Method for improving plant callus induction, transformation, and regeneration efficiency

Abstract

Provided is a method for plant callus induction or improving plant callus induction rate, wherein the method comprises: overexpressing trehalose-6-phosphate synthase in cells of a plant, or culturing the cells of the plant in the presence of a product of a trehalose-6-phosphate synthase-involved synthetic pathway. By means of overexpressing the TPS1 gene in the plant or exogenous addition of the final product trehalose of the TPS1-involved synthetic pathway into a culture medium, the callus induction efficiency and regeneration efficiency of different crops can be improved, and the genetic transformation efficiency of the plant is improved.

Description

A method to improve the efficiency of plant callus induction, transformation and regeneration

[0001] Priority and related applications

[0002] This invention claims priority to Chinese Patent Application No. 202410704397.6, filed on May 31, 2024, entitled "A method for improving the efficiency of plant callus induction, transformation and regeneration", the entire contents of which, including the appendices, are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of genetic engineering, and to a method for improving the efficiency of plant callus induction, transformation and regeneration. Background Technology

[0004] CRISPR gene editing technology offers significant advantages over traditional breeding methods in major food and strategic crops. Directed evolution of key traits based on gene editing technology has led to the development of crops that are higher-yielding, more nutritious, more resistant to extreme weather, and require less fertilizer and pesticides. Simultaneously, the integration of multiple technologies powered by CRISPR has spearheaded the creation of new concept crops through the de novo domestication or re-domestication of wild species, propelling plant species revitalization initiatives into a race-like era. In the decade of rapid development of gene editing technology, its derivative and upgraded technologies, such as single-base editing and guided editing, have also been established. The core technology for effectively addressing agricultural challenges lies in how to efficiently, targetedly, and safely deliver existing gene editing tools into plant target cells to maximize their editing efficiency. However, the low regeneration efficiency of edited plants has kept the application of gene editing technology in plants in its early stages, severely delaying its ability to utilize the natural diversity of crops in new ways to address global food challenges.

[0005] Rice (Oryza sativa L.) is an important food crop and a typical model crop for plant research, holding a vital position in agricultural production and basic research. Rice is mainly divided into indica rice (Oryza sativa L. subsp. indica Kato) and japonica rice (Oryza sativa L. subsp. japonica Kato). In contrast, most indica rice varieties have low embryogenic callus induction efficiency, easily exhibiting browning, hydration, and subsequent callus death, making them difficult to culture. In genetic transformation, low embryogenic callus induction efficiency is a fundamental bottleneck in the tissue culture and genetic improvement of most indica rice varieties. Theoretically, improving callus induction efficiency in indica rice can be achieved by using specific growth-regulating genes or optimizing the culture medium composition during transformation. Besides rice, a considerable proportion of plant species also face difficulties in callus induction, transformation, and regeneration, posing significant obstacles to plant science research and crop breeding. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] In view of the above-mentioned problems in the prior art, the main objective of the present invention is to provide a method for improving the efficiency of plant callus induction, transformation and regeneration.

[0008] Solution for solving the problem

[0009] This invention primarily comprises a key callus-inducing gene, trehalose-6-phosphate synthase (TPS1), obtained through single-cell RNA sequencing screening. It also includes methods to enhance callus induction, transformation, and regeneration efficiency in various plants by overexpressing this gene and exogenously adding its synthetic end product, trehalose, thereby improving genetic transformation efficiency. Experimental results show that overexpression of this gene in Agrobacterium can improve callus induction and transformation efficiency in crops such as rice (e.g., various indica rice varieties), and also enhance regeneration efficiency. Furthermore, by analyzing the synthetic pathway of TPS1 in plants, this invention found that trehalose content significantly increased while sucrose content decreased. Therefore, by exogenously regulating the ratio of trehalose to sucrose, this invention found that exogenously added trehalose could also mimic TPS1 overexpression and promote regeneration. The results indicate that this enhancement is effective for various plants, including rice (indica rice), sugarcane, sorghum, and strawberry, and can be extended to a wider variety of plants.

[0010] In some aspects of the present invention, a method for inducing plant callus or improving the plant callus induction rate is provided, wherein the method includes:

[0011] (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the synthetic pathway involving trehalose-6-phosphate synthase.

[0012] In some aspects of the present invention, a method for plant regeneration or improving plant regeneration efficiency is provided, wherein the method includes:

[0013] (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the trehalose-6-phosphate synthase-mediated synthetic pathway; and,

[0014] (ii) Regenerate a complete plant from the plant cells.

[0015] In some aspects of the present invention, a method for plant genetic transformation or improving the efficiency of plant genetic transformation is provided, wherein the method includes:

[0016] (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the synthetic pathway involving trehalose-6-phosphate synthase;

[0017] (ii) Introducing at least one expression construct containing at least one nucleic acid sequence of interest into the plant cells; and,

[0018] (iii) Regenerate a complete plant from the plant cells.

[0019] In some embodiments, the method is a method for performing gene editing in plants or for improving the efficiency of gene editing in plants, wherein the at least one nucleic acid sequence of interest encodes a component of the gene editing system.

[0020] In some implementations, the method is a method for transforming a nucleic acid sequence of interest into a plant or for improving the transformation efficiency of a nucleic acid sequence of interest in a plant.

[0021] In some embodiments of the above aspects, in step (i), the plant cells are somatic cells or embryonic cells.

[0022] In some optional embodiments, the plant cells are derived from plant explants or portions thereof.

[0023] In some alternative embodiments, the plant cells are derived from plant callus tissue.

[0024] In some embodiments of the above aspects, the synthetic pathway products involved by the trehalose-6-phosphate synthase include trehalose.

[0025] In some alternative embodiments, the culture medium for culturing the plant cells also contains a carbon source other than trehalose, wherein the weight ratio of trehalose to the carbon source other than trehalose is 0.01 to 99:1.

[0026] In some embodiments, trehalose-6-phosphate synthase is overexpressed in the cells of the plant by introducing into the cells an expression construct containing a nucleic acid sequence encoding trehalose-6-phosphate synthase or a polypeptide of trehalose-6-phosphate synthase.

[0027] In some implementations, methods for introducing the expression construct into plant cells include protoplast electroporation, gene gun method, PEG-mediated transformation, and Agrobacterium-mediated transformation.

[0028] In some embodiments of the above aspects, the trehalose-6-phosphate synthase is a rice-derived trehalose-6-phosphate synthase.

[0029] In some specific embodiments, the trehalose-6-phosphate synthase comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

[0030] In some embodiments of the above aspects, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0031] In some embodiments of the above, the plant is selected from wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, or potato.

[0032] In some preferred embodiments, the plant is a non-renewable plant.

[0033] In some optional embodiments, the plant is selected from rice, sorghum, sugarcane, or strawberry.

[0034] In some aspects of the present invention, any of the following uses (i) to (iii) are also provided for trehalose-6-phosphate synthase, expression constructs comprising a nucleic acid sequence encoding trehalose-6-phosphate synthase, or products of synthetic pathways involving trehalose-6-phosphate synthase:

[0035] (i) Use in callus induction or to improve the callus induction rate in plants;

[0036] (ii) Uses in plant regeneration or in improving the efficiency of plant regeneration;

[0037] (iii) Use in plant genetic transformation or in improving the efficiency of plant genetic transformation.

[0038] In some embodiments, the synthetic pathway products involved by the trehalose-6-phosphate synthase include trehalose.

[0039] In some implementations, the genetic transformation includes gene editing in plants and / or transforming nucleic acid sequences of interest into plants.

[0040] The effects of the invention

[0041] This invention discloses trehalose-6-phosphate synthase and a method for improving callus induction, transformation, and regeneration efficiency in plants by exogenously adding the synthetic final product trehalose. This invention improves callus induction and regeneration efficiency in different crops and enhances plant genetic transformation efficiency by overexpressing a TPS1 gene in Agrobacterium and exogenously adding trehalose, the final product of the TPS1 synthesis pathway, to the culture medium. Attached Figure Description

[0042] Figure 1A: Schematic diagram of the vector used for overexpressing TPS1 and the control vector.

[0043] Figure 1B: Growth of 93-11 callus after transformation and regeneration in the control (left) and TPS1 overexpression (right) groups.

[0044] Figure 2A: Callus induction of indica rice variety 93-11 on callus induction media with different proportions of trehalose and sucrose applied externally.

[0045] Figure 2B: Callus induction of indica rice varieties 93-11, Huanghuazhan, and R498 on callus induction media with and without trehalose.

[0046] Figure 2C: Regeneration of indica rice varieties 93-11, Huanghuazhan, and R498 on regeneration media with and without trehalose.

[0047] Figure 2D: Callus induction of sorghum cultivar E048 on callus induction media with different proportions of trehalose and sucrose.

[0048] Figure 2E: Callus induction and regeneration of strawberry cultivar 'Hongyan' on callus induction media with different proportions of trehalose and sucrose applied externally; in the figure, DAT represents the number of treatment days.

[0049] Figure 2F: Callus induction and regeneration of sugarcane cultivars “Huangpi Guozhe” and “Liucheng 05-136” on culture media with different proportions of trehalose and sucrose. Detailed Implementation

[0050] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0051] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0052] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0053] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0054] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0055] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0056] In this instruction manual, when "room temperature" or "room temperature" is used, the temperature can be 15-25℃.

[0057] As used herein, the term "plant" includes the whole plant and any offspring, plant cells, tissues, or parts. The term "plant part" includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without a seed coat, and immature seeds); plant cuttings; plant cells; plant cell cultures; plant organs (e.g., pollen, embryo, flower, fruit, bud, leaf, root, stem, and related explants). Plant tissues or plant organs can be seeds, callus, or any other group of plant cells organized into structural or functional units.

[0058] Plant "offspring" includes any subsequent generations of a plant.

[0059] The terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases.

[0060] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0061] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0062] Sequence “identity” has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence similarity can be measured along the full length of the polynucleotide or polypeptide or along a region of the molecule. Although many methods exist for measuring the similarity between two polynucleotides or polypeptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0063] In peptides or proteins, suitable conserved amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0064] As used in this invention, "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in plants. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein. The "expression construct" of this invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (e.g., mRNA). The "expression construct" of this invention can comprise expression regulatory sequences and nucleotide sequences of interest from different sources, or expression regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from those typically found naturally.

[0065] "Expression regulatory sequence" and "expression regulatory element" are used interchangeably, referring to nucleotide sequences located upstream (5' non-coding sequence), in the middle, or downstream (3' non-coding sequence) of a coding sequence that affect the transcription, RNA processing, stability, or translation of the relevant coding sequence. Plant expression regulatory elements refer to nucleotide sequences that can control the transcription, RNA processing, stability, or translation of nucleotide sequences of interest in plants.

[0066] Expression regulatory sequences may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences. A "promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, the promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it originates from a plant cell. The promoter may be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter.

[0067] As used herein, the term "operably linked" refers to the linking of a regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., coding sequences or open reading frames) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0068] In relation to a sequence, “exogenous” means a sequence that originates from a foreign species, or, if from the same species, a sequence whose composition and / or loci have been significantly altered from its natural form through deliberate human intervention.

[0069] "Introducing" a nucleic acid molecule (such as an expression construct) into a plant cell means presenting the nucleic acid molecule to the plant cell so that it enters the interior of the plant cell.

[0070] "Regeneration" refers to the process of growing a complete plant from one or more plant cells (e.g., plant protoplasts, callus, or explants).

[0071] Callus is the new tissue that grows on the surface of a wound after a localized injury to the original plant. It is composed of living parenchyma cells and can originate from living cells in various tissues within any organ of the plant.

[0072] "Plant genetic transformation" or "genetic transformation" refers to the process of introducing exogenous or endogenous target genes into the genome of a recipient plant using certain genetic transformation methods, or modifying specific target genes in the genome of a recipient plant using gene editing tools.

[0073] Invention Details

[0074] The inventors have surprisingly discovered that overexpressing trehalose-6-phosphate synthase in plant cells, or culturing plant cells in the presence of trehalose-6-phosphate synthase-involved synthetic pathway products, can enhance the callus induction and regeneration efficiency of different plants.

[0075] In some aspects of the present invention, a method for inducing plant callus or improving the plant callus induction rate is provided, wherein the method includes:

[0076] (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the synthetic pathway involving trehalose-6-phosphate synthase.

[0077] In some aspects of the present invention, a method for plant regeneration or improving plant regeneration efficiency is provided, wherein the method includes:

[0078] (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the trehalose-6-phosphate synthase-mediated synthetic pathway; and,

[0079] (ii) Regenerate a complete plant from the plant cells.

[0080] In some aspects of the present invention, a method for plant genetic transformation or improving the efficiency of plant genetic transformation is provided, wherein the method includes:

[0081] (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the synthetic pathway involving trehalose-6-phosphate synthase;

[0082] (ii) Introducing at least one expression construct containing at least one nucleic acid sequence of interest into the plant cells; and,

[0083] (iii) Regenerate a complete plant from the plant cells.

[0084] In the method for plant regeneration or improving plant regeneration efficiency, in some embodiments, steps (i) and (ii) are performed in parallel (simultaneously). In some embodiments, step (i) is performed first, followed by step (ii). In some embodiments, step (ii) is performed first, followed by step (i).

[0085] Similarly, in the method for plant genetic transformation or improving the efficiency of plant genetic transformation, steps (i) to (iii) can be performed in any order or in parallel.

[0086] In some specific implementations, the genetic transformation can be callus transformation, or callus tissue transformation, which refers to the process of introducing exogenous or endogenous DNA into plant callus cells using Agrobacterium tumefaciens infection or gene gun method.

[0087] In some exemplary embodiments, in the method of plant genetic transformation or improving the efficiency of plant genetic transformation, at least one nucleic acid sequence of interest may be a nucleic acid sequence encoding trehalose-6-phosphate synthase, that is, "overexpressing trehalose-6-phosphate synthase in the cells of the plant" and "introducing at least one expression construct containing at least one nucleic acid sequence of interest into the plant cells" may be a single step.

[0088] Methods for regenerating complete plants (transformed complete plants) by culturing transformed protoplasts, callus, immature embryos, or explants are known in the art. During this regeneration process, transformants can also be screened based on selectivity markers carried on the introduced expression construct.

[0089] In some exemplary embodiments, the method for plant regeneration or improving plant regeneration efficiency includes:

[0090] (i) Inducing callus formation by culturing cells of the plant in the presence of products of the trehalose-6-phosphate synthase-mediated synthesis pathway; and,

[0091] (ii) Regenerate a complete plant from the callus obtained in step (i).

[0092] In some exemplary embodiments, the method for plant regeneration or improving plant regeneration efficiency includes:

[0093] (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, wherein the plant cells are derived from callus tissue, culturing the plant cells, and,

[0094] (ii) Regenerate a complete plant from the callus tissue of step (i).

[0095] In these exemplary embodiments, the callus tissue in step (i) can be obtained by explant induction using methods known in the art.

[0096] In some exemplary embodiments, the method for plant genetic transformation or improving the efficiency of plant genetic transformation includes:

[0097] (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the synthetic pathway involving trehalose-6-phosphate synthase, to induce callus formation.

[0098] (ii) Introducing at least one expression construct containing at least one nucleic acid sequence of interest into cells of the callus; and,

[0099] (iii) Regenerate a complete plant from the callus obtained in step (ii).

[0100] In other aspects of the invention, any of the following uses (i) to (iii) are provided for trehalose-6-phosphate synthase, expression constructs comprising a nucleic acid sequence encoding trehalose-6-phosphate synthase, or products of synthetic pathways involving trehalose-6-phosphate synthase:

[0101] (i) Use in callus induction or to improve the callus induction rate in plants;

[0102] (ii) Uses in plant regeneration or in improving the efficiency of plant regeneration;

[0103] (iii) Use in plant genetic transformation or in improving the efficiency of plant genetic transformation.

[0104] (Plants, plant cells)

[0105] In some embodiments, the plant cells described in this invention are cells suitable for inducing callus formation, genetic transformation (e.g., callus transformation), and / or regeneration into complete plants through tissue culture. Examples of suitable plant cells include, but are not limited to, protoplast cells, callus cells, immature embryonic cells, and explant cells.

[0106] In some specific implementations, plant cells suitable for inducing callus formation (and subsequently genetic transformation, such as callus transformation) include embryonic plant cells (embryonic cells) and somatic cell line plant cells (somatic cells). Plant cells can be used in isolated form or as part of plant tissue. For example, explants isolated from a plant can provide embryonic or somatic cell line plant cells. Cells are isolated from the explant or used directly to induce callus (and subsequently genetic transformation, such as callus transformation). Which part of the plant is suitable for obtaining explants depends on the specific plant species. Generally, suitable plant cells can be obtained from the hypocotyl, seedling, leaf, bud, flower, petiole, and root of the plant.

[0107] In some implementations, in step (i), the plant cells are somatic cells or embryonic cells, preferably derived from plant explants or parts thereof, such as various explants, such as stem segments, leaves, roots, cell suspensions, etc.

[0108] In some embodiments, the plants suitable for the methods described above in this invention can be monocotyledonous or dicotyledonous plants. In some specific embodiments, examples of the plants include, but are not limited to, wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, and potato.

[0109] In some preferred embodiments, the plant is a non-renewable plant.

[0110] In this invention, the term "difficult-to-regenerate plant" refers to a plant that is difficult to induce, transform, or regenerate callus tissue, such as plants with low callus induction efficiency, low transformation efficiency, or low regeneration efficiency.

[0111] In some optional embodiments, examples of the plant (difficult-to-regenerate plant) include rice, sorghum (e.g., sorghum cultivar E048), sugarcane (e.g., sugarcane cultivar Huangpi Guozhe, Liucheng 05-136), and strawberry (e.g., octoploid strawberry cultivar Hongyan strawberry).

[0112] In some more specific embodiments, the rice is indica rice, such as indica rice variety 93-11, indica rice variety Huanghuazhan, indica rice variety R498, indica rice variety Yuehesimiao, and wild tetraploid rice variety Gaogan Wild Rice.

[0113] (Trehalose-6-phosphate synthase)

[0114] In some embodiments, the trehalose-6-phosphate synthase is a plant-derived trehalose-6-phosphate synthase or a functional variant thereof, such as trehalose-6-phosphate synthase or a functional variant thereof from rice, sorghum, sugarcane, or strawberry. In some embodiments, the trehalose-6-phosphate synthase is a trehalose-6-phosphate synthase or a functional variant thereof from a plant species to which callus is to be induced, regenerated, or genetically transformed. In some embodiments, the trehalose-6-phosphate synthase is a trehalose-6-phosphate synthase or a functional variant thereof from a plant species different from the plant species to which callus is to be induced, regenerated, or genetically transformed.

[0115] In some embodiments, the trehalose-6-phosphate synthase is rice trehalose-6-phosphate synthase or a functional variant thereof. Exemplary rice trehalose-6-phosphate synthase or a functional variant thereof comprises, for example, the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

[0116] (Products involved in the synthetic pathway of trehalose-6-phosphate synthase)

[0117] Trehalose-6-phosphate synthase (TPS) catalyzes the condensation reaction of UDP-glucose with glucose-6-phosphate to form trehalose-6-phosphate (T6P), which can be further dephosphorylated by trehalose-6-phosphate phosphatase (TPP) to generate trehalose. In some embodiments, the synthetic pathway products involved by the trehalose-6-phosphate synthase include trehalose.

[0118] In some embodiments, the culture medium for culturing the plant cells further includes a carbon source other than trehalose, wherein the weight ratio of trehalose to the carbon source other than trehalose is 0.01 to 99:1, preferably 0.1 to 10:1, more preferably 0.5 to 8:1, and even more preferably 0.5 to 6:1, for example 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, or 6:1.

[0119] In some implementations, carbon sources other than trehalose include sugars such as sucrose, fructose, maltose, and glucose.

[0120] In some preferred embodiments, the carbon source other than trehalose includes sucrose.

[0121] In some other preferred embodiments, the culture medium in which the plant cells are cultured does not contain a carbon source other than trehalose (e.g., sucrose).

[0122] In some embodiments, the content of trehalose or the content of trehalose and carbon sources other than trehalose in the culture medium is 10-50 g / L, preferably 20-40 g / L, more preferably 25-35 g / L, for example 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L.

[0123] Those skilled in the art can select appropriate culture media based on factors such as callus induction, transformation, and regeneration, as well as the types of plant cells.

[0124] (Overexpression of trehalose-6-phosphate synthase)

[0125] In some embodiments of the present invention, “overexpression of trehalose-6-phosphate synthase in the cells of the plant” refers to the artificial manipulation (e.g., genetic manipulation) that upregulates the expression and / or activity of trehalose-6-phosphate synthase in the plant (plant cells) relative to the corresponding wild-type plant (plant cells) that has not undergone said manipulation.

[0126] In some embodiments of the present invention, trehalose-6-phosphate synthase is overexpressed in the cells of the plant by introducing an expression construct containing a nucleic acid sequence encoding trehalose-6-phosphate synthase or a polypeptide of trehalose-6-phosphate synthase into the plant cells. Methods for introducing the expression construct into the plant are known in the art, including but not limited to protoplast electroporation, gene gun method, PEG-mediated transformation, and Agrobacterium-mediated transformation.

[0127] In other embodiments of the invention, overexpression of trehalose-6-phosphate synthase in the plant cells can also be achieved by modifying the expression regulatory sequence of the endogenous trehalose-6-phosphate synthase gene (TPS1 gene) in the plant cells. For example, overexpression of trehalose-6-phosphate synthase in the plant cells can be achieved by gene editing the expression regulatory sequence of the endogenous trehalose-6-phosphate synthase gene in the plant cells. Therefore, in some embodiments, the method includes introducing a gene editing system (including the introduction of one or more expression constructs encoding components of the gene editing system) targeting the expression regulatory sequence of the endogenous trehalose-6-phosphate synthase gene into the plant cells, resulting in overexpression of the endogenous trehalose-6-phosphate synthase gene in the plant cells.

[0128] In some embodiments of the invention, overexpression of trehalose-6-phosphate synthase in the plant cells further includes increasing the biological activity of endogenously expressed trehalose-6-phosphate synthase in the plant cells. In some embodiments, a mutation leading to increased biological activity may be introduced into the endogenous trehalose-6-phosphate synthase, for example, by gene editing. Therefore, in some embodiments, the method includes introducing a gene editing system (including the introduction of one or more expression constructs encoding components of the gene editing system) targeting the endogenous trehalose-6-phosphate synthase coding sequence into the plant cells, resulting in increased biological activity of endogenously expressed trehalose-6-phosphate synthase in the plant cells.

[0129] The present invention also provides a kit for carrying out the method of the present invention, comprising at least an expression construct encoding a nucleic acid sequence of trehalose-6-phosphate synthase, an expression construct encoding one or more components of a gene editing system, or a product of a synthetic pathway involving trehalose-6-phosphate synthase (e.g., trehalose). The gene editing system targets the expression regulatory sequence or coding sequence of endogenous trehalose-6-phosphate synthase and can lead to overexpression of the endogenous trehalose-6-phosphate synthase gene in plant cells or increased biological activity of endogenously expressed trehalose-6-phosphate synthase protein in plant cells.

[0130] The present invention also provides an expression construct comprising a nucleic acid sequence encoding trehalose-6-phosphate synthase, an expression construct comprising one or more components of a gene editing system encoding a regulatory sequence or coding sequence for the expression of endogenous trehalose-6-phosphate synthase, or a synthetic pathway product involving trehalose-6-phosphate synthase (e.g., trehalose), for use in improving plant callus induction rate, plant regeneration efficiency, and / or plant genetic transformation efficiency, wherein the gene editing system for the regulatory sequence or coding sequence for the expression of endogenous trehalose-6-phosphate synthase can lead to overexpression of the endogenous trehalose-6-phosphate synthase gene in plant cells or increased biological activity of endogenously expressed trehalose-6-phosphate synthase protein in plant cells.

[0131] In some embodiments, the encoding nucleic acid sequence of the trehalose-6-phosphate synthase and / or the at least one nucleic acid sequence of interest are operatively linked to a transcriptional regulatory element.

[0132] (At least one nucleic acid sequence of interest)

[0133] The "at least one nucleic acid sequence of interest" can be any nucleic acid sequence that needs to be transformed into a plant. For example, the nucleic acid sequence of interest can encode nucleic acid sequences that encode traits important to agronomy, insect resistance, disease resistance, herbicide resistance, sterility, and commercial products. The nucleic acid sequence of interest can also include those nucleic acid sequences involved in the metabolism of oil, starch, carbohydrates, or nutrients, as well as those affecting fruit size, sucrose load, etc. That is, the method (use) of plant genetic transformation or improving the genetic transformation efficiency of plants is a method (use) of transforming a nucleic acid sequence of interest into a plant or improving the transformation efficiency of a nucleic acid sequence of interest in a plant.

[0134] In some preferred embodiments, the "at least one nucleic acid sequence of interest" encodes a component of a gene editing system, thereby enabling gene editing in plants. That is, the method (use) for plant genetic transformation or improving the efficiency of plant genetic transformation is a method (use) for performing gene editing in plants or improving the efficiency of gene editing in plants, wherein the at least one nucleic acid sequence of interest encodes a component of a gene editing system.

[0135] The gene editing system can target endogenous genes or their expression regulatory sequences that are important for traits important to agronomy, insect resistance, disease resistance, herbicide resistance, sterility, and commercial products, thereby modifying the expression or activity of the endogenous genes. For example, the gene editing system can target endogenous genes or their expression regulatory sequences involved in the metabolism of oil, starch, carbohydrates, or nutrients, or endogenous genes or their expression regulatory sequences that affect fruit size, sucrose load, etc., thereby modifying the expression or activity of the endogenous genes.

[0136] In some specific implementations, at least one nucleic acid sequence of interest may also be a nucleic acid sequence encoding trehalose-6-phosphate synthase.

[0137] Gene editing, also known as genome editing, involves the insertion, deletion, or substitution of nucleotides in an organism's genome using sequence-specific nucleases or their derivatives. Gene editing typically works by inducing site-specific double-strand breaks (DSBs) at desired locations in the genome, followed by the introduction of the desired DNA insertion, deletion, or substitution during DSB repair. However, gene editing can also encompass base editing techniques that do not involve DSBs, transcriptional activation or repression, and epigenetic modification techniques, provided they are sequence-specific.

[0138] This invention does not particularly limit the gene editing system used. For example, gene editing systems suitable for use in this invention include, but are not limited to, zinc finger nucleases (ZFNs), large-scale nucleases (MGNs), transcription activator-like effector nucleases (TALENs), and CRISPR (Clustered regularly interspaced short palindromic repeats) systems.

[0139] Zinc finger nucleases (ZFNs) are artificial restriction enzymes prepared by fusing a zinc finger DNA-binding domain with a DNA-cutting domain. A single ZFN typically contains 3-6 individual zinc finger repeats, each of which can recognize a unique sequence, for example, 3 bp. By combining different zinc finger repeats, different genomic sequences can be targeted.

[0140] Meganucleases are typically homing endonucleases that recognize nucleic acid sequences of 14-40 bases in length. The long recognition sequence gives meganucleases high specificity, thus reducing off-target effects.

[0141] "Transcription activator-like effector nucleases" are restriction enzymes that can be engineered to cleave specific DNA sequences. They are typically prepared by fusing the DNA-binding domain of a transcription activator-like effector (TALE) with its DNA-cleaving domain. Once engineered, TALEs can bind to almost any desired DNA sequence.

[0142] A CRISPR system typically comprises two components that can form a sequence-specific complex: a CRISPR nuclease or a variant thereof, and a corresponding guide RNA. Therefore, for a CRISPR system, the "at least one nucleic acid sequence of interest" described in this invention may include the nucleic acid sequence encoding a CRISPR nuclease or a variant thereof, and / or the nucleic acid sequence encoding the corresponding guide RNA.

[0143] In some preferred embodiments, the gene editing system is a CRISPR system. A large number of different CRISPR gene editing systems are known in the art and can all be used in this invention. For example, suitable CRISPR gene editing systems can be found at http: / / www.addgene.org / crispr / . CRISPR gene editing systems encompass systems that alter genome sequences, as well as systems used for transcriptional regulation that do not alter genome sequences.

[0144] As used herein, the term "CRISPR nuclease" generally refers to a nuclease present in the naturally occurring CRISPR system. "CRISPR nuclease variants" include modified forms of natural CRISPR nucleases, artificial mutants (including nicking enzyme mutants), catalytically active fragments, or fusions with other functional proteins / peptides. Various artificial functional variants of CRISPR nucleases are known in the art, such as highly specific variants or nicking enzyme variants, or fusion proteins of them with cytidine deaminases or adenosine deaminases. CRISPR nucleases or their variants can recognize, bind to, and / or cleave target nucleic acid structures by interacting with corresponding guide RNAs. Those skilled in the art will understand how to select suitable CRISPR nucleases or their variants to achieve the objectives of this invention.

[0145] The CRISPR nuclease or variant thereof used in the CRISPR gene editing system of the present invention may be selected, for example, from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1 (Cas12a), C2c1, C2c3 or C2c2 proteins, or functional variants of these nucleases.

[0146] In some embodiments, the CRISPR nuclease or a variant thereof includes the Cas9 nuclease or a variant thereof. CRISPR gene editing systems based on the Cas9 nuclease or a variant thereof are also referred to herein as CRISPR-Cas9 gene editing systems. The Cas9 nuclease may be a Cas9 nuclease from a different species, such as spCas9 from *Streptococcus pyogenes*.

[0147] Cas9 nuclease variants may include Cas9 nickase (nCas9), in which one of the two subdomains (HNH nuclease subdomain and RuvC subdomain) of the DNA cleavage domain of the Cas9 nuclease is inactivated to form the nickase. In some embodiments, the Cas9 nickase can be combined with two gRNAs targeting upstream and downstream of the sequence to be edited to achieve deletion of the sequence to be edited, or to achieve replacement of the sequence to be edited in the presence of a donor sequence.

[0148] In some embodiments, the CRISPR nuclease or a variant thereof may further include a Cpf1 (Cas12a) nuclease or a variant thereof, such as a highly specific variant. The Cpf1 nuclease may be a Cpf1 nuclease from different species, such as Cpf1 nucleases from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacterium ND2006. CRISPR gene editing systems based on Cpf1 nucleases or variants thereof are also referred to herein as CRISPR-Cpf1 systems.

[0149] In some implementations, the CRISPR nuclease variant may also include a base editor. The base editor is typically a fusion protein containing a deaminase and a CRISPR nuclease variant lacking DNA cleavage activity.

[0150] As used in this invention, "CRISPR nuclease variants lacking DNA cleavage activity" include, but are not limited to, Cas9 nick nuclease (nCas9), cas9 nuclease with dead nuclease (dCas9), or casf1 nuclease with dead nuclease (dCpf1). Cas9 nuclease with dead nuclease (dCas9) or casf1 nuclease with dead nuclease (dCpf1) completely lacks DNA cleavage activity. Various CRISPR nuclease variants lacking DNA cleavage activity are known in the art.

[0151] As used in this invention, "deaminase" refers to an enzyme that catalyzes deamination reactions. In some embodiments of this invention, the deaminase refers to a cytosine deaminase, which accepts single-stranded DNA as a substrate and catalyzes the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments of this invention, the deaminase refers to an adenine deaminase, which accepts single-stranded DNA as a substrate and catalyzes the formation of inosine (I) from adenosine or deoxyadenosine (A). Various suitable cytosine deaminases or adenine deaminases that accept single-stranded DNA as a substrate are known in the art. Suitable cytosine deaminases include, but are not limited to, for example, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, and human APOBEC3A deaminase. In some preferred embodiments, the cytosine deaminase is human APOBEC3A. Examples of suitable adenine deaminases include, but are not limited to, the DNA-dependent adenine deaminases disclosed by Nicole M. Gaudelli et al. (doi: 10.1038 / nature24644, 2017).

[0152] By fusing a CRISPR nuclease variant lacking DNA cleavage activity with a deaminase (forming a so-called "base editor"), base editing of target nucleotide sequences, such as C-to-T or A-to-G conversions, can be achieved. Various base editors are known in the art, and those skilled in the art know how to select a suitable base editor to achieve the objectives of this invention. CRISPR gene editing systems based on base editors are also called base editing systems.

[0153] As used herein, "guide RNA" and "gRNA" are used interchangeably and refer to RNA molecules capable of forming a complex with a CRISPR nuclease or a variant thereof and targeting the target sequence by means of a certain degree of similarity to the target sequence. For example, the gRNA used by the Cas9 nuclease or a variant thereof typically consists of partially complementary crRNA and tracrRNA molecules forming a complex, wherein the crRNA contains a guide sequence that is sufficiently similar to the target sequence to hybridize with the complementary strand of the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence. However, it is known in the art that single guide RNAs (sgRNAs) can be designed that contain features of both crRNA and tracrRNA. The gRNA used by the Cpf1 nuclease or a variant thereof typically consists only of mature crRNA molecules, which may also be referred to as sgRNA. Designing a suitable gRNA based on the CRISPR nuclease or a variant thereof and the target sequence to be edited is within the capabilities of those skilled in the art.

[0154] The sequence-specific nucleases used for gene editing in this invention, such as zinc finger nucleases, transcription activator-like effector nucleases, or CRISPR nucleases or variants thereof, may also include subcellular localization signals (such as nuclear localization signals), peptide linkers, detectable tags, and other elements. For example, the base editor in a CRISPR base editing system typically includes one or more nuclear localization signals (NLS) to facilitate its entry into the cell nucleus and achieve editing of chromosomal DNA.

[0155] In order to achieve effective expression in plants, in some embodiments of the present invention, the coding nucleic acid sequence or the nucleic acid sequence of interest is codon-optimized for the plant species.

[0156] Codon optimization refers to the modification of nucleic acid sequences to enhance expression in host cells of interest by replacing at least one codon of the natural sequence with codons that are used more frequently or most frequently in the gene in the host cell (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the natural amino acid sequence). Different species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used for peptide synthesis. Therefore, genes can be customized to achieve optimal gene expression in a given organism based on codon optimization. Codon utilization tables are readily available, for example, in the Codon Usage Database (“Codon Usage Database”) available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See Nakamura. Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0157] In some implementations, the nucleic acid sequence encoding trehalose-6-phosphate synthase is selected from SEQ ID NO:2.

[0158] (Import Method)

[0159] In this invention, cells can be transformed by introducing nucleic acid molecules (e.g., nucleic acid constructs) into cells to induce the stable or transient presence of a nucleic acid sequence (preferably stable or transient expression of the nucleic acid sequence) or by introducing polypeptides into cells in a manner that induces transient presence. For example, the stable presence of a DNA sequence means that the DNA sequence is stably integrated into the cell's genome. Stable expression refers to, for example, the expression of a DNA sequence stably integrated into the cell's genome. Transformation of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most suitable transformation technique will be determined by the experimenter. The choice of method varies depending on the type of plant to be transformed; those skilled in the art will recognize the applicability of a particular method to a given plant type. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG)-mediated transformation; transformation using viruses; microinjection of plant cells; micro-ballistic bombardment of plant cells (gene gun method); vacuum permeation; and Agrobacterium-mediated transformation.

[0160] In some practical embodiments, as described above, the expression construct of the present invention can be introduced into plant cells by one of a variety of methods known in the art, including but not limited to gene gun method, PEG-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation.

[0161] In some exemplary embodiments, taking Agrobacterium-mediated transformation as an example, the method for obtaining gene-edited or transgenic plants of interest, or improving the efficiency of gene-editing or transgenic transformation of interest, may include the following steps:

[0162] i) Obtain activated Agrobacterium, wherein the Agrobacterium contains at least one overexpression vector encoding TPS1 and at least one overexpression vector for introducing gene editing and / or transgenic modification of interest;

[0163] ii) Infect the plant cells to be modified with the Agrobacterium from step i);

[0164] iii) Co-culture of Agrobacterium with plant cells infected in step ii);

[0165] iv) Induction and regeneration to obtain gene-edited or transgenic plants of interest.

[0166] In some embodiments, the expression constructs of the present invention are transiently transformed into plant cells. Transient transformation refers to introducing the construct into the cell to function but without integrating it into the cellular genome. This is particularly useful for gene editing because it can produce non-GMO modified plants.

[0167] In some aspects of the invention, the invention also provides plants and their offspring obtained by the method of the invention.

[0168] Example

[0169] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0170] Example 1: Improvement of rice regeneration efficiency based on overexpression of trehalose-6-phosphate synthase (TPS1).

[0171] 1. Carrier Construction

[0172] In this embodiment, a control plasmid pUBI-mCherry and a plasmid pUBI-OsTPS1-P2A-mCherry-Term for overexpressing TPS1 were set up. pUBI-mCherry contains the mCherry protein expressed by the maize UBI promoter and is used to indicate a successful transformation event. pUBI-OsTPS1-P2A-mCherry-Term contains the OsTPS1 protein and mCherry protein expressed by the maize UBI promoter, linked by the coding sequence of the self-cleaving peptide P2A for post-translational separation of the two proteins. mCherry is used to indicate a successful transformation event. The construction of the above expression vector is shown in Figure 1A.

[0173] 2. Determination of the regeneration efficiency of indica rice variety 93-11 after plasmid 1 was transformed into Agrobacterium.

[0174] The recipient material used in this embodiment is callus induced from mature embryos of the indica rice variety 93-11. The mature embryos used for callus induction in 93-11 were derived from mature seeds of 93-11 harvested from field planting.

[0175] (1) Selection and sterilization of explants: 200 mature seeds were placed in 50ml clean centrifuge tubes and sterilized in a shaker for 20min with 3.5% sodium hypochlorite solution and 1μl Tween 20. After sterilization, the seeds were thoroughly washed 3-4 times with sterile water and dried on sterile filter paper in a clean operating table.

[0176] (2) Callus induction: After drying, the seeds were inoculated into the callus induction culture medium at a density of 14 seeds / plate and cultured in the dark at 28 degrees Celsius for 30 days to induce callus formation. The callus induction culture medium formula was N6B5 + 2 mg / L 2,4-D + 30 g / L sucrose + 0.5 g / L glutamine + 2.8 g / L proline + 0.5 g / L acid-hydrolyzed casein + 3 g / L plant gel, with a pH of 5.8, and sterilized by high temperature and high pressure.

[0177] (3) Preparation of Agrobacterium bacterial suspension: Agrobacterium cultured overnight on kanamycin and rifampicin resistant LB medium was centrifuged at 5000 rpm for 5 min. The collected bacterial suspension was resuspended in MS solution containing 40 g / L sucrose and 18 g / L glucose. Acetyl eugenol was added to the resuspended bacterial suspension to a final concentration of 200 mM.

[0178] (4) Agrobacterium infection and co-culture: Sterile, relatively firm, and light yellow viable callus tissue was selected for transformation. The selected callus was infected in bacterial solution for 15 min, and then excess bacterial solution was removed with several sterile filter papers. The callus was then transferred to co-culture medium or sterile filter paper and co-cultured at 22-25℃ for 2 days. The co-culture medium formula was: N6B5 + 2 mg / L 2,4-D + 30 g / L sucrose + 0.5 g / L glutamine + 2.8 g / L proline + 0.5 g / L acid-hydrolyzed casein + 3 g / L plant gel, pH 5.8. After high temperature and high pressure sterilization, 200 mM acetylsyl syringone was added.

[0179] (5) Screening culture: The co-cultured callus was transferred to the screening medium for 8 weeks of growth under the dark culture conditions of 28℃. The callus was subcultured every 14 days and transferred to a new screening medium. The screening medium formula was: N6B5 + 2mg / L 2,4-D + 30g / L sucrose + 0.5g / L glutamine + 2.8g / L proline + 0.5g / L acid hydrolyzed casein + 3g / L plant gel, pH 5.8. After high temperature and high pressure sterilization to 50℃, 50mg / L hygromycin B and 200mg / L termethin were added.

[0180] (6) Regeneration and Rooting Culture: Positive calluses obtained after transformation were transferred to regeneration medium for regeneration. After 45 days of culture, all regenerated seedlings were transferred to rooting medium for rooting culture. The regeneration medium formula was: MS + 2 mg / L 6-BAP + 0.2 mg / L kinetin + 30 g / L sucrose + 20 g / L sorbitol + 0.5 g / L acid-hydrolyzed casein + 30 mg / L hygromycin B + 3 g / L plant gel, pH 5.8. After autoclaving, 200 mg / L termethin was added. The rooting medium formula was: MS + 30 g / L sucrose + 0.1 g / L inositol + 3 g / L plant gel, pH 5.8. After autoclaving, 200 mg / L termethin was added.

[0181] The statistical method for the regeneration rate is: the total number of regenerated green seedlings produced after 45 days of culture on the regeneration medium / the total number of transformed callus (i.e., the number of explants in Table 1).

[0182] As shown in Figure 1B and Table 1, the regeneration efficiency of 93-11 was higher than that of the control group (pUBI-mCherry) after TPS1 overexpression. This indicates that the TPS1 gene can promote the regeneration of indica rice 93-11.

[0183] Table 1:

[0184] Example 2: The exogenous addition of trehalose, the final product of the TPS1 synthesis pathway, improves the callus induction and regeneration efficiency of indica rice.

[0185] Trehalose-6-phosphate synthase (TPS) catalyzes the condensation reaction of UDP-glucose with glucose-6-phosphate to form product T6P, which can be further dephosphorylated by trehalose-6-phosphate phosphatase (TPP) to produce trehalose.

[0186] Therefore, this embodiment tested the sugar content of various sugars in rice 93-11 after TPS1 overexpression and found that this gene significantly increased the trehalose content in the plant while decreasing the sucrose and other fructose content. This embodiment simulated the sugar content level of TPS1 overexpression by replacing exogenously added sucrose in the callus induction medium with different mass ratios of trehalose and sucrose for gradient testing. It was found that when trehalose and sucrose were used together as carbon sources, with a total amount of 30 g / L and a trehalose:sucrose mass ratio of 5:1, the callus induction efficiency and callus formation state were optimal, as shown in Figure 2A. The callus induction medium formula was: N6B5 + 2 mg / L 2,4-D + 0.5 g / L glutamine + 2.8 g / L proline + 0.5 g / L acid-hydrolyzed casein + 3 g / L plant gel. Four different proportions of trehalose and sucrose were added as carbon sources, specifically trehalose:sucrose = 0:1, 1:2, 5:1, and 1:0. pH value is 5.8, sterilized by high temperature and high pressure.

[0187] Subsequent tests were conducted on more non-regenerating indica rice varieties, and it was found that using trehalose and sucrose together as carbon sources in the callus induction medium, with a total amount of 30 g / L and a trehalose:sucrose mass ratio of 5:1, could improve the callus induction rate and regeneration efficiency of indica rice (as shown in Figures 2B and 2C, and Tables 2-1 to 2-3).

[0188] Table 2-1:

[0189] Table 2-2:

[0190] Table 2-3: * indicates a significant difference analysis. A two-tailed unpaired t-test was used to determine statistical significance. *P < 0.05

[0191] Example 3: Exogenous application of trehalose can promote callus induction and regeneration processes in a variety of recalcitrant plants.

[0192] To test whether the effect of exogenous application of trehalose on improving plant callus induction and regeneration efficiency is universal across species and genotypes, this embodiment further tested sorghum, sugarcane, and octoploid strawberries.

[0193] (1) Effect of exogenous trehalose on callus induction in sorghum culture medium: Grains of sorghum cultivar E048 were harvested 10 to 12 days after flowering. After sterilization with 3.5% sodium hypochlorite solution for 25 min, the grains were washed 3 to 4 times with sterile water. After absorbing the residual moisture with sterile filter paper, immature embryos were separated with a scalpel blade and transferred to four callus induction culture media with trehalose and sucrose as carbon sources, totaling 30 g / L, and the specific ratios of trehalose:sucrose = 0:1, 1:2, 5:1 and 1:0. The callus induction was observed after 10 days. The callus induction medium was formulated as follows: MS medium + 1–3 g / L proline + 0.5 g / L LMES + 1.5 mg / L 2,4-D + 200–400 mg / L vitamin C + 3 g / L plant gel, with different proportions of trehalose and sucrose added. The pH was adjusted to 5.8, and after high-temperature and high-pressure sterilization, 0.1–2 g / L asparagine, 0.1–5 mg / L copper sulfate pentahydrate, 0.5 g / L glutamine, and 200 mg / L termethin were added. Experimental results showed that increasing the trehalose content effectively alleviated browning during sorghum callus induction and promoted callus formation (Figure 2D).

[0194] (2) Effect of exogenous trehalose on callus induction and regeneration in octoploid strawberry: After 4 weeks of growth in a seedling culture medium, sterile seedlings of the octoploid strawberry cultivar Hongyan were cut into leaf discs with a length and width of approximately 7 mm using a scalpel. The leaf discs were then inoculated onto callus induction medium with 10 explants per dish, with the back of the leaf disc facing upwards, at a density of sucrose or trehalose as the carbon source. After 50 days of culture, the regeneration rate (regeneration callus rate in Table 3) was calculated as: number of leaf discs producing regenerated seedlings (regenerated callus in Table 3) / total number of leaf discs (number of explants in Table 3). The callus induction medium was formulated as follows: MS medium + 0.5 g / L MES + 3 g / L plant gel, with 30 g / L sucrose or trehalose added as the carbon source, pH = 5.8. After autoclaving, 1–5 mg / L TDZ + 0.2–2 mg / L IAA + 200 mg / L termethin were added. Subculture was performed every 14 days. The test results showed that replacing sucrose with trehalose in the callus induction medium significantly improved callus formation and regeneration in octoploid strawberries, increasing the regeneration efficiency by nearly 25 times (Table 3, Figure 2E).

[0195] (3) Effect of exogenous trehalose on callus induction and regeneration in the test medium: Sugarcane cultivars Huangpi and Liucheng 05-136 were planted in a 30℃ greenhouse. After picking new tillers, the outer leaves were removed and the tillers were wiped with 75% ethanol. The leaves were further removed in a clean workbench using a scalpel and sterile forceps until the tender new meristem was exposed. Under sterile conditions, the meristem was cut off from the top 0.5-3 cm with a scalpel and further transversely cut into slices about 2 mm thick. The cut surfaces were then transferred to callus induction medium with a carbon source of trehalose:sucrose ratio of 0:1 or 5:1 for 8-10 weeks. After the embryogenic callus tissue with a firm texture and yellowish color was induced, it was transferred to a regeneration medium for culture. The regeneration was observed after 4 weeks. The callus induction medium was formulated as follows: MS medium + 3 mg / L 2,4-D + 1.2 mg / L copper sulfate pentahydrate + 3.5 g / L plant gel, with 30 g / L sucrose or 25 g / L trehalose plus 5 g / L sucrose as the carbon source, pH = 5.8, and autoclaved. The regeneration medium was formulated as follows: MS medium + 0.2 mg / L KT + 3.5 g / L plant gel, pH = 5.8, and after autoclaving, 0.5 mg / L NAA, 0.2 mg / L 6-BA, and 200 mg / L vitamin C were added. The test results showed that partially replacing sucrose with trehalose in the callus induction medium significantly improved the callus induction and regeneration process in sugarcane. For *Saccharomyces cerevisiae*, the optimal callus formation and regeneration were observed when 30 g / L trehalose completely replaced 30 g / L sucrose. For *Saccharomyces cerevisiae* var. *huangpi*, the optimal callus formation and regeneration were observed when 25 g / L trehalose + 5 g / L sucrose were used (Figure 2F).

[0196] Table 3:

[0197] The above results indicate that overexpression of endogenous TPS1 in rice can significantly improve rice regeneration efficiency. In addition, the exogenous addition of trehalose, the final synthetic product of TPS1, to the culture medium can also significantly improve callus induction and regeneration efficiency in a wide range of plant species, providing a simpler and more controllable way to replace transgenic plants with exogenous compounds.

[0198] Example 4: Exogenous application of trehalose can promote the improvement of genetic transformation efficiency in a variety of recalcitrant plants.

[0199] 1. The genetic transformation efficiency of the DR5::RUBY(Yang,H.,Zhang,Y.,Fu,Q.,Jia,X.,Zhao,T.,Xu,X.,...Li,J.(2023).The DR5 and E8 reporters are suitable systems for studying the application of the Ruby reporter gene in tomato.Vegetable Research,3(1).doi:10.48130 / VR-2023-0012) plasmid was determined by transforming it into Agrobacterium tumefaciens for the genetic transformation of indica rice variety 93-11 and wild tetraploid rice variety tall wild rice.

[0200] The recipient materials used in this embodiment were calluses induced from mature embryos of the indica rice variety 93-11 and the wild tetraploid rice variety tall wild rice. The mature embryos were derived from mature seeds harvested from field cultivation.

[0201] (1) Selection and sterilization of explants: 200 mature seeds were placed in 50ml clean centrifuge tubes and sterilized in a shaker for 20min with 3.5% sodium hypochlorite solution and 1μl Tween 20. After sterilization, the seeds were thoroughly washed 3-4 times with sterile water and dried on sterile filter paper in a clean operating table.

[0202] (2) Callus induction: After drying, the seeds were inoculated into the callus induction culture medium at a density of 14 seeds / plate and cultured in the dark at 28 degrees Celsius for 30 days to induce callus production. The callus induction culture medium formula was N6B5 + 2 mg / L 2,4-D + 0.5 g / L glutamine + 2.8 g / L proline + 0.5 g / L acid-hydrolyzed casein + 3 g / L plant gel, with carbon source of 30 g / L sucrose or 25 g / L trehalose plus 5 g / L sucrose, pH value of 5.8, and sterilized by high temperature and high pressure.

[0203] (3) Preparation of Agrobacterium bacterial suspension: Agrobacterium cultured overnight on kanamycin and rifampicin resistant LB medium was centrifuged at 5000 rpm for 5 min. The collected bacterial suspension was resuspended in MS solution containing 40 g / L sucrose and 18 g / L glucose. Acetyl eugenol was added to the resuspended bacterial suspension to a final concentration of 200 mM.

[0204] (4) Agrobacterium infection and co-culture: Sterile, relatively firm, and light yellow viable callus tissue was selected for transformation. The selected callus was infected in bacterial solution for 15 min, and then excess bacterial solution was removed with several sterile filter papers. The callus was then transferred to co-culture medium or sterile filter paper and co-cultured at 22-25℃ for 2 days. The co-culture medium formula was: N6B5 + 2 mg / L 2,4-D + 30 g / L sucrose + 0.5 g / L glutamine + 2.8 g / L proline + 0.5 g / L acid-hydrolyzed casein + 3 g / L plant gel, pH 5.8. After high temperature and high pressure sterilization, 200 mM acetylsyl syringone was added.

[0205] (5) Screening culture: The co-cultured callus was transferred to the screening medium for 8 weeks of growth under the dark culture conditions of 28℃. The callus was subcultured every 14 days and transferred to a new screening medium. The screening medium formula was: N6B5 + 2mg / L 2,4-D + 30g / L sucrose + 0.5g / L glutamine + 2.8g / L proline + 0.5g / L acid hydrolyzed casein + 3g / L plant gel, pH 5.8. After high temperature and high pressure sterilization to 50℃, 50mg / L hygromycin B and 200mg / L termethin were added.

[0206] (6) Regeneration and Rooting Culture: Positive calluses obtained after transformation were transferred to regeneration medium for regeneration. After 45 days of culture, all regenerated seedlings were transferred to rooting medium for rooting culture. The regeneration medium formula was: MS + 2 mg / L 6-BAP + 0.2 mg / L kinetin + 30 g / L sucrose + 20 g / L sorbitol + 0.5 g / L acid-hydrolyzed casein + 30 mg / L hygromycin B + 3 g / L plant gel, pH 5.8. After autoclaving, 200 mg / L termethin was added. The rooting medium formula was: MS + 30 g / L sucrose + 0.1 g / L inositol + 3 g / L plant gel, pH 5.8. After autoclaving, 200 mg / L termethin was added.

[0207] Positive callus: The number of positive callus tissues of DR5::RUBY plasmid.

[0208] The conversion rate was calculated as follows: the total number of positive calluses generated after 30 days of culture in callus induction medium (i.e., the positive calluses in Tables 4 and 5) / the total number of transformed calluses (i.e., the number of explants in Tables 4 and 5).

[0209] The statistical method for regenerated callus rate is: the total number of regenerable seedling callus produced after 45 days of culture on regeneration medium (i.e., the regenerated callus in Tables 4 and 5) / the total number of transformed callus (i.e., the number of explants in Tables 4 and 5).

[0210] As shown in Tables 4 and 5, the conversion and regeneration efficiency of the indica rice variety 93-11 and the wild tetraploid rice variety tall wild rice were significantly improved.

[0211] Table 4: Conversion and Regeneration Efficiency of Indica Rice Variety 93-11

[0212] Table 5: Conversion and Regeneration Efficiency of Wild Tetraploid Rice Varieties (Tall Wild Rice)

[0213] 2. The effect of exogenous trehalose in the culture medium on the genetic transformation efficiency and regeneration of octoploid strawberries:

[0214] (1) Treatment of explants: After the octoploid strawberry cultivar Hongyan has grown in the seedling culture medium for 4 weeks, the first to third newly grown leaves are cut into leaf discs with a length and width of about 5 mm, ready for Agrobacterium infection.

[0215] (2) Preparation of Agrobacterium tumefaciens containing pHUE-GFP plasmid (Xing,S.,Chen,K.,Zhu,H.,Zhang,R.,Zhang,H.,Li,B.,&Gao,C.(2020).Fine-tuning sugar content in strawberry.Genome Biology,21(1),230.doi:10.1186 / s13059-020-02146-5): Agrobacterium tumefaciens cultured overnight in kanamycin and rifampicin resistant LB medium was centrifuged at 5000 rpm for 5 min, and the collected bacterial cells were resuspended in MS solution. Acetyl eugenol was added to the resuspended bacterial solution to a final concentration of 200 mM.

[0216] (3) Agrobacterium transformation and co-culture: Strawberry leaf discs were immersed in the bacterial solution for 15 min, and then excess bacterial solution was removed with several sterile filter papers. The samples were then transferred to a co-culture medium and co-cultured at 25°C in the dark for 2 days. The co-culture medium formula was: MS + 0.5 g / L MES + 3 g / L plant gel, with 30 g / L sucrose or trehalose added as the carbon source, pH = 5.8, and 200 mM acetylsyl syringone added after high temperature and high pressure sterilization.

[0217] (4) Plant regeneration: After co-culturing, the leaf discs were washed twice with sterile water containing 200 mg / L termethin, and excess water was absorbed with sterile filter paper. The leaf discs were then inoculated with the underside facing up onto callus induction medium containing sucrose or trehalose as the carbon source at a density of 10 explants / plate. Subcultures were performed every 14 days, and the plants were transferred to fresh callus induction medium. No selection agent was used throughout the process. After approximately 50 days of culture, the number of positive calluses, conversion rate, and regeneration rate were calculated. The callus induction medium formula was: MS + 0.5 g / L LMES + 3 g / L plant gel, with 30 g / L sucrose or trehalose added as the carbon source, pH = 5.8. After high-temperature and high-pressure sterilization, 1–5 mg / L TDZ + 0.2–2 mg / L IAA + 200 mg / L termethin were added.

[0218] Positive callus count: The number of positive callus tissues exhibiting GFP fluorescence.

[0219] The conversion rate was calculated as follows: the total number of positive calluses generated after 30 days of culture in callus induction medium (i.e., the positive calluses in Table 6) / the total number of transformed calluses (i.e., the number of explants in Table 6).

[0220] The statistical method for the regeneration callus rate is as follows: the total number of regenerable seedling calluses produced after 45 days of culture on the regeneration medium (i.e., the regenerated calluses in Table 6) / the total number of transformed calluses (i.e., the number of explants in Table 6); where, in this experiment, the regeneration medium is the callus induction medium.

[0221] The test results showed that replacing sucrose with trehalose in the callus induction medium could significantly improve the conversion and regeneration efficiency of octoploid strawberries (Table 6).

[0222] Table 6: Conversion and Regeneration Efficiency of Octoploid Strawberry Red Face

[0223] Rice TPS1 amino acid sequence (SEQ ID NO:1)

[0224] Rice TPS1 CDS sequence (SEQ ID NO:2)

[0225] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0226] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for inducing plant callus or increasing the rate of plant callus induction, wherein, The method includes: (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the synthetic pathway involving trehalose-6-phosphate synthase.

2. A method of regenerating a plant or improving the efficiency of plant regeneration, wherein, The method includes: (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the trehalose-6-phosphate synthase-mediated synthetic pathway; and, (ii) Regenerate a complete plant from the plant cells.

3. A method for genetic transformation of a plant or for increasing the efficiency of genetic transformation of a plant, wherein, The method includes: (i) Overexpressing trehalose-6-phosphate synthase in the cells of the plant, or culturing the cells of the plant in the presence of products of the synthetic pathway involving trehalose-6-phosphate synthase; (ii) Introducing at least one expression construct containing at least one nucleic acid sequence of interest into the plant cells; and, (iii) Regenerate a complete plant from the plant cells.

4. The method of claim 3, wherein, The method is a method for performing gene editing in plants or improving the efficiency of gene editing in plants, wherein the at least one nucleic acid sequence of interest encodes a component of the gene editing system.

5. The method of claim 3, wherein, The method described is a method for transforming nucleic acid sequences of interest into plants or improving the transformation efficiency of nucleic acid sequences of interest in plants.

6. The method according to any one of claims 1 to 5, wherein, In step (i), the plant cells are somatic cells or embryonic cells; Optionally, the plant cells are derived from plant explants or portions thereof, or from plant callus tissue.

7. The method according to any one of claims 1 to 6, wherein, The products of the synthetic pathway involving trehalose-6-phosphate synthase include trehalose; Optionally, the culture medium for culturing the plant cells further includes a carbon source other than trehalose, wherein the weight ratio of trehalose to the carbon source other than trehalose is 0.01 to 99:

1.

8. The method according to any one of claims 1 to 7, wherein, Overexpression of trehalose-6-phosphate synthase in the cells of the plant is achieved by introducing into the cells an expression construct containing a nucleic acid sequence encoding trehalose-6-phosphate synthase or a polypeptide of trehalose-6-phosphate synthase.

9. The method of claim 8, wherein, Methods for introducing the expression construct into plant cells include protoplast electroporation, gene gun method, PEG-mediated transformation, and Agrobacterium-mediated transformation.

10. The method according to any one of claims 1 to 9, wherein, The trehalose-6-phosphate synthase is a rice-derived trehalose-6-phosphate synthase.

11. The method according to any one of claims 1 to 10, wherein, The trehalose-6-phosphate synthase comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:

1.

12. The method of any one of claims 1-11, wherein, The plant is a monocotyledonous plant or a dicotyledonous plant.

13. The method of any one of claims 1-12, wherein, The plants are selected from wheat, strawberry, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava or potato; Preferably, the plant is a non-renewable plant; Optionally, the plant is selected from rice, sorghum, sugarcane or strawberry.

14. Any of the following (i) to (iii) uses of trehalose-6-phosphate synthase, an expression construct containing a nucleic acid sequence encoding trehalose-6-phosphate synthase, or a product of a synthetic pathway involving trehalose-6-phosphate synthase: (i) Use in callus induction or to improve the callus induction rate in plants; (ii) Uses in plant regeneration or in improving the efficiency of plant regeneration; (iii) Use in plant genetic transformation or in improving the efficiency of plant genetic transformation.

15. The use according to claim 14, wherein, The synthetic pathway products involved by the trehalose-6-phosphate synthase include trehalose.

16. The use according to claim 14 or 15, wherein, The genetic transformation includes gene editing in plants and / or the conversion of nucleic acid sequences of interest into plants.

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