Gene regulating soybean plant type having tolerance to high density, and use thereof

Abstract

Provided are a gene regulating a leguminous plant plant type having tolerance to high density, and a use thereof. The nucleotide sequence of the gene is as shown in SEQ ID NO: 1, the amino acid sequence of a protein encoded thereby is as shown in SEQ ID NO: 2, and the genome sequence is as shown in SEQ ID NO: 3. Transgenic leguminous plants having the gene have a reduced branch number, a reduced plant height, a reduced node number, a shortened internode length, and / or a shortened petiole length compared to control or wild-type plants.

Description

Genes regulating soybean dense-tolerance plant type and uses thereof Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to genes for regulating soybean dense-tolerance plant types and uses thereof. Background Art

[0002] Soybeans provide a rich source of plant protein and oil, making them an important grain and oil crop. However, my country's long-standing dependence on soybean imports remains unchanged, and the gap between supply and demand continues to widen year by year. Furthermore, soybean yields per unit area differ significantly from the international average. Therefore, given the declining availability of arable land in my country, increasing soybean yield per unit area while meeting the growing demand for plant protein amidst a shifting dietary structure is a significant concern. Designing ideal soybean plant types, optimizing soybean planting density per unit area, and reducing field light loss provide the biological basis for addressing this issue.

[0003] As early as 1968, Donald proposed using ideal plant architecture (ideotype) to guide crop breeding (Donald, 1968). This approach involves deeply understanding the molecular regulatory mechanisms of existing high-yield plant traits and, based on this understanding, optimizing the combination of various desirable traits to design crops with ideal plant architectures, aiming to achieve higher grain yields. During the Green Revolution, the cultivation of semi-dwarf wheat and rice, at the expense of straw biomass, not only increased grain yield but also demonstrated superior lodging resistance (Peng et al., 1999). The rice genotype IPA1 (Ideal Plant Architecture 1) reduces tiller number and increases spikelet branch number, thereby increasing yield (Jiao et al., 2010; Miura et al., 2010; Wang et al., 2018). The wheat genotype TaCOL-B5 increased yield by 11.9% by increasing spikelet branch number and spikelet node number (Zhang et al., 2022).

[0004] To achieve a green revolution in the soybean industry, an ideal soybean plant type has been proposed: one suitable for high-density planting, characterized by suitable plant height, short internode length, numerous nodes, few or no branches, a moderate number of pods per node, a high pod-setting rate, a high ratio of four pods per node, a moderate 100-grain weight, a small petiole angle, and short petioles (Liu et al., 2020). Previous research on increasing soybean yield has primarily focused on achieving higher grain weight and a larger number of pods, often resulting in a small "source" and insufficient "sink," making it difficult to achieve significant improvements. Therefore, ensuring a balance between "source" and "sink," ensuring the proper spatial positioning of lateral organs, optimizing plant architecture, and improving overall plant efficiency in the field for light energy utilization, ultimately increasing soybean yield, has become an ideal approach to address this issue. Designing a branched soybean plant type offers a promising approach.

[0005] Therefore, to reduce light energy loss in the field and increase soybean planting density, the first thing to consider is a suitable branching structure. Among the many traits that affect soybean yield, branching traits include the length, number, and angle of branches with the main stem, which affect the ventilation and light energy utilization of the soybean population. They are important factors in constructing an ideal soybean plant type and affecting soybean yield. Among them, the number of branches is an important agronomic trait that affects crop yield. In the grass plant rice, reducing the number of tillers and increasing the number of spikelet branches ultimately increases yield. In the dicotyledonous plant soybean, the branch number trait, which is a quantitative trait, is controlled by major and minor effect polygenes, and is also affected by environmental factors such as planting density and light conditions. This makes the study of soybean branch number traits particularly complicated. At present, there are no reports on genes related to the shaping of soybean dense tolerance plant type.

[0006] Summary of the Invention

[0007] This study, based on functional studies of the soybean Ln gene, found that a naturally occurring variant of Ln, ln, significantly reduced branch number. Transgenic functional validation revealed that ln shortened petioles and internodes, shortened plant height, and significantly reduced branch number to a single stem, resulting in a highly tolerant plant type. This not only lays a genetic foundation for understanding the molecular mechanisms of soybean plant architecture but also provides genetic resources for subsequent increases in soybean yield per unit area.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] On the one hand, the present invention provides an isolated genome sequence for regulating the dense-tolerant plant type of legume plants, preferably plants of the genera Glycine max, Pisum sativum, and Garbanzo pea, such as soybean, pea, mung bean, broad bean, black bean, and chickpea, characterized in that the genome sequence is shown in SEQ ID NO: 3.

[0010] On the other hand, the present invention provides a use of ln in regulating the dense-tolerance plant type of legumes, preferably plants of the genera Glycine max, Pisum sativum, and Garbanzo pea, such as soybean, pea, mung bean, broad bean, black bean, and chickpea, characterized in that the coding sequence of ln is shown in SEQ ID NO: 1.

[0011] In some embodiments, the amino acid sequence of ln is as shown in SEQ ID NO:2.

[0012] In some embodiments, the genomic sequence of ln is shown as SEQ ID NO:3.

[0013] In some embodiments, the use is manifested as one or more of the following:

[0014] a. Reduce the number of branches;

[0015] b. Reduce plant height;

[0016] c. Reduce the number of sections;

[0017] d. Shorten internode length;

[0018] e. Shorten the length of petiole.

[0019] On the other hand, the present invention provides an expression vector, characterized in that the expression vector comprises the genomic sequence as described above.

[0020] In some embodiments, the expression vector has an antibiotic marker and / or a chemical resistance marker.

[0021] On the other hand, the present invention provides a host cell, characterized in that the host cell comprises the genomic sequence or expression vector as described above.

[0022] On the other hand, the present invention provides a method for regulating the dense-density-tolerant plant type of legumes, preferably plants of the genus Glycine max, Pisum sativum, or Garbanzo pea, such as soybean, pea, mung bean, broad bean, black bean, or chickpea, characterized in that the method comprises the step of introducing the expression vector or host cell as described above into a legume, preferably a plant of the genus Glycine max, Pisum sativum, or Garbanzo pea, such as soybean, pea, mung bean, broad bean, black bean, or chickpea plant or cell or tissue.

[0023] In some embodiments, the dense-tolerant plant type exhibits one or more of the following: relative to wild-type or control plants: a. reduced number of branches; b. reduced plant height; c. reduced number of nodes; d. shortened internode length; e. shortened petiole length, and still grows well when planted at high density.

[0024] definition

[0025] High-density planting: Dongnong 50 is defined as 80-120 plants per square meter as high-density planting, for example, the planting density is 5cm plant spacing and 50cm row spacing.

[0026] Low-density planting: 26-40 plants per square meter of Dongnong 50 is defined as low-density planting, for example, the planting density is 15cm plant spacing and 50cm row spacing. Beneficial effects

[0027] The soybean plant type shaping-related proteins, encoding nucleic acid sequences and genomic nucleic acid sequences (including promoters) provided by the present invention are all discovered for the first time by the applicant, and phenotypic analysis of transgenic plants and wild-type plants has verified that the expression of the plant type shaping protein of the present invention can significantly affect the soybean plant type.

[0028] The present invention will have great theoretical and practical value for the construction of ideal soybean plant types, the breeding and creation of dense planting-resistant varieties, and related basic and applied research. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows that ln plants are compact under low density, indicating that ln can shape a dense-tolerant soybean plant. A shows that ln resulted in a compact plant; B shows that ln significantly reduced branch number, plant height, and internode distance; C shows that ln shortened petioles; and D shows the relative expression level of ln in leaves of the ln-overexpressing lines.

[0030] Figure 2 shows the performance of transgenic materials at different planting densities. A shows the performance of DN50 at low planting density (15 cm plant spacing; 50 cm row spacing); B shows the performance of DN50 at high planting density (5 cm plant spacing; 50 cm row spacing); C shows the performance of the ln line at low planting density (15 cm plant spacing; 50 cm row spacing); and D shows the performance of the ln line at high planting density (5 cm plant spacing; 50 cm row spacing).

[0031] FIG3 shows a map of the construction of the ln genome expression vector.

[0032] Figure 4 shows the statistical results of plant height (n ≥ 30), number of branches, number of nodes, petiole length, and internode length between the transgenic ln accessions and the wild-type Dongnong 50 accession at low density. These include the sample size, sample mean, sample standard deviation, sample extreme value, and the significance level of the difference between the transgenic ln accessions and the wild-type Dongnong 50 accession. The significance level was tested using a one-tailed T-test, P < 0.01, n = 30.

[0033] Figure 5 shows the yield of the wild-type Dongnong 50 transgenic material at different planting densities (LD: low density, 15 cm plant spacing, 50 cm row spacing; HD: high density, 5 cm plant spacing, 50 cm row spacing). A shows the yield per plant at different planting densities (n = 30); B shows the yield per mu of plots at different planting densities (n = 3). One-way analysis of variance was used with Tukey's multiple comparisons, and P < 0.05 or P < 0.01 was used. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0035] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The quantitative tests in the following examples were all repeated three times, and the results were averaged to have statistical significance.

[0036] In the following examples, the transformation recipient was Dongnong 50 (DN50, Heishendou 2007022), which can be purchased from the market. The vector pTF101.1 and Agrobacterium strain EHA101 were purchased from Biovector Science Lab, Inc., China.

[0037] Homologous recombination kits and other consumables were purchased from Novozymes Biotechnology Co., Ltd. and Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0038] Example 1 Discovery of ln protein and its encoding gene

[0039] Our research team previously identified the gene ln, which controls pointed leaves and four-pod traits in soybean, through map-based cloning (Fang et al., 2013). Its CDS sequence is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2. To further investigate the function of ln, we amplified the approximately 8 kb full-length ln gene (SEQ ID NO:3) from the soybean cultivar DN50 (ln / ln). This gene, comprising a 2.6 kb upstream promoter, 1.8 kb ln genomic DNA sequence, and 3.2 kb ln downstream sequence (the present invention selectively introduces pre-existing genomic DNA sequences to more closely resemble the in vivo transcriptional and translational landscape). Transfection into DN50 revealed significant improvements in soybean plant architecture, resulting in a compact plant shape, shorter petioles, a lack of branching, and shortened internodes, suggesting that it is suitable for dense planting and contributes to higher yield per unit area.

[0040] Example 2 Functional verification of ln protein

[0041] 1. Construction of recombinant plasmid

[0042] Genomic DNA was extracted from Dongnong 50 (DN50) and used as a template to amplify the ln gene by KOD high-fidelity PCR. The gene was then homologously recombined into the pTF101.1 vector to obtain the recombinant plasmid pTF101.1-pGmln-gGmln. The specific steps are as follows:

[0043] 1. Extract DNA from leaves of soybean variety DN50.

[0044] 2. Using the total DNA obtained in step 1 as a template, perform PCR amplification using the primer pair consisting of F1 and R1 to obtain a PCR amplification product.

[0045] F1:5'-ATGTTGACCTGCAGGCATGCAAGCTTGGCGTCAAACTTTACCGAT-3'(SEQ ID NO:4)

[0046] R1:5'-AACAGCTATGACATGATTACGAATTCACTTTTAAGCACACCTGACC-3'(SEQ ID NO:5)

[0047] 3. The pTF101.1 vector (purchased from China Plasmid Vector, Strain and Cell Gene Collection Center) was double-digested with restriction endonucleases Hind III and EcoR I, and the linearized vector of about 8 kb was recovered by 1% agarose gel electrophoresis.

[0048] 4. The PCR product of step 2 and the vector backbone of step 3 were connected by homologous recombination to obtain the recombinant plasmid pTF101.1-pGmln-gGmln (the map of which is shown in Figure 3).

[0049] 2. Obtaining Ln-overexpressing transgenic plants

[0050] 1. The recombinant plasmid pTF101.1-pGmln-gGmln was introduced into the Agrobacterium tumefaciens strain EHA101 (purchased from the China Plasmid Vector, Strain, and Cell Gene Collection Center) to obtain recombinant Agrobacterium.

[0051] 2. The recombinant Agrobacterium obtained in step 1 was used for soybean genetic transformation using the cotyledonary node Agrobacterium infection transformation method (Li et al., 2017) to transform the recipient plant DN50.

[0052] (1) Sterilization of soybean seeds: Select soybean seeds that are large and full, with a smooth surface without disease spots and without scratches on the seed coat, and divide them into petri dishes. Open the petri dishes and put them into a desiccator. Chlorine fumigation sterilization is used for seed disinfection. Place a small beaker in the desiccator, add 100 mL of sodium hypochlorite, then add 5 mL of concentrated hydrochloric acid, quickly cover the lid, and sterilize for 10-14 hours. After sterilization is completed, take the soybeans out of the desiccator and blow them in the clean bench for more than 30 minutes to eliminate excess chlorine.

[0053] (2) Soybean seed imbibition germination: Place the sterilized seeds with the hilum facing downward into the germination medium and place them in a dark environment at 23°C for 16-18 hours.

[0054] (3) Preparation of Agrobacterium: Take out the bacteria stored at -80℃ and add them to 5mL of YEB medium containing rifampicin and kanamycin resistance. Incubate at 28℃ and 250rpm for 24h to recover. Then, take 200μL of the recovered bacteria solution and add it to 200mL of YEB medium containing rifampicin and kanamycin resistance. Incubate at 28℃ and 250rpm until OD 600 =0.6 Collect the bacterial solution. Centrifuge at 22°C, 5,000 rpm for 10 min, and then resuspend the bacteria in liquid infection medium to OD 600 =0.5, 22°C, 70 rpm, incubate for 0.5 h and set aside.

[0055] (4) Preparation of explants: Remove the imbibed seed coat and use a scalpel to cut off the part of the hypocotyl away from the cotyledons, leaving about 2-3 mm of the hypocotyl. Then, split the soybean seeds in half and use a small iron brush to remove the embryo. This is the prepared explant.

[0056] (5) Infection of explants: Place the prepared explants into the Agrobacterium suspension, making sure the suspension covers the explants, shake gently, and incubate for 0.5 h.

[0057] (6) Co-cultivation of explants and Agrobacterium: The infected explants were blotted on sterile filter paper to remove excess bacterial liquid, and a sterile filter paper was placed on the co-cultivation medium. The blotted explants were placed on the culture medium and cultured at 23°C in the dark for 3-5 days.

[0058] (7) Callus induction culture: After the explants are co-cultured with Agrobacterium, the explants are transferred to the callus induction medium. At this time, the explants are tilted at 45 degrees and the bottom is gently inserted into the culture medium. The culture is carried out at 23°C with 16 hours of light and 8 hours of darkness for 14 days.

[0059] (8) Bud induction culture: The induced callus was cut from the explant and placed in bud induction medium at 23°C, 16 h light / 8 h dark, for 14 days, and transferred to new bud induction medium every 14 days;

[0060] (9) Bud elongation culture: The explants that have induced new buds are transferred to bud elongation culture medium, and transferred to new bud elongation culture medium every 2 weeks until the buds elongate to more than 3 cm.

[0061] (10) Rooting culture: The elongated buds were cut off and inserted into the rooting medium until the root length exceeded 3 cm. Then the seedlings were trained and transplanted into the greenhouse at 24°C with 16 h light / 8 h dark until they matured. During this period, Basta was sprayed and positive strains were identified by PCR.

[0062] Table 1 Formula of 1L germination medium

[0063] Table 2 Formula of 1L YEB medium

[0064] Table 3 Formula of 1L infection solution

[0065] Table 4 Formula of 1L co-culture medium

[0066] Table 5 Formula of 1L callus induction medium

[0067] Table 6 Formula of 1L bud induction medium

[0068] Table 7 Formula of 1L shoot elongation medium

[0069] Table 8 Formula of 1L rooting medium

[0070] Soybean leaves were coated with 0.1% Basta herbicide (Coollebo, CB2471 - 100 mL). Plants showing no yellowing after three days were considered transgenic plants. T0 seeds were harvested. Subsequent T1 and subsequent generations of transgenic lines were sprayed with 0.1% Basta herbicide for screening. All of these lines showed Basta resistance, resulting in successful ln-overexpressing transgenic plants. Three transgenic lines, ln-1, ln-2, and ln-3, were obtained (Figure 1).

[0071] Total RNA was extracted and reverse transcribed into cDNA, and the expression of ln transcripts was detected by fluorescence quantitative PCR. The RT-qPCR primers were designed as follows:

[0072] ln gene

[0073] F2:5'-GTGTTTGCCTCACATCATTTTTCC-3'(SEQ ID NO:6)

[0074] R2:5'-TTGTGCAGCAATGTTATGATCACA-3'(SEQ ID NO:7)

[0075] Internal reference gene ACTIN

[0076] F3:5'-CGGTGGTTTCTATCTTGGCATC-3'(SEQ ID NO:8)

[0077] R3:5'-GTCTTTCGCTTCAATAACCCTA-3'(SEQ ID NO:9)

[0078] The instrument model is Roche Light Cycler 480, and the reagent is 480 SYBR Green I Master, reaction system is as follows (20 μL):

[0079] Table 9 RT-qPCR reaction system

[0080] RT-qPCR parameters were set as follows:

[0081] Hot start at 95°C for 5 minutes; denaturation at 95°C for 10 seconds, annealing at 59°C for 10 seconds, and extension at 72°C for 7 seconds, for 45 cycles; after the amplification cycle, a melting curve was performed: denaturation at 95°C for 5 seconds, 65°C for 1 minute, and then heating from 65°C to 97°C at a heating rate of 0.11°C / s.

[0082] Each amplification reaction was repeated at least 3 times. -ΔΔCt The relative expression level was calculated by the method.

[0083] The results showed that in the overexpression strain, the expression level of ln increased by 100-fold and the expression level was significantly upregulated (see Figure 1D).

[0084] Example 3 Statistics of transgenic phenotypes of ln

[0085] This example demonstrates that transgenic plants overexpressing the ln gene in a DN50 background exhibited a compact plant shape, significantly reduced branching, and a single stem. Plant height was significantly shortened (plant height was reduced to 56.8%, 49.24%, and 46.04% of the original value); the number of nodes was reduced by 7.01%, 11.06%, and 11.37%; internodes were significantly shortened (internode length was reduced to 39.94%, 38.84%, and 37.71% of the original value); and petioles were significantly shortened (petiole length was reduced to 13.3%, 10.35%, and 6.75% of the original value) (Figures 1A-C, Figure 4). Furthermore, compared to low-density planting conditions (15 cm plant spacing; 50 cm row spacing), transgenic ln plants performed well under high-density sowing conditions (5 cm plant spacing; 50 cm row spacing) (see Figure 2). This example demonstrates that overexpression of the ln gene can significantly improve plant shape, making the plant structure more compact, and plays an important role in tolerance to dense planting.

[0086] To this end, we further compared the yield under different densities. The results showed that high-density planting reduced the yield of DN50 plants by 68.2%, while the yield of ln transgenic plants was only reduced by 22.9% (Figure 5A), suggesting that ln transgenic plants can increase the overall yield by increasing the number of plants per unit area. We then identified the yield of plots under different planting densities, and compared the yield per mu, we found that ln transgenic plants can significantly increase the yield per mu. Under high-density planting conditions, ln transgenic plants increased yield by 38.5% compared to DN50, and under low-density planting conditions, they increased yield by 27.9% compared to DN50 (Figure 5B). This result once again confirmed that ln transgenic plants can increase soybean yield by increasing planting density, reducing field light loss, and increasing light energy utilization per unit area.

[0087] sequence

[0088] SEQ ID NO:1ln protein CDS sequence

[0089] SEQ ID NO:2ln protein amino acid sequence

[0090] SEQ ID NO: 3ln genomic sequence (including promoter and 5'-UTR (underlined portion), exons, introns, 3'-UTR sequence, downstream sequence)

[0091] SEQ ID NO: 4ln primer amplified sequence F1

[0092] SEQ ID NO:5ln primer amplified sequence R1

[0093] SEQ ID NO:61n Fluorescence quantitative PCR primer F2

[0094] SEQ ID NO:71n fluorescent quantitative PCR primer R2

[0095] SEQ ID NO:8 Internal reference gene ACTIN fluorescence quantitative PCR primer F3

[0096] SEQ ID NO:9 Internal reference gene ACTIN fluorescence quantitative PCR primer R3

[0097] References

[0098] Donald CM(1968)The breeding of crop ideotypes.Euphytica 17:385-403.

[0099] Fang C, Li WY, Li GQ, Wang Z, Zhou ZK, Ma YM, Shen YT, Li CC, Wu YS, Zhu BG, et al. (2013) Cloning of Ln gene through combined approach of map-based cloning and association study in soybean. J Genet Genomics 40:93-96.

[0100] Jiao YQ, Wang YH, Xue DW, Wang J, Yan MX, Liu GF, Dong GJ, Zeng DL, Lu ZF, Zhu XD, et al. (2010) Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nat Genet 42:541-544.

[0101] Li SX,Cong YH,Liu YP,Wang TT,Shuai Q,Chen NN,Gai JY,Li Y(2017).Optimization of Agrobacterium-mediated transformation in soybean.Front Plant Sci 8:246.

[0102] Liu SL,Zhang M,Feng F,Tian ZX(2020)Toward a“green revolution”for soybean.Mol Plant 13:688-697.

[0103] Miura K,Ikeda M,Matsubara A,Song XJ,Ito M,Asano K,Matsuoka M,Kitano H,Ashikari M(2010)OsSPL14 promotes panicle branching and higher grain productivity in rice.Nat Genet 42:545-549.

[0104] Peng J,Richards DE,Hartley NM,Murphy GP,Devos KM,et al.(1999)‘Green revolution’genes encode mutant gibberellin response modulators.Nature 400:256-261.

[0105] Wang J,Zhou L,Shi H,Chern M,Yu H,Yi H,He M,Yin JJ,Zhu XB,Li Y,et al.(2018)A single transcription factor promotes both yield and immunity in rice.Science 361:1026-1028.

[0106] Zhang XY,Jia HY,Li T,Wu JZ,Nagarajan R,Lei L,Powers C,Kan CC,Hua W,Liu Z,et al.(2022)TaCol-B5 modifies spike architecture and enhances grain yield in wheat.Science 376:180-183.

[0107] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Isolated genomic sequences regulating the dense-planting type of leguminous plants, preferably plants of the genus Glycine, Pisum, or Cicer, such as soybean, pea, mung bean, broad bean, black bean, or chickpea, Characterized in that, The genomic sequence is as shown in SEQ ID NO:

3.

2. Use of ln in regulating the dense-planting type of leguminous plants, preferably plants of the genus Glycine, Pisum, or Cicer, such as soybean, pea, mung bean, broad bean, black bean, or chickpea, Characterized in that, The coding sequence of ln is as shown in SEQ ID NO:

1.

3. The use according to claim 2, Characterized in that, The amino acid sequence of ln is as shown in SEQ ID NO:

2.

4. The use according to claim 2, Characterized in that, The genomic sequence of ln is as shown in SEQ ID NO:

3.

5. The use according to any one of claims 2-4, Characterized in that, The use is manifested as one or more of the following: a. Reducing the number of branches; b. Reducing the plant height; c. Reducing the number of nodes; d. Shortening the internode length; e. Shortening the petiole length.

6. An expression vector, Characterized in that, The expression vector contains the genomic sequence according to claim 1.

7. The expression vector according to claim 6, Characterized in that, The expression vector has an antibiotic marker and / or an anti-chemical reagent marker.

8. A host cell, Characterized in that, The host cell contains the genomic sequence according to claim 1 or the expression vector according to claim 6 or 7.

9. A method for regulating the dense-planting type of leguminous plants, preferably plants of the genus Glycine, Pisum, or Cicer, such as soybean, pea, mung bean, broad bean, black bean, or chickpea, Characterized in that, The method includes the step of introducing the expression vector according to claim 6 or 7 or the host cell according to claim 8 into leguminous plants, preferably plants of the genus Glycine, Pisum, or Cicer, such as soybean, pea, mung bean, broad bean, black bean, or chickpea plants or cells or tissues.

10. The method according to claim 9, Characterized in that, The dense-planting type is manifested as one or more of the following: compared with the control or wild-type plants, a. reducing the number of branches; b. reducing the plant height; c. reducing the number of nodes; d. shortening the internode length; e. shortening the petiole length.

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