Studies On Transferring Leafy And Hs1～(pro-1) Into Soybean

In the field of plant genetic transformation, soybean is one of the crops whose transformaton is difficult. It is because that tissue regeneration of soybean, repetition of experiment is not ideal, and the results among genotypes are very different, and the transformation system entirely depends on the soybean genotype.In this experiment, the cotyldonary node regeneration system was used, and one genotype with high regeneration frequency was screened from 10 different Fujian geo-ecological soybean genotypes, and the major factors influencing the regeneration system and transformation system for agrobacterium-mediated method were optimized. We established a high-effciency Soybean Cotyledonary node receptor system. LFY and Hs1^pro-1 were introduced into soybean, and gained the positive plants determined by PCR and Southern blot. The main results were showed as follows:1．Screening of the soybean genotypes and basic culture mediums10 varieties from Fujian were screened on MS and B5 respectively. The regeneration frequence is same between two different culture medium. Among the varieties, the regenetion of Shanglyp was higher with 70%. So it was used as the receptor material.2．Screening of the hormone concentration(l)The concentration of BA and IBA was designed as an alternation changing at the differentiation stage of the adventitious shoot from cotyledonary node. The suitable proportion between BA and IBA was screened. When the concentration of BA was 1．4mg/L, and the concentration of IBA was 0．2mg/L, the differentiation frequence is 78．95%.(2) The frequence of root regeneration was remarkably increaded by the addition of IBA at the root regeneration stage. The basic medium for root regeneration was 1/2MS, and the concentration of sugar was 2%. When the concentration of IBA was 2mg/L, the concentration of BA was 0．2mg/L, the root regeneration frequencies for Shanghlyp was 75．56%.3．Screening of the factors influencing the transformation effiency of Agrobaterium-mediated method(l)The selection genes of LEAFY and Hs1^pro-1 in the were both NPTⅡ,and the selective regeant was Kan. The shoots were transferred to the screening medium before the root regeneration. The suitable concentration of Kan is 150 mg/L.(2) Timentin was used as antibiotic. When the concentration of Timentin between 0 and 250 mg/L, it didn't influence the differentiation frequence of the adventitious shoot from Cotyledonary node. When the concentration of Timentin in the liquid medium was 75 mg/L and in the solid medium was 200mg/L, the growth of Agrobactium was suppressed. So the suitable concentration of Timentin in the solid medium was selected with 200 mg/L.(3) GUS transient expression was used to determine the suitable time of infection and co-culture. When Shanghlyp was infected 15 min, and the co-culture time was 80 hours, the GUS transient expression was the highest with 23．5%. So the suitable proportion for infection and co-culture was 15 min and 80h.4．PCR test of the positive plantsFrom this experiment, the positive plants with LEAFY gene and Hs1^pro-1 gene were tested by PCR. There were 2 positive plants with LEAFY gene, and 3 positive plants with Hs1 gene. The transformation frequence of gene were 0．76% and 1．11% respectively.5．Southern blot hydridisation of the positive plantsThe positive plants were tested by Southern blot hydridisation with the probe of LEAFY gene and Hs1^pro-1 gene. The modification plants tested primarily ingrated the foreign gene.