A Preliminary Study On The Regeneration System Of Soybean Petiole In Vitro And The Study Of GmBZL2* Gene Transferred Form Embryo Tip System

Soybeans(Glycin max(Linn)Merr)also known as yellow beans and called"Shu" in ancient times,is a dicotyledonous leguminous soybean genus diploid(2n =40)annual herbaceous plants.Soybeans are both important high-protein grain and feed crops and oil crops,in addition to high quality raw materials for feed and food industrial processing of finished products,but also functional genome research model plants.With the increasing reduction of cultivated land,the damage of pathogens and pests,environmental stress and difficult to cross pollination and other issues highlighted,the market urgently need to call for new varieties of soybean with high yield,stable production,high quality.However,traditional breeding techniques and methods have encountered technical bottlenecks in the creation of new soybean germplasm.With the completion of the whole genome sequencing of soybean and the vigorous development of gene editing technology,genetic transformation technology provides a quick and effective way for the creation of new soybean germplasm resources.Therefore,the construction of soybean genetic transformation system is necessary.At present,the use of Agrobacterium tumefaciens-mediated method to soybean embryo and cotyledon as the donor system,the soybean genetic engineering research reports are more common.However,both transgenic systems have the disadvantages of high false positive rate,chimeric existence and low conversion efficiency.It can be seen that exploring the new soybean genetic transformation system is imminent.Reliable genetic transformation systems must rely on efficient in vitro regeneration systems.In order to overcome the false positive phenomenon of genetic transformation caused by budding,our lab carried out the research on the soybean petiole regeneration in order to construct a new soybean transgenic system.BZR1 is a key transcription factor in BR signaling pathway of Arabidopsis thaliana,which can increase the number of konjac seeds.Its homologous in soybean is GmBZL2,which may regulate the seed number of soybean pods.The GmBZL2*gene is obtained by cloning the GmBZL2 gene and subjecting the gene to a protein spot mutation(bzr1-1D protein).Subsequent BZR1 transformation into Arabidopsis showed that the number of seeds per pod was increased.Therefore,this study validates that this gene is expected to be transferred into soybeans through the embryo tip transformation system and efficiently expressed in soybean to increase the number of seeds of single fruit pods and realize the new germplasm of soybean yield.In this study,we used soybean varieties with Jiaoda green beans and "K06-82"to successfully explore the in vitro regeneration of soybean leaf petals,and it was suggested that regenerated plants could be obtained by using petiole as explants.The results show that:petiole explants can be obtained by 15 d enlargement of embryos stripped from sterile seed and placed in MS B5 medium added with 3 mg/L 6-BA;3 mg/L and 3.5 mg/L NAA treatment for 45 d can make the differentiation rate of Jiaoda green bean and "K06-82" callus reached 89.2%and 88.9%;The optimum medium for the differentiation of Jiaoda green beans was 2 mg/L 6-BA and 0.2 mg/L NAA,“K06-82”was 1 mg/L 6-BA and 0.1 mg/L NAA,and the differentiation efficiency was 58.3%and 52.6%;Added 2 mg/L TDZ can make the green beans of Jiaotong University adventitious buds increased 6.3%,and 1.5 mg/L TDZ to"K06-82" adventitiousshoots incidence increased by 2.3%,and 2 mg/L AgNO3 can improve the green beans of Jiaotong University and K06-82 adventitious bud differentiation rate of.The differentiation process needed about 30 days;The addition of 1 mg mg/L GA3 can get more and normal elongated seedlings in about 15 days;The rooting medium was 1/2MS B5,and the addition of 0.8 mg/L IBA could lead to the rooting efficiency of 86.67%after 14 days.The establishment of soybean petiole regeneration system provides a new donor regeneration system for future genetic transformation of soybean in this laboratory.In the study of GmBZL2*gene transfermation in soybeans,we used Tianlong No.1 embryos tip for transgenic study.The results showed that the sterile soybean embryo was preincubated for 1 day or 2 day,and the bacteria culture liquid medium was shaken to OD600 = 0.8.The tansfection solution and co-cultured medium were treated with 300 μmol/L medium were treated with 300 mg/L and under dark condition for 2 days,300 mg/L cephalosporin was added for 2 days,and then transferred to 2 mg/L Hyg B and 300 mg/L cephalosporin for 10 days;buds were induced by 3 mg/L Hyg B and 200 mg/L cephalosporin for 10 days and then transferred to 4 mg/L Hyg B and 100 mg/L cephalosporin buds for 5 days,and finally resistant buds could be obtained by transferred to 3 mg/L Hyg B and 100 mg/L cephalosporin for 5 days;then buds were incubated with 5 mg/L Hyg B and 50 mg/L cephalosporin for elongation.Finally,the rooting rate and the average number of adventitious roots of resistant buds reached 67.35%and 2.65,respectively,on 1/2MS B5 medium containing 5 mg/L Hyg B,50 mg/L Cef and 0.5mg/L IBA.The GUS staining and PCR results showed that 20 positive strains of soybean seedlings were identified from 25 rooting seedlings.The positive rate was 80%.