Method for developing de novo transgenic and gene-edited shoots without tissue culture

Abstract

The present disclosure pertains to methods of altering a plant or seed by introducing a reagent into the plant or seed. The reagent includes (1) an altering component operable to introduce or edit one or more nucleotide sequences in the plant or seed, and (2) one or more developmental-related genes. Thereafter, the introduction of the reagent results in the introduction or editing of one or more nucleotide sequences in the plant or seed, and the expression of the developmental related genes. The present disclosure also pertains to a plant or seed that expresses one or more developmental-related genes. The present disclosure also pertains to reagents that include: (1) an altering component operable to introduce or edit one or more nucleotide sequences in a plant or seed; and (2) one or more developmental-related genes.

Description

PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031TITLEMETHOD FOR DEVELOPING DE NOVO TRANSGENIC AND GENE-EDITED SHOOTS WITHOUT TISSUE CULTURECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 687,694, filed on August 27, 2024. The entirety of the aforementioned application is incorporated herein by reference.SEQUENCE DISCLOSURE STATEMENT

[0002] Pursuant to 37 C.F.R. § 1.834, Applicant has submitted a sequence listing in XML format (“Sequence Listing”). The name of the file containing the Sequence Listing is “AF13368.P075WO.xml”. The date of the creation of the Sequence Listing is August 27, 2025. The size of the Sequence Listing is 58,000 bytes. Applicant hereby incorporates by reference the material in the Sequence Listing.BACKGROUND

[0003] A need exists for more effective methods and systems for genetically editing plants and seeds. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to a method of altering a plant or seed by introducing a reagent into the plant or seed. In some embodiments, the reagent includes (1) an altering component operable to introduce or edit one or more nucleotide sequences in the plant or seed, and (2) one or more developmental-related genes. Thereafter, the introduction of the reagent results in the introduction or editing of one or more nucleotide sequences in the plant or seed, and the expression of the developmental related genes. In some embodiments, the methods of the present disclosure also include a step of removing the developmental-related genes.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0005] In alternative embodiments, the methods of the present disclosure include introducing a reagent with an altering component into a plant or seed, where the plant or seed expresses one or more developmental-related genes. Thereafter, the introduction of the reagent results in the introduction or editing of one or more nucleotide sequences in the plant or seed. In some embodiments, the methods of the present disclosure also include a step of removing the developmental-related genes.

[0006] Additional embodiments of the present disclosure pertains to a plant or seed that expresses one or more developmental-related genes. Further embodiments of the present disclosure pertain to reagents that include: (1) an altering component operable to introduce or edit one or more nucleotide sequences in a plant or seed; and (2) one or more developmental-related genes.

[0007] The methods and reagents of the present disclosure may have various altering effects on plants and seeds. For instance, in some embodiments, the methods and reagents of the present disclosure induce formation of somatic embryos on a plant or seed. In some embodiments, the methods and reagents of the present disclosure induce formation of shoots (e.g., de novo transgenic and / or gene-edited shoots) on a plant or seed.

[0008] In some embodiments, the developmental-related genes include, without limitation, Wound Induced Dedifferentiation 1 (WIND I). Enhancer of Shoot Regeneration 1 (ESRI). Isopentenyl transferase (IPT), or combinations thereof. In some embodiments, the altering component includes, without limitation, a nucleotide sequence, a gene, a nucleotide editing system, or combinations thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A-1B provide methods of altering a plant or seed in accordance with various embodiments of the present disclosure.

[0010] FIGS. 2A-2E illustrate experimental validation of gene constructs designed to assess pESRI -driven gene activation in response to AtWINDl expression. Tobacco shoot segmentsPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 transformed with Agrobacterium tumefaciens (strain GV3101) containing pCmYLCV (SEQ ID NO: 1A)::RUBY (SEQ ID NO: 15) (FIG. 2A) and pAtESRl -..RUBY (FIG. 2B) showed limited or no callus formation. Whereas co-expression of p35S; AtWINDl-pAtESRl'..RUBY (FIG. 2C) showed significantly more callus formation and activation of the AtESRl promoter. FIG. 2D shows a quantitative analysis of callus formation, shown in Area (mm2). Explants transformed with p35S AtWINDl showed approximately 3-4-fold more callus formation. FIG. 2E shows the expression of developmental regulator genes like ipt under the AtESRl promoter and its activation by AtWINDl induced rapid callus induction, and formation of shoot apical meristems, leading to the induction of de novo meristems in phytohormone-free media.

[0011] FIGS. 3A-3D illustrate schematic diagrams showing constructs and in planta transfection strategies. FIG. 3A shows a construct showing a guide RNA targeting the tobacco PHYTOENE DESATURASE (PDS) gene driven by Arabidopsis U6-snRNA26 (U6-26) promoter (SEQ ID NO: 45), AtWINDl driven by the CaMV 35S promoter (SEQ ID NO: 46), an AtESRl promoter driving different developmental regulators (one DR in each construct), and a visible reporter gene, RUBY driven by the Cistrum Yelow Leaf Curl Vims (CmYLCV) promoter within the T-DNA border. FIG. 3B shows three different in planta transformation strategies in tobacco. After testing three different transfection strategies in tobacco, only the third strategy led to de novo organogenesis. Therefore, Applicant employed only the third strategy in tomato (FIG. 3C) and soybean (FIG. 3D). FIG. 3C shows the construct for in planta transfection and regeneration strategy in tomato. FIG. 3D shows the construct for in planta transfection and regeneration strategy in soybean.

[0012] FIGS. 4A-4F provide data illustrating the efficiencies of various constructs for de novo shoot induction, regeneration, transgenesis, and gene editing. FIG. 4A provides efficiencies of various constructs for de novo shoot induction, regeneration, transgenesis, and gene editing in N. benthamiana. Students’ t-test; ****P < 0.0001. ND: no significance detected. FIG. 4B shows the stages of de novo shoot regeneration: Left, initiation of transgenic shoots expressing RUBY; Center, transgenic branches; Right, transgenic flowers expressing the RUBY gene. FIG. 4C showsPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 regeneration outcomes showing transgenic shoots (red), biallelic mutations in the PDS gene (white), and non-transgenic shoots (green). FIG. 4D shows a gel image showing PCR analysis and confirmation of the presence of the RUBY gene in progeny plants. FIG. 4E shows the DNA sequence analysis for a subset of edited NbPDS alleles at the target sites. The single guide RNA (sgRNA) target region, the PAM site, and deleted nucleotides (dashes) are indicated. The Sequences are disclosed in SEQ ID NOS: 13-26. FIG. 4F provides transmission of transgenes to the next generation: Left, a representative plant showing RUBY expression in red flowers. Center (top), the zoomed in transgenic flower in TO generation, center (bottom), segregation of transgenic (red seedlings) and non-transgenic (green seedlings) progeny germinated on Vi MS media. Right, transgenic and wild-type (WT) plants grown to maturity in pots show fading RUBY expression in late developmental stages, although flowers of transgenic plants retained a red phenotype compared to the white flowers in WT plants.

[0013] FIGS. 5A-5D illustrate in planta transformation and de novo .shoot regeneration in tomato. FIG. 5A shows stages of de novo shoot regeneration in tomato, showing representative transgenic shoots (red tissues) emerging from wound sites transformed with the WEipt construct. FIG. 5B shows quantification of transgenic frequencies in de novo regenerated shoots, highlighting transformation efficiency. FIG. 5C shows a gel image showing PCR analysis and confirmation of the transgene presence in representative TO plants. FIG. 5D shows flowers from transgenic red shoots with high betalain accumulation.

[0014] FIGS. 6A-6G illustrate semi tissue-culture-free transformation in soybean using embryogenic axis. FIG. 6A shows embryo axis (EA) explants used for soybean transformation. Scanning electron microscopy images show the initiation of callus formation after 5 days of transformation. FIG. 6B shows various stages of de novo shoot formation, expression of the RUBY gene in EA explants transformed with the \VEipt2 construct. FIG. 6C provides representative de novo gene-edited shoots (semi-albino / albino shoots) regenerated from EA explants transformed with the ~WEipt2 construct. FIG. 6D shows maturing transgenic plant -120 days after transformation. Note the visible RUBY expression in young leaves. FIG. 6E shows pods andPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 seeds of transgenic plants expressing RUBY in the early stage of pod development, whereas RUBY expression faded in mature pods and seeds. FIG. 6F shows transgenesis and gene-editing frequencies in de novo regenerated shoots. FIG. 6G provides genotyping of edited shoots in GmPDSl and GmPDS2 in TO generation. Five plants (TO-4, T0-5, T0-6, TO-7, and T0-8) showed biallelic mutations of the PDS. The guide RNA (gRNA) target region, the PAM site, and deleted nucleotides (dashes) are indicated. The sequences are shown in SEQ ID NOS: 27-44.

[0015] FIGS. 7A-7C illustrate a schematic model illustrating de novo shoot regeneration (shoot at site). Intact shoot or embryo axis is shown as an explant. The dotted line indicates the pruning of the shoot apical meristem. FIG. 7A shows transformation with an empty vector (no DR gene) at the pruned site triggers wound healing, leading to callus formation. FIG. 7B shows that Callus formation is enhanced by the activity of the WIND1 gene, which promotes cellular reprogramming. FIG. 7C shows de novo shoot initiation is driven by the synergistic action of WIND! and ipt (WEipt). The WIND1 reprograms somatic cells to initiate differentiation (callus formation) at the injection site, while simultaneously activating ipt and producing cytokinin (CKs) under the control of the ESRI promoter. This coordinated expression accelerates differentiation through WINDl, while ipt facilitates the transition from differentiated cells to organogenesis, resulting in the formation of de novo shoot primordia.DETAILED DESCRIPTION

[0016] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0017] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0018] Plant tissue culture or in vitro regeneration is a cornerstone in genetic engineering and plant biotechnology. However, only a limited number of plant species exhibit amenable characteristics for this process, often requiring several months to achieve successful regeneration. Additionally, the hurdle in obtaining transgenic and gene-edited progeny not only relies on their regeneration plasticity but also the successful transformation of the transgene and gene-editing reagents which significantly impede the efficient development of transgenic and gene-edited plants. Somatic cell regeneration is a highly complex process and success in transformation and regeneration is influenced by several pivotal factors, encompassing the application of plant growth regulators, the formulation of the basal media, the nature of the explant, selectable markers and transformation methods. Significantly, this process depends on the species and genotype, underscoring the importance of precision in understanding and manipulating these variables.

[0019] Considerable efforts have been dedicated to investigating innovative approaches for regeneration and transformation, aiming to overcome the conventional procedures dependent on tissue culture-based transformation. Early endeavors to adopt a “tissue culture-free” transformation approach involved the use of Agrobacterium to transform germinating seeds and in planta transformation and regeneration of apical shoots in Arabidopsis. Later, a simple floral dip transformation method was developed in Arabidopsis to successfully generate transgenic events. However, this method is suitable to only a few species, mainly belonging to the Brassicaceae family.

[0020] In parallel to these endeavors, several researchers have identified that the ectopic expression of morphogenic genes or developmental regulators (DRs) such as isopentenylPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 pyrophosphate transferase (ipt), WUSCHEL (WUS), BABY-BOOM (BBM), SHOOT MERISTEMLESS (STM), among others, dramatically enhanced the efficiency of plant transformation and regeneration. More recently, with the advent of gene-editing technology, the concept of tissue culture-free transformation in plants became even more critical for the general application of this technology in agriculture. Tissue culture-free plant gene editing has been achieved by the co-delivery of gene editing reagents (CRISPR / Cas9 and guide RNAs) with DRs that induce de novo organogenesis, thereby obtaining stable targeted gene-edited plants.

[0021] However, oftentimes, expression of DRs alone is not enough for direct organogenesis. They must collaborate with endogenous or exogenous phytohormones, wound stress, plant developmental stages and several other factors, thereby establishing and maintaining meristematic activity and reprogramming of differentiated somatic cells.

[0022] Among several regeneration pathways, developing de novo organs induced by wounding stress is one of the primary triggers of plant regeneration. This property of wounding and wound healing has long been utilized for clonal propagation, such as cutting and grafting, and direct and indirect somatic cell regeneration. In nature, plants are constantly challenged by harsh environments, predators, diseases, and physical injury, but they overcome these diverse challenges by repairing local wounds and reconstructing tissues or organs.

[0023] The wound-healing process induces the transcriptional activation of molecular signals necessary for reprogramming, allowing local cells to heal, survive, and initiate new growth and development. Additionally, it triggers the biosynthesis of endogenous hormonal pathways, crucial for determining cellular fate during regeneration. Although the development of masses of undifferentiated cells or callus after wounding is the first step of tissue healing, appropriate biosynthesis of endogenous hormones and activation of molecular signals can induce cell fate reprogramming and initiate meristematic activity.

[0024] Notably, during this process, a wound-induced tissue repair pathway gets activated involving transcriptional regulators belonging to APETAL2 / ETHYLENE RESPONSE EACTORPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031(AP2 / ERF) gene family; WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), ENHANCER OF SHOOT REGENERATION 1 (ESRI), PLETHORA3 (PLT3 and TYPE-B ARABIDOPSIS RESPONSE REGULATORS (ARRs). which reprogram cell fate and initiation of new developmental pathways. Among these regulators, WIND1 is a master regulator of wound-induced cellular reprogramming in plants. It promotes callus formation, formation of tracheary elements, and pluripotency acquisition as a primary step for plant regeneration. Following wounding, the expression of WIND1 is upregulated within 30 minutes and peaks to about ten-fold one hour after wounding, which in turn acts as a transcriptional activator of ESRI belonging to the AP2 / ERF gene family. Furthermore, ectopic expression of ESRI in Arabidopsis induces callus formation and initiates shoot regeneration under optimal in vitro conditions.

[0025] Previously, several studies have employed various hormone biosynthesis genes and DRs to enhance the efficiency of genetic transformation and plant regeneration. Ectopic expression of WUS and BBM significantly improved transformation efficiency in recalcitrant maize inbreds. Similarly, researchers have demonstrated enhanced in vitro transformation of rice, wheat, and citrus by expressing a GRF-GIF chimeric protein. However, these approaches still depend on long, laborious, and complex tissue-culture protocols.

[0026] In recent advancements, development of tissue-culture-free methods for plant regeneration and gene editing rely on overexpressing combinations of DR genes in various plant species. Similarly, viral delivery methods have been employed for heritable, tissue-culture-free gene editing in plants like tomatoes and tobacco. However, these approaches often require plants to mature, involve DR delivery at axillary meristems, and demand extended periods to achieve de novo shoot formation. Additionally, regenerated plants frequently exhibit severe pleiotropic effects or depend on viral vectors specific to plant species, highlighting the need for improved strategies.

[0027] As such, a need exists for more effective methods and systems for genetically editing plants and seeds. Numerous embodiments of the present disclosure aim to address the aforementioned need.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0028] In some embodiments, the present disclosure pertains to a method of altering a plant or seed. In some embodiments illustrated in FIG. 1A, the methods of the present disclosure include introducing a reagent into the plant or seed (step 10). The reagent includes (1) an altering component operable to introduce or edit one or more nucleotide sequences in the plant or seed, and (2) one or more developmental-related genes. Thereafter, the introduction of the reagent results in the introduction or editing of one or more nucleotide sequences in the plant or seed (step 12), and the expression of the developmental related genes (step 14). In some embodiments, the methods of the present disclosure also include a step of removing the developmental-related genes (step 16). In some embodiments, the methods of the present disclosure may have various altering effects on plants and seeds (step 17), such as formation of de novo shoots (step 18) and / or somatic embryos (step 19).

[0029] In alternative embodiments illustrated in FIG. IB, the methods of the present disclosure include introducing a reagent with an altering component into a plant or seed, where the plant or seed expresses one or more developmental-related genes (step 20). Thereafter, the introduction of the reagent results in the introduction or editing of one or more nucleotide sequences in the plant or seed (step 22). In some embodiments, the methods of the present disclosure also include a step of removing the developmental-related genes (step 24). In some embodiments, the methods of the present disclosure may have various altering effects on plants and seeds (step 26), such as formation of de novo shoots (step 27) and / or somatic embryos (step 29).

[0030] Additional embodiments of the present disclosure pertains to a plant or seed that expresses one or more developmental-related genes. Further embodiments of the present disclosure pertain to reagents that include: (1) an altering component operable to introduce or edit one or more nucleotide sequences in a plant or seed; and (2) one or more developmental -related genes.

[0031] As set forth in more detail herein, the methods, plants, seeds and reagents of the present disclosure can have numerous embodiments.

[0032] ReagentsPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0033] The methods of the present disclosure can utilize various reagents to alter plants and seeds. Additionally, the reagents of the present disclosure can include various components. For instance, in some embodiments, the reagents of the present disclosure include an altering component operable to introduce or edit one or more nucleotide sequences in a plant or seed. In some embodiments, the reagents of the present disclosure include one or more developmental-related genes. In some embodiments, the reagents of the present disclosure include an altering component and one or more developmental-related genes.

[0034] The reagents of the present disclosure can be in various forms. For instance, in some embodiments, the reagents of the present disclosure include an expression vector. In some embodiments, the expression vector is in the form of a plasmid. In some embodiments, a single expression vector includes an altering component and developmental-related genes. In some embodiments, a first expression vector includes an altering component and a second expression vector includes developmental-related genes.

[0035] In some embodiments, the reagents of the present disclosure include an expression cassette. In some embodiments, a single expression cassette includes an altering component and developmental-related genes. In some embodiments, a first expression cassette includes an altering component and a second expression cassette includes developmental-related genes.

[0036] Altering component

[0037] Altering components of the reagents of the present disclosure are generally operable to introduce or edit one or more nucleotide sequences in a plant or seed. The reagents and methods of the present disclosure can include or utilize various altering components. For instance, in some embodiments, the altering component includes, without limitation, a nucleotide sequence, a gene, a nucleotide editing system, or combinations thereof.

[0038] In some embodiments, the altering component includes a nucleotide editing system. In some embodiments, the nucleotide editing system includes a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system). In somePCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 embodiments, the CRISPR / Cas system includes at least one Cas nuclease and at least one guide RNA. In some embodiments, the Cas nuclease includes, without limitation, class 2 of Cas nucleases, Cas 9, Cas . CasQ2, Cpfl, or combinations thereof. In some embodiments, the CRISPR / Cas system includes a CRISPR / Cas9 system.

[0039] In some embodiments, the altering component includes a nucleotide sequence. In some embodiments, the nucleotide sequence is operable to be expressed in a plant or seed. In some embodiments, the altering component includes a gene operable to be expressed in a plant or seed.

[0040] The altering components of the present disclosure may be operable to edit one or more nucleotide sequences of a plant or seed. In some embodiments, the methods of the present disclosure include a step of editing one or more nucleotide sequences of a plant or seed through the utilization of the altering components. In some embodiments, the editing includes, without limitation, introducing a mutation to a gene, introducing a deletion to a gene, introducing an insertion to a gene, removing a portion of a gene, changing a base of a gene, removing a gene, inserting a gene, partially or fully replacing a gene, deleting a nucleotide, inserting a nucleotide, or combinations thereof. In some embodiments, the editing occurs without the use of in vitro tissue culture techniques.

[0041] Developmental-related genes

[0042] In some embodiments, the developmental-related genes of the present disclosure are involved in stem cell activity, tissue differentiation, regeneration, or combinations thereof. The reagents and methods of the present disclosure can include or utilize various developmental-related genes. For instance, in some embodiments, the developmental-related genes include, without limitation, Wound Induced Dedifferentiation 1 (WIND / ). Enhancer of Shoot Regeneration 1 (ESRI ), Isopentenyl transferase (IPT). or combinations thereof.

[0043] In some embodiments, the developmental-related genes include Wound Induced Dedifferentiation 1 (WIND1). In some embodiments, WIND1 includes SEQ ID NO: 1. In some embodiments, WIND1 includes a sequence with at least 65% sequence identity to SEQ ID NO: 1.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031In some embodiments, WIND1 includes a sequence with at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, WIND1 includes a sequence with at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, WIND1 includes a sequence with at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, WIND1 includes a sequence with at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, WIND1 includes a sequence with at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, WIND1 includes a sequence with at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, WIND! includes a sequence with at least 99% sequence identity to SEQ ID NO: 1.

[0044] In some embodiments, WIND1 includes SEQ ID NO: 4. In some embodiments, WIND! includes a sequence with at least 65% sequence identity to SEQ ID NO: 4. In some embodiments, WIND1 includes a sequence with at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, WIND1 includes a sequence with at least 75% sequence identity to SEQ ID NO: 4. In some embodiments, WIND1 includes a sequence with at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, WIND1 includes a sequence with at least 85% sequence identity to SEQ ID NO: 4. In some embodiments, WIND! includes a sequence with at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, WIND1 includes a sequence with at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, WIND1 includes a sequence with at least 99% sequence identity to SEQ ID NO: 4.

[0045] In some embodiments, the developmental-related genes include Isopentenyl transferase (IPT). In some embodiments, IPT includes SEQ ID NO: 2. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, IPT includes a sequence with at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, IPT includes a sequence with at least 95% sequence identity toPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031SEQ ID NO: 2. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 2.

[0046] In some embodiments, IPT includes SEQ ID NO: 5. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 5. In some embodiments, IPT includes a sequence with at least 70% sequence identity to SEQ ID NO: 5. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 5. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO:5. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, IPT includes a sequence with at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 5.

[0047] In some embodiments, IPT includes SEQ ID NO: 6. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 6. In some embodiments, IPT includes a sequence with at least 70% sequence identity to SEQ ID NO: 6. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 6. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO:6. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, IPT includes a sequence with at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 6.

[0048] In some embodiments, IPT includes SEQ ID NO: 7. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 7. In some embodiments, IPT includes a sequence with at least 70% sequence identity to SEQ ID NO: 7. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 7. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 7. InPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO:7. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, IPT includes a sequence with at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 7.

[0049] In some embodiments, IPT includes SEQ ID NO: 8. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 8. In some embodiments, IPT includes a sequence with at least 70% sequence identity to SEQ ID NO: 8. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 8. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO:8. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, IPT includes a sequence with at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 8.

[0050] In some embodiments, IPT includes SEQ ID NO: 9. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 9. In some embodiments, IPT includes a sequence with at least 70% sequence identity to SEQ ID NO: 9. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 9. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO:9. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, IPT includes a sequence with at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 9.

[0051] In some embodiments, IPT includes SEQ ID NO: 10. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 10. In some embodiments, IPTPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 includes a sequence with at least 70% sequence identity to SEQ ID NO: 10. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 10. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO:10. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, IPT includes a sequence with at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 10.

[0052] In some embodiments, IPT includes SEQ ID NO: 11. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 11. In some embodiments, IPT includes a sequence with at least 70% sequence identity to SEQ ID NO: 11. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 11. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 11. In some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO:11. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 11. In some embodiments, IPT includes a sequence with at least 95% sequence identity to SEQ ID NO: 11. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 11.

[0053] In some embodiments, IPT includes SEQ ID NO: 12. In some embodiments, IPT includes a sequence with at least 65% sequence identity to SEQ ID NO: 12. In some embodiments, IPT includes a sequence with at least 70% sequence identity to SEQ ID NO: 12. In some embodiments, IPT includes a sequence with at least 75% sequence identity to SEQ ID NO: 12. In some embodiments, IPT includes a sequence with at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, IPT includes a sequence with at least 85% sequence identity to SEQ ID NO:12. In some embodiments, IPT includes a sequence with at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, IPT includes a sequence with at least 95% sequence identityPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 to SEQ ID NO: 12. In some embodiments, IPT includes a sequence with at least 99% sequence identity to SEQ ID NO: 12.

[0054] In some embodiments, the developmental-related genes include an isoform of IPT. In some embodiments, the IPT isoform includes, without limitation, ADP-TPT, ATP-IPT, iRNA-IPT, or combinations thereof.

[0055] The reagents and methods of the present disclosure can include or utilize different variations of developmental-related genes. For instance, in some embodiments, the developmental-related genes include a single developmental-related gene. In some embodiments, the developmental-related genes include multiple developmental-related genes.

[0056] In some embodiments, the developmental-related genes include Wound Induced Dedifferentiation 1 (WIND1) and Isopentenyl transferase (IPT). In some embodiments, IPT is operatively linked to a promoter for Enhancer of Shoot Regeneration 1 (ESRI). In some embodiments, the ESRI promoter includes SEQ ID NO: 3. In some embodiments, the ESRI promoter includes a sequence with at least 65% sequence identity to SEQ ID NO: 3. In some embodiments, the ESRI promoter includes a sequence with at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the ESRI promoter includes a sequence with at least 75% sequence identity to SEQ ID NO: 3. In some embodiments, the ESRI promoter includes a sequence with at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the ESRI promoter includes a sequence with at least 85% sequence identity to SEQ ID NO: 3. In some embodiments, the ESRI promoter includes a sequence with at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the ESRI promoter includes a sequence with at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the ESRI promoter includes a sequence with at least 99% sequence identity to SEQ ID NO: 3.

[0057] Expression of developmental-related genes

[0058] In some embodiments, the introduction of the reagents of the present disclosure into a plant or seed results in the expression of one or more developmental-related genes in the plant or seed.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031The plants and seeds of the present disclosure may express developmental-related genes in various manners. For instance, in some embodiments, a plant or seed of the present disclosure endogenously expresses one or more developmental-related genes. In some embodiments, a plant or seed of the present disclosure exogenously expresses one or more developmental-related genes. In some embodiments, a plant or seed of the present disclosure ectopically expresses one or more developmental-related genes. In some embodiments, a plant or seed of the present disclosure overexpresses one or more developmental-related genes.

[0059] The expression of developmental-related genes can have various effects on plants and seeds. For instance, in some embodiments, the expression of the developmental-related genes results in the differentiation of plants and seeds. In some embodiments, the expression of the developmental-related genes results in the regeneration of shoots directly on a plant. In some embodiments, the shoots include, without limitation, genetically engineered shoots, transgenic shoots, de novo shoots, gene-edited shoots, or combinations thereof. In some embodiments, the expression of developmental-related genes results in the regeneration of a plant or seed.

[0060] Introduction of reagents into plants and seeds

[0061] The reagents of the present disclosure may be introduced to plants and seeds in various manners. For instance, in some embodiments, the reagents of the present disclosure are introduced into a plant or seed by a method that includes, without limitation, transfection, electroporation, particle bombardment, agroinfiltration, or combinations thereof.

[0062] In some embodiments, the reagents of the present disclosure are introduced into a plant or seed through a bacterial host strain carrying the reagent. In some embodiments, the bacterial host strain is A. tumefaciens. In some embodiments, the bacterial host strain is R. rhizogenes.

[0063] In some embodiments, the reagents of the present disclosure are introduced into a plant or seed in vivo. In some embodiments, the reagents of the present disclosure are introduced into a seed. In some embodiments, the seed includes, without limitation, a mature seed, an immature seed, a germinating seed, or combinations thereof.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0064] In some embodiments, the reagents of the present disclosure are introduced into a plant. In some embodiments, the reagents of the present disclosure are introduced through in planta transformation. In some embodiments, the reagents of the present disclosure are introduced into leaves, stems, cotyledons, shoot apical meristem, floral meristem, inflorescence meristems, root apical meristems, lateral meristems, or combinations thereof.

[0065] In some embodiments, the altering component and the developmental-related genes of a reagent of the present disclosure are introduced into a plant or seed at the same time. In some embodiments, the altering component and the developmental-related genes of a reagent of the present disclosure are introduced into a plant or seed at different times. For instance, in some embodiments, developmental-related genes may be introduced into a plant or seed. Thereafter, the altering component of a reagent may be introduced into a plant or seed.

[0066] Removal of developmental-related genes

[0067] In some embodiments, the methods of the present disclosure also include a step of removing developmental-related genes from a plant or seed. For instance, in some embodiments, removal occurs after the plants or seeds have been altered by an altering component. In some embodiments, the removal occurs after the plants or seeds have been regenerated. In some embodiments, the removal occurs through gene editing, such as through the utilization of a CRISPR / Cas system.

[0068] Altering effects

[0069] The methods and reagents of the present disclosure may have various altering effects on plants and seeds. For instance, in some embodiments, the methods and reagents of the present disclosure improve plant or seed regeneration. In some embodiments, the methods and reagents of the present disclosure improve plant or seed transgenesis. In some embodiments, the methods and reagents of the present disclosure induce formation of somatic embryos on a plant or seed.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0070] In some embodiments, the methods and reagents of the present disclosure induce formation of shoots on a plant or seed. For instance, in some embodiments, the methods and reagents of the present disclosure induce formation of de novo shoots, transgenic shoots, gene-edited shoots, chimeric shoots, shoot primordia and / or in planta shoots on a plant or seed.

[0071] Plants and seeds

[0072] The methods and reagents of the present disclosure may be utilized to alter various plants and seeds. Additionally, the plants and seeds of the present disclosure can include various species and varieties. For instance, in some embodiments, the plants and seeds of the present disclosure include, without limitation, crops, weeds, maize, rice, soybean, cotton, wheat, N. benthamiana, Arabiclopsis, Amaranthus palmeri, tobacco, tomato, lettuce, common beans, pinto beans, coms, potato, grapes, citrus, blueberry, sorghum, sugar cane, snapdragon, Bok choy, chickpea, flowering crops, marigold, chrysanthemum, vegetables, ornamental plants, horticultural crops, varieties thereof, or combinations thereof.

[0073] In some embodiments, the plant or seeds of the present disclosure includes a plant. In some embodiments, the plant is less than one month old. In some embodiments, the plant is less than two months old.

[0074] In some embodiments, the plants and seeds of the present disclosure include soybean. In some embodiments, the plants and seeds of the present disclosure include cotton. In some embodiments, the plants and seeds of the present disclosure include green-house grown plants.

[0075] Additional Embodiments

[0076] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0077] Example 1. Shoot at Site: Advancing in planta transformation, regeneration, and gene-editing through a cascade of wounding-mediated developmental regulators

[0078] Developing transgenic and / or gene-edited plants largely depends on tedious, lengthy, and costly in vitro regeneration protocols. While plants have remarkable regeneration ability, not all species, genotypes, or even explants exhibit the same transformation and regeneration potential under in vitro conditions. To tackle this bottleneck, Applicant developed a seamless and user- friendly system to induce transgenic and gene-edited de novo meristems via a synthetic cascade comprising a wound-induced regeneration pathway, plant developmental regulators (DRs), and gene-editing reagents. WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) was used as a transcriptional regulator to control the expression of various DR genes through ENHANCER OF SHOOT REGENERATION 1 (ESRI) promoter. This cascade was strategically applied in planta to the non-meristematic internode of N. benthamiana to induce meristematic activity and regenerate de novo shoots with knock-out mutations of the phytoene desaturase (PDS) gene. Among the DR genes tested, the strategic expression of isopentenyl transferase (ipt) proved most effective for efficient regeneration in tobacco, and this synthetic toolkit was subsequently applied with success to both tomato and soybean. AtWINDl served as a key cellular reprogramming factor, initiating differentiation, while ipt complemented this process by promoting organogenesis through cytokinin biosynthesis. This methodology offers a transformative approach to overcome barriers in plant biotechnology, potentially accelerating the generation of transgenic and gene- edited plants without reliance on conventional tissue-culture intermediates.

[0079] In particular, Applicant developed an innovative approach leveraging the WIND1-ESR1 cascade to accelerate de novo shoot regeneration. Applicant successfully developed transgenic and gene-edited plants by transfecting young tobacco and tomato plants (under one month old) at internodes, and embryo axes of soybeans lacking meristematic activity. This method enabled shoot induction as early as two weeks post-transfection, significantly enhancing regeneration efficiency.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0080] Applicant utilized the wound-response pathway to overcome limitations associated with labor-intensive tissue culture, prolonged regeneration times, and pleiotropic effects. Applicant engineered robust synthetic cascade coordinating genes involved in stem cell maintenance, rapid tissue differentiation, and regeneration under wound-induced regulatory control. Important DR genes, including ipt, WUS, STM, BBM, and the GRF-GIF chimera, were individually tested to assess de novo shoot formation in tobacco, tomato, and soybean. Additionally, this system was combined with gene-editing reagents to target the phytoene desaturase (PDS) gene in tobacco and soybeans. This streamlined approach offers a promising framework for advancing efficient, scalable, and cost-effective plant regeneration and gene editing.

[0081] Example 1.1. Results

[0082] Assessment of WIND1 :ESR1 response'. The WIND1 plays a central role in promoting wound-induced cellular reprogramming in plants, a process crucial for callus formation through the activation of cytokinin response. One of the critical outcomes of WIND1 activity is transcriptional activation of the ESRI gene, guiding somatic cells toward a dedifferentiated state (wound healing) and, in some cases, initiation of organogenesis. The transactivation assay of ESR] has demonstrated that WIND! binds directly to the vascular tissue and wound-responsive cis- element-like motifs in the ESRI promoter and activates the expression of ESRI. Based on this rationale, Applicant designed a series of cascade constructs to test the regulatory influence of AtWINDl on the AtESRl promoter (proAlESR) in tobacco (N. benthamiana) (FIGS. 2A-2E and 3A-3D).

[0083] The RUBY gene was used to visualize transgene expression non-invasively. The ectopic expression of the RUBY gene results in betalain accumulation, causing bright red pigmentation in plant tissues. The constructs proCmYLCV '..RUBY and proAtESRl : :RUBY were used as controls to assess callus formation and gene induction under the control of AtESRl, respectively. Finally, to verify if AtWINDl binds and activates the AtESRl promoter and induces callus formation, Applicant developed a construct carrying pro35S::WINDl-proESRl::RUBY cassettes. As expected, relatively low callus formation was observed when the RUBY gene was expressed underPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 proCmYLCV or the AtESRl promoter in tobacco explants, due to the natural response of wounding during explant preparation (FIG. 2A and FIG. 2B). However, explants transformed with pro35S: A / AT) / demonstrated a 3-4-fold increase in callus formation (area) in phytohormone- free MS media (FIG. 2C).

[0084] These results validated that the overexpression of AtWINDl in tobacco accelerates the callus formation, activating the gene underlying the AtESRl promoter. Therefore, Applicant reasoned that driving the expression of DR genes under the AtESRl promoter could enhance de novo shoot formation at the wound site.

[0085] To test this hypothesis, Applicant designed and constructed several vectors. In these constructs, DR genes were driven by the AtESRl promoter, and AtWINDl was driven by the CaMV 35S promoter, alongside RUBY driven by CmYLCV promoter as a visible reporter gene. Given ipt has been identified as a key cytokinin biosynthesis gene capable of promoting de novo shoot formation, Applicant first tested the WEipt construct under tissue culture conditions using internode and petiole explants of tobacco. Within three weeks, the WEipt significantly enhanced callus formation and induced the formation of multiple de novo shoot meristems on phytohormone- free I / 2 strength MS media (FIG. 2E). This successful in vitro regeneration confirmed that controlled expression of ipt under pmAtESRl mediated by AtWINDl, can effectively induce de novo shoot regeneration (FIG. 2E). This outcome provided a robust foundation for further testing various DR genes for in planta transformation experiments.

[0086] In planta shoot formation, transgenesis and gene editing: To evaluate the combined effect of AtWINDl and pm A I ESRI with various DRs on de novo meristem formation and in planta transgenesis, several well characterized DRs including ZmWUS, AtSTM, ipt, GmBBM, and the GmGRF-GIF chimera were cloned under AtESRl promoter in modular vectors. Additionally, modular vectors containing AtWINDl, RUBY, and gRNA targeting the tobacco Phytoene Desaturase (NbPDS) gene were constructed. The Agrobacterium carrying these constructs were then introduced into young, Cas9-cxprcssing transgenic tobacco plants by targeting three simplePCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 delivery sites: (1) multiple internode sites, (2) intemodal areas proximal to the shoot apical meristem, and (3) pruned shoot sites (FIG. 3B).

[0087] Following the Agrobacterium delivery, the injection sites were carefully observed for callus formation and initiation of de novo buds. Since the RUBY gene was incorporated with all DRs genes, the expression of RUBY (red pigment) and the development of callus and de novo shoot formation were recorded after 1 week of transformation. Notably, all AtWINDl -expressing constructs, regardless of the delivery site, accelerated callus formation at the injection site compared to constructs without AtWINDl. The de novo shoot formation did not occur when constructs were delivered at multiple internode sites (delivery site 1) or areas proximal to the apical shoot (delivery site 2).

[0088] For instance, injections at multiple sites along each internode led to callus formation, but the calli at these sites were more compact and exhibited signs of wound healing. Similarly, injections proximal to the shoot apical meristem resulted in compact callus formation, albeit relatively larger than multiple injection sites. In contrast, constructs carrying AtWINDl and ipt genes injected into pruned young shoots (delivery site 3) of N. benthamiana led to not only callus formation but dramatically accelerated the pace and frequency of de novo shoot formation at the wound site, as early as 12 days after transformation.

[0089] Applicant followed an in planta transfection strategy that involved injecting older plants (over 2 months old). However, these plants have already developed mature, woody stems, limiting the availability of actively dividing somatic cells and resulting in low efficiency in transformation and regeneration. Applicant shifted to using younger (about one month old) plants to address this issue and targeted the internode region (which is non-meristematic) after pruning. This approach created a more conducive cellular environment for transformation, leading to significantly improved regeneration of transgenic and gene-edited meristems (FIGS. 4A-4F).

[0090] Implementing this method, Applicant consistently achieved an average regeneration frequency of -71% across three independent experiments, using the ipt gene in tobacco.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031Expression of GmGRF-GIF on pruned shoots resulted in the induction of de novo meristems at the injection sites. However, the efficiency was less than 4%, and no transgenic shoots were obtained (FIG. 4A). Constructs with ZmWUS. AtSTM, and GmBBM did not result in de novo shoot formation, and their response was similar to the empty vector. Upon testing a construct WINDruby (35SAVIND1) or RUBY-Empty (CrnYLCV :RUBY) without any DR genes also failed to regenerate shoots. Following the successful regeneration of de novo shoots at site of injection in tobacco with the third method, Applicant employed similar delivery sites and in planta transformation strategy for tomato and soybean (FIG. 3C and FIG. 3D). Previous in planta transformation methods used mature plants (over 2 months old), and they also involved repeated axillary shoot removal, or multiple Agrobacterium injections. This approach used younger, healthy seedlings (22-28 days old) to accelerate de novo shoot regeneration efficiency.

[0091] It has been well-studied that the ectopic expression of ipt gene from a tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is sufficient to induce shoot primordia in dicots in in vitro conditions. Notably, Applicant observed that expression of DRs (especially ipt) regulated by AtWINDl-ESRl showed formation of callus followed by de novo shoot formation via indirect organogenesis, whereas previous studies showed that expression of DRs under constitutive promoters resulted in direct organogenesis with pleiotropic effects. Since the indirect organogenesis pathway involves formation of calli, de novo shoot induction in the present Example was more uniform due to a rapid but short callus phase, resulting in -71% shoots regeneration between 12-18 days (FIG. 4A). Hence, the AtWIND-ESRl-ipt (WEipt) combination was used for downstream investigations and testing for in planta transformation in tomato (a close relative of tobacco) and soybean (distantly related species to tobacco).

[0092] Applicant noted that despite stable integration of the RUBY gene (confirmed by PCR), the betalain accumulation (red pigmentation) was not always apparent in developing shoots, and in most cases, the red color was faint and variegated in transgenic shoots (FIG. 4B). For instance, five days after transformation, most injection sites displayed red foci until shoot emergence. However, this red coloration faded during shoot elongation, and it was variegated in mature leaves,PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 but flowers retained bright red pigment (FIG. 4B and FIG. 4F). Similarly, in the subsequent generations, young tobacco seedlings germinated on petri dishes remained red (FIG. 4F) but faded over time.

[0093] Applicant also noticed that intense red pigmentation was occasionally linked to the development of fruits (in the case of tomato; discussed in sections below). Previous reports have shown that despite stable integration of the RUBY gene in maize, some transgenic seeds did not accumulate the apparent phenotype. Similarly, it has been reported that the betalain accumulation patterns were highly variable across different tissues and developmental stages of maize, but heavily pigmented plants showed stunted growth.

[0094] Nevertheless, Applicant conducted genotyping on all regenerated shoots to validate transgene insertion. Two weeks after shoot initiation, irrespective of RUBY expression, tissue samples were collected from each shoot to confirm transgenesis and assess gene editing outcomes. Genotyping (PCR analysis) revealed that 35% of the total regenerated shoots were transgenic, a notable improvement over previously reported transgenesis rates of 5-10% using single or combined DRs, including ipt.

[0095] In parallel, the efficacy of the WEipt on gene editing was evaluated. About 10% of de novo shoots exhibited mutations in either or both PDS homologs, producing mono and biallelic mutations often accompanied by semi-albino and complete albino phenotypes (FIG. 4C and FIG. 4E). Some transgenic RUBY red) shoots showed mutations in the PDS gene. However, the albino phenotype was not visible due to the masking effect of red coloration from RUBY expression. Notably, Applicant identified a subset of albino shoots in the TO generation, indicating biallelic mutations in both PDS homologs in tobacco (noted as plant #7 in FIG. 4C). A range of 1 - 37 bp deletions were observed in PDS genes (FIG. 4E). This observation suggests that the co-expression of WEiptl and gene-editing reagents was effective enough to knockout PDS genes and induce meristematic cells simultaneously.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0096] Assessment of pleotropic effects: In this Example, Applicant also hypothesized that overexpression of AfWINDl and ipt, particularly ipt would lead to pleiotropic effects on regenerated plants similar to previous studies that used DR genes for regeneration. As expected, Applicant observed some plants with developmental defects, including short stature and profuse branching, and bushy leaves. However, compared to previous studies, frequency of pleiotropic effects in the method was less severe and average 4% of the plants (9 out of 205 regenerated tobacco shoots) in the TO generations showed unusual phenotypes.

[0097] Furthermore, tobacco plants in the T1 generation remained relatively normal except for short stature but exhibited higher shoot biomass and flower settings than those of the wild type. Notably, the seed yield per plant in the T1 generation was more than 54% in the plants expressing WEiptl constructs. However, in subsequent generations, normal phenotypes were restored. It has been shown that AfWINDl overexpression in Arabidopsis has relatively milder effects on the plant morphology, and transcriptome profiling indicated an upregulation of auxin and cytokinin biosynthesis genes and ectopic formation of tracheary elements. The gene expression analysis (RT- qPCR) of stably expressing DR genes (AfWINDl and ipt) across three generations (T1-T3) showed relatively higher expression of WIND1 in de novo shoots of Tl, which declined marginally (by 0.5 to 0.8-fold) in the subsequent generations. A similar trend was observed with ipt since ESRI promoter regulated its expression in WIND1 dependent manner (rather than constitutive expression) and the reduction in gene expression correlated with the observed recovery of pleiotropy and normal plant growth. This suggests that overexpression of AtWINDl in tobacco (this Example) has milder effect compared to Arabidopsis.

[0098] Likewise, it has been shown that overexpression of cytokinin biosynthesis genes significantly alters plant morphology, enhances yield, and reduces senescence. In the present Example, the pleiotropic effects of expressing ipt under the control of AtWIND / AtESRl (WEiptl) cascade in tobacco were lower than those observed when expressing under constitutive or strong promoters.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0099] WEipt mediated in planta transformation in tomato and soybean'. The transformation of WEiptl with “shoot at site” method proved to be an efficient and seamless approach, resulting in higher regeneration, transformation, and gene-editing efficiencies in tobacco. To assess the broader applicability of this strategy, these reagents were systematically tested in crop species, including tomato and soybean. In general, conventional tissue culture-based transformation in tomato and soybean requires 4-5 and 7-9 months, respectively. To streamline this process, Applicant adopted a strategy similar to the tobacco experiments (FIGS. 3A-3D). Tomato plants were pruned after three weeks and inoculated with Agrobacterium strain GV3101, carrying constructs with or without WEipt. As expected, injection with constructs lacking WEipt did not result in callus or de novo meristem formation. In contrast, rapid callus formation and shoot emergence (red and green shoots) were observed at injection sites treated with WEipt constructs (FIG. 5A).

[0100] For tomato, gene-editing was not evaluated since no tomato gene-specific gRNA and Cas9 constructs were incorporated in WEipt transformation vector. Following transformation, within 3 weeks, about 53% of injection sites developed de novo shoots. Among these regenerated shoots, 40% were confirmed to be transgene -positive through PCR analysis, resulting in an overall 21% transformation efficiency (FIG. 5B and FIG. 5C). Transformation frequency was calculated as the number of injection sites regenerating de novo shoots that are transgenic (based on PCR analysis). If multiple shoots were regenerated from the same injection site, it was considered one event. Based on findings from the tobacco experiments, Applicant hypothesized that the regeneration of non-transgenic shoots (-60%) might result from the transient activity of WEipt, influence of apical dominance at the injection site, or the absence of selection pressure during these experiments.

[0101] Previous studies demonstrated that in planta transformation efficiency of PLT5 and VITAS' in tomato was 13% and 3%, respectively, while the constitutive overexpression of WIND1 failed to develop de novo shoots. This finding suggests that while AtWINDl activity promotes callus formation, the subsequent activity of a potent DR gene, such as ipt, is essential to induce de-PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 differentiation and drive the de novo shoot formation in plants (FIGS. 7A-7C). This evidence underscores the synergistic role of DRs in orchestrating differentiation and de-differentiation processes, ultimately promoting effective shoot regeneration. It also highlighted the importance of combining AtWINDl-ESRl with complementary DRs like ipt to achieve robust and reproducible outcomes in plant transformation and regeneration. Similar to tobacco, a few ~2% (1 out of 52-55 regenerated shoots per experiment) regenerated tomato plants showed pleotropic effects (mainly a bushy phenotype) in the TO generation.

[0102] While this method successfully regenerated de novo shoots that produced otherwise morphologically normal tomato flowers expressing betalain, they failed to develop into fertile fruits (FIG. 5D). A similar observation was noted in maize, where excessive accumulation of betalain adversely impacts plant growth and development and proposed using weaker constitutive promoters as a strategy to mitigate growth retardation.

[0103] In the present Example, intense accumulation of betalain might have negatively affected fertilization or related processes in tomato flowers. Interestingly, transgenic red shoots of tomato regenerated from the injection site were affected, while the non-transgenic shoots from the same plant remain fertile and developed fruits. Additionally, compared to tobacco and soybean, Applicant observed highly intense red pigmentation in tomato throughout plant development (FIG. 5A). This observation led Applicant to assume the negative impact of betalain pigmentation in the plant development.

[0104] To further demonstrate the utility and versatility of wounding pathway-induced de novo shoot formation, Applicant next targeted soybean, which is relatively more recalcitrant for genetic transformation and regeneration. In this experiment, 5-day-old soybean seedlings were germinated in soil and subsequently used for in planta transformation. Like tobacco and tomato, soybean shoots were pruned and used for injecting WEipt2 (including CRISPR components targeting the soybean PDS gene) and Rt / BT-Empty construct. The injection sites were covered with cotton plugs, as was done in the tobacco and tomato experiments.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0105] As expected, Applicant observed accelerated callus formation in 97% of shoots within 5- 10 days after transformation with the WEipt2 construct. The injection sites also showed the accumulation of red foci in developing calli. However, despite several attempts, these plants with proliferated calli failed to develop de novo shoots and the callus remained compact and hard, suggesting more recalcitrant injection sites in soybean compared to tomato and tobacco. Previous soybean transformation studies have relied on laborious plant tissue culture methods and primarily using half-seed, hypocotyl, and embryogenic axis explants for transformation.

[0106] Applicant tested WEipt2 using embryogenic axis explants due to the ease of explant preparation and the potential to develop a semi-tissue-culture-free transformation method in soybean (FIGS. 6A-6G). This approach involved removing the apical meristem of the embryogenic axis to prevent the growth of untransformed primary shoots or leaf primordia (FIG. 6A).

[0107] These explants were subsequently subjected to co-cultivation. Approximately five days after co-cultivation, all of the embryogenic axes displayed red pigment accumulation, indicating transient expression of RUBY. The EAs transformed with 7?UBF-empty vector failed to initiate callus formation and proliferation, while those transformed with WEipt2 exhibited short but rapid callus initiation without growth hormones (FIG. 6A, SEM image). In 28-30 days after transformation, when explants were maintained on Gamborg B5 basal media without selection and hormones, initiation of green, red, chimeric, and albino de novo shoots (-80%) from the injection sites were observed (FIG. 6B and FIG. 6C).

[0108] Approximately 54% of EAs transformed with the control vector (RUBY Empty) regenerated green shoots, likely due to the inherent meristematic activity of the EA tissue and the absence of selection pressure. However, none of these regenerated shoots were transgenic, as expected. Similarly, in the absence of selection, explants transformed with the WEipt2 construct also exhibited vigorous regeneration of non-transgenic shoots.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0109] However, the presence of betalain pigmentation enabled clear visual differentiation between transgenic and non-transgenic shoots and facilitated the precise identification and successive pruning of non-transgenic shoots, ultimately allowing the selective propagation of confirmed transgenic lines. Since the root primordia of the explants remained intact, explants with well-established non-transgenic roots and transgenic shoots were easily transferred to soil between 35-45 days after transformation and grown to maturity. Within approximately 120 days, transgenic plants matured, producing flowers and seeds in some transgenic lines (FIG. 6D and FIG. 6E).

[0110] Although the regeneration frequency exceeded 80%, approximately 28% of the regenerated shoots were confirmed as transgenic via PCR, with most showing betalain accumulation (FIG. 6F). Similar to observations in tomato, soybean flowers experienced delays in pod formation. Nevertheless, two transgenic lines successfully set pods, with pods exhibiting complete or partial red pigmentation during early development that later turned green before final maturation (FIG. 6D and FIG. 6E).

[0111] The gene-edited, homozygous albino plants did not survive longer and failed to advance to maturation. Deep amplicon sequencing analysis for GmPDSl and GmPDS2 genes in TO plants showed reads with varying mutations in a single event, suggesting that the pool of cells with multiple mutations existed in the tissue collected for DNA extraction. These mutations were achieved mainly by deletions and insertion events resulting in chimeric and albino phenotypes (FIG. 6C). The gene-editing efficiency achieved in soybean was 12% confirmed using deep amplicon sequencing, In some embodiments, five plants (TO-4, T0-5, TO-6, TO-7, and T0-8) exhibited a biallelic mutation in the PDS gene in TO shoots (FIG. 6F and FIG. 6G). The remaining shoots displayed monoallelic or no mutations in the PDS gene (FIG. 6G). This method demonstrates a promising approach for achieving transgenesis and gene editing in soybean using WEipt2 with minimal reliance on traditional tissue culture processes.

[0112] Example 1.2. DiscussionPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0113] Regeneration is a broad term commonly used in biology that refers to a highly complex, reparative, and restorative process. Reparative signifies the repair of local wounding, while restorative indicates the reconstruction of a tissue, organ, or whole individual. In simple terms, regeneration provides the ability to heal from injury, survive, and initiate new life from a previous state, and it is the most crucial process of multicellular organisms to overcome harsh environments, diseases, and physical injury.

[0114] The regeneration process generally hinges on responses to growth hormones, tissue repair after wounding, or environmental factors. Importantly, these triggers collaborate with developmental regulator genes to initiate distinct responses that facilitate differentiation, dedifferentiation, and regeneration. Following the identification of pathways and molecular components downstream of different plant hormones, researchers significantly improved methods to accelerate plant regeneration, both through traditional tissue culture and emerging in planta techniques.

[0115] While tissue culture remains the dominant approach for regenerating transgenic or gene- edited plants, it is time-intensive and laborious. In contrast, in-planta transformation provides a rapid alternative, bypassing tissue culture and accelerating de novo regeneration for transgenic and gene-edited plant production. In this Example, leveraging the WIND1-ESR1 genes involved in the wounding pathway, Applicant designed a synthetic cascade that enhances tissue differentiation via the wounding pathway and promotes de novo transgenic and gene-edited shoots by activating a specific DR gene (ipt).

[0116] The WIND1 acts as a central regulator in promoting wound-induced cellular reprogramming in plants via systemin-independent local wound signal from REGENERATION FACTOR1 (REFI ) and PERP 1 / 2 ORTHOLOG RECEPTOR-KIN ASE1 (PORK1 ), which regulates defense and regeneration in response. Following the activation, WIND1 initiates a complex cascade of molecular events, regulating downstream genes, such as ESRI, guiding somatic cells toward a dedifferentiated state (FIG. 2C). By taking advantage of WIND1 in cellularPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 differentiation and activating ESRI (FIGS. 2A-2E), Applicant tested the suite of DR genes that are known to play a role in organogenesis and different developmental processes in plants.

[0117] For example, the WUS gene plays a central role in the maintenance of stem cell populations; STM is required for maintaining undifferentiated cells; Agrobacterium ipt produces isopentenyl AMP, a precursor of cytokinin production; BBM promotes cell proliferation and embryo development; and the GRF-GIF complex promotes organ development. These combinations were delivered to three different sites in tobacco plants (FIG. 3B), In some embodiments, delivery at the pruned site resulted in the successful development of de novo shoots.

[0118] Researchers have explored diverse strategies for delivering DR genes using in planta transformation, each exhibiting varying efficiencies in regeneration and transgenesis . Researchers in a previous report implemented a technique involving the introduction of DR genes to over 2- month-old mature tobacco plants by selectively removing all visible shoot meristems, preserving only 2-3 nodes and supporting leaves. This approach facilitated the development of shoot-like outgrowths 38-48 days post-Agrobacterium transfection. Similarly, another group utilized snapdragons, cultivating them for 70 days and removing primary and axillary stems at flower bud initiation to prepare for Agrobacterium transfection and transgenic shoots regeneration. In citrus, DR gene application in 4-month-old seedlings induced regeneration in 3-8 weeks. Additionally, researchers used a viral delivery system that introduces DR genes into latent axillary meristematic cells in tomato, enabling gene editing via de novo shoot formation.

[0119] While the above-mentioned methodologies induce de novo shoots bypassing tissue culture, they require extended durations, meticulous pruning to activate axillary meristems, or use viral vector delivery systems in meristematic cells. Additionally, in these methods, initial regenerating shoots were often non-transgenic and required removal during the first 20 days posttransformation. Notably, delivering constructs at pruned apical shoots (shoot at site) method significantly reduced the time required for preparing plants for transformation as well as recovering transgcnic / gcnc-cditcd shoots (FIG. 3B).PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0120] The transformation frequency achieved were 35% in tobacco, 21% in tomato, and 28% in soybean, demonstrating significant improvements compared to other in planta transformation methods. Additionally, successful gene-editing was achieved in tobacco and soybean, In some embodiments, several de novo shoots showed biallelic mutation in TO shoots. Among various DRs tested, Applicant observed a synergistic and complementary effect of WEipt in improving de novo shoot regeneration in tobacco, tomato, and soybean. During this process, WIND! acted as a cellular reprogramming factor, primarily facilitating the process of differentiation, whereas ipt played a crucial role in indirect organogenesis via cytokinin biosynthesis. This observation suggests that while AtWINDl can initiate differentiation, it does not promote the maintenance or growth of plant tissue in a developmental context, and hence, coordinated interaction of ipt is required for de novo shoot induction and regeneration (FIGS. 7A-7C).

[0121] Transcription factors and DR genes in multicellular organisms are often expressed with precise spatial and temporal specificity, appearing in specific tissues or cell types at defined developmental stages. These proteins orchestrate complex networks of gene expression, influencing multiple downstream targets that govern key processes such as growth, differentiation, and morphogenesis. While acting as primary drivers of developmental timing and spatial organization, even minor changes or deviations in their expression (compared to their normal state) can significantly impact plant morphology and physiology.

[0122] For example, DR genes such as Clavata3 (Clv3), BBM, WUS, and STM play critical roles in initiating de novo tissue regeneration, shifting floral meristem identity, or inducing the transition from vegetative to embryonic growth. However, ectopic or altered expression of these genes can also lead to pronounced abnormalities in plant development, underscoring the need for precise control over their expression to avoid unintended developmental phenotypes. The ectopic expression of BBM and WUS2 genes enhances regeneration in recalcitrant monocot species. However, constitutive expression of these genes also resulted in pleiotropic effects in transgenic plants, necessitating CRE-mediated excision of BBM and WUS2 to achieve normal transgenicPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 plants. Similarly, constitutive overexpression of various DR genes resulted in a significant proportion of abnormal, shoot-like structures across different DR combinations.

[0123] Other studies involving the application of DR genes have reported similar outcomes, with regenerated transgenic plants constitutively expressing WUS and PLT5 often exhibiting abnormal morphology. Another significant study showed that, although constitutive expression of the GRF- GIF chimera markedly improved transformation and regeneration efficiencies in wheat, it also led to a decrease in grain yield per spikelet and increase in grain weight. Collectively, these findings underscore that deviations from normal DR gene expression may disrupt plant growth, yield traits, and possibly reproductive development. Therefore, carefully controlled expression strategies are necessary to overcome the unintended phenotypic consequences.

[0124] This Example showed distinct response of AtWINDl-ESREipt combination on pleiotropic effect as compared to constitutive overexpression of other DR genes in in vitro regeneration of maize, sorghum, and sugarcane and in planta transformation of tobacco, potato, snapdragon, Bok choy, and tomato. This approach demonstrated a relatively low (4%) pleiotropic effect of ectopic expression of WEipt on plant growth and development in TO and subsequent generation of tobacco (FIGS. 4A-4F). Among approximately 4% of abnormal regenerated plants, several plants showed profuse branching, abnormal leaf shape and stature. Although the lower frequency of pleiotropic effects observed in this Example warrants further investigation, Applicant hypothesized that the activity of the WEipt construct was largely confined to the shoot regeneration phase rather than affecting broader developmental processes.

[0125] Specifically, the ectopic expression of AtWINDl at the injection site reprogrammed somatic cells to initiate differentiation. Simultaneously, through AtESRl promoter, it activated the ipt gene, promoting a transition from differentiated to pluripotent states and enabling de novo shoot formation (FIGS. 7A-7C). This coordinated interplay between differentiation and dedifferentiation may have contributed to a self-regulating mechanism, effectively limiting the duration and extent of gene expression, and reduced pleiotropic effects.PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031

[0126] At its current stage, this system is particularly well-suited for gene-editing applications, as the transgene can be effectively segregated from the desired edits in subsequent generations. However, for applications requiring stable overexpression of specific genes, especially in commercial settings, additional steps to remove DR genes remain essential for regulatory compliance and minimizing unintended effects.

[0127] In conclusion, this Example highlights the synergistic action and transformative potential of the WEipt to accelerate de novo shoot regeneration with minimal pleiotropic effects. By activating a wound-induced cellular reprogramming cascade, WIND! facilitated somatic cell differentiation, while ipt mediated cytokinin biosynthesis, enabling a seamless transition to pluripotent states and subsequent shoot formation (FIGS. 7A-7C). Importantly, co-expression of gene-editing reagents with WEipt facilitated precisely targeted mutations, establishing a scalable and efficient platform for crop improvement. The indirect organogenesis pathway improved regeneration rates and also established a consistent and swift timeline for shoot induction, overcoming the variability and inefficiencies observed in earlier methodologies. Instead of relying on constitutive overexpression of DR genes, which often result in developmental abnormalities, the targeted and localized expression of ipt achieved consistent and efficient regeneration across tobacco, tomato, and soybean. This approach holds a significant promise for in planta transformation, reducing the dependency on tissue culture and accelerating the generation of transgenic or gene-edited plants.

[0128] Example 1,3. Methods

[0129] Plant materials and growth conditions'. Tomato seeds (Solanum lycopersicum var money maker) were purchased from the marketplace and used to evaluate various constructs. For the in vitro assessment of various AtWINDl -ESRl constructs, surface sterilized seeds of N. benthamiana (sterilized with 70% ethanol for 1 min, rinsed with sterile water and surface sterilized with 10% commercial bleach for 10 min and subsequently rinsed with sterile water for 5 times) were germinated in ' / 2-strcngth MS media with 3% sucrose in magenta vessels under long-day conditions at 22°C, exposed to 16 hours of white light and 8 hours of darkness at 20°C. The petiolesPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 and internodes of about one month old aseptically grown plants were taken for Agrobacterium transformation. The transformed explants were placed in co-cultivation media (fi-strcngth MS media) and incubated in dark for 3 days. Following co-cultivation, the explants were rinsed with sterile water containing Timentin (50mg / L). The blot-dried explants were placed in phytohormone-free fi-strcngth MS media supplemented with Timentin (50mg / L) to eliminate excessive Agrobacterial growth. The callus area was measured 4 weeks post-transfection to assess WINDl and its role in callus induction. A similar method was applied for the in vitro assessment of the WEipt construct.

[0130] Vector design and construction-. All DNA constructs were created following plant genome engineering toolkit. The DNA cassettes included CRISPR components (Cas9 and guide RNAs), DRs, and reporter genes cloned in one of the modular vectors that are finally assembled on a T- DNA destination vector by the Golden Gate cloning strategy. RUBY as a visible reporter gene under the Cestrum Yellow Leaf Curling Virus (CmYLCV) promoter was cloned into module A. Guide RNAs targeting PDS genes in tobacco and soybean were cloned in module B under Arabidopsis U6-snRNA26 (U6-26) and CmYLCV promoters, respectively. The Arabidopsis ESRI (AT1G12980) promoter (1046 bp) was amplified with primers flanking BamHI and Kpnl from the Arabidopsis CoLO genotype and cloned into pUC19 vector. DR gene particularly ipt was amplified from Addgene repository #127211 with the forward primer flanking Kpnl and the reverse primer from Pinll terminator flanking EcoRI / SacI sites and cloned under AtESRl promoter in pUC19 through restriction-digestion cloning. The whole cassette (pESRl ::zpt::PinII ter) was subsequently cloned to module C’. All other DR genes were cloned from crop specific cDNA and cloned under AtESRl promoter. Similarly, WIND1 (AT1G78080) from Arabidopsis CoLO was cloned in module D under the CaMV 35S promoter. For soybean transformation, Cas9 protein was cloned in Module A driven by soybean ubiquitin (GmUbi) promoter and visible reporter gene RUBY was cloned to module E under CmYLCV promoter. All the modules were finally assembled into T-DNA transformation backbone using Golden Gate Assemblies. These assembled constructs were transformed into the Agrobacterium tumefaciens strain GV3101 to transform tobacco and tomato, and strain 18rl2 for soybean transformation. Before plant transformation, confirmation ofPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 successful delivery of T-DNA carrying the RUBY gene was visualized through Agroinfiltration of tobacco leaves.

[0131] Assessment of WIND1-ESR1 cascade: To confirm the role of AtWlNDl in tissue differentiation, three different constructs were generated. The construct Rl / BF-Empty (proCmYLCV::R 7BF) and ESRruby (proAtESRl::RUBY) were used as negative controls for callus formation and induction of ESRI activity, respectively. To verify if AtWlNDl activates the AtESRl promoter and induces callus formation, construct WE ruby comprising pro35S::WAD / and proAtESRl ::RUBY were developed. These constructs were transformed into Agrobacterium strain GV3101. The overnight grown culture was resuspended in a transformation buffer containing lOmM MES, 150pM acetosyringone, and lOmM MgCh until the ODeoo was between 0.2-0.5 and tobacco explants (petioles and internodes) were used for transformation. The transformed explants were cultured in Vi strength hormone-free MS media for 4 weeks. Areas of calli were measured to assess the role of AtWlNDl in callus induction.

[0132] In planta transformation in tobacco, tomato, and soybean: For in planta transformation, both tobacco and tomato seeds were germinated under long-day conditions at 22°C, exposed to 16 hours of white light and 8 hours of darkness at 20°C. Approximately one month old plants were used for in planta transformation. Similarly, for soybean in planta transformation, mature and dried soybean seeds (genotype ‘Williams 82’) were sown in soil and germinated at 25°C, exposed to 16 hours of white light and 8 hours of darkness. Specific constructs were utilized to assess in planta shoot formation, transgenesis and gene editing. For in planta transformation of tobacco, Cas9 positive transgenic seeds were germinated and grown for approximately 4 weeks and then injected with the Agrobaclerium carrying the construct with one of the DRs at various sites that do not have meristematic activity. A single colony of Agrobacterium harboring synthetic cascade (WEipt and CRISPR components) was inoculated in 30mL yeast extract peptone (YEP) broth with antibiotics (Kanamycin, 50mg / L, Rifampicin, lOmg / L, and Gentamicin, 30mg / L for GV3101) and grown (shaking at 160rpm / 12 hours) at 28°C. After 12 hours, the culture was pelleted byPCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 centrifuging at 4500 rpm for 10 minutes. The pellet was resuspended with the transfection buffer (lOmM MES, 150pM acetosyringone, and lOmM MgCh) until the ODeoo was between 0.2-0.8.

[0133] Three different wounding strategies or locations were used for in planta transformation (FIGS. 3A-3D). First, multiple wounding sites in the internode of the plant; second, a single wounding site at the internode; and third, pruning the young shoot (4-6 cm from top) at the internode (FIGS. 3A-3D). These sites were used to inoculate the Agrobacterium with respective DR cascades vectors. After inoculation, the wounding area was covered with a sterile cotton plug soaked in the same Agrobacterium solution, creating a localized microenvironment conducive to co-cultivation. Next, plants were placed in the dark in a high humidity (80%) growth chamber at 22°C for 3 days. After 3 days, the cotton plugs were carefully removed, and plants were grown in a normal condition at 16 / 8-hour day / night cycle at 22 / 20°C. Plants were routinely observed for shoot-like growth at the site of injection. For tomato, only one of the three strategies was adapted, based on the successful N. benthamiana experiments. The tomato plants between 3 and 4 weeks old were pruned and inoculated with Agrobacterium carrying WEipt at the wound site. Pruning was performed by removing the shoot meristem (4-6 cm from top). The gene-editing experiment was not performed in tomato; therefore, the WEipt construct did not harbor gene-editing reagents. The rest of the protocol followed the same as the tobacco experiment described above.

[0134] For soybean in planta transformation, healthy soybean seedlings were excised above the cotyledon before they formed the first true leaf. The WEipt2 constructs harboring gene-editing reagents targeting soybean PDS gene and ipt was injected following wounding, and the wound sites were plugged in with cotton plugs soaked with Agrobacterium solution. Plants were incubated in the dark for 3 days in high humidity (80%). After 3 days, cotton plugs were removed, and the plants were placed in a growth chamber with similar growth conditions as they were germinated. The infusion sites were observed for callus (transgenic; red or normal) formation and regeneration.

[0135] Next, for transformation using soybean cmbryogcnic axis, mature and healthy seeds of soybean genotype ‘Williams 82’ were surface sterilized. After imbibition, seeds were rinsed twicePCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 with sterile water and prepared for isolation of the embryogenic axis. The shoot apical meristem of the embryogenic axis was carefully removed, and these decapitated embryos were used for cocultivation (FIG. 6A). The embryogenic axes were transformed by sonicating them with Agrobacterium (strain 18rl2) solution for 30 seconds and shaking them in a rotary shaker at 75RPM for 30 minutes. The Agrobaclerium solution was drained by pipetting after shaking, and the explants were placed in petri dishes containing sterile filter paper and presoaked in Vi strength MS media. The plates were sealed with parafilm and placed in a growth chamber with 23°C under 16hr light / 8hr dark photoperiod for 5 days. After 5 days of co-cultivation, the explants were aseptically transferred to Vi strength plant growth regulator (hormone)-free Gamborg B5 basal media supplemented with antibiotic (Timcntin, 50mg / L) to eliminate the excessive Agrobacterium growth. The regeneration of non-transgenic (green), transgenic (red) and gene-edited shoots (white) was observed, and the count of each was recorded. After 35-45 days, regenerated plants were transferred to the soil in Jeffy peat pellets and covered with plastic domes for a week to maintain humidity. After plants were established in Jeffy pellet, they were transplanted to the soil.

[0136] Phenotypic analysis of regeneration, transgenesis, and gene editing'. Any shoot- like growth at the site of injection was considered as a de novo shoot induced by developmental regulators. The de novo meristems were visually observed for white (indicating possible loss of function of PDS by gene editing), red betalain pigment (indicating expression of the RUBY transgene), and green (functional PDS and no transgene expression).

[0137] Genotypic analysis for gene editing and transgenesis: Genomic DNA was extracted from the leaf tissues collected from white, red, and green meristems using CTAB (cetyl trimethyl ammonium bromide). A fragment of RUBY gene was amplified for transgene PCR analysis. For analysis of gene-editing events, both the copies of the PDS genes were amplified (464bp) with primers. These amplicons were used for capillary gel-based electrophoresis using seqstudio genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s guidelines. Peak intensity and mutation size were detected by microsatellite analysis (MSA) software (Thermo Fisher Scientific, Waltham, MA, USA). Based on the fragment analysis results,PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 lines were further selected for deep amplicon sequencing. Deep amplicon sequencing and analysis were performed.

[0138] RT-qPCR assays: Total RNA was isolated from leaf tissue of transgenic N. benthamiana from three different generations (T1-T3) using RNesay Plant Mini Kit (Qiagen). Reverse transcription reactions were conducted using a Verso cDNA Synthesis Kit (Thermo Fisher Scientific) and PCR assay was conducted in a real-time PCR machine (CFX96 Real-Time System, BioRad) using SsoAdvanced Universal SYBR Green Supermix (Biorad) following manufacturer’s guidelines. An Elongation Factor 1 (EF-I) gene was used as reference gene for expression normalization. The assay was performed in three biological replicates and two technical replicates for each gene. Specific primers were used for the EF-l, AtWINDl, and ipt.

[0139] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031WHAT IS CLAIMED IS:

1. A method of altering a plant or seed, said method comprising: introducing a reagent into the plant or seed, wherein the reagent comprises: an altering component operable to introduce or edit one or more nucleotide sequences in the plant or seed, wherein the altering component is selected from the group consisting of a nucleotide sequence, a gene, a nucleotide editing system, or combinations thereof, and one or more developmental-related genes, wherein the developmental-related genes are selected from the group consisting of Wound Induced Dedifferentiation 1 (WIND / ). Enhancer of Shoot Regeneration 1 (ESRI), Isopentenyl transferase (IPT), or combinations thereof; and wherein the introducing of the reagent results in the introduction or editing of one or more nucleotide sequences in the plant or seed, and the expression of the developmental related genes.

2. The method of claim 1, wherein the reagent comprises an expression vector.

3. The method of claim 2, wherein a single expression vector comprises the altering component and the developmental-related genes.

4. The method of claim 1, wherein the altering component comprises a nucleotide editing system.414823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-0315. The method of claim 4, wherein the altering component edits one or more nucleotide sequences of the plant or seed.

6. The method of claim 5, wherein the editing is selected from the group consisting of introducing a mutation to a gene, introducing a deletion to a gene, introducing an insertion to a gene, removing a portion of a gene, changing a base of a gene, removing a gene, inserting a gene, partially or fully replacing a gene, deleting a nucleotide, inserting a nucleotide, or combinations thereof.

7. The method of claim 4, wherein the editing occurs without the use of in vitro tissue culture techniques.

8. The method of claim 4, wherein the nucleotide editing system comprises a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system), wherein the CRISPR / Cas system comprises at least one Cas nuclease and at least one guide RNA.

9. The method of claim 1, wherein the altering component comprises a nucleotide sequence that is introduced in the plant or seed.

10. The method of claim 9, wherein the nucleotide sequence comprises a gene that is expressed in the plant or seed.

11. The method of claim 1 , wherein the developmental-related genes comprise Wound Induced Dedifferentiation 1 (WIND1).424823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-03112. The method of claim 11, wherein WIND! comprises SEQ ID NO: 1 or a sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 4 or a sequence with at least 85% sequence identity to SEQ ID NO: 4, or combinations thereof.

13. The method of claim 1, wherein the developmental-related genes comprise Isopenlenyl transferase (I PT).

14. The method of claim 13, wherein the IPT comprises SEQ ID NO: 2 or a sequence with at least 85% sequence identity to SEQ ID NO: 2, SEQ ID NO: 5 or a sequence with at least 85% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6 or a sequence with at least 85% sequence identity to SEQ ID NO: 6, SEQ ID NO: 7 or a sequence with at least 85% sequence identity to SEQ ID NO: 7, SEQ ID NO: 8 or a sequence with at least 85% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9 or a sequence with at least 85% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10 or a sequence with at least 85% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11 or a sequence with at least 85% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12 or a sequence with at least 85% sequence identity to SEQ ID NO: 12, or combinations thereof.

15. The method of claim 1, wherein the developmental-related genes comprise Wound Induced Dedifferentiation 1 (WIND1) and Isopentenyl transferase (IPT).

16. The method of claim 15, wherein the IPT is operatively linked to a promoter for Enhancer of Shoot Regeneration 1 (ESRI).

17. The method of claim 16, wherein the ESRI promoter comprises SEQ ID NO: 3 or a sequence with at least 85% sequence identity to SEQ ID NO: 3.434823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-03118. The method of claim 1, wherein the reagent is introduced into the plant or seed by a method selected from the group consisting of transfection, electroporation, particle bombardment, agroinfiltration, or combinations thereof.

19. The method of claim 1, wherein the reagent is introduced into a plant through in planta transformation.

20. The method of claim 1, wherein the altering component and the developmental-related genes are introduced into the plant or seed at the same time.

21. The method of claim 1, wherein the altering component and the developmental-related genes are introduced into the plant or seed at different times.

22. The method of claim 1, wherein the method results in the formation of a de novo shoot on a plant or seed.

23. The method of claim 1, wherein the method results in the formation of somatic embryos on a plant or seed.

24. The method of claim 1, further comprising a step of removing the developmental-related genes from the plant or seed.444823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-03125. The method of claim 1, wherein the plant or seed is selected from the group consisting of crops, weeds, maize, rice, soybean, cotton, wheat, N. benthamiana, Arabidopsis, Amaranthus palmeri, tobacco, tomato, lettuce, common beans, pinto beans, corns, potato, grapes, citrus, blueberry, sorghum, sugar cane, snapdragon, Bok choy, chickpea, flowering crops, marigold, chrysanthemum, vegetables, ornamental plants, horticultural crops, varieties thereof, or combinations thereof.

26. The method of claim 1, wherein the plant or seed comprises a plant.

27. The method of claim 26, wherein the plant is less than one month old.

28. The method of claim 26, wherein the plant is less than two months old.

29. The method of claim 1, wherein the plant or seed comprises cotton.

30. A method of altering a plant or seed, said method comprising: introducing a reagent into the plant or seed, wherein the reagent comprises: an altering component operable to introduce or edit one or more nucleotide sequences in the plant or seed, wherein the altering component is selected from the group consisting of a nucleotide sequence, a gene, a nucleotide editing system, or combinations thereof, wherein the introducing of the reagent results in the introduction or editing of one or more nucleotide sequences in the plant or seed, and wherein the plant or seed expresses one or more developmental-related genes, wherein the developmental-related genes are selected from the group consisting of Wound454823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031Induced Dedifferentiation 1 ( WIND I). Enhancer of Shoot Regeneration 1 (ESRI). Isopentenyl transferase (IPT), or combinations thereof.

31. The method of claim 30, wherein the altering component comprises a nucleotide editing system.

32. The method of claim 31, wherein the altering component edits one or more nucleotide sequences of the plant or seed.

33. The method of claim 32, wherein the editing is selected from the group consisting of introducing a mutation to a gene, introducing a deletion to a gene, introducing an insertion to a gene, removing a portion of a gene, changing a base of a gene, removing a gene, inserting a gene, partially or fully replacing a gene, deleting a nucleotide, inserting a nucleotide, or combinations thereof.

34. The method of claim 32, wherein the editing occurs without the use of in vitro tissue culture techniques.

35. The method of claim 32, wherein the nucleotide editing system comprises a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system), wherein the CRISPR / Cas system comprises at least one Cas nuclease and at least one guide RNA.

36. The method of claim 30, wherein the altering component comprises a nucleotide sequence that is introduced in the plant or seed.

37. The method of claim 36, wherein the nucleotide sequence comprises a gene that is expressed in the plant or seed.464823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-03138. The method of claim 30, wherein the developmental-related genes comprise Wound Induced Dedifferentiation 1 (WIND1).

39. The method of claim 38, wherein WIND1 comprises SEQ ID NO: 1 or a sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 4 or a sequence with at least 85% sequence identity to SEQ ID NO: 4, or combinations thereof.

40. The method of claim 30, wherein the developmental-related genes comprise Isopentenyl transferase (IPT).

41. The method of claim 40, wherein the IPT comprises SEQ ID NO: 2 or a sequence with at least 85% sequence identity to SEQ ID NO: 2, SEQ ID NO: 5 or a sequence with at least 85% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6 or a sequence with at least 85% sequence identity to SEQ ID NO: 6, SEQ ID NO: 7 or a sequence with at least 85% sequence identity to SEQ ID NO: 7, SEQ ID NO: 8 or a sequence with at least 85% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9 or a sequence with at least 85% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10 or a sequence with at least 85% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11 or a sequence with at least 85% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12 or a sequence with at least 85% sequence identity to SEQ ID NO: 12, or combinations thereof.

42. The method of claim 30, wherein the developmental-related genes comprise Wound Induced Dedifferentiation 1 (WIND1) and Isopentenyl transferase (IPT).

43. The method of claim 42, wherein the IPT is operatively linked to a promoter for Enhancer of Shoot Regeneration 1 (ESRI).

44. The method of claim 43, wherein the ESRI promoter comprises SEQ ID NO: 3 or a sequence with at least 85% sequence identity to SEQ ID NO: 3.474823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-03145. The method of claim 30, wherein the reagent is introduced into the plant or seed by a method selected from the group consisting of transfection, electroporation, particle bombardment, agroinfiltration, or combinations thereof.

46. The method of claim 30, wherein the reagent is introduced into a plant through in planta transformation.

47. The method of claim 30, wherein the method results in the formation of a de novo shoot on a plant or seed.

48. The method of claim 30, wherein the method results in the formation of somatic embryos on a plant or seed.

49. The method of claim 30, further comprising a step of removing the developmental-related genes from the plant or seed.

50. The method of claim 30, wherein the plant or seed is selected from the group consisting of crops, weeds, maize, rice, soybean, cotton, wheat, N. benthamiana, Arabidopsis, Amaranthus palmeri, tobacco, tomato, lettuce, common beans, pinto beans, corns, potato, grapes, citrus, blueberry, sorghum, sugar cane, snapdragon, Bok choy, chickpea, flowering crops, marigold, chrysanthemum, vegetables, ornamental plants, horticultural crops, varieties thereof, or combinations thereof.

51. The method of claim 30, wherein the plant or seed comprises cotton.

52. A reagent comprising:484823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-031 an altering component operable to introduce or edit one or more nucleotide sequences in a plant or seed, wherein the altering component is selected from the group consisting of a nucleotide sequence, a gene, a nucleotide editing system, or combinations thereof; and one or more developmental-related genes, wherein the developmental-related genes are selected from the group consisting of Wound Induced Dedifferentiation 1 (WIND I). Enhancer of Shoot Regeneration 1 (ESRI). Isopentenyl transferase (JPT), or combinations thereof.

53. The reagent of claim 52, wherein the reagent comprises an expression vector.

54. The reagent of claim 53, wherein a single expression vector comprises the altering component and the developmental-related genes.

55. The reagent of claim 52, wherein the altering component comprises a nucleotide editing system.

56. The reagent of claim 55, wherein the nucleotide editing system comprises a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Cas) system (CRISPR / Cas system), wherein the CRISPR / Cas system comprises at least one Cas nuclease and at least one guide RNA.

57. The reagent of claim 52, wherein the altering component comprises a nucleotide sequence.

58. The reagent of claim 57, wherein the nucleotide sequence comprises a gene that is operable to be expressed in the plant or seed.494823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-03159. The reagent of claim 52, wherein the developmental-related genes comprise Wound Induced Dedifferentiation 1 (WIND1).

60. The reagent of claim 59, wherein WIND1 comprises SEQ ID NO: 1 or a sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 4 or a sequence with at least 85% sequence identity to SEQ ID NO: 4, or combinations thereof.

61. The reagent of claim 52, wherein the developmental-related genes comprise Isopentenyl transferase (IPT).

62. The reagent of claim 61, wherein the IPT comprises SEQ ID NO: 2 or a sequence with at least 85% sequence identity to SEQ ID NO: 2, SEQ ID NO: 5 or a sequence with at least 85% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6 or a sequence with at least 85% sequence identity to SEQ ID NO: 6, SEQ ID NO: 7 or a sequence with at least 85% sequence identity to SEQ ID NO: 7, SEQ ID NO: 8 or a sequence with at least 85% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9 or a sequence with at least 85% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10 or a sequence with at least 85% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11 or a sequence with at least 85% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12 or a sequence with at least 85% sequence identity to SEQ ID NO: 12, or combinations thereof.

63. The reagent of claim 52, wherein the developmental-related genes comprise Wound Induced Dedifferentiation 1 (WIND1) and Isopentenyl transferase (IPT).

64. The reagent of claim 63, wherein the IPT is operatively linked to a promoter for Enhancer of Shoot Regeneration 1 (ESRI).

65. The reagent of claim 64, wherein the ESRI promoter comprises SEQ ID NO: 3 or a sequence with at least 85% sequence identity to SEQ ID NO: 3.504823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-03166. The reagent of claim 52, wherein the reagent is operable to form a de novo shoot on a plant or seed.

67. The reagent of claim 52, wherein the reagent is operable to form somatic embryos on a plant or seed.

68. A plant or seed, wherein the plant or seed expresses one or more developmental-related genes, wherein the developmental-related genes are selected from the group consisting of Wound Induced Dedifferentiation 1 (WIND]), Enhancer of Shoot Regeneration 1 (ESRI ), Isopentenyl transferase (IPT), or combinations thereof.

69. The plant or seed of claim 68, wherein the plant or seed ectopically expresses the developmental-related genes.

70. The plant or seed of claim 68, wherein the plant or seed is selected from the group consisting of crops, weeds, maize, rice, soybean, cotton, wheat, N. benthamiana, Arabidopsis, Amaranthus palmeri, tobacco, tomato, lettuce, common beans, pinto beans, corns, potato, grapes, citrus, blueberry, sorghum, sugar cane, snapdragon, Bok choy, chickpea, flowering crops, marigold, chrysanthemum, vegetables, ornamental plants, horticultural crops, varieties thereof, or combinations thereof.

71. The plant or seed of claim 68, wherein the plant or seed comprises cotton.

72. The plant or seed of claim 68, wherein the developmental-related genes comprise Wound Induced Dedifferentiation 1 (WIND1).514823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P075WOTexas Tech No. 2024-03173. The plant or seed of claim 72, wherein WIND1 comprises SEQ ID NO: 1 or a sequence with at least 85% sequence identity to SEQ ID NO: 1, SEQ ID NO: 4 or a sequence with at least 85% sequence identity to SEQ ID NO: 4, or combinations thereof.

74. The plant or seed of claim 68, wherein the developmental-related genes comprise Isopentenyl transferase (IPT).

75. The plant or seed of claim 74, wherein the IPT comprises SEQ ID NO: 2 or a sequence with at least 85% sequence identity to SEQ ID NO: 2, SEQ ID NO: 5 or a sequence with at least 85% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6 or a sequence with at least 85% sequence identity to SEQ ID NO: 6, SEQ ID NO: 7 or a sequence with at least 85% sequence identity to SEQ ID NO: 7, SEQ ID NO: 8 or a sequence with at least 85% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9 or a sequence with at least 85% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10 or a sequence with at least 85% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11 or a sequence with at least 85% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12 or a sequence with at least 85% sequence identity to SEQ ID NO: 12, or combinations thereof.

76. The plant or seed of claim 75, wherein the developmental-related genes comprise Wound Induced Dedifferentiation 1 (WIND1 ) and Isopentenyl transferase (IPT).

77. The plant or seed of claim 76, wherein the IPT is operatively linked to a promoter for Enhancer of Shoot Regeneration 1 (ESRI).

78. The plant or seed of claim 77, wherein the ESRI promoter comprises SEQ ID NO: 3 or a sequence with at least 85% sequence identity to SEQ ID NO: 3.524823-4406-5785V.3 13368-42

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