Soybean plants characterized by high drought resistance

Abstract

The invention provides a method for improving drought resistance of soybean plants by introducing site specific mutation into stomata open genes. The invention also relates to a soybean plant, an isolated part thereof or a seed thereof, wherein the gene has been inactivated.

Description

Technical Field

[0001] The present invention relates to a method for improving drought resistance of soybean plants by inactivating one or both of the genes Glyma.03g006600 and Glyma.19g119300. Other aspects of the present invention relate to soybean plants, isolated parts thereof or seeds thereof, wherein the genes Glyma.03g006600 and Glyma.19g119300 have been inactivated. Background Art

[0002] Soybeans are one of the most widespread agricultural crops in the world, with a cultivated area equivalent to 6% of the global agricultural area and a global annual production of more than 330 million tons. From an economic point of view, soybeans are of strategic importance to many manufacturing industries. In the food industry, soybeans are a cheap source of protein and fat, and the cheapest meat substitute for vegetarians and vegans. Soybeans are also widely used in industries such as lubricants, waxes and coatings.

[0003] Soybean is a crop with high water requirements and its yield is closely related to water availability. Even short-term water deficiency can lead to a significant reduction in soybean yield, with yield losses as high as 40-50%. Low water intake adversely affects various physiological processes, including symbiotic nitrogen fixation, photosynthetic efficiency, pod formation, and seed development. Plants that are under water stress usually have fewer pods per plant and fewer seeds per pod. In addition, individual seeds are generally smaller.

[0004] In recent decades, worrying drought periods have recurred, even in particularly suitable agricultural areas, with serious consequences for soybean yields. Ongoing climate change will lead to an intensification of such events in the future, with serious implications for global soybean production. In this context, the selection of new varieties with low water requirements and low yield losses under water stress conditions is a necessary and urgent goal for the genetic improvement of the species.

[0005] In this regard, genome editing technology represents an innovative and strategic tool to improve soybean drought resistance by modulating the activity of specific target genes.

[0006] Genetic improvement methods

[0007] A variety of approaches have been used to improve drought resistance in soybean. These approaches include classical or assisted breeding strategies that pre-select genotypes with resistance traits. However, introgression of favorable alleles in the above genotypes into marketable elite varieties requires a lot of time and extensive analysis of many individuals over several generations.

[0008] The development of protocols for the regeneration of multiple soybean varieties and the optimization of transformation methods mediated by Agrobacterium tumefaciens have paved the way for the possibility of engineering novel soybean resistance traits. Most biotechnological applications developed to date are based on the overexpression of single genes involved in the plant response to drought using the constitutive viral promoter CaMV35S. Such techniques often produce undesirable pleiotropic effects on plant development and growth. For example, overexpression of MYB14 in soybean improves drought resistance, but at the same time significantly reduces plant height and leaf area.

[0009] The recent design and dissemination of methods for editing the soybean genome offers an alternative approach for selecting resilient new varieties that are able to maintain high production standards even under stress conditions.

[0010] Editing techniques

[0011] Editing technologies can modify the soybean genome in a precise, specifically targeted manner. Three main editing technologies can be used to modify the sequence and activity of specific target genes: (i) zinc finger nucleases (ZFNs); (ii) transcription activator-like effector nucleases (TALENs); and (iii) clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9. CRISPR / Cas9 technology is more efficient and cheaper than ZFNs and TALENs and has become the preferred method for editing plant genomes.

[0012] The CRISPR / Cas9 system consists of three main elements: (i) CRISPR sequence, which is characterized by short repetitive DNA sequences alternating with spacer sequences; (ii) Cas9 protein, which contains two domains with nuclease activity (RuvC-like and HNH); and (iii) RNA guide sequence (sgRNA), which is complementary to the region of the target gene and can guide the Cas9 protein to the target. Therefore, the sgRNA sequence is an element that determines the specificity of CRISPR / Cas9 for a given gene. When a specific sgRNA is used, the Cas9 protein can be targeted to a given target gene. The binding of the composite sgRNA-CRISPR / Cas9 to the target gene causes the DNA sequence to be cut by Cas9, thereby forming a "double-strand break" (DSB). The repair of double-strand breaks usually involves the introduction of errors compared to the original gene sequence, such as insertions, deletions, or base substitutions. The new mutations induced by CRISPR / Cas9 can change the coding sequence of the gene and impair its normal function.

[0013] Target Gene

[0014] In order to apply CRISPR / Cas9 technology to adapt soybean to water stress, it is necessary to identify target genes involved in regulating the plant's response to water deficiency. The completion of soybean whole genome sequencing has made it possible to identify various genes that regulate plant adaptation to water stress conditions. Among the genes, transcription factors belonging to gene families NAC, MYB, MYC, WRKY, AREB and DREB have been identified as essential elements regulating the cellular, metabolic and developmental mechanisms that cause soybean stress responses.

[0015] When overexpressed in transgenic lines using the promoter CaMV35S or other constitutive promoters, most of these genes can improve the ability of plants to tolerate drought periods. However, their inactivation by CRIPR / Cas9 generally makes plants more sensitive to water stress. For example, soybean plants that overexpress the gene Glyma.NAC8 show higher drought resistance and better stress recovery. In contrast, plants in which the Glyma.NAC8 gene has been inactivated by genome editing show a lower response to water deficiency and a high mortality rate at the end of stress.

[0016] Gene MYB60

[0017] AtMYB60 is a gene encoding an R2R3MYB transcription factor that is specifically expressed in stomata, where it controls their opening in response to light and water deficiency. Stomata are small openings in the surface of the aerial parts of land plants that are surrounded by two highly specialized cells called guard cells. The opening and closing of stomata allows plants to optimize CO 2 , the ratio between the intake required for photosynthesis and the loss of water through transpiration. Closure of stomata represents the first basic adaptive response of plants to water stress conditions, enabling them to limit tissue dehydration.

[0018] The loss of AtMYB60 function in the mutant allele atmyb60-1 resulted in a constitutive reduction in stomatal opening. Even when mutant plants were kept under ideal growth conditions (high water availability, light), their stomata remained partially closed. Under water-deficient conditions, the reduced stomatal aperture opening resulted in a significantly improved drought resistance in atmyb60-1 compared to wild-type plants.

[0019] It should be emphasized that the beneficial effects of AtMYB60 inactivation on plant water balance were independent of adverse effects on plant growth and productivity. In fact, under optimal growth conditions, the mutant atmyb60-1 did not show any growth or developmental abnormalities, or a reduction in photosynthetic efficiency, compared to wild-type plants.

[0020] Prior Art

[0021] WO 2005 / 085449 discloses gene constructs for selectively expressing nucleic acid sequences in stomatal guard cells, in particular sequences involved in intracellular signaling pathways regulated by abscisic acid and in the regulation of stomatal opening.

[0022] Cominelli E. et al., Current Biology vol. 15, 1196-1200 (2005) describe the characterization of the Arabidopsis gene AtMYB60, a transcription factor involved in regulating stomatal movement.

[0023] Cominelli E. et al., BMC Plant Biology 2011 (11: 162) describe the analysis of mutagenesis and deletion of the AtMYB60 promoter using the GUS reporter promoter system.

[0024] The article by Galbiati M. et al., BMC Plant Biology 2011, 11: 142 reports the identification of the VvMYB60 gene as a functional ortholog of the Arabidopsis gene AtMYB60 and its regulatory role in response to abscisic acid and water stress conditions.

[0025] Rusconi F. et al., Journal of Experimental Botany Advance (2013) reported the use of reporter gene method to study the activity of AtMYB60 promoter in rice, tobacco and tomato.

[0026] Simeoni F. et al., Scientific Report (12: 533) 2022 demonstrated that AtMYB60 regulates stomatal opening by regulating oxylipin biosynthesis in guard cells.

[0027] Simeoni F. et al., Agronomy (12: 694) 2022 reported the expression of the gene VvMYB60 in stomatal guard cells of grapevine and the correlation between its expression level and stomatal conductance in various grapevine genotypes. Summary of the invention

[0028] It has now been discovered that inactivation of one or both of the soybean genes Glyma.19g119300 and Glyma.03g006600, by introducing site-specific mutations therein, constitutively reduces stomatal opening, enabling plants to limit tissue dehydration and adapt to water stress conditions.

[0029] Therefore, a first aspect of the present invention relates to a method for improving drought resistance in soybean plants, comprising inactivating at least one target gene selected from the group consisting of Glyma.19g119300 and Glyma.03g006600.

[0030] Although two genes are combined to control stomatal opening, it is preferred to inactivate the gene Glyma.19g119300, whose expression is mainly localized to stomata. In any case, the inhibitory effect on stomatal opening can be adjusted by introducing mutations into one or both target genes.

[0031] According to the invention, inactivation may involve one or both alleles of each gene and may involve total or partial loss of function of the corresponding encoded protein (transcription factor R2R3MYB).

[0032] In a preferred embodiment, the genes Glyma.19g119300 and Glyma.03g006600 are inactivated by site-specific mutagenesis using the CRISPR / Cas9 system.

[0033] Site-specific mutation is achieved by the action of the nuclease Cas9 in combination with the target gene-specific sgRNA. The sgRNA sequences specific for the genes Glyma.03g006600 and Glyma.19g119300 are preferably selected from SEQ ID NO: 1-SEQ ID NO: 30 and SEQ ID NO: 31-SEQ ID NO: 60, respectively.

[0034] In one embodiment, the method according to the invention comprises the following steps:

[0035] (i) constructing a vector for expressing Cas9 and sgRNA in soybean plant cells;

[0036] (ii) introducing the expression vector into soybean plant cells or parts or isolated tissues thereof, particularly cotyledon explants, by contacting said cells with a culture of Agrobacterium bacteria containing the expression vector.

[0037] The method according to the invention preferably comprises the following further steps:

[0038] (iii) planting plants containing the Cas9 / sgRNA vector and obtaining seeds;

[0039] (iv) propagating and subsequently selecting plants with low stomatal patency;

[0040] (v) further selecting plants with higher drought resistance as measured under water stress conditions.

[0041] In a preferred embodiment, the vector comprises an sgRNA coding sequence functionally bound to soybean promoter U6, an expression cassette of a Cas9 gene comprising a sequence encoding an enzyme Cas9 (preferably SpCas9) functionally bound to promoter CaMV35S, a nuclear localization sequence of protein Cas9, and a DNA transfer (T-DNA) region. The expression vector can be inserted into Agrobacterium bacteria, preferably Agrobacterium tumefaciens, using techniques known to the skilled person, such as electroporation.

[0042] In step (iv), the openness of the stomata can be measured by stomatal conductance analysis (gs) or optical microscopic analysis of the pore size.

[0043] In step (v), the response of plants to water stress conditions can be assessed by measuring biometric, physiological and production parameters at various stages of the biocycle, the plants being subjected to different growth conditions wherein the water content of the medium is varied.

[0044] Another aspect of the invention relates to a soybean plant or part thereof or seed, wherein at least one of the genes Glyma.19g119300 and Glyma.03g006600, preferably the gene Glyma.19g119300, has been inactivated. The gene is preferably inactivated by introducing a mutation into the corresponding sequence that is capable of inhibiting or reducing the function of the encoded protein. The resulting plant may be homozygous or heterozygous for a given mutation. In a preferred embodiment, the gene is inactivated by site-specific mutagenesis using the CRISPR / Cas9 system. DETAILED DESCRIPTION

[0045] Homology analysis

[0046] The amino acid sequence of the Arabidopsis protein AtMYB60 ( Figure 1 A-SEQ ID NO: 77) to identify homologous proteins in the soybean genome. Two proteins were identified and named Glyma.03g006600 ( Figure 1 B-SEQ ID NO: 78) and Glyma.19g119300 ( Figure 1 C-SEQ ID NO: 79), which are highly homologous to Arabidopsis proteins ( Figure 2 ).

[0047] The genomic loci encoding these two proteins are located on soybean chromosome 3 (Glyma.03g006600, position 601548-602971, sequence included in GenBank Gene ID: 100802204) and soybean chromosome 19 (Glyma.19g119300, position 37492051-37493530, sequence included in GenBank Gene ID: 100817854), respectively. Figure 3 shows the sequence of each coding region (CDS). Comparative analysis of the CDS of the Arabidopsis gene AtMYB60 and the soybean genes Glyma.03g006600 and Glyma.19g119300 demonstrated the conservation of the gene structure in terms of introns, exons and untranslated regions (UTRs) ( Figure 4 ).

[0048] Analysis of the upstream genomic regions of Glyma.03g006600 and Glyma.19g119300 containing the putative promoters of both genes demonstrated the presence of various nucleotide sequences [A / T]AAAG, which correspond to DNA binding sites for DOF transcription factors. Said sequences, also present in the promoter of AtMYB60, represent important regulatory elements capable of directing gene expression specifically in stomata. In particular, cis-elements, which are necessary and sufficient to regulate gene expression in stomata, consist of clusters of at least three DOF elements distributed on the same strand of a region not exceeding 100 bp. Three DOF clusters were found in the putative promoter of Glyma.03g006600, while six clusters were located in the promoter of Glyma.19g119300 (Figure 5).

[0049] Functional analysis

[0050] To verify the conservation of biological functions between AtMYB60 and the two soybean genes, complementation tests were performed by inserting the gene Glyma.03g006600 or Glyma.19g119300 into the Arabidopsis atmyb60-1 mutant. The coding regions of the two genes were amplified from cDNA obtained from soybean leaves (cv Williams 82) using the following primers:

[0051] -Glyma.F35'-AAAAAGCAGGCTATGGGGAGGCCTCCTT-3'SEQ ID NO: 61

[0052] -Glyma.R35'-AGAAAGCTGGGTTGAACATCGGAGACAACTC-3'SEQ ID NO: 62

[0053] The obtained fragment was sequenced to confirm the identity of the two genes and then cloned into the downstream of the constitutive promoter CaMV35S in the vector pB7FWG2 using Gateway technology ( Figure 6 A). Two vectors containing the CDS of Glyma.03g006600 or Glyma.19g119300 were introduced into the genome of mutant atmyb60-1 by floral dipping using Agrobacterium tumefaciens GV3101. Transformed lines were selected with the herbicide phosphinothricin (PPT), and resistant individuals were used for further analysis. As expected, independent lines transformed with genes Glyma.03g006600 or Glyma.19g119300 showed much higher expression levels of the two soybean genes than control plants ( Figure 6 B and C). Stomatal opening was then analyzed in two independent lines for each gene. Ectopic expression of the two soybean genes in the Arabidopsis mutants rescued the stomatal opening defect exhibited by atmyb60-1 ( Figure 7 ). This demonstrates that both soybean genes are active in stomata, where they act as positive regulators of stomatal opening, like AtMYB60. Taken together, the results confirm that Glyma.03g006600 and Glyma.19g119300 represent functional orthologs of AtMYB60 in soybean and support their role as targets for editing approaches designed to reduce stomatal opening in the species.

[0054] Promoter activity in heterologous systems

[0055] A particular feature of AtMYB60 is that its expression is specifically localized to stomatal guard cells of Arabidopsis. To evaluate the cell specificity of expression of Glyma.03g006600 and Glyma.19g119300, the respective promoters were cloned from the soybean genome (cv Williams82), fused to the reporter genes GUS and GFP, and the resulting constructs were used for transient expression experiments in tobacco and for the construction of stable transgenic lines in Arabidopsis.

[0056] The putative promoter of gene Glyma.03g006600, corresponding to the genomic sequence 1848 bp upstream of the translation start codon ( FIG. 5A ), was amplified using the following primers:

[0057] -Glyma.3F25'-CACCCTCAGCATTGACTGCACA-3'SEQ ID NO: 63

[0058] -Glyma.3R25'-CCTCTCAACTCACTAACTCACTC-3'SEQ ID NO: 64

[0059] The putative promoter of gene Glyma.19g119300, corresponding to the genomic sequence 1912 bp upstream of the translation start codon ( FIG. 5A ), was amplified using the following primers:

[0060] -Glyma.19F25'-CACCTATGTGACTCTCAAGTCAC-3'SEQ ID NO: 65

[0061] -Glyma.19R25'-TCACTCACTCCACCTTTCTTCCT-3'SEQ ID NO: 66

[0062] Using Gateway technology, the amplified product was cloned into the downstream of two reporter genes GUS and GFP in the vector pBGWFS7 ( Figure 8 ). The vector thus obtained was used for transient expression assays in tobacco leaves (Nicotiana benthamiana) infiltrated with Agrobacterium tumefaciens and GUS histochemical staining was performed 48 hours after Agrobacterium infiltration. Nicotiana benthamiana leaves infiltrated with constructs carrying the Glyma.03g006600 promoter showed GUS activity mainly in trichomes. In contrast, leaves infiltrated with vectors containing the Glyma.19g119300 promoter showed GUS activity mainly in stomatal guard cells.

[0063] Stable Arabidopsis lines were generated using the same vector and transformed by floral dip. The obtained lines were screened with the herbicide PPT and their progeny were analyzed by GUS histochemical assay. A total of 22 independent lines for each construct were analyzed. All lines containing the Glyma.03g006600 promoter showed GUS activity in trichomes (100%). 14 of them (63.6%) also showed activity in vascular tissue, while only two (9.1%) showed stomatal staining. 21 (95.5%) lines containing the Glyma.19g119300 promoter showed stomatal staining. 18 of them (81.1%) also showed GUS activity in trichomes, while no staining was shown in vascular tissue. Interestingly, the Glyma.19g119300 promoter activity in Arabidopsis trichomes was very high in young leaves and gradually decreased during leaf development, then was completely localized in the stomata of mature leaves.

[0064] In summary, the expression results in heterologous systems (tobacco and Arabidopsis) indicate that the promoter of gene Glyma.03g006600 is mainly active in trichomes, while the promoter of Glyma.19g119300 is preferentially active in stomata. This finding suggests that although both genes are able to compensate for the loss of AtMYB60 function in stomata, Glyma.19g119300 may play a more general role in regulating stomatal activity than Glyma.03g006600.

[0065] Expression in soybean

[0066] Then, expression profiling of Glyma.19g119300 and Glyma.03g006600 in soybean organs and tissues was performed using qPCR. This analysis demonstrated that both genes were expressed in leaves but not in roots ( Fig. 9 A). The specific expression in green tissues of plants is consistent with the observations of AtMYB60 in Arabidopsis. The expression of the two genes in whole leaves and trichomes dissected from the leaf epidermis was then compared. This comparison demonstrated that the two genes were expressed at comparable levels in soybean trichomes ( Fig. 9 B). Finally, the expression of Glyma.03g006600 and Glyma.19g119300 in leaves and stomata purified from soybean leaves by successive cycles of mechanical disruption and filtration was analyzed according to a method commonly referred to as ice mixing. Glyma.03g006600 showed comparable expression levels between whole leaves and isolated stomata, whereas Glyma.19g119300 showed considerably higher expression in purified stomata than in whole leaves ( Fig. 9 C).

[0067] In summary, analysis of endogenous gene expression in soybean tissues demonstrated that both are expressed in trichomes and that Glyma.19g119300 is preferentially expressed in stomata, consistent with expression data obtained in heterologous systems.

[0068] Soybean gene editing

[0069] The results obtained from the analysis of the genes Glyma.03g006600 and Glyma.19g119300 in heterologous systems and soybean plants showed a high degree of homology with the Arabidopsis gene AtMYB60 in terms of sequence (nucleotide and amino acid), biological function and expression profile. In particular, Glyma.19g119300, like AtMYB60, showed preferential expression in stomata.

[0070] Therefore, in order to reduce stomatal opening and improve drought resistance in soybean plants, a method for inactivating the Glyma.03g006600 and Glyma.19g119300 genes was designed. The method is based on the CRISPR / Cas9 editing system, which uses sgRNAs specific to the genes Glyma.03g006600 and Glyma.19g119300, and involves transforming soybean by integrating T-DNA into the genome through Agrobacterium tumefaciens, wherein the T-DNA contains: (i) a bar selection marker that confers resistance to the herbicide PPT; (ii) sgRNAs specific to Glyma.03g006600 and Glyma.19g119300; and (iii) a Cas9 gene.

[0071] From an operational point of view, the method includes the following steps: (i) selection and synthesis of sgRNA, (ii) construction of pCRISPR35SCas9_BAR_sgRNA vector; (iii) transformation of the vector into the soybean genome by Agrobacterium tumefaciens; (iv) selection of T0 edited lines; and (v) generation and analysis of T1 lines.

[0072] Selection of sgRNA

[0073] The guide sequences for CRISPR / Cas9-mediated editing were selected using the CRISPOR analysis program (http: / / crispor.tefor.net / ). The complete CDS of Glyma.03g006600 and Glyma.19g119300 were used as targets for identifying sgRNAs. The trinucleotide NGG, commonly used in applications involving the Streptococcus pyogenes CRISPR / Cas9 system, was used as a protozoan adjacent site (PAM). The length of the target sequence and the corresponding sgRNA was set to 20 base pairs (bp). The CRISPR / Cas9 vectors selected for the editing experiments (pCRISPR35SCas9_BAR, Fig.10) used the U6 soybean promoter for sgRNA expression. Although the promoter prefers sgRNAs that begin with a G nucleotide, no filter was used to select the first nucleotide in the guide sequence. Subsequently, when the synthetic guide sequence was used for cloning into the sgRNA-CRISPR-Cas9 vector, the starting nucleotide G was inserted into the guide sequence. Among the possible sgRNAs identified by the software, 30 guide sequences were selected for each gene based on three main criteria: (i) GC content; (ii) specificity; (iii) efficiency; and (iv) induction of out-of-frame mutations. Only guide sequences with a GC content of more than 20% and less than 80% were selected to ensure higher cutting efficiency. The specificity of each guide sequence to its target was evaluated by the CFD specificity scoring method, and guide sequences with a specificity coefficient greater than 70 were selected. According to the method developed by Doench et al., the guide sequences expressed by the U6 promoter were optimized to estimate the degree of efficiency of the guide sequence in determining the target DNA cutting. Guide sequences with a degree of efficiency greater than 50 were selected. Since the editing strategy was aimed at inactivation of the genes Glyma.03g006600 and Glyma.19g119300, guide sequences were selected with out-of-frame scores greater than 50, i.e. guide sequences characterized by a high probability of inducing inactivating mutations in both targets.

[0074] Tables 1 and 2 list the selected guide sequences, corresponding DNA sequences, and related information for the editing experiments.

[0075]

[0076]

[0077]

[0078]

[0079] Construction of pCRISPR35SCas9_BAR_sgRNA vector

[0080] To edit Glyma.03g006600and Glyma.19g119300, the pCRISPR35SCas9_BAR vector was constructed, which contains a transfer DNA (T-DNA) region surrounded by two inverted terminal repeat sequences, namely the “right border” (RB T-DNA repeat sequence) and the “left border” (LB T-DNA repeat sequence). The following is contained between the two borders: (i) an expression cassette of the bar gene, which confers herbicide PPT resistance and consists of the bar coding sequence, the CaMV35S promoter, and the poly-ACaMV terminator signal; (ii) an expression cassette of the Cas9 gene, which is formed by a sequence encoding the SpCas9 gene optimized for expression in plants, the CaMV35S promoter, the SV40 NLS sequence for nuclear localization of the Cas9 protein, and the poly-ACaMV terminator signal; and (iii) a scaffold-gRNA region for expressing sgRNA specific for Glyma.03g00890 or Glyma.19g29750 under the control of the soybean promoter U6-10. Oligonucleotides corresponding to sgRNAs were directly synthesized and cloned into the pCRISPR35SCas9_BAR plasmid using Gateway technology to generate the pCRISPR35SCas9_BAR_sgRNA vector.

[0081] Transformation of soybean

[0082] The method includes introducing pCRISPR35SCas9_BAR_sgRNA into the soybean genome by Agrobacterium-mediated transformation. The vector is introduced into Agrobacterium tumefaciens strain EHA105 by electroporation, and the transformed bacteria are selected on solid LB medium supplemented with kanamycin. A single colony of Agrobacterium is used as an inoculum. The Agrobacterium culture is maintained in YEB liquid medium until the optical density OD650 reaches 0.7. The bacteria are then centrifuged and resuspended in a liquid co-culture medium (CCM).

[0083] Mature soybean seeds were sterilized with sodium hypochlorite-hydrochloric acid for about 16 hours and allowed to swell in water for about 24 hours. The seeds were then cut longitudinally to separate the two cotyledons and the outer coating was removed. The cotyledons were contacted with the Agrobacterium suspension for 30 minutes.

[0084] After co-cultivation with Agrobacterium, the cotyledons were transferred to a petri dish containing CCM solid medium, and PPT was added to select resistant explants and kept at 24°C for five days with a 16-hour light / 8-hour dark photoperiod. The cotyledons were then transferred to SIM (+PPT) solid medium for a total of four weeks, and the explants were subsequently transferred to SEM (+PPT) medium. When the resistant shoots reached a length of about three centimeters, they were transferred to RIM medium to promote rooting.

[0085] The seedlings obtained from the transformation / regeneration process (T0 generation) were transferred to plant pots, grown in a greenhouse under controlled conditions, and analyzed to select individuals with mutations in the genes Glyma.03g00890 and Glyma.19g29750.

[0086] 1. Agrobacterium LB medium

[0087]

[0088] 2. Agrobacterium YEB medium

[0089]

[0090] 3. Liquid co-culture medium (CCM1)

[0091]

[0092] 4. Solid co-culture medium (CCM)

[0093]

[0094] 5. Liquid germination medium (SIM1)

[0095]

[0096] 6. Solid sprout medium (SIMs)

[0097]

[0098]

[0099] 7. Shoot elongation medium (SEM)

[0100]

[0101] 8. Rooting medium (RIM)

[0102]

[0103] Selection of T0 editing lines

[0104] Molecular analysis was performed on T0 plants grown in the greenhouse to verify the presence of mutations in two genes, Glyma.03g00890 and Glyma.19g29750, and to verify the presence of the genome in the vector pCRISPR35SCas9_BAR_sgRNA. DNA samples were extracted from each plant, and the target region complementary to the sgRNA used for editing was amplified with the following specific primers:

[0105] Glvma.03g00890

[0106] -Glyma.3F3: 5'-ATGGGCAGCCATAGCTTCCTATCTTCCA-3'SEQ ID NO: 67

[0107] -Glyma.3R3: 5'-TCGGAGACAACTCCTTCATCTCCT-3'SEQ ID NO: 68

[0108] Glyma.19g29750

[0109] -Glyma.19F3: 5'-TTGTGAAGTTGTTGACTTTTGGAGCAGAT-3'SEQ ID NO: 69

[0110] -Glyma.19R3: 5'-TGACAATTCCTTGTTAATTAGAACAT-3'SEQ ID NO: 70

[0111] The obtained PCR product is then cloned by TA cloning into a vector optimized for PCR fragment sequencing (e.g., The sequences thus obtained were analyzed to verify the presence of mutations in the target regions of Glyma.03g00890 and Glyma.19g29750 and to verify the nature of the mutations.

[0112] The method comprises selecting TO plants carrying mutations that inactivate the activity of the genes Glyma.03g00890 and Glyma.19g29750 in whole or in part, such as frameshift mutations, introduction of stop codons, amino acid deletions and substitutions in the relevant domains of the proteins encoded by Glyma.03g00890 and Glyma.19g29750.

[0113] The selected T0 strains were then analyzed using primers specific for the Cas9 gene to verify the presence of the vector pCRISPR35SCas9_BAR_sgRNA in their genome:

[0114] -Cas9F1:5'-AGACCGTGAAGGTTGTGGAC-3'SEQ ID NO:71

[0115] -Cas9R1:5'-TAGTGATCTGCCGTGTCTCG-3'SEQ ID NO:72

[0116] Generation and analysis of T1 lines

[0117] TO plants selected for the presence of mutations with a high impact on the activity of the genes Glyma.03g00890 and Glyma.19g29750 were propagated by self-pollination to produce T1 seeds.

[0118] Molecular analysis of T1 lines

[0119] The molecular analysis of T0 plants was repeated on T1 individuals to verify the presence of the selected mutation and the heterozygous or homozygous state of the individual mutation, as well as the presence or absence of the vector sgRNA-CRISPR / Cas9. In addition, T1 individuals with inactivating mutation signatures were analyzed by qPCR to verify the transcript levels of the genes Glymca.03g00890 and Glyma.19g29750 using specific primers:

[0120] Glvma.03g00890

[0121] -Glyma.3F4:5'-AACAAGGGATCAATAATATCCCA-3'SEQ ID NO:73

[0122] -Glyma.3R4:5'-CCATGTTATTATCTACTCCACC-3'SEQ ID NO:74

[0123] Glyma.19g29750

[0124] -Glyma.19F4:5'-CCATGTTATTATCTACTCCACC-3'SEQ ID NO:75

[0125] -Glyma.19R4: 5'-GATGATCAACAGAATACTCAG-3'SEQ ID NO: 76

[0126] T1 plants carrying the inactivating mutation (in the homozygous or heterozygous state) were self-pollinated to produce T2 seeds and plants. T1 individuals with the genomic vector sgRNA-CRISPR / Cas9 were crossed with untransformed soybean plants (cv Williams 82) to facilitate transgene segregation in the F2 progeny.

[0127] Stomatal activity analysis

[0128] The selected T1 lines were subjected to a series of physiological analyses to determine the effect of inactivation of Glyma.03g00890 and Glyma.19g29750 on stomatal activity. The analyses included measurement of stomatal conductance (gs) using a SC-1 portable porometer manufactured by Decagon Device. Measurements were made on five leaves per plant, using the center portion of the upper and lower surfaces of the leaf, with six replicates in each leaf. Untransformed soybean plants (Cv Williams 82) were used as controls for the measurements. After exposure to light (light intensity 400 μM m- 2 s -1 ) for at least four hours, with measurements taken in the middle part of the day (approximately between 11 a.m. and 1 p.m.).

[0129] Stomatal opening was measured in parallel in epidermal fragments obtained from leaves of edited and control plants. These fragments were taken from leaves of dark-adapted plants, incubated in a solution of KCl 30 mM, MES-KOH 10 mM, pH 6.5, and kept in the dark or exposed to light (400 μM m- 2 s -1 ) for four hours. Stomata were then photographed under an optical microscope (40× magnification), and the images were analyzed using ImageJ software (https: / / imagej.net / ) to measure the width and length of the stomata. The degree of stomatal opening was calculated as the ratio between the width and length of the stomata.

[0130] Finally, the water loss rate in the cut leaves was evaluated to assess the transpiration rate of T1 plants. Four leaves of comparable developmental stage and size were removed from the control and edited plants and air-dried in a controlled environment at a temperature of 25 °C and a relative humidity of 50%. The weight of each leaf was determined every four hours, and the water loss was expressed as a percentage of the initial fresh weight.

[0131] Overall, the described analysis allowed the selection of T1 individuals characterized by low stomatal patency, low stomatal conductance and low transpiration. Said individuals were further selected to produce T2 seeds and plants by self-pollination.

[0132] Analysis of drought response in selected T2 lines

[0133] Each T2 line was analyzed again to confirm the presence of the selected mutation in the ancestors T0 and T1 and to confirm the absence of vector sgRNA-CRISPR / Cas9.

[0134] T2 lines were then subjected to physiological analysis to assess plant responses to water stress conditions. Untransformed plants and T2 lines were grown in plant pots under three different growth conditions: control conditions, in which the relative water content of the soil was maintained at 80% of field capacity (FC); (ii) moderate stress conditions, in which the soil was maintained at 50% FC; and (iii) high stress conditions, in which the soil was maintained at 30% FC.

[0135] The performance of the various T2 lines (and non-transformed control plants) under the three water conditions was determined by measuring various biological characteristics, physiological and production parameters during various stages of the biocycle.

[0136] The biometric parameters considered included the number of internodes produced, internode length, total height of mature stems and average leaf size.

[0137] Physiological parameters include (i) measurement of stomatal conductance and photosynthetic efficiency (A n ,mmol CO 2 m -2 s -1 ), measured using a LI-6400 portable system (Li-Cor Inc., Lincoln, NE, USA), and (ii) leaf water potential (Ψleaf, MPA), measured using a Scholander pressure chamber (Model PMS-1000, PMS Instruments, Corvallis, OR, USA).

[0138] Production parameters included flowering time, number of pods produced, number of seeds per pod, average weight of seeds and total weight of seeds produced.

[0139] The T2 lines that responded best to water deficit were selected based on the evaluation of the parameters listed above and used as genetic starting material for a breeding program aimed at introducing mutations in genes Glyma.03g00890 and Glyma.19g29750 into elite soybean varieties to develop new varieties with high drought resistance.

Claims

1. A method for improving drought resistance of soybean plants, comprising inactivating at least one target gene selected from Glyma.19g119300 and Glyma.03g006600 in the plants.

2. The method according to claim 1, wherein the target gene is Glyma.19g119300.

3. The method according to claim 1, wherein the target gene is inactivated by inserting a site-specific mutation through the combination of Cas9 enzyme nuclease activity and sgRNA specific for the target gene. 4 . The method according to claim 3 , wherein the sgRNA specific to the target gene Glyma.19g119300 is encoded by a nucleotide sequence selected from SEQ ID NO: 31 to SEQ ID NO:

60. 5 . The method according to claim 3 , wherein the sgRNA specific to the target gene Glyma.03g006600 is encoded by a nucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO:

30.

6. The method according to claims 3-5, comprising the following steps: (i) constructing an expression vector capable of expressing Cas9 enzyme and sgRNA in soybean plant cells; (ii) introducing the expression vector into cells or parts or isolated tissues thereof from soybean plants, particularly cotyledon explants thereof, by contacting the cells with a culture of bacteria of the genus Agrobacterium containing the expression vector.

7. The method according to claim 6, wherein the expression vector comprises: - a sgRNA coding sequence functionally linked to the U6 soybean promoter; - An expression cassette of the Cas9 gene, which comprises a sequence encoding the Cas9 enzyme, a CaMV35S promoter, and a sequence for nuclear localization of the Cas9 protein; -DNA transfer zone.

8. The method according to claim 6-7, further comprising the following steps: (iii) planting plants containing the Cas9 / sgRNA expression vector and obtaining seeds; (iv) growing plants generated from said seeds and subsequently selecting plants having reduced levels of stomatal aperture; (v) Further selecting plants with increased drought resistance by testing the plants under conditions of water stress.

9. A soybean plant, or part or seed thereof, wherein at least one of the Glyma.19g119300 gene and the Glyma.03g006600 gene has been inactivated.

10. The soybean plant, part thereof or seed according to claim 9, wherein The gene is Glyma.19g119300.

11. The soybean plant, part thereof or seed according to claim 9 or 10, wherein the inactivation of at least one of the Glyma.19g119300 gene and the Glyma.03g006600 gene is obtained by site-specific mutagenesis using the CRISPR / Cas9 system.

12. The soybean plant, part thereof or seed of claim 11 which is homozygous or heterozygous for said mutation.

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