Establishment Of Soybean Genetic Transformation System And Study On Bt Gene Transfer With Agrobacterium-mediated Method

In this research, ten varieties of soybean were chosen and the cotyIedonny nodes ofthese varieties were used to be the targat explantS of Bt gene trgnsformation. The factorsaffecting the Agrobacterium-mediated transformation and the shoot regeneration werestudied. The resistut plrt were obtained from the two steps selection. Four positive plantswas obtained. Southern analysis show that Bt gene was integrated into soybean's genome.The main resuItS of this research as folIows:l. There were lnye differences of regeneration frequency between soybean genotypes.Regeneration frequency was betWeen 0-65.04% in l0 soybean genotypes that were used inthis research. The results in this research has shown that regeneration frequency was high ofHeinong 35, Hefeng 35 and Hefeng 25, which were 65.04%, 64.02 and 60.82% respectively.So the 3 genetypes were chosen to be the research materials in this study.2. The concentraion of BA and IBA was designed as an altemation changing at thedifferentiation stage of the adventitious shoot from cotyledonary node. The suitableproportion betWeen BA and IBA fOr different genotyPes was sceened. For Heinong 35, whenthe concentraon of BA was l.50mg/I., and the concentraion of IBA was 0.20mg/L, thedifferentiation frequence is the highest with 78.57%. For Hefeng 25, when the concentraionof BA was l.30mg/L, and the concentraion of IBA was 0.25mg/L, the differentiationfrequency is the highest 48.57%. With the suitable proPOrtion, the shoots of each explantswere 7 tO l0, not over l0, which is benefit for elongation. FOr Hefeng 35, when theconcentration of BA was l.20mg/L, and the concetaion of IBA was 0.25mg/L, thedifferentiation frequency is the highest with 74.07 %. T'he shootS of each explants were 4 to9.3. The frequency of root regeneraion was remarkably increased by the addition of lBA atthe root regeneration stage. The basic medium fOr root regenefation was l/2 MSB, and theconcentraion of sugar was 2%. When the concentraion of IBA was 2.0mg/L, the rootregeneration frequency fOr Heinong35 and Hefeng 25 were higher with 7l .88 % and 72.4l %resPectively. When the concentheion of IBA was l.5mg/L, the root tegeneration frequencyfor Hefeng 35 were higher with 68.97 %.4. The selectfon genes of Bt were NPTII, and the selective regeant was Kan. The lateselection was used tO overcome the difficulty of adventitious shoo differentiation and theroot regeneration. When the shootS were 0.3-Icm, and before being transferred to rootregeneration medium, the shoOts were screened with Kan, and the suitable concefltration ofKan for Heinong35, Hefeng35 and Hefeng25 was l00mg/L.5. The Cef was used as amibiotic. When the concentration of Cef between 0 and700mg/L, it didn't influence the differentiation frequency of the adventitious shoots frorn22002^11^cotyledoary 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 .6. GUS transient expression were used to determine the suitable time of pre-culture, infection , and co-culture . When the Heinong35 , Hefeng35 . and Hefeng25 were precultured forabout 24 hours, and the infection time were 20min, 25min and 20min respectively, and co-culture time were 70 hours, 80 hours and TOhours, GUS transient expression were the highest with 25.00 % , 2 1 .80 % , 23.81 % . So for different genotypes , the suitable proportion for pre-culture, infection, co-culture was different.7. From this experiment the kanamycin resistant plants with Bt gene were tested by PCR. Among 4 positive plants with Bt gene, 2 were from Heinong35, 1 was from Hefeng25 , and 1 wasfrom Hefeng35, the transformation frequency was 0.52%. Southern analysis show that Bt gene was integrated into soybean's genome.