Establishment Of The Receptor System For Soybean Cotyledonary Node And Study For The Transformation With Agrobacterium-mediated Method

Soybean is one of the crops whose tusformaton is difficult. It is because that tissueregeneration of sOybeq repetition of exPeriment is not ideal, and the resultS amonggenoboes are very differenL and the toformation system is not good enough.In Ans experiment the cotyldomp node regeneration system was used, and threegenotwe with high regeneration frequency were screened, and the major factorsinfluencing the regeneration system and transfOrmation system for agrobarterium-mediated method were oPtimized. We etallished a high-efficiency receptor system Btand chi were introduced into soybean, and gained the positive plantS determined by PCR.The main resultS were showed as following fl .' Screening of the soybean genotypesl2 varieties from Heilongiiang were screened at the sa!ne conclusion. Theregeneration frequence is very different. Among them, the regeneration frequences ofHeinong 35, Hefeng 25, Hefeng 35 were higher with 64.02%, 65.04%, and 60.82%respectively. So they were used as the receptor material.2. Screening of the hormonc concentration(1 ) The concentraion of BA and mA was designed as an altemation changing at the'differentiation stage of the adventitious shoot from cotyledonny node. Thesuitable proportion betWeen BA and IBA for different genotwe was sceened. ForHeinong 35, when the concentalon of BA was l .50mg/L, and the concentraionof IBA was 0.20mg/L, the differentiation frequence is the highest with 78.57%.For Hefeng 25, when the concentraion of BA was l.30mg/L, and theconcentraion of IBA was 0.25mg/L, the differentiation frequence is the higheStwith 48.57O/. Wth the suitable proportion, the shoots of each exPlantS were 7 tol0, not over 10, which is benefit for elongation. For Hefeng 35, when theconcentraion of BA was l .20mglL, and the concentraion of IBA was 0.25mglL,the differentiation frequence is the highest with 74.07%. The shootS of eachexPlantS were 4 to 9.(2) The frequence of rOO regeneration was remarkably increased by the addihon ofIBA at the root regenndion Stag. The basic medium for root regeneration waslQ MSB, and the concentheion of sugar was 2%. W'hen the concentraion of IBAwas 2.0mg/L, the root regeneration frequencies fOr Heinong 35 and Hefeng 25were higher with 7l.88% and 72.41% respectiveIy. When the concentraion ofIBA was l.5mg/L, the root regeneration frequencies for Hefeng 35 were higherwith 68.97%.Illm  2002^ 53. Factors influencing the transformation effiency of Agrobaterium-mediated method( 1 ) The selection genes of Bt and chi were both npt II, and the selective regeant was Kan. The late selection was used to overcome the difficulty of adventitious shoot differentiation and the root regeneration. When the shoots were 0.3- IcM, and before being transferred to root regeneration medium, the shoots were screened with Kan, and the suitable concentration of Kan for Heinong 35, Hefeng 35, and Hefeng 25 was 125mg/L.(2) The Cef was used as antibiotic. When the concentration of Cef between 0 and 700mg/L, it didn't influence the differentiation frequence of the adventitious shoot from Cotyledonary node. When the concentration of Cef was 500mg/L, the growth of Agrobactium was suppressed. So the suitable concentration of Cef were selected with 500mg/L.( 3 ) GUS transient expression were used to determine the suitable time of pre-culture, infection, and co-culture. When the Heinong 35, Hefeng 35, and Hefeng 25 were precultured for about 24 hours, and the infection time were 20min, 25min, 20min respectively, and the co-culture time were 70 hours, 80 hours, 70 hours, the GUS transient repression were the highest with 25.00%, 21.80%, 23.81%. So for different genotypes, the suitable proportion for pre-culture, infection, co-culture was different.4. PCR testFrom this experiment, the positive plants with Bt gene and chi gene were tested by PCR. Among 5 positive plants with Bt gene, 2 were from Heinong 35, 2 were from Hefeng 25, a...