Search

CN-121992030-A - Method for improving genetic transformation efficiency of soybean based on glyphosate screening system

CN121992030ACN 121992030 ACN121992030 ACN 121992030ACN-121992030-A

Abstract

The invention discloses a method for improving soybean genetic transformation efficiency based on a glyphosate screening system, which is characterized in that aromatic amino acid with proper concentration is added in an elongation stage for the first time to obviously improve the soybean genetic transformation efficiency taking glyphosate as a screening mark, and the transformation efficiency is improved by more than 8%. Meanwhile, compared with a transformation technical system which uses glufosinate as a screening marker, the non-chimeric proportion and the positive rate proportion are improved by more than 50%.

Inventors

  • ZHANG XIANWEN
  • LUO BIAO

Assignees

  • 浙江省农业科学院

Dates

Publication Date
20260508
Application Date
20260330

Claims (9)

  1. 1. A method for improving soybean genetic transformation efficiency based on a glyphosate screening system is characterized in that in the soybean genetic transformation process mediated by agrobacterium with glyphosate as a screening agent, aromatic amino acids are added into a bud elongation culture medium in a clustered bud elongation culture stage, so that the soybean genetic transformation efficiency is improved, wherein the aromatic amino acids comprise tryptophan, phenylalanine and tyrosine, and the addition final concentration is 300-600 mu mol/L respectively.
  2. 2. The method of claim 1, wherein the aromatic amino acid consists of 400 to 600 μmol/L tryptophan, 300 to 500 μmol/L phenylalanine, 300 to 500 μmol/L tyrosine, and the concentration is the final concentration added to the shoot elongation medium.
  3. 3. The method of claim 2, wherein the aromatic amino acid consists of 500 μmol/L tryptophan, 400 μmol/L phenylalanine, 400 μmol/L tyrosine.
  4. 4. The method of claim 1, wherein the soybeans comprise granular soybeans and fresh soybeans.
  5. 5. The method of claim 4, wherein the variety of the granular soybeans includes but is not limited to Tianlong No. 1, weilan 82 and Zhongbean 43, and the variety of the fresh soybeans includes but is not limited to Zhejiang No. 8, huning 95-1 and Nannong 30.
  6. 6. The method of claim 1, wherein the final bud elongation medium concentration comprises MS basal salt mixture 4.33 g/L, 2-morpholinoethanesulfonic acid 1.0 g/L, sucrose 30 g/L, ferric salt solution 3.5 mL/L, L-glutamine 75 mg/L, L-aspartic acid 75 mg/L, B5 vitamin 1 mL/L, zeatin riboside 1 mg/L, timetin 400 mg/L, gibberellin 0.2 mg/L, indoleacetic acid 0.1 mg/L, and glyphosate 10 mg/L,7.5 g/L agar, deionized water as solvent, pH 5.6, and ferric salt solution 13g/L EDTA-FeNa aqueous solution.
  7. 7. The method of claim 1, wherein the soybean genetic transformation method comprises using a glyphosate-tolerant gene as a selectable marker gene and using glyphosate as a selectable agent.
  8. 8. The method of claim 1, wherein the glyphosate-tolerant gene is selected from one of OsmEPSPS genes, GAT genes, GOX genes, G10 genes, CP4 genes.
  9. 9. The method of claim 1, wherein the genetic transformation of soybean is performed as follows: (1) Sterilizing by selecting healthy, full and mature soybean, placing in a dryer filled with chlorine gas, and sterilizing for 16 hr; (2) Germinating, namely sowing sterilized soybeans in a super clean workbench, and culturing for 1 day at 25 ℃, wherein the soybean sprouts with hypocotyls removed are cut into five or five pieces in length, so that two pieces of explants are provided with cotyledons and epicotyls, and the explants are cut at 3-5 positions at the joints of the cotyledons and the epicotyls, wherein the final concentration of the germination culture medium comprises 3.21 g/L of B5 basal culture medium, 30 g/L of sucrose and 3.2 g/L of plant gel, and the solvent is deionized water with pH of 5.8; (3) Immersing the prepared explant in agrobacterium tumefaciens bacteria liquid containing target genes for co-culture at 28 ℃ for 10-30 minutes, absorbing excessive bacteria liquid on the explant with absorbent paper, transferring the bacteria liquid to a CCM (continuous culture) medium for dark culture at 22 ℃ for 3-5 days, wherein the final concentration of the CCM medium comprises 3.21 g/L of B5 basal medium, 2.5 g/L of 2-morpholinoethanesulfonic acid, 20 g/L of sucrose, 10 g/L, B5 vitamin 1 mL/L of glucose, 80mg/L of acetosyringone, 300 mg/L of dithiothreitol and deionized water as a solvent with the pH of 5.4; (4) Transferring the explant cultured in the step (3) into a recovery medium, culturing for 7 days at 26 ℃ with 16 h/8: 8 h light per day, wherein the final concentration of the recovery medium comprises 3.21: 3.21 g/L of B5 basal medium, 1.0: 1.0 g/L of 2-morpholinoethanesulfonic acid, 30: 30 g/L of sucrose, 4: 4 mL/L, L-glutamine 125 mg/L, L-aspartic acid 125: 125 mg/L, B5 vitamin 1:1 mL/L, 6.8: 6.8 g/L of agar, 2:2 mg/L of zeatin, 400mg/L of termet, deionized water as solvent and pH 5.6, and the ferric salt solution is an aqueous solution of EDTA-FeNa of 13 g/L; (5) Transferring the explant subjected to co-culture in the step (4) into a screening culture medium containing 20 mg/L glyphosate, culturing at 26 ℃ for 21 weeks under illumination, wherein the culture medium is replaced every two weeks every day with 16 h/8/h of dark light, the final concentration of the screening culture medium comprises 3.21/g/L of B5 basal medium, 1.0 g/L of 2-morpholinoethanesulfonic acid, 30 g/L of sucrose, 4 mL/L, L-glutamine 125 mg/L, L-aspartic acid 125 mg/L, B5 vitamin 1 mL/L, 6.8 g/L of agar, 6-benzyl adenine 1 mg/L, 400mg/L of timentin and 20 mg/L of glyphosate, the solvent is deionized water, the pH is 5.6, and the ferric salt solution is an aqueous solution of EDTA-FeNa of 13 g/L; (6) Elongating, namely transferring the embryo tissues screened in the step (5) into a bud elongation culture medium added with aromatic amino acid, and culturing at 26 ℃ until the embryo tissues grow into seedlings; (7) Transplanting, transplanting directly into aseptic nutrient soil, moisturizing, culturing at 26 deg.C and 80% humidity under 16 h/h g/g for 1 month, washing to remove agar, and transplanting in greenhouse.

Description

Method for improving genetic transformation efficiency of soybean based on glyphosate screening system Field of the art The invention belongs to the field of plant genetic transformation, and relates to a method for improving soybean genetic transformation efficiency based on a glyphosate screening system. (II) background art Soybean is a global important crop with both oil and protein, and the stress resistance, quality, yield and other characters of the soybean are improved by genetic transformation technology, so that the soybean is one of the core directions of modern agricultural biotechnology research. The current soybean genetic transformation mainstream technology mainly adopts an agrobacterium-mediated cotyledonary node/hypocotyl transformation system, but the technology system still has the key problems of low transformation efficiency, long regeneration period, insufficient elongation of resistant buds and the like, and severely restricts the industrialized application of soybean gene editing and transgenic breeding. The glyphosate is used as a most commonly used screening agent in soybean genetic transformation (depending on the glyphosate-tolerant EPSPS gene as a screening marker), and the action mechanism of the glyphosate is to inhibit the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in a shikimate pathway of plants so as to further block endogenous synthesis of tryptophan, phenylalanine and tyrosine three aromatic amino acids. In actual transformation operation, although the glyphosate in the screening stage can effectively eliminate untransformed cells, the untransformed cells can be damaged in a sublethal way, even if the transformed cells carry glyphosate-tolerant genes, the shikimic acid pathway of the transformed cells is slightly inhibited by the glyphosate, so that the synthesis efficiency of aromatic amino acids is reduced. The aromatic amino acid is an essential nutrient for soybean cell division and bud elongation, and is also a synthesis precursor of key metabolites such as auxin (IAA, tryptophan is taken as a direct precursor), lignin, phenol antioxidant substances and the like. In the bud elongation stage after the screening stage, the transformation buds are deficient in endogenous aromatic amino acid, so that the problems of low elongation rate, high browning rate, low survival rate and the like are frequently caused, which become core bottlenecks for restricting soybean genetic transformation from 'resistance screening' to 'regenerated seedling obtaining', and the problem is not solved pertinently in the prior art system, so that the soybean transformation efficiency is generally lower than 10 percent and is far lower than that of crops such as corn, rice and the like. In the existing soybean genetic transformation technical scheme, optimization aiming at an elongation stage is mainly focused on aspects of hormone proportion (such as auxin/cytokinin proportion adjustment), culture medium salt concentration optimization, culture environment (illumination/temperature) regulation and the like, and research is not focused on the specificity problem of lack of aromatic amino acids after glyphosate screening, wherein (1) a conventional elongation culture medium only contains basic nitrogen sources (such as nitrate nitrogen and ammonium nitrogen) and cannot directly supplement aromatic amino acids which cannot be quickly synthesized by plants, so that the inhibition effect of glyphosate on shikimic acid paths is difficult to alleviate, (2) part of research is carried out on completely removing the glyphosate in the elongation stage to reduce injury, but the untransformed cells are easy to "escape" and reduce the purity of resistant buds, and (3) few researches are carried out blindly to add amino acid mixtures, so that the variety and concentration of the aromatic amino acids are not optimized for soybean transformation characteristics, or the concentration is too low to have no effect, or the amino acid toxicity is generated due to the too high concentration, and the bud elongation is inhibited. Therefore, there is a need to develop a soybean genetic transformation optimization technology suitable for a glyphosate screening system, which can make up for the shortage of endogenous synthesis by precisely adding aromatic amino acid with specific concentration in the elongation stage, and simultaneously consider the continuous screening effect of glyphosate, and finally improve the elongation efficiency and survival rate of resistant buds, so as to construct a high-efficiency and stable soybean genetic transformation technology system. (III) summary of the invention The invention aims to provide a method for improving soybean genetic transformation efficiency based on a glyphosate screening system, which is based on a soybean genetic transformation technology of the glyphosate screening system, and aims to make up for the inhibition effect of glyphosate on shikimic a