Genetic transformation method of leguminous plants

Abstract

The invention provides a leguminous plant genetic transformation method or a method for improving the leguminous plant genetic transformation efficiency, and particularly relates to a leguminous plant genetic transformation method by using the screening culture medium, and the method can realize high-efficiency and stable genetic transformation without genotype dependence, and can be used for improving the leguminous plant genetic transformation efficiency. And the problem that some soybean varieties are difficult to carry out genetic transformation can be solved, and the method has a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic transformation, and in particular relates to a screening culture medium for improving the genetic transformation efficiency of leguminous plants and an application thereof. Background Art

[0002] Soybean [Glycine max (L.) Merr.] is a dicotyledonous plant of the genus Glycine max in the family Leguminosae. It is widely cultivated around the world and is an important economic and food crop. At present, rice, corn, wheat and soybean are the four major crops in my country. Soybean production can no longer meet domestic demand, and more than 80% of soybeans are imported each year. In addition, genetically modified soybeans are high-yielding and economically profitable. In 2020, genetically modified soybean seeds accounted for 37% of the global genetically modified seed market share. my country urgently needs to quickly cultivate high-yield, high-quality, disease-resistant and stress-resistant soybean varieties. The cultivation of new soybean varieties mostly uses traditional breeding and genetic engineering technology. Traditional breeding technology has many disadvantages such as long cycle and inability to achieve cross-species distant hybridization. Genetic engineering breeding technology overcomes these difficulties and is an important way to quickly cultivate new soybean strains.

[0003] Soybean genetic engineering breeding mostly uses Agrobacterium-mediated genetic transformation system, so the quality of genetic transformation system is crucial to soybean breeding. At present, soybean is still one of the most difficult crops to transform. During the transformation process, low infection efficiency, difficult regeneration, high genotype dependence, early flowering, lack of repeatability, etc., lead to significant differences in transformation efficiency between different genotypes. In addition, some varieties are prone to early flowering, making it difficult to achieve efficient transformation of some excellent varieties (such as Heihe 43) that have been widely promoted and applied.

[0004] Based on the current status of soybean genetic modification, we optimized the soybean genetic transformation system in order to overcome the problems of high genotype dependence, low transformation efficiency, long cycle, difficulty in regeneration and replication, etc. Summary of the invention

[0005] The present invention aims to provide a method for genetic transformation of legumes, or a method for improving the genetic transformation efficiency of legumes, wherein the method can achieve efficient and stable genetic transformation without genotype dependence.

[0006] On the one hand, the present invention provides a screening medium for genetic transformation of legumes, or a screening medium for improving the genetic transformation efficiency of legumes, wherein the screening medium contains 6-benzylaminopurine (6-BA), 6-furfurylaminopurine (KT), indoleacetic acid (IAA) and Timentin.

[0007] In one embodiment, the selection medium contains 6-benzylaminopurine (6-BA), 6-furfurylaminopurine (KT), indoleacetic acid (IAA), Timentin and spectinomycin.

[0008] In one embodiment, the screening culture medium comprises: MS culture medium, asparagine, glutamine, sucrose, agar powder, 6-benzylaminopurine (6-BA), Timentin, vitamin B1, vitamin B6, niacin, inositol, 6-furfurylaminopurine (KT), indoleacetic acid (IAA), and spectinomycin.

[0009] In one embodiment, the concentration of MS medium in the screening medium is 4.43 g / l.

[0010] In one embodiment, the asparagine concentration in the screening medium is 50-100 mg / l, preferably, 100 mg / l.

[0011] In one embodiment, the glutamine concentration in the screening medium is 50-100 mg / l, preferably, 100 mg / l.

[0012] In one embodiment, the sucrose concentration in the screening medium is 30 g / l.

[0013] In one embodiment, the concentration of agar powder in the screening medium is 6.2 g / l.

[0014] In one embodiment, the concentration of 6-benzylaminopurine (6-BA) in the screening medium is 0.1-2 mg / l; preferably, 1 mg / l.

[0015] In one embodiment, the concentration of Timentin in the screening medium is 200 mg / l.

[0016] In one embodiment, the concentration of vitamin B1 in the screening medium is 5-15 mg / l; preferably, 10 mg / l.

[0017] In one embodiment, the concentration of vitamin B6 in the screening medium is 0.5-1.5 mg / l; preferably, 1 mg / l.

[0018] In one embodiment, the niacin concentration in the screening medium is 0.5-1.5 mg / l; preferably, 1 mg / l.

[0019] In one embodiment, the inositol concentration in the screening medium is 5-15 mg / l; preferably, 15 mg / l.

[0020] In one embodiment, the concentration of 6-furfurylaminopurine (KT) in the screening medium is 0.1-1 mg / l; preferably, 0.5 mg / l.

[0021] In one embodiment, the concentration of indoleacetic acid (IAA) in the screening medium is 0.02-0.2 mg / l; preferably, 0.1 mg / l.

[0022] In one embodiment, the concentration of spectinomycin in the screening medium is 5-24 mg / l; preferably, the concentration of spectinomycin in the screening medium is 24 mg / l, 20 mg / l, 15 mg / l, 10 mg / l or 5 mg / l.

[0023] In one embodiment, the concentration of spectinomycin in the screening medium is 24 mg / l; and the soybean is Handan 16, Qihuang 34 or Andou 5240.

[0024] In one embodiment, the concentration of spectinomycin in the screening medium is 20 mg / l; and the soybean is Zhonghuang 302.

[0025] In one embodiment, the concentration of spectinomycin in the screening medium is 15 mg / l; and the soybean is Guangda 103, Dongsheng 19, Gm06, Dongsheng 1, Heihe 43, or Donnong 252.

[0026] In one embodiment, the concentration of spectinomycin in the screening medium is 10 mg / l; and the soybean is Henong 95 or Dongsheng 119.

[0027] In one embodiment, the concentration of spectinomycin in the screening medium is 5 mg / l; and the soybean is Jiadou 30.

[0028] In one embodiment, the culture medium for screening culture includes: MS medium (4.43 g / l), asparagine (100 mg / l), glutamine (100 mg / l), sucrose (30 g / l), agar powder (6.2 g / l), 6-benzylaminopurine (1 mg / l), timentin (200 mg / l), vitamin B1 (10 mg / l), vitamin B6 (1 mg / l), niacin (1 mg / l), inositol (15 mg / l), 6-furfurylaminopurine (0.5 mg / l), indoleacetic acid (0.1 mg / l), and spectinomycin (5-24 mg / l).

[0029] In another aspect, the present invention provides use of the screening medium in genetic transformation of legumes, or use of the screening medium in improving the genetic transformation efficiency of legumes.

[0030] On the other hand, the present invention provides a method for genetic transformation of legumes, or a method for improving the efficiency of genetic transformation of legumes, the method comprising the step of genetically transforming the legumes using the screening medium.

[0031] In one embodiment, the method comprises the following steps:

[0032] (1) Seed disinfection and germination;

[0033] (2) explant treatment;

[0034] (3) Infection;

[0035] (4) co-culture;

[0036] (5) Recovery culture;

[0037] (6) Screening and culture;

[0038] (7) elongation culture;

[0039] (8) Rooting;

[0040] The method comprises the step of using the screening culture medium to perform screening culture on leguminous plants.

[0041] In one embodiment, the disinfection and germination of seeds in step (1) are conventional methods; specifically, the disinfection of seeds is chlorine disinfection; the germination method of seeds is to inoculate the disinfected seeds on a germination medium and culture them in the dark at 22°C for 16 hours; in one embodiment, the germination medium comprises: B5 medium (3.21 g / l), sucrose (30 g / l), and agar powder (6.2 g / l).

[0042] In one embodiment, the cotyledon node is selected as the explant; and the treatment of the explant in step (2) does not require acupuncture treatment of the growing point.

[0043] In one embodiment, the treatment of the explant in step (2) comprises the following steps: retaining the entire hypocotyl, peeling off the seed coat, gently separating the two cotyledons, and removing the two true leaves.

[0044] In one embodiment, the infection method in step (3) is a conventional method; specifically, the explant is placed in an infection solution containing Agrobacterium for infection.

[0045] In one embodiment, the method of infection in step (3) is to evenly spread the Agrobacterium on a YEP plate and culture it in a 28°C incubator overnight; then wash the Agrobacterium with an infection solution and culture it in a shaking incubator at room temperature for 0.5-2 hours; then place the treated explants in the infection solution containing Agrobacterium for infection.

[0046] In one embodiment, the Agrobacterium is EH105.

[0047] In one embodiment, the infection solution includes: B5 culture medium, vitamin B1, vitamin B6, niacin, inositol, sucrose, MES (2-morpholineethanesulfonic acid), cysteine, dithiothreitol (DTT), acetosyringone (AS), and 6-benzylaminopurine (6-BA).

[0048] In one embodiment, the concentration of B5 culture medium in the infection solution is 0.321-3.21 g / l; preferably, 0.321 g / l.

[0049] In one embodiment, the concentration of vitamin B1 in the infection solution is 5-15 mg / l; preferably, 10 mg / l.

[0050] In one embodiment, the concentration of vitamin B6 in the infection solution is 0.5-1.5 mg / l; preferably, 1 mg / l.

[0051] In one embodiment, the niacin concentration in the infection solution is 0.5-1.5 mg / l; preferably, 1 mg / l.

[0052] In one embodiment, the inositol concentration in the infection solution is 5-15 mg / l; preferably, 15 mg / l.

[0053] In one embodiment, the sucrose concentration in the infection solution is 30 g / l.

[0054] In one embodiment, the concentration of MES (2-morpholineethanesulfonic acid) in the infection solution is 3.9 g / l.

[0055] In one embodiment, the cysteine ​​concentration in the infection solution is 0.15-0.5 g / l; preferably, 0.4 g / l.

[0056] In one embodiment, the concentration of dithiothreitol (DTT) in the infection solution is 0.15-0.5 mg / l; preferably, 0.2 mg / l.

[0057] In one embodiment, the concentration of acetosyringone (AS) in the infection solution is 20-40 mg / l; preferably, 30 mg / l.

[0058] In one embodiment, the concentration of 6-benzylaminopurine (6-BA) in the infection solution is 1-2 mg / l; preferably, 1 mg / l.

[0059] In one embodiment, the infection solution includes: B5 culture medium (0.321 g / l), vitamin B1 (10 mg / l), vitamin B6 (1 mg / l), niacin (1 mg / l), inositol (15 mg / l), sucrose (30 g / l), MES (3.9 g / l), cysteine ​​(0.4 g / l), dithiothreitol (0.2 mg / l), acetosyringone (30 mg / l), and 6-benzylaminopurine (1 mg / l).

[0060] In one embodiment, the infection in step (3) further comprises an ultrasonic treatment step.

[0061] In one embodiment, the co-cultivation in step (4) is a conventional method; specifically, the explants are inoculated onto the co-culture medium and cultured in the dark at 22° C. for 2-5 days (eg, 2 days, 3 days, 4 days, 5 days).

[0062] In one embodiment, the co-cultivation culture medium has the same components as the infection solution.

[0063] In one embodiment, the recovery culture in step (5) is a conventional method; specifically, the cotyledons after co-cultivation in step (4) are taken out, the excess hypocotyls are cut off at a distance of 3-5 mm from the cotyledons, and the cotyledons are obliquely inserted into the recovery culture medium and cultured under light at 26° C. for 2-5 days (for example, 2 days, 3 days, 4 days, 5 days).

[0064] In one embodiment, the recovery medium comprises: MS medium, vitamin B1, vitamin B6, niacin, inositol, asparagine, glutamine, sucrose, agar powder, and 6-furfurylaminopurine (KT).

[0065] In one embodiment, the concentration of MS medium in the recovery medium is 4.43 g / l.

[0066] In one embodiment, the concentration of vitamin B1 in the recovery medium is 5-15 mg / l; preferably, 10 mg / l.

[0067] In one embodiment, the concentration of vitamin B6 in the recovery medium is 0.5-1.5 mg / l; preferably, 1 mg / l.

[0068] In one embodiment, the niacin concentration in the recovery medium is 0.5-1.5 mg / l; preferably, 1 mg / l.

[0069] In one embodiment, the inositol concentration in the recovery medium is 5-15 mg / l; preferably, 15 mg / l.

[0070] In one embodiment, the asparagine concentration in the recovery medium is 50-100 mg / l, preferably, 100 mg / l.

[0071] In one embodiment, the glutamine concentration in the recovery medium is 50-100 mg / l, preferably, 100 mg / l.

[0072] In one embodiment, the sucrose concentration in the recovery medium is 30 g / l.

[0073] In one embodiment, the concentration of agar powder in the recovery medium is 6.2 g / l.

[0074] In one embodiment, the concentration of 6-furfurylaminopurine (KT) in the recovery medium is 0.1-1 mg / l; preferably, 1 mg / l.

[0075] In one embodiment, the recovery medium comprises: MS medium (4.43 g / l), vitamin B1 (10 mg / l), vitamin B6 (1 mg / l), niacin (1 mg / l), inositol (15 mg / l), asparagine (100 mg / l), glutamine (100 mg / l), sucrose (30 g / l), agar powder (6.2 g / l), and 6-furfurylaminopurine (1 mg / l).

[0076] In one embodiment, the method for screening and culturing in step (6) is to remove the cotyledons recovered from the step (5), cut off the hypocotyl at 3-5 mm of the ionic leaf node, insert them into the screening culture medium for screening and culturing for 10-18 days (preferably, 14-16 days; for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days), and when the screening and culturing lasts for 3-5 days (for example, 3 days, 4 days, 5 days), cut off the apical meristem region of about 0.5-1 cm, and culture at 26°C under light.

[0077] In one embodiment, the screening culture does not require subculture.

[0078] In one embodiment, the method for elongation culture in step (7) is to remove the cotyledons of the material screened and cultured in step (6) and transfer it into the elongation medium for culture under 26°C light for 15-30 days (preferably, 20-25 days; for example, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 day, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days).

[0079] In one embodiment, the elongation medium contains indoleacetic acid (IAA), indolebutyric acid (IBA), Timentin, spectinomycin, vitamin B1, vitamin B6, niacin, and inositol.

[0080] In one embodiment, the elongation medium comprises: MS medium, asparagine, glutamine, sucrose, agar powder, indoleacetic acid (IAA), indolebutyric acid (IBA), Timentin, spectinomycin, vitamin B1, vitamin B6, niacin, and inositol.

[0081] In one embodiment, the concentration of MS medium in the elongation medium is 4.43 g / l.

[0082] In one embodiment, the asparagine concentration in the elongation medium is 50-100 mg / l, preferably, 100 mg / l.

[0083] In one embodiment, the glutamine concentration in the elongation medium is 50-100 mg / l, preferably, 100 mg / l.

[0084] In one embodiment, the sucrose concentration in the elongation medium is 30 g / l.

[0085] In one embodiment, the concentration of agar powder in the elongation medium is 6.2 g / l.

[0086] In one embodiment, the concentration of indoleacetic acid (IAA) in the elongation medium is 0.2-1 mg / l; preferably, 0.5 mg / l.

[0087] In one embodiment, the concentration of indolebutyric acid (IBA) in the elongation medium is 0.2-1 mg / l; preferably, 0.5 mg / l.

[0088] In one embodiment, the concentration of Timentin in the elongation medium is 200 mg / l.

[0089] In one embodiment, the concentration of spectinomycin in the elongation medium is 5-24 mg / l; preferably, the concentration of spectinomycin in the elongation medium is 24 mg / l, 20 mg / l, 15 mg / l, 10 mg / l or 5 mg / l.

[0090] In one embodiment, the concentration of spectinomycin in the elongation medium is 24 mg / l; and the soybean is Handou 16, Qihuang 34 or Andou 5240.

[0091] In one embodiment, the concentration of spectinomycin in the elongation medium is 20 mg / l; and the soybean is Zhonghuang 302.

[0092] In one embodiment, the concentration of spectinomycin in the elongation medium is 15 mg / l; and the soybean is Guangda 103, Dongsheng 19, Gm06, Dongsheng 1, Heihe 43, or Donnong 252.

[0093] In one embodiment, the concentration of spectinomycin in the elongation medium is 10 mg / l; and the soybean is Henong 95 or Dongsheng 119.

[0094] In one embodiment, the concentration of spectinomycin in the elongation medium is 5 mg / l; and the soybean is Jiadou 30.

[0095] In one embodiment, the concentration of vitamin B1 in the elongation medium is 5-15 mg / l; preferably, 10 mg / l.

[0096] In one embodiment, the concentration of vitamin B6 in the elongation medium is 0.5-1.5 mg / l; preferably, 1 mg / l.

[0097] In one embodiment, the niacin concentration in the elongation medium is 0.5-1.5 mg / l; preferably, 1 mg / l.

[0098] In one embodiment, the inositol concentration in the elongation medium is 5-15 mg / l; preferably, 15 mg / l.

[0099] In one embodiment, the culture medium for the elongation culture includes: MS medium (4.43 g / l), asparagine (100 mg / l), glutamine (100 mg / l), sucrose (30 g / l), agar powder (6.2 g / l), indoleacetic acid (0.5 mg / l), indolebutyric acid (0.5 mg / l), timentin (200 mg / l), spectinomycin (5-24 mg / l), vitamin B1 (10 mg / l), vitamin B6 (1 mg / l), niacin (1 mg / l), and inositol (15 mg / l).

[0100] In one embodiment, the rooting in step (8) is a conventional method; specifically, the seedlings are cut after they grow to 3-4CM and placed in nutrient soil for rooting, and after rooting, they are moved to a climate chamber for cultivation under light at 26°C.

[0101] In one embodiment, the legumes include soybeans, peanuts, broad beans, peas, adzuki beans, mung beans, cowpeas, green beans, and lentils.

[0102] In one embodiment, the legume is soybean, cowpea or mung bean.

[0103] In one embodiment, the leguminous plant is soybean.

[0104] In one embodiment, the legume or soybean is Handou 16, Qihuang 34, Andou 5240, Zhonghuang 302, Guangda 103, Dongsheng 19, Gm06, Dongsheng 1, Heihe 43, Henong 95, Dongnong 252, Dongsheng 119 or Jiadou 30.

[0105] In one embodiment, the improvement in genetic transformation efficiency means that, compared with conventional genetic transformation methods (or compared with genetic transformation methods using other screening media), the genetic transformation efficiency of legumes is improved by the genetic transformation method described in the present invention (or the method of genetic transformation using the screening medium described in the present invention) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500%.

[0106] In one embodiment, using the genetic transformation method of the present invention (or the method for genetic transformation using the screening medium of the present invention), the genetic transformation efficiency of leguminous plants is not less than 1%, not less than 2%, not less than 3%, not less than 4%, not less than 5%, not less than 6%, not less than 7%, not less than 8%, not less than 9%, not less than 10%, not less than 13%, not less than 15%, not less than 18%, not less than 20%, not less than 22%, not less than 25%, or not less than 27%.

[0107] In another aspect, the present invention provides a method for preparing a genetically transformed legume plant, the method comprising the steps of:

[0108] (a) genetically transforming and rooting leguminous plant explants using the above method; and

[0109] (b) planting and culturing the rooted legume plant of step (a) into a plant body, thereby obtaining the genetically transformed plant.

[0110] In another aspect, the present invention provides a genetically transformed legume plant, wherein the legume plant is prepared by the above method.

[0111] General Definition

[0112] Unless defined in this application, the scientific terms or professional terms used in the present invention have the meanings understood by those skilled in the art. When the meanings understood by those skilled in the art conflict with the meanings defined in this application, the meanings defined in this application shall prevail.

[0113] The term "Agrobacterium" refers to a class of Gram-negative bacteria commonly found in the soil that live on the surface of plant roots and survive on nutrients that penetrate from the root tissue. There are two main types of Agrobacterium: Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens can chemotactically infect the injured parts of more than 140 dicotyledonous plants or gymnosperms under natural conditions and induce crown galls. Agrobacterium rhizogenes induces the production of hairy roots, which are characterized by a large number of highly branched root systems. There is a T-DNA on the Ti plasmid of Agrobacterium tumefaciens and the Ri plasmid of Agrobacterium rhizogenes. After Agrobacterium enters the cells by infecting plant wounds, it can insert the T-DNA into the plant genome. Therefore, Agrobacterium is a natural plant genetic transformation system. The target gene can be inserted into the modified T-DNA region, and the transfer and integration of exogenous genes into plant cells can be achieved by infection with Agrobacterium, and then genetically engineered or transgenic plants can be obtained through cell and tissue culture techniques. Among them, Agrobacterium tumefaciens can be divided into Agrobacterium tumefaciens with C58 gene background, Agrobacterium tumefaciens with Ach5 gene background, and so on. Agrobacterium tumefaciens with C58 gene background include EHA105, GV3101, C58C1, and AGL1.

[0114] The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), bok choy, yellow taro, melons (e.g., melon, watermelon, Crenshaw melon, honeydew melon, cantaloupe), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, pak choi), cardoon, carrot, napa, okra, onion, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, cabbage, cauliflower, zucchini ... onions, rutabagas, eggplant (also called eggplant), salsify, lettuce, shallots, endive, garlic, spinach, green onions, squash, greens, beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, horseradish, tomatoes, turnips, and spices; fruits and / or vines such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, y), cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomegranates, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, silver grass, corn / maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans) , oil plants (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut), Arabidopsis, fiber plants (cotton, flax, hemp, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plant; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, as well as trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0115] In the present invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue culture, plant callus, plant pieces, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0116] In the present invention, "plant cell" is understood to be any cell from or found in a plant, which is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, plant components, plants or seeds.

[0117] The main advantages of the present invention are:

[0118] The invention provides a method for improving the genetic transformation efficiency of legume plants. The method comprises the step of genetically transforming the legume plants using the screening culture medium. The method has the advantages of short transformation cycle, high positive rate, low dependence on genotype, and controllable early flowering phenomenon. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 .Images of soybean at different stages of genetic transformation.

[0120] Figure 2 .The growth status of Heihe 43 after 15-30 days of elongation culture in different screening media. DETAILED DESCRIPTION

[0121] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0122] The following experimental content is combined with embodiment examples to further explain the present invention.All methods and operations described in these embodiments are provided by way of example, and they should not be construed as restrictive.The method for the operation of relevant DNA can refer to Current Protocols in Molecular Biology, the 1st and 2 volumes, Ausubel FM Greene Publishing Associates and Wiley Interscience, 1989, Molecular Cloning, T.Maniatis et al., 1982, or Sambrook J. and Russell D., 2001, Molecular Cloning: alaboratory manual, version 3.

[0123] Example 1. Genetic transformation method of soybean

[0124] 1. Preparation of Agrobacterium infection solution

[0125] (1)Bacteria coated plate

[0126] The Agrobacterium strain EH105 was selected. The Agrobacterium was taken out from -80°C, 200ul of bacterial solution was sucked up with a pipette tip, and evenly spread on a YEP (kanamycin 50 mg / L, rifampicin 20 mg / L) plate, sealed with tape and placed in a 28°C incubator for overnight culture.

[0127] (2) Washing plate

[0128] Take 10ml of infection solution and add it to the bacterial plate. Use a 1000ul pipette to blow back and forth until all the plaques are washed off. Then transfer it to a 50ml centrifuge tube and culture it in a shaking incubator at room temperature for 0.5-2h. Adjust OD600 to 0.5 before infection.

[0129] Infectious solution ingredients:

[0130] B5 medium (phytotech, product number G398, 0.321 g / l) + vitamin B1 (10 mg / l) + vitamin B6 (1 mg / l) + niacin (1 mg / l) + inositol (15 mg / l) + sucrose (30 g / l) + MES (2-morpholineethanesulfonic acid, 3.9 g / l) + cysteine ​​(0.4 g / l) + dithiothreitol (DTT, 0.2 mg / l) + acetosyringone (AS, 30 mg / l) + 6-benzylaminopurine (6-BA, 1 mg / l).

[0131] 2. Explant Preparation and Infection

[0132] (1) Chlorine disinfection

[0133] Open the lid of the disposable petri dish containing soybean seeds and place it in a sealed desiccator. Place a 250ml glass beaker containing 150ml sodium hypochlorite solution in the middle of the desiccator, then slowly add 10ml of concentrated hydrochloric acid along the wall of the beaker to gradually react and produce chlorine. Cover the lid of the desiccator tightly and seal it with vaseline, place it in a fume hood, and set the sterilization time to 8-16h. After the chlorine sterilization is completed, place it in a 4℃ refrigerator for later use.

[0134] (2) Germination

[0135] Inoculate on germination medium and germinate in the dark at 22°C for 16 hours.

[0136] Composition of germination medium: B5 medium (phytotech, product number G398, 3.21 g / l) + sucrose (30 g / l) + agar powder (6.2 g / l).

[0137] (3) Explant treatment

[0138] In conventional explant processing, one cotyledon and two true leaves need to be removed, and 0.5 cm of the hypocotyl needs to be retained. The growth point needs to be pierced 2-3 times with the tip of a knife (stabbing the growth point). The effect of the piercing will affect the later germination rate.

[0139] In the present invention, an optimized explant (cotyledon node) processing method is adopted without puncturing the growth point. The specific method is: retain the entire hypocotyl, peel off the seed coat, gently separate the two cotyledons, remove the two true leaves, and then put them into 25 ml of infection solution without Agrobacterium for use.

[0140] (4) Ultrasonic treatment

[0141] Pour out the infection solution, add it into the infection solution containing Agrobacterium culture solution, treat it with ultrasound for 30 seconds, and then pour out the infection solution.

[0142] (5) Dyeing

[0143] Pour the prepared Agrobacterium solution (OD600 = 0.2-1.0) into the cut beans, infect at 100r-110r horizontally for 0.5-3h, vacuum for 5min, and then take out to dry.

[0144] 3. Co-cultivation

[0145] The explants were plated on co-culture medium and cultured at 22°C in the dark for 2-5 days.

[0146] The composition of the co-culture medium is the same as that of the infection solution.

[0147] 4. Resume training

[0148] After co-cultivation, the cotyledons were removed, the excess hypocotyls were cut off at a distance of 3-5 mm from the cotyledons, and the cotyledons were inserted obliquely into the recovery medium for 2-5 days. The culture was carried out at 26°C under light.

[0149] Recovery medium composition:

[0150] MS medium (phytotech, product number M524, 4.43 g / l) + vitamin B1 (10 mg / l) + vitamin B6 (1 mg / l) + niacin (1 mg / l) + inositol (15 mg / l) + asparagine (100 mg / l) + glutamine (100 mg / l) + sucrose (30 g / l) + agar powder (6.2 g / l) + 6-furfurylaminopurine (KT, 1 mg / l).

[0151] 5. Screening and cultivation

[0152] The cotyledons that have been restored to culture were taken out, and the hypocotyls were cut off at 3-5 mm from the ion leaf node. Then, the culture medium was inserted and the screening was carried out for 10-18 days, with 10 explants per dish. When the screening lasted for 3-5 days, the apical meristem area of ​​about 0.5-1 cm was cut off, and the culture was carried out at 26°C under light.

[0153] There is no need for subculture during the screening stage, and only one screening culture medium is sufficient.

[0154] The novel screening culture medium (experimental group) used in the present invention comprises the following components:

[0155] MS medium (phytotech, product number M524, 4.43g / l) + vitamin B1 (10mg / l) + vitamin B6 (1mg / l) + niacin (1mg / l) + inositol (15mg / l) + asparagine (100mg / l) + glutamine (100mg / l) + sucrose (30g / l) + agar powder (6.2g / l) + 6-benzylaminopurine (6-BA, 1mg / l) + 6-furfurylaminopurine (KT, 0.5mg / l) + indoleacetic acid (IAA, 0.1mg / l) + Timentin (200mg / l) + spectinomycin (5-24mg / l, too high concentration of spectinomycin will cause problems of whitening and over-screening).

[0156] Among them, the optimum concentration of spectinomycin is different for different soybean varieties. The optimum concentration of Handou 16, Qihuang 34, and Andu 5240 is 24 mg / l; the optimum concentration of Zhonghuang 302 is 20 mg / l; the optimum concentration of Guangda 103, Dongsheng 19, Gm06, Dongsheng 1, Heihe 43, and Donnong 252 is 15 mg / l; the optimum concentration of Henong 95 and Dongsheng 119 is 10 mg / l; the optimum concentration of Jiadou 30 is 5 mg / l.

[0157] The components of the control group screening medium (conventional screening medium) are:

[0158] B5 medium (phytotech, product number G398, 3.1 g / l) + vitamin B1 (10 mg / l) + vitamin B6 (1 mg / l) + niacin (1 mg / l) + inositol (100 mg / l) + MES (2-morpholineethanesulfonic acid, 0.98 g / l) + asparagine (50 mg / l) + glutamine (50 mg / l) + sucrose (30 g / l) + agar powder (6.2 g / l) + 6-benzylaminopurine (6-BA, 1 mg / l) + Timentin (200 mg / l) + spectinomycin (5-24 mg / l).

[0159] Compared with conventional screening culture medium, the novel screening culture medium used in the present invention has different hormone types and proportions. Generally, only 6-benzylaminopurine is used as hormone, while the hormones used in the novel screening culture medium include 6-benzylaminopurine, 6-furfurylaminopurine and indoleacetic acid.

[0160] 6. Elongation training

[0161] After the screening and culture is completed, the cotyledons are removed and the materials are transferred to the elongation medium. After 15-30 days, all the resistant seedlings basically elongate at once. The green elongated resistant buds are basically positive seedlings, and the non-positive seedlings are mostly white or cannot elongate normally. Culture at 26℃ light.

[0162] There is no need for subculture during the elongation stage, and elongation can be achieved in one go, significantly shortening the transformation cycle.

[0163] Elongation medium composition:

[0164] MS medium (phytotech, product number M524, 4.43g / l) + vitamin B1 (10mg / l) + vitamin B6 (1mg / l) + niacin (1mg / l) + inositol (15mg / l) + asparagine (100mg / l) + glutamine (100mg / l) + sucrose (30g / l) + agar powder (6.2g / l) + indoleacetic acid (IAA, 0.5mg / l) + indolebutyric acid (IBA, 0.5mg / l) + Timentin (Timentin, 200mg / l) + spectinomycin (5-24mg / l).

[0165] Among them, the optimum concentration of spectinomycin is different for different soybean varieties. The optimum concentration of Handou 16, Qihuang 34, and Andu 5240 is 24 mg / l; the optimum concentration of Zhonghuang 302 is 20 mg / l; the optimum concentration of Guangda 103, Dongsheng 19, Gm06, Dongsheng 1, Heihe 43, and Donnong 252 is 15 mg / l; the optimum concentration of Henong 95 and Dongsheng 119 is 10 mg / l; the optimum concentration of Jiadou 30 is 5 mg / l.

[0166] 7. Rooting

[0167] When the seedlings grow to 3-4CM, cut them and place them in nutrient soil to take root. After taking root, move them to the climate room and culture them under light at 26℃.

[0168] 8. Detection

[0169] After the seedlings are planted and survive, samples are taken and sent for sequencing, and the transformation is completed.

[0170] Soybean in different stages of genetic transformation Figure 1 shown.

[0171] 9. Conclusion

[0172] The transformation data of multiple soybean varieties were completed using this system, as shown in the following table (the control group is soybeans genetically transformed using conventional screening culture media; the experimental group is soybeans genetically transformed using new screening culture media. Except for the different screening culture media, the other steps of the control group and the experimental group are exactly the same):

[0173]

[0174] In addition, soybean Heihe 43 was genetically transformed using the above method, and it was found that after 15-30 days of elongation culture in the control group (conventional screening medium), the positive seedlings were difficult to elongate quickly and had poor growth potential. Figure 2 As shown in A; after 15-30 days of elongation culture in the experimental group (new screening culture), the positive seedlings can elongate rapidly and grow well, as shown in Figure 2 As shown in B.

[0175] The above method was used to complete the genetic transformation of 13 soybean varieties. The results showed that this method had a high positive rate, with an average positive rate of more than 74% (the positive rate of conventional soybean genetic transformation methods was about 10%), as shown in the following table:

[0176] Soybean varieties Number of explants Number of positive seedlings / number of resistant seedlings Positive rate Transformation efficiency Handan Bean 16 126 34 / 48 71％ 27％ Qi Huang 34 135 30 / 42 71％ 22％ Andou5240 97 13 / 20 65％ 13％ Zhonghuang 302 116 21 / 27 78％ 18％ Guangda 103 400 72 / 85 85％ 18％ Dongsheng19 450 45 / 61 74％ 10％ Gm06 813 122 / 186 66％ 15％ Dongsheng 1 1162 93 / 131 71％ 8％ Black River 43 638 51 / 65 78％ 8％ Henong95 260 13 / 18 72％ 5％ Dongnong 252 171 12 / 16 75％ 7％ Dongsheng 119 267 16 / 20 80％ 6％ Jiadou 30 240 12 / 14 86％ 5％

[0177] By using the method described in the present invention to carry out genetic transformation of soybean, transgenic or gene-edited plants can be obtained more stably and efficiently, and for different soybean varieties (Handou 16, Qihuang 34, Andou 5240, Zhonghuang 302, Guangda 103, Dongsheng 19, Gm06, Dongsheng 1, Heihe 43, Henong 95, Donnong 252, Dongsheng 119 or Jiadou 30), higher genetic transformation efficiency can be obtained (including the difficult-to-transform variety Heihe 43), genotype dependence is low, and the transformation efficiency of different varieties is between 5-27%; the positive rate is high, averaging 74%; no subculture is required in the screening and elongation stages, so that the transformation cycle is shortened from 3-4 months to 1.5-2 months; due to the short transformation cycle, the early flowering phenomenon of early flowering varieties can be reduced.

[0178] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A screening medium for genetic transformation of leguminous plants, or a screening medium for improving the genetic transformation efficiency of leguminous plants, characterized in that: The screening culture medium contains 6-benzylaminopurine, 6-furfurylaminopurine, indoleacetic acid and timentin.

2. The screening medium according to claim 1, characterized in that The screening medium includes MS medium (4.43 g / l), asparagine (100 mg / l), glutamine (100 mg / l), sucrose (30 g / l), agar powder (6.2 g / l), 6-benzylaminopurine (1 mg / l), timentin (200 mg / l), vitamin B1 (10 mg / l), vitamin B6 (1 mg / l), nicotinic acid (1 mg / l), inositol (15 mg / l), 6-furfurylaminopurine (0.5 mg / l), indoleacetic acid (0.1 mg / l) and spectinomycin (5-24 mg / l).

3. Use of the screening medium according to any one of claims 1 to 2 in the genetic transformation of leguminous plants, or in improving the genetic transformation efficiency of leguminous plants.

4. A method for genetic transformation of leguminous plants, or a method for improving the genetic transformation efficiency of leguminous plants, characterized in that: The method comprises the step of genetically transforming a leguminous plant using the screening medium according to any one of claims 1-2.

5. The method according to claim 4, characterized in that The method comprises the following steps: (1) Seed disinfection and germination; (2) explant treatment; (3) Infection; (4) co-cultivation; (5) Recovery culture; (6) Screening and culture; (7) elongation culture; (8) Rooting; The method comprises the step of screening and culturing leguminous plants using the screening culture medium described in any one of claims 1-2.

6. The method according to claim 5, characterized in that The cotyledon node is selected as the explant; the treatment of the explant in step (2) does not require acupuncture treatment of the growing point.

7. The method according to claim 5, characterized in that The method for screening and culturing in step (6) is as follows: taking out the cotyledons recovered and cultured in step (5), cutting off the hypocotyls at 3-5 mm from the ion leaf node, inserting the cotyledons into the screening culture medium for screening and culturing for 10-18 days, cutting off the apical meristem region of about 0.5-1 cm after 3-5 days of screening and culturing, and culturing at 26° C. under light; Preferably, the screening culture in step (6) does not require subculture.

8. The method according to claim 5, characterized in that The method of elongation culture in step (7) is to remove the cotyledons of the material screened and cultured in step (6) and transfer it to the elongation culture medium for culturing at 26° C. under light for 15-30 days; Preferably, the elongation culture in step (7) does not require subculture.

9. The method according to claim 5, characterized in that The culture medium for the elongation culture in step (7) contains MS culture medium, asparagine, glutamine, sucrose, agar powder, indoleacetic acid, indolebutyric acid, timentin, spectinomycin, vitamin B1, vitamin B6, niacin, and inositol.

10. The method according to any one of claims 4 to 9, wherein the leguminous plant is soybean, cowpea or mung bean.

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