeptides, and / or expression cassettes, to promote microspore embryogenesis, chromosome doubling, and optionally genome-editing.

[0455] Example 7A

[0456] In a first method, immature embryos having an Fl genome are transformed with one or more expression cassettes expressing morphogenic developmental polypeptide(s) for microspore embryogenesis induction, a genetic chromosome doubling polypeptide, a transgene, and genomeediting components, for example a Cas polypeptide and guide polynucleotide.

[0457] Immature embryos are isolated from ears and placed in Agrobacterium infection medium to be transformed with cargo. Following transformation, embryos are cultured, grown into plants, and microspores are isolated and cultured therefrom.

[0458] Robotic arms, automated liquid handlers, and / or manipulative tooling are used for media changes during microspore culture, transfer of microspores to solid medium, and / or transfer of microspore-derived regenerated plantlets to a greenhouse.

[0459] Example 7B

[0460] In a second method, microspores are isolated and transfected with polynucleotides encoding morphogenic developmental polypeptide(s) for microspore embryogenesis induction, a genetic chromosome doubling polypeptide, a transgene, and / or genome-editing components.

[0461] Isolated microspores are cultured in induction medium from a single-cell microspore to a microspore-derived multicellular structure or a microspore-derived embryo-like structure. Transfection reagents and polynucleotide cargo is added to the culture medium of isolatedDocket No.: 214105-WO-SEC-l microspores. Microspores are then incubated until development of multicellular structures (MCS) and embryo-like structures (ECS). Transfection reagents can be chemical (e.g., cell -penetrating peptides) or physical (e.g., nano-needle delivery).

[0462] Automated liquid handlers are used for addition and removal of transfection reagents and polynucleotide cargo. Amicrofluidics system is used for automated nano-needle delivery. Robotic arms, automated liquid handlers, and / or manipulative tooling are used for media changes during microspore culture, transfer of microspores to solid medium, and / or transfer of microspore-derived regenerated plantlets to a greenhouse.

[0463] Example 7C

[0464] In a third method, microspore-derived multicellular structures and embryo-like structures are transformed with one or more expression cassettes encoding morphogenic developmental polypeptide(s) for microspore embryogenesis induction, a genetic chromosome doubling polypeptide, a transgene, and / or genome-editing components.

[0465] Isolated microspores are cultured in induction medium from a single-cell microspore to a microspore-derived multicellular structure or a microspore-derived embryo-like structure. Microspore-derived structures are subsequently placed in Agrobacterium infection medium to be transformed with plasmids encoding morphogenic developmental polypeptide(s) for microspore embryogenesis induction, a genetic chromosome doubling polypeptide, a transgene, and / or genome-editing components.

[0466] Robotic arms, automated liquid handlers, and / or manipulative tooling are used for media changes during microspore culture, transfer of microspores to solid medium, and / or transfer of microspore-derived regenerated plantlets to a greenhouse.Example 8: Automated method and apparatus for image-based characterization and selection of microspores and microspore-derived structures

[0467] Example 8A

[0468] In a first image-based method for sampling, sorting, and / or selecting, image segmentation, morphometry, and color / spectral analysis of tassels, anthers, immature flowers, or reproductive plant parts are used to determine microspore quality, potential for development, and developmental stage is used. Automated steps include image acquisition of tassels, anthers, immature flowers, orDocket No.: 214105-WO-SEC-l reproductive plant parts, programming image acquisition software, and processing images to determine microspore health and developmental stage.

[0469] Example 8B

[0470] In a second image-based method for sampling, sorting, and / or selecting, microspore segmentation and is used for fluorescent label-free measurement of the count, viability, and average values of key morphological properties of microspores. In this method, microspores can be isolated and diluted, imaged by brightfield microscopy, and the resulting images processed to determine microspore count and viability. Automated steps include microspore image acquisition, programming image acquisition software, and processing microspore images to determine count and viability (e.g., binarization and segmentation, morphology and intensity analysis, viability analysis).

[0471] Example 8C

[0472] In a third image-based method for sampling, sorting, and / or selecting, an algorithm for label-free nuclei segmentation and morphometry is used for label-free microspore ploidy determination. In this method, microspore are imaged by z-stack, multi -position brightfield microscopy and the resulting images processed to determine microspore ploidy and chromosome doubling. Automated steps include microspore image acquisition, programming image acquisition software, and processing microspore images to determine ploidy (e.g., microspore segmentation, viability analysis, Z-stack edge detection and filling, identification and segmentation of nuclei, and signal processing to optimize nuclei detection). In addition to determining ploidy and chromosome doubling, this method enables deriving correlations between microspore developmental stage and potency for embryogenesis induction.

[0473] Example 8D

[0474] In a fourth image-based method for sampling, sorting, and / or selecting, microspore segmentation is used for cargo delivery analysis. In one example, automated measurement of the relative concentration of cargo in individual microspores and germination pore can be used to assess cargo delivery using fluorescence-based delivery assays. In another example, automated bright field / phase contrast screening of microspore, germination pore, and cell wall can be used to detect defects caused by physical or digestion-based delivery assays. In this method, microspores are imaged, simultaneously or sequentially, by brightfield and wide-field fluorescence / confocal microscopy (automated z-stack multi-position image sequence acquisition) and the resultingDocket No.: 214105-WO-SEC-l images processed to determine whether or not microspores contain cargo. Automated steps can include microspore image acquisition, programming image acquisition software, and processing microspore images to determine cargo presence (e.g., segmentation, z-stack / 3D edge detection, pore identification and 3D segmentation, and normalization steps and intensity analysis).

[0475] Example 8E

[0476] In a fifth image-based method for sampling, sorting, and / or selecting, colorimetric or morphometry analysis of microspore structural development from microspore to microspore- derived multicellular structure (MCS) to microspore-derived embryo-like structure (ELS) using z- stack brightfield microscopy is used for sampling, sorting, and / or selecting. In this method, following microspore isolation, embryogenesis induction, and plating / embedding, microspores / MCS / ELS are imaged by are imaged by wide-field fluorescence / confocal microscopy (automated z-stack multi-position image sequence acquisition) at multiple time points, and the resulting microspores / MCS / ELS z-stack / 3D images processed to determine morphometry at different time points. Automated steps can include microspore / MCS / ELS image acquisition, programming image acquisition software, and processing microspore / MCS / ELS images to determine morphometry.

[0477] Example 8F

[0478] In a sixth image-based method for sampling, sorting, and / or selecting, analysis of microspore-derived macroscopic structures is used for sampling, sorting, and / or selection. More specifically, images of macroscopic structures are segmented and analyzed to determine count, color matrix, and morphology of macroscopic structures. In this method, following microspore isolation, embryogenesis induction, and plating / embedding, upright color images of macroscopic structures are captured with a camera and image segmentation, morphometry, and RGB analysis are performed. Automated steps can include microspore-derived macroscopic structure image acquisition, programming image acquisition software, and processing microspore-derived macroscopic structure images to perform segmentation, morphometry, and color analysis.

[0479] Example 8G

[0480] Microspore sampling can also include automated selection methods following image analysis.

[0481] In a first selection method for sampling, sorting, and / or selecting, robotic handling is used to collect microspore-derived multicellular structures and microspore-derived embryo-likeDocket No.: 214105-WO-SEC-l structures in liquid medium based on analyzed images. Highly specific selection of desired structures early in embryogenesis can improve paternal DH-based production efficiency. In this method, microspore-derived multicellular structures and / or microspore-derived embryo-like structures are imaged, the images processed and analyzed, and the desired, selected structures collected via robotic handling. Automated steps can include image acquisition, programming image acquisition software, processing images, programming and control of robotic handling, and selection of microspore-derived structures.

[0482] Example 8H

[0483] In a second selection method for sampling, sorting, and / or selecting, a robotic system is used to collect microspore-derived macroscopic structures in solid medium based on analyzed images. In this method, macroscopic structures are imaged, the images processed and analyzed, and the desired, selected structures collected via the robotic system. Automated steps can include image acquisition, programming image acquisition software, processing images, programming and control of robotic system, and selection of microspore-derived macroscopic structures.

[0484] Example 9: Methods for microspore extraction

[0485] This example compares methods for extracting microspores from sterilized immature florets or flower buds of a plant, including mechanical disruption (ball mill, blender, juicer) and manual slicing.

[0486] Following sterilization and drying, immature anther-containing florets were removed and gathered into sterile containers. Florets were evenly distributed into sample tubes or cups to a single tassel equivalent, and isolation media was added to submerge the florets.

[0487] A first group of florets were sliced into 1-2 mm sections using a scalpel. Sliced material was transferred to a sieve and dipped in isolation media. A second group of florets underwent mechanical disruption using a ball mill. Beads of various sizes (small BBs, large (’A”) BBs, and UFO-shaped BBs) were loaded into each tube and isolation media added. Tubes were transferred to a ball mill, which agitated samples at 1500 rpm for 120-240 seconds. A third group of florets underwent mechanical disruption using a blender with a programmable controller. Sample cups containing florets suspended in isolation media were capped and loaded onto a blender with a preset speed and duration (speed 6 for 3-6 seconds, with tamping between cycles). A fourth group of florets underwent mechanical disruption using a juicer. Samples were placed in the input sideDocket No.: 214105-WO-SEC-l of the juicer with collection cups on the dry and liquid outlets. Isolation media was added during juicing to facilitate flow and extraction.

[0488] Samples were tracked individually. Microspores were plated and counted to determine yield. Results are shown in Table 3. These data demonstrate mechanical disruption by blending or milling yielded the highest microspore counts.Table 3; Microspore yield based on extraction method

[0489] Example 10: Mesh microspore culture system

[0490] Example 10A.

[0491] This example describes a mesh culture system for haploid induction and germination of microspore-derived structures.

[0492] Following maize microspore isolation, microspores were plated on mesh inserts having a pore size of 35 microns. The pore size of the mesh insert, which is smaller than the size of maize microspores, traps the microspores on top of the insert. The microspore-containing mesh insert was placed into a petri dish with liquid media (FTG. 13A). After 5 days of 32°C heat stress, the mesh insert was lifted from the petri dish, with the microspores being retained in the mesh, and reinserted into a new petri dish with fresh induction media (FIG. 13B).

[0493] Microspores were incubated under dark conditions for 25 days at 28°C to derive macroscopic structures on the mesh (FIG. 13C). To germinate macroscopic structures, the mesh was reversed and the macroscopic structures deposited onto a solid media plate by gently shaking the mesh (FIG. 13D). Macroscopic structure-derived plantlets regenerated on the solid media (FIG. 13E)

[0494] FIG. 14A is an image of microspore-derived macroscopic structures growing on the mesh insert in liquid media after 30 days in culture (i.e., 30 days from microspore isolation). FIG. 14B is an image of the inverted mesh insert for transfer of macroscopic structures to solid media. FIG. 14C is an image of macroscopic structures transferred to solid germination media.

[0495] Example 10B.Docket No.: 214105-WO-SEC-l

[0496] This example describes a mesh culture system for haploid induction, chromosome doubling, and germination of microspore-derived structures.

[0497] Microspores were isolated, plated, and cultured in liquid media as described in Example 10B. Microspores were incubated under dark conditions for 25 days at 28°C to derive macroscopic structures on the mesh insert. The macroscopic structure-containing mesh insert was placed into a petri dish with liquid media and a chromosome doubling agent. After 24 hours, the mesh insert was reversed and the macroscopic structures deposited onto a solid media plate for germination and plantlet regeneration. Macroscopic structure-derived plantlets regenerated on the solid media.

[0498] FIG. 15A is an image of microspore-derived macroscopic structures growing on the mesh insert in liquid media after 30 days in culture (i.e., 30 days from microspore isolation). FIG. 15B is an image of the mesh insert with macroscopic structures in liquid media and a chromosome doubling agent. FIG. 15C is an image of the inverted mesh insert for transfer of macroscopic structures to solid media. FIG. 15D is an image of macroscopic structures transferred to solid germination media.

[0499] Example 11: Microfluidic dielectrophoresis of microspores and microspore-derived structures

[0500] This example describes a microfluidic system utilizing dielectrophoresis (DEP) for culturing, enrichment, sorting, and / or characterization of microspores and microspore-derived structures.

[0501] Example 11A

[0502] FIG. 16 is a diagram of microfluidic system 70 utilizing dielectrophoresis (DEP) for sorting cells and multicellular structures. The flow cell chip 72 was placed on a microscope stage for process viewing by microscope 74. The flow cell contained three input ports 76 and three output ports 78. The outer two input ports were connected to syringe pumps 80a, 80b operating in infusion mode in a range of flow rates (400 uL / min - 700 uL / min). The outer syringe pumps 80a, 80b provided the laminar flow stream of carrier liquid to focus the input stream of cells. The middle input port was connected to an elevated reservoir 82 containing the cells in suspension. The cell suspension was passively drawn into the flow cell by gravity, and the flow rate difference between the input and output. The three output ports 84a, 84b, 84c were connected to syringe pumps operating in withdraw mode in a range of flow rates (500 uL / min to 1200 uL / min). All syringeDocket No.: 214105-WO-SEC-l pumps connected to the flow cell using 20 gauge blunt end needles and FEP tubing with an I.D. of 0.8 mm. The reservoir-containing cells connected to the flow cell using FEP tubing with an I.D. of 0.5 mm. Cells in the reservoir were maintained in suspension through continuous mixing with an overhead stir probe

[0503] Interaction of microspores within the dielectric field gradient is described, e.g., in US20230191427A1, incorporated hereinby reference. The effective range includes a frequency of 50 kHz - 1.5 MHz and a voltage of 1 Vpp - 100 Vpp. To specifically sort the microspores by viability and size, a narrower frequency and voltage range of 800 kHz - 1.3 MHz and 20 Vpp - 50 Vpp was utilized.

[0504] The DEP system, capable of processing 30,000 cells / minute, was applied to the enrichment of maize microspore-derived multicellular structures 5 days into the culture process (5 days postmicrospore isolation). DEP selection of the multicellular structures was based on cell size with the multicellular structures being greater than 120 pm.

[0505] The percentage of multicellular structures (MCS) present in 5-day old microspore cultures before and after DEP sorting are shown in FIG. 17. The flowthrough population are the MCS that experienced negative DEP selection and passed through the middle channel. Input cultures, on average, started at 11% MCS. The flowthrough average was 8% MCS and the DEP selected average was 54%. The maximum MCS enrichment reached through DEP selection was 94% MCS. These data demonstrate that DEP selection provided an average 6-fold enrichment of multicellular structures as compared to the input population (n=30).

[0506] FIG. 18A is a representative image of 5-day old microspore cultures before DEP sorting (input; circled cells indicate multicellular structures). Images of MCS microspore cultures after DEP sorting are shown in FIG. 18B (flowthrough) and FIG. 18C (DEP selected).

[0507] Example 11B

[0508] In a second experiment, a COMSOL 2D simulation of AC DEP in a microfluidic channel demonstrated that a large difference between nucleus electric conductivity of live and dead maize microspores is a primary factor in sorting of live and dead microspores.

[0509] FIG. 19 shows components of the two-shell model used for simulating the movement of a plant cell under AC electric field using COMSOL. In this model, the electric conductivity of a live plant nucleus is 25 times greater than the dead cell nucleus resulting in di electrophoretic separation.Docket No.: 214105-WO-SEC-l

[0510] Example 11C

[0511] In a third experiment, an AmphaZ32 impedance flow cytometer demonstrated that electrical impedance to AC field varies by cell viability and developmental stage of maize microspores. The clustering by developmental stage in amplitude-phase response graphs of the cytometer mirrored the number of nuclei present in a microspore. Impedance flow cytometry phase measurements showed a noticeable shift in the microspore phase distribution between the unsorted input population and the DEP sorted output population, which experience positive or negative DEP forces (FIGS. 20A- 20D and FIGS. 21A- 21D).

[0512] FIGS. 20A - 20D show Amphasys impedance flow cytometry measurements of maize micropores before and after DEP sorting using flow cell chips. R values on each graph show the percent of viable cells highlighted in black. Dark grey cells were excluded from the population and light grey cell are the non-viable microspores. The bottom (FIG. 20D) and top (FIG. 20B) channel outlets that collect microspores experiencing positive DEP are enriched for viable cells as compared to the input population (FIG. 20A) and microspores that do not experience DEP and pass through the middle outlet (FIG. 20C).

[0513] FIGS. 21A - 21D are violin plots showing the phase distribution of viable microspores from FIGS. 20A - 20D. The median phase for viable sorted microspores that experience strong attractive (positive) DEP (FIG. 21B and FIG. 21D) is shifted upward compared to the input population (FIG. 21A) and microspores that do not experience attractive DEP and pass through the middle outlet (FIG. 21C).

[0514] Collectively, the data of Examples 11B and 11C confirm electrical impedance differs with cell viability and developmental stage and thus supports viability and developmental stage population enrichment via high throughput DEP separation.Example 12: Non-destructive sampling of microspore-derived structures

[0515] This example describes methods of non-destructively obtaining the genotype of microspore-derived structures at one or more steps in the automated workflow.

[0516] Example 12A: Sampling of macroscopic structures based on isolation buffer

[0517] Microspore culture:

[0518] Microspores were isolated from tassels and cultured in induction media for development of macroscopic structures for 40 days.Docket No.: 214105-WO-SEC-l

[0519] Macroscopic structure selection:

[0520] Individual macroscopic structures (~2-3 mm) were removed from bulk culture and transferred into wells of a 96-well plate containing 100 pL of fresh induction media per well, with 6 macroscopic structures being selected per treatment condition. After selection and transfer, macroscopic structures were rinsed three times with 100 pL of fresh induction media to remove extraneous debris or DNA cross-contamination from the bulk culture.

[0521] DNA collection:

[0522] After the final rinse, the induction media was removed from each well and replaced with 100 pL of isolation buffer. In this Example, five isolation buffers were tested: (1) Tri s-EDTA buffer pH 8.0; (2) NaOH extraction buffer; (3) 20% NaOH extraction buffer / 80% Tris-EDTA; (4) 50% NaOH extraction buffer / 50% Tris-EDTA; and (5) microspore induction media. The 96-well plates were closed, wrapped in foil, placed on a VarioMag shaker plate at -60% max speed, and shaken for 60 minutes. After agitation, isolation buffer was transferred to a 96-well PCR plate and the macroscopic structures were plated onto solid media for continued culture and germination. Macroscopic structures that did not undergo shed cell collection served as a negative control.

[0523] Molecular analysis:

[0524] Immediately after collection and transfer, the PCR plate containing isolation buffer with macroscopic structure shed cellular material was incubated at 95°C for 20 minutes, and then used for a genotyping assay via TAQMAN assays (Life Technologies). A 384-well plate containing DNA and assay was loaded onto a Quantstudio 7 real-time PCR thermocycler and run with the following program: preincubation: 1 cycle of 95°C for 10 minutes; amplification: 45 cycles of 95°C for 15 seconds followed by 60°C for 60 seconds. Two PCR replicates were performed per sample (three for samples isolated via microspore induction media). Calls were read using Quantstudio 7 software.

[0525] Results:

[0526] The percent genotyping data return of DNA isolated from macroscopic structures based on isolation buffers is shown in FIG. 22. The Tris-EDTA (TE) and NaOH isolation buffers showed consistent genotyping data return while the induction media showed lower data return. These data demonstrate successful sampling of macroscopic structures for genotyping analysis.

[0527] Example 12B: Sampling of macroscopic structures based on agitation conditions

[0528] Microspore culture:Docket No.: 214105-WO-SEC-l

[0529] Microspores were isolated from tassels and cultured in induction media for development of macroscopic structures for 40 days.

[0530] Macroscopic structure selection:

[0531] Individual macroscopic structures (~2-3 mm) were removed from bulk culture and transferred into wells of a 96-well plate containing 100 pL of fresh induction media per well, with 48 macroscopic structures being selected per treatment condition. After selection and transfer, macroscopic structures were rinsed three times with 100 pL of fresh induction media to remove extraneous debris or DNA cross-contamination from the bulk culture.

[0532] DNA collection:

[0533] After the final rinse, the induction media was removed from each well and replaced with 100 pL of Tris-EDTA buffer. The 96-well plates were closed, wrapped in foil, placed on a VarioMag shaker plate at -60% max speed. In this Example, four agitation durations were tested: 15, 30, 45, or 60 minutes. After agitation, TE buffer was transferred to a 96-well PCR plate and the macroscopic structures were plated onto solid media for continued culture and germination. Macroscopic structures that did not undergo shed cell collection served as a negative control.

[0534] Molecular analysis:

[0535] Immediately after collection and transfer, the PCR plate containing TE buffer with macroscopic structure shed cellular material was incubated at 95°C for 20 minutes, and then used for a genotyping assay via TAQMAN assays (Life Technologies). A 384-well plate containing DNA and assay was loaded onto a Quantstudio 7 real-time PCR thermocycler and run with the following program: preincubation: 1 cycle of 95°C for 10 minutes; amplification: 45 cycles of 95°C for 15 seconds followed by 60°C for 60 seconds. Two PCR replicates were performed per sample. Calls were read using Quantstudio 7 software.

[0536] Continued macroscopic structure culture:

[0537] After sampling, macroscopic structures were transferred to solid germination media plates for further culture and development. Plates were incubated for ~ 6 weeks, and structures were observed for further growth and root / shoot formation.

[0538] Results:

[0539] The percent genotyping data return of DNA isolated from macroscopic structures based on agitation duration is shown in FIG. 23. Each agitation duration condition showed similarDocket No.: 214105-WO-SEC-l genotyping data return (89.58% - 97.92%). These data demonstrate all agitation duration conditions resulted in successful sampling of macroscopic structures for genotyping analysis.

[0540] FIG. 24 shows the percent of macroscopic structures having no observed further growth after solid media culture. FIG. 25 shows the percent of macroscopic structures showing root or shoot formation after solid media culture. Collectively, these data demonstrate successful root and shoot formation of macroscopic structures following DNA collection for genotyping.

[0541] Example 12C: Sampling of macroscopic structures without agitation

[0542] Microspore culture:

[0543] Microspores were isolated from tassels and cultured in induction media for development of macroscopic structures for 40 days.

[0544] Macroscopic structure selection:

[0545] Individual macroscopic structures (~2-3 mm) were removed from bulk culture and transferred into wells of a 96-well plate containing 100 pL of fresh induction media per well, with 48 macroscopic structures being selected per treatment condition. After selection and transfer, macroscopic structures were rinsed three times with 100 pL of fresh induction media to remove extraneous debris or DNA cross-contamination from the bulk culture.

[0546] DNA collection - with agitation

[0547] After the final rinse, the induction media was removed from each well and replaced with 100 pL of Tris-EDTA buffer. The 96-well plates were closed, wrapped in foil, placed on a VarioMag shaker plate at -60% max speed. The plates were shaken for 15 minutes. After agitation, TE buffer was transferred to a 96-well PCR plate and the macroscopic structures were plated onto solid media for continued culture and germination. Macroscopic structures that did not undergo shed cell collection served as a negative control.

[0548] DNA collection - no agitation

[0549] After the final rinse, the induction media was removed from each well and replaced with 100 pL of Tris-EDTA buffer. The plates sat without agitation for 15 minutes before TE buffer was transferred to a 96-well PCR plate and the macroscopic structures were plated onto solid media for continued culture and germination.

[0550] Molecular analysis:

[0551] Immediately after collection and transfer, the PCR plates containing TE buffer with macroscopic structure shed cellular material was incubated at 95°C for 20 minutes, and then usedDocket No.: 214105-WO-SEC-l for a genotyping assay via TAQMAN assays (Life Technologies). A 384-well plate containing DNA and assay was loaded onto a Quantstudio 7 real-time PCR thermocycler and run with the following program: preincubation: 1 cycle of 95°C for 10 minutes; amplification: 45 cycles of 95°C for 15 seconds followed by 60°C for 60 seconds. Two PCR replicates were performed per sample. Calls were read using Quantstudio 7 software.

[0552] Results:

[0553] Data return from samples without agitation dropped to 46.32%, compared to 94.85% data return with a 15 minute agitation step.Example 13: Early singulation and non-destructive sampling of microspore-derived structures

[0554] This example describes early singulation of microspore-derived structures.

[0555] Microspore culture:

[0556] Microspores were isolated from tassels and cultured in induction media for development of macroscopic structures for 22, 28, 35, or 40 days.

[0557] Macroscopic structure selection:

[0558] At 22, 28, 35, and 40 days after microspore isolation, individual visible macroscopic structures (1-3 mm) were removed from bulk culture and transferred into wells of a 96-well plate containing 100 pL of fresh induction media per well, with 96 macroscopic structures being selected per treatment condition. After selection and transfer, macroscopic structures were rinsed three times with 100 pL of fresh induction media to remove extraneous debris or DNA crosscontamination from the bulk culture. As a control, additional plates were prepared at 22 days and 40 days after microspore isolation that singulated the macroscopic structures, but did not undergo any agitation or DNA collection.

[0559] DNA collection

[0560] After the final rinse, the induction media was removed from each well and replaced with 100 pL of Tris-EDTA buffer. The 96-well plates were closed, wrapped in foil, placed on a VarioMag shaker plate at -60% max speed. The plates were shaken for 15 minutes. After agitation, TE buffer was transferred to a 96-well PCR plate and the macroscopic structures were plated onto solid media for continued culture and germination. Macroscopic structures that did not undergo shed cell collection served as a negative control.Docket No.: 214105-WO-SEC-l

[0561] Molecular analysis:

[0562] Immediately after collection and transfer, the PCR plates containing TE buffer with macroscopic structure shed cellular material was incubated at 95°C for 20 minutes, and then used for a genotyping assay via TAQMAN assays (Life Technologies). A 384-well plate containing DNA and assay was loaded onto a Quantstudio 7 real-time PCR thermocycler and run with the following program: preincubation: 1 cycle of 95°C for 10 minutes; amplification: 45 cycles of 95°C for 15 seconds followed by 60°C for 60 seconds. Two PCR replicates were performed per sample. Calls were read using Quantstudio 7 software.

[0563] Continued macroscopic structure culture

[0564] After sampling, fresh induction media was added to each of the sampled macroscopic structures and plates returned to culture for the remainder of the 40 day incubation period. At the end of the incubation period, macroscopic structures were transferred to solid germination media plates for further culture and development. Plates were incubated for ~ 6 weeks, and structures were observed for further growth and root / shoot formation.

[0565] Results:

[0566] The percent genotyping data return of DNA isolated from macroscopic structures based on sampling date post-microspore isolation is shown in FIG. 26. Data return from macroscopic structures sampled as early as 22 days after isolation were comparable to those sampled at 40 days post-isolation. These data demonstrate macroscopic structures successfully undergo singulation and sampling / genotyping before 40 days of culture in induction media.

[0567] FIG. 27 shows the percent of macroscopic structures having no observed further growth after solid media culture. FIG. 28 shows the percent of macroscopic structures showing root or shoot formation after solid media culture. Comparable rates of macroscopic structure death and further root and shoot development were observed at the earlier sampling dates, as well as those that did not undergo sample collection (“22 DAI no shed” and “40 DAI no shed” in FIGS. 27 and 28). A representative image of macroscopic structures A, B, and C 76 days after microspore isolation and 54 days after sampling are shown in FIG. 29. Collectively, these data demonstrate successful root and shoot formation of macroscopic structures following early singulation and DNA collection for genotyping. Continued growth and development of macroscopic structures post-sampling / genotyping was observed, with development being comparable to macroscopic structures that did not undergo sampling.Docket No.: 214105-WO-SEC-l

[0568] Example 14: Methods for microspore classification by developmental stage and ploidy

[0569] Example 14A:

[0570] Immature tetrapioid tassels producing diploid microspores and immature diploid tassels producing haploid microspores were harvested from greenhouse grown maize plants and stored at 10°C for five to ten days. Microspores were isolated from spikelets via anther squashes.

[0571] Z-stack bright field images of microspore training dataset comprising microspores were captured using Nikon Eclipse JI inverted microscope equipped with a CFi60 Plan Apochromat Lambda D 20x objective lens, a 7.8 MP monochrome CMOS camera, and Perfect Focusing System. DIC-like high resolution bright field images using 20x objective lens depict cytoplasmic contents.

[0572] Automated label-free nuclei detection for developmental stage quantification of viable microspores includes nucleus morphometry analysis (diameter, perimeter, area, aspect ratio, and circularity) for classifying microspores by developmental stage (i.e., uninucleate, early binucleate, late binucleate) and health (viable or nonviable).

[0573] After Z-stacks were captured, the file directory containing the Z-stacks, the objective magnification, Z-interval, and intensity / morphology thresholds were input into an image analysis program. The following intensity / morphology thresholds were used:

[0574] First and last image numbers within Z-stack sequence to be processed. Default values were 1 and the last image number in folder.

[0575] The ratio of average to maximum microspore pixel intensity was measured for viability determination. The default value was set to 0.7.

[0576] Microspore area for viability determination. The default value was set to 2700 pm2, below which microspores are considered unviable.

[0577] Normalized intensity threshold for elimination of high intensity pixels from corresponding binary images in Z-stack. These signals are normally located on the cell pore and wall, and interfere with nuclei detection. Default value is set to 0.7.

[0578] After user inputs were complete, the image analysis program was run on a computer. Z- stack images were assembled, inverted, and stored in 3D array and / or MATLAB cells. High- intensity pixels from raw microspore images were eliminated from the data.Docket No.: 214105-WO-SEC-l

[0579] Once initial processing was complete, microspores were segmented. The magnified images required additional image transformation and filling operations to achieve precise segmentation. The step-by-step operations for image segmentation are listed below:

[0580] Image binarization

[0581] Object filling

[0582] Removing small outlier objects

[0583] Erase object on imager borders

[0584] Distance transform and object-object contact detections

[0585] Isolation (segmentation) of touching objects

[0586] Closing gaps in object (microspore) boundary

[0587] Microspore pixel filling

[0588] Distance transform and microspore-microspore contact detections

[0589] Isolation of touching microspores

[0590] Labeling isolated (segmented) microspores

[0591] Counting the number of microspores by counting the number of labels

[0592] Additional threshold parameters were needed for binary classification of microspores by viability. If at least one of the 4 thresholds were not met, the microspore is unviable:

[0593] Parameter 1 : normalized intensity threshold as metric for microspore whiteness under brightfield in monochromatic images. Dark microspores are known to be unviable.

[0594] Parameter 2: the standard deviation of microspore pixel intensities as metric for cytoplasm intactness. Collapsed cytoplasm is one of the main signs of microspore death.

[0595] Parameter 3 : microspore elliptical aspect ratio threshold as the metric for microspore shape. There were a large number of highly deformed unviable microspores in the suspension.

[0596] Parameter 4: distance-transformed pixel intensity threshold as a metric for optically focused objects. Some shrunken, unviable microspores were suspended out of the optical plane of focus for imaging and appear blurry with significantly higher distance-transformed pixel intensity.

[0597] Z-stacks of viable microspores were then processed for nuclei segmentation and counting as described below:

[0598] Nuclei edge detection image analysisDocket No.: 214105-WO-SEC-l

[0599] After segmentation was performed, a first binary detection of the edges of the microspore walls, the germination pores, and the nuclei was performed for all image planes. A second binary detection was also performed for the edges of the microspore walls and the germination pores in all image planes, without detection of the nuclei. Logical subtraction of dilated edges from the first and second binary detections was performed to provide nuclei edges in all image planes.

[0600] In order to provide binary nuclei edges in all image panes, secondary dilations of edges in the second binary detection were calculated using morphological structuring elements with specific radii. These secondary dilations were also subtracted from the first binary detection in order to provide binary nuclei edges in all image planes.

[0601] Binary nuclei edges were dilated and filled resulting in filled binary nuclei edges in all image planes. The filled edges were then multiplied by the corresponding bright field image at each focal plane to get intensity matrix of the detected nuclei. The output stack was then concatenated to get 3D intensity matrix of nuclei edges. Maximum intensity projection as well as dilation was performed on the concatenated matrix for displaying the output or performing k- means clustering for nuclei classification.

[0602] Line scanning and intensity signal processing

[0603] Pixel values (intensity), rows (y-coordinate), and columns (x-coordinate) of segmented microspores for all images within the Z-stack were vectorized and tabulated.

[0604] The pixel columns (vertical pixel lines) which cross filled binary nuclei edges were identified.

[0605] Pixel values of each column were normalized to the focal plane’s maximum microspore intensity. The normalized pixel values and associated row numbers (y-coordinate) were stored as vector pairs.

[0606] Gaussian curve fitting was performed on vector pairs at all focal planes of the segmented microspores.

[0607] Peak value, peak thickness, and peak prominence for fitted curves were identified and stored as 3 -component groups.

[0608] Certain peak groups with specific ranges for group components belong to a nucleus signal. If all 3 components of a group fall within these ranges and their x and y coordinates belong to a nucleus signal detected from the edge filling operation, the group along with its coordinates are stored as a true nucleus signal.Docket No.: 214105-WO-SEC-l

[0609] Example 14B

[0610] Microspore size is used to determine microspore ploidy, thereby enabling the identification of chromosome-doubled microspores. As shown in FIG. 30A, diploid microspores of a tetrapioid line are significantly larger than haploid microspores from a diploid line (FIG. 30B).

[0611] Tetrapioid plants are generated and the area as a measure of size of the diploid microspores are compared with the area of haploid microspores of the same parent line via a student’s t test to determine if microspore size differs significantly between haploid and diploid cells of the same genetic background. In addition to area, measures such as circularity, aspect ratio, and eccentricity are analyzed to determine significant differences in morphology between haploid and diploid cells of the same genetic background.

[0612] It is expected there will be a significant difference between haploid and diploid size as measured by area, and that this difference will be of sufficient size relative to the variation in area within the haploid and diploid microspores to determine a threshold for microspore ploidy determination.

[0613] Example 14C

[0614] Diploid and haploid microspores of the same tetrapioid parent line are affixed in media using a low melting point agarose and their growth patterns monitored over the course of several days to determine growth trends between the two groups.

[0615] It is expected that diploid microspores will grow at the same rate as the paired haploid controls while maintaining the difference in size and morphology throughout the time course.

Claims

Docket No.: 214105-WO-SEC-lCLAIMS1. A high-throughput, automated method for generating microspore-derived fertile doubled haploid crop plants, the method comprising:(a) selecting tassels, anthers, immature flowers, tillers, or reproductive plant parts at an appropriate growth stage and harvesting tassels, anthers, immature flowers, or reproductive plant parts from crop plants with an automated selection and harvesting assembly, the automated selection and harvesting assembly comprising a first computer vision processing system or image processing system and a computer-vision guided robotic assembly, the first computer vision processing system or image processing system images the tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts and the computer- vision guided robotic assembly removes the tassels, anthers, immature flowers, or reproductive plant parts from the crop plants, wherein the crop plants are optionally located in a field or greenhouse or have been removed from the field using agricultural harvesting equipment, wherein selected tassels, anthers, immature flowers, or reproductive plant parts are optionally stored in an automated storage assembly;(b) sterilizing the selected tassels, anthers, immature flowers, tillers, or reproductive plant parts with an automated sterilization assembly that receives the tassels, anthers, immature flowers, or reproductive plant parts in one or more containers either singularly or in batches, dispenses and removes one or more sterilization agents from the one or more containers after a pre-set duration, and optionally provides mechanical agitation to increase surface contact of the tassels, anthers, immature flowers, or reproductive plant parts with the one or more sterilization agents to obtain sterilized tassels, anthers, immature flowers, or reproductive plant parts;(c) extracting microspores from the sterilized tassels, anthers, immature flowers, tillers, or reproductive plant parts with an automated extraction assembly that receives the sterilized tassels, anthers, immature flowers, tillers, or reproductive plant parts in one or more containers or optionally in a larger batch-process container, dispenses and removes media from the containers or optionally the larger batch-process container, and extracts a substantial portion of the microspores from the sterilized tassels, anthers, immature flowers, tillers, or reproductive plant parts by applying a mechanical force to the one or more containers;Docket No.: 214105-WO-SEC-l(d) separating extracted microspores from non -microspore plant material by fdtering microspore-containing media through one or more fdtering assemblies to collect the extracted microspores in one or more containers or pooled in a large-batch processing container and optionally the one or more containers or the large-batch processing container comprising the extracted microspores are subjected to a centrifugal force to further separate remaining nonmicrospore plant material and concentrating the extracted microspores;(e) providing separated microspores with a culture media that promotes microspore embryogenesis induction such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores are distributed in a plurality of containers, the plurality of containers capable of being handled by a robotic handler for a high-throughput, continuous or a semi-continuous workflow, optionally wherein the microspores are dispensed into the plurality of containers at a target concentration or density;(f) optionally providing cargo to the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures for introducing (i) one or more sitespecific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing to the microspores, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex;(g) optionally providing the culture media with a chromosome doubling agent;(h) culturing the microspores, multicellular structures, embryo-like structures, and / or macroscopic structures in an automated incubation assembly;(i) sorting the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures with an automated sorting assembly comprising a second computer vision processing system or image processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the second computer vision processing system or image processing system;Docket No.: 214105-WO-SEC-l(j) selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures with an automated selection assembly, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model;(k) growing the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures in a growing station adapted to work with an automated handling and sampling system that samples and tracks the multicellular structures, the embryolike structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for genetic determination, and further culturing the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to generate microspore- derived doubled haploid plantlets; and(l) transferring the microspore-derived doubled haploid plantlets to growth medium for regeneration of doubled haploid crop plants.

2. The method of claim 1, wherein the automated extraction assembly extracts microspores from the sterilized tassels, anthers, immature flowers, tillers, or reproductive plant parts by blending, milling, mashing, slicing, chopping, or shredding.

3. The method of claim 1, wherein selected tassels, anthers, immature flowers, tillers, or reproductive plant parts comprise a substantial number of microspores that are in the uninucleate to binucleate stage.

4. The method of claim 1, wherein the microspores are extracted from the sterilized tassels, anthers, immature flowers, tillers, or reproductive plant parts by a mechanical blending process.Docket No.: 214105-WO-SEC-l5. The method of claim 1, wherein the microspores within the tassels, anthers, immature flowers, tillers, or reproductive plant parts comprise a pre-existing heterologous genetic element that promotes microspore embryogenesis.

6. The method of claim 1, wherein the cargo comprises a site-specific genome editing agent or system.

7. The method of claim 1, wherein the culture media comprises an embryogenesis inducing agent.

8. The method of claim 1, wherein the chromosome doubling agent is a chemical agent.

9. The method of claim 1, wherein the method further comprises singulating the multicellular structures, the embryo-like structures, and / or the macroscopic structures from batch culture.

10. The method of claim 1, further comprises: sampling the multicellular structures, the embryo-like structures, and / or the macroscopic structures at step (h), (i), (j), (k), or a combination of foregoing; and / or sampling the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures at step (k).

11. The method of claim 1, wherein the crop plant is maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola.

12. The method of claim 1, wherein a fluorescent dye is added to the culture media for enhanced imaging.

13. The method of claim 1, wherein the first computer vision processing system or image processing system and / or the second image processing system obtain a series of images at various intervals to develop a time-dependent growth stage for the tassels, anthers, immature flowers, or reproductive plant parts and / or the single-cell microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures.Docket No.: 214105-WO-SEC-l14. The method of claim 1, wherein the one or more filtering assemblies comprise a reverse flow apparatus to separate the microspores from the non-microspore plant material.

15. The method of claim 1, wherein the selected tassels, anthers, immature flowers, or reproductive plant parts are stored in a temperature-controlled chamber prior to sterilization.

16. The method of claim 1, wherein the tassels, anthers, immature flowers, or reproductive plant parts are harvested after the crop plants are exposed to an embryogenesis inducing agent and / or to a site-specific genome editing agent or system such that the embryogenesis inducing agent and / or the site-specific genome editing agent or system acts upon the microspores within the tassels, anthers, immature flowers, or reproductive plant parts.

17. The method of claim 1, wherein the second computer vision processing system or image processing system is under the control of an artificial intelligence model capable of classifying or determining the developmental stage, viability, ploidy, and / or predicted fertility of the single-cell microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures based on captured images and the determined developmental stage or maturation state is used to facilitate further processing of the single-cell microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures selected therefrom in an automated manner.

18. The method of claim 1, wherein containers in which the multicellular structures, embryo-like structures, and / or macroscopic structures are distributed are robotically transferred to an imaging station as part of the second computer vision processing system or image processing system or alternatively, an imaging modality of the second computer vision processing system or image processing system is brought in close proximity to the containers in which the multicellular structures, embryo-like structures, and / or macroscopic structures are distributed to enable image capture of the multicellular structures, the embryo-like structures, and / or the macroscopic structures.Docket No.: 214105-WO-SEC-l19. The method of claim 1, wherein sorting the multicellular structures, the embryo-like structures, and / or the macroscopic structures comprises sorting by developmental stage, viability, ploidy, and / or predicted fertility.

20. The method of claim 1, wherein sorting the multicellular structures and / or the embryo-like structures comprises introducing a suspension of multicellular structures or embryo-like structures into a microfluidic device and applying a di electrophoretic field to separate the multicellular structures or the embryo-like structures based on developmental stage, viability, or ploidy.

21. The method of claim 1, wherein the genetic determination of the multicellular structures, the embryo-like structures, and / or the macroscopic structures comprises transferring individual multicellular structures, embryo-like structures, and / or the macroscopic structures to a container with a non-destructive medium, agitating the container to collect shed cellular material from the multicellular structures, the embryo-like structures, and / or the macroscopic structures, and genotyping DNA obtained from the shed cellular material.

22. The method of claim 1, wherein the genetic determination of the macroscopic structures occurs 20-50 days after extracting the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts.

23. The method of claim 1, wherein selecting the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures comprises:(a) acquiring an image of each multicellular structure, each embryo-like structure, or each macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same using an imaging modality;(b) segmenting or masking the image of each multicellular structure, each embryo-like structure, or each macroscopic structure;(c) extracting one or more colorimetric features or one or more wavelength -based features from each segmented or masked image; andDocket No.: 214105-WO-SEC-l(d) classifying each multicellular structure, each embryo-like structure, or each macroscopic structure as having a positive predicted fertility outcome or a negative predicted fertility outcome based on extracted colorimetric features or extracted wavelength-based features using the artificial intelligence model.

24. The method of claim 1, wherein selecting the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures comprises:(a) acquiring an image of each multicellular structure, each embryo-like structure, or each macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same using an imaging modality; and(b) classifying each multicellular structure, each embryo-like structure, or each macroscopic structure as having a positive predicted fertility outcome or a negative predicted fertility outcome based directly on the image of each multicellular structure, each embryo-like structure, or each macroscopic structure using a deep learning transformer model.

25. The method of claim 1, wherein the microspore-derived doubled haploid plantlets obtained from the diploidized macroscopic structures are maize, soy, wheat, rice, barley, sorghum, sunflower, brassica, millet, or canola.

26. An end-to-end, high-throughput, automated system for generating microspore-derived doubled haploid crop plants, the system comprising:(a) an automated selection and harvesting assembly for selecting tassels, anthers, immature flowers, tillers, or reproductive plant parts at an appropriate growth stage and removing the tassels, anthers, immature flowers, or reproductive plant parts from crop plants, wherein the automated selection and harvesting assembly comprises a first computer vision processing system or image processing system that images the tassels, anthers, immature flowers, or reproductive plant parts to determine characteristics of the tassels, anthers, immature flowers, or reproductive plant parts and comprises a computer-vision guided robotic assembly to remove the tassels, anthers, immature flowers, or reproductive plant parts from the crop plants, wherein the crop plants are optionallyDocket No.: 214105-WO-SEC-l located in the field or greenhouse or have been removed from the field using agricultural harvesting equipment;(b) an optional automated storage assembly for storing the selected tassels, anthers, immature flowers, or reproductive plant parts;(c) an automated sterilization assembly for sterilizing the selected tassels, anthers, immature flowers, or reproductive plant parts, wherein the automated sterilization assembly receives the tassels, anthers, immature flowers, or reproductive plant parts in one or more containers either singularly or in batches, dispenses and removes one or more sterilization agents from the one or more containers after a pre-set duration, and optionally provides mechanical agitation to increase surface contact of the tassels, anthers, immature flowers, or reproductive plant parts with the one or more sterilization agents;(d) an automated extraction assembly for extracting microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts, wherein the automated extraction assembly receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one more containers or optionally in a larger batch-process container, dispenses and removes media from the one or more containers or optionally the larger batch-process container, and extracts the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by application of a mechanical force to the one or more containers;(e) one or more filtering assemblies that separate the extracted microspores from nonmicrospore plant material by filtering microspore-containing media to collect the separated microspores in one or more containers or pooled in a larger batch-processing container and optionally the one or more containers or the larger batch-processing container comprising the extracted microspores are subjected to a centrifugal force to further separate remaining nonmicrospore plant material and concentrate the extracted microspores;(f) a robotic handler that provides the separated microspores with a culture media, wherein the robotic handler distributes the separated microspores in a plurality of containers for a high- throughput continuous or a semi-continuous workflow;(g) an automated incubation assembly that cultures the microspores such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures areDocket No.: 214105-WO-SEC-l optionally provided cargo for introducing (i) one or more site-specific genomic changes, (ii) one or more transgenes for integration into the genomic DNA, (iii) a morphogenic developmental polypeptide or a polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex, and wherein the automated incubation assembly optionally provides a chromosome doubling agent to the culture media;(h) an automated sorting assembly that sorts the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly comprises a second computer vision processing system or image processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the second computer vision processing system or image processing system;(i) an optional artificial intelligence model that selects the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures predicted to regenerate into fertile that are more likely to germinate into fertile plantlets, wherein the artificial intelligence model has been trained on a dataset comprising microspores, multicellular structures, embryo-like structures, macroscopic structures, and / or plantlets that had positive fertility outcomes (e.g., flowered or set seed);(j) an automated selection assembly that selects diploidized multicellular structures, embryo-like structures, and / or macroscopic structures for a high-throughput continuous or a semi- continuous workflow, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model;(k) a growing station that grows the multicellular structures, the embryo-like structures, and / or the macroscopic structures;Docket No.: 214105-WO-SEC-l(l) an automated handling and sampling system adapted to work with the growing station, wherein the automated handling and sampling system samples and tracks the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for genetic determination, and further cultures the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to obtain microspore-derived doubled haploid plantlets; and(m) a robotic arm that transfers the microspore-derived doubled haploid plantlets to growth medium for regeneration of doubled haploid crop plants.

27. A high-throughput, automated method for generating genome-edited microspore-derived doubled haploid structures, plantlets, crop plants, the method comprising:(a) extracting microspores from sterilized tassels, anthers, immature flowers, tillers, or reproductive plant parts with an automated extraction assembly that receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one or more containers or optionally in a larger batch-process container, dispenses and removes media from the containers or optionally the larger batch-process container, and separates the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by applying a mechanical force to the one or more containers;(b) separating extracted microspores from non -microspore plant material by filtering microspore-containing media through one or more fdtering assemblies to collect the extracted microspores in one or more containers or pooled in a large-batch processing container and optionally the one or more containers or the large-batch processing container comprising the extracted microspores are subjected to a centrifugal force to further separate remaining nonmicrospore plant material and concentrating the extracted microspores;(c) providing separated microspores with a culture media that promotes microspore embryogenesis induction such that the separated microspores progress to multicellular structures, from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores are distributed in a plurality of containers, the plurality of containers capable of being handled by a robotic handler for a high-throughput,Docket No.: 214105-WO-SEC-l continuous or a semi-continuous workflow, optionally wherein the microspores are dispensed into the plurality of containers at a target concentration or density;(d) providing cargo to the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures for introducing (i) one or more site-specific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing to the microspores, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex, wherein introducing the one or more site-specific genomic modifications comprises providing to the microspores, the multicellular structures, the embryolike structures, and / or the macroscopic structures a site-specific genome editing agent or system, optionally wherein the a site-specific genome editing agent or system comprises a Cas polypeptide having DNA binding activity or a polynucleotide sequence encoding the Cas polypeptide and a guide polynucleotide that comprises a region of complementarity to a double-stranded DNA target site in the multicellular structures, the embryolike structures, and / or the macroscopic structures, wherein the Cas polypeptide and the guide polynucleotide form a complex that binds the double-stranded DNA target site and optionally induces a double-strand or single-strand break at the double-stranded DNA target site, wherein the one or more site-specific genomic modifications is insertion, deletion, single nucleotide polymorphism, inversion, or translocation;(e) optionally providing the culture media with a chromosome doubling agent;(f) culturing the microspores, the multicellular structures, the embryo-like structures, and or the macroscopic structures in an automated incubation assembly;(g) sorting the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures with an automated sorting assembly comprising a second computer vision processing system or image processing system that images the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures, wherein the automated sorting assembly selects and transfers the multicellular structures, the embryo-like structures, and / or the macroscopic structures for culture based on images from the second computer vision processing system or image processing system;Docket No.: 214105-WO-SEC-l(h) selecting diploidized multicellular structures, embryo-like structures, and / or macroscopic structures with an automated selection assembly for a high-throughput continuous or a semi-continuous workflow, optionally wherein selecting a diploidized multicellular structure, a diploidized embryo-like structure, and / or a diploidized macroscopic structure comprises selecting a multicellular structure, an embryo-like structure, and / or a macroscopic structure that was contacted with the chromosome doubling agent and / or provided the genetic chromosome doubling polypeptide or polynucleotide encoding the same predicted to regenerate into fertile plantlets based on an artificial intelligence model;(i) growing the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures in a growing station adapted to work with an automated handling and sampling system that samples and tracks the multicellular structures, the embryolike structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures for genetic determination, and further culturing the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures to obtain genome-edited microspore- derived doubled haploid plantlets; and(j) transferring the genome-edited microspore-derived doubled haploid plantlets to growth medium for regeneration of doubled haploid crop plants.

28. A high-throughput, automated method of genotyping microspore-derived doubled haploid structures, the method comprising:(a) extracting microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts with an automated extraction assembly that receives the sterilized tassels, anthers, immature flowers, or reproductive plant parts in one or more containers or optionally in a larger batch-process container, dispenses and removes media from the containers or optionally the larger batch-process container, and separates the microspores from the sterilized tassels, anthers, immature flowers, or reproductive plant parts by applying a mechanical force to the one or more containers;(b) providing separated microspores with a culture media that promotes microspore embryogenesis induction such that the separated microspores progress to multicellular structures,Docket No.: 214105-WO-SEC-l from multicellular structures to embryo-like structures, and from embryo-like structures to macroscopic structures, wherein the microspores are distributed in a plurality of containers, the plurality of containers capable of being handled by a robotic handler for a high-throughput, continuous or a semi-continuous workflow, optionally wherein the microspores are dispensed into the plurality of containers at a target concentration or density;(c) optionally providing cargo to the microspores, the multicellular structures, the embryo-like structures, and / or the macroscopic structures for introducing (i) one or more sitespecific genomic changes, (ii) one or more transgenes for integration into genomic DNA, (iii) a morphogenic developmental polypeptide or polynucleotide encoding the same, (iv) an embryogenesis inducing agent, (v) a genetic chromosome doubling polypeptide or polynucleotide encoding the same, or (vi) a combination of the foregoing to the microspores, wherein the cargo comprises a polypeptide, DNA, RNA, a small molecule, a chemical, and / or a ribonucleoprotein complex;(d) optionally providing the culture media with a chromosome doubling agent;(e) culturing the multicellular structures, the embryo-like structures, and / or the macroscopic structures in an automated incubation assembly to obtain diploidized multicellular structures, diploidized embryo-like structures, and / or diploidized multicellular structures; and(f) genotyping the multicellular structures, the embryo-like structures, the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures with an automated handling sampling system that samples and tracks the multicellular structures, the embryo-like structures, and / or the macroscopic structures, the diploidized multicellular structures, the diploidized embryo-like structures, and / or the diploidized macroscopic structures, wherein genotyping comprises: transferring individual multicellular structures, embryo-like structures, macroscopic structures, diploidized multicellular structures, diploidized embryo-like structures, or diploidized macroscopic structures to a container with a non-destructive medium, agitating the container to collect shed cellular material from the individual multicellular structure, embryo-like structure, macroscopic structure, diploidized multicellular structure, diploidized embryo-like structure, or diploidized macroscopic structure, and genotyping DNA obtained from the shed cellular material.

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