| Bacillur thuringienszs c7yL4 gene was introduced into soybean successfi~lly with Agrobactenwncotyledonaiy node transformation system. Cotyledonaiy node explants were prepared from 5-7 day old seedlings, infected and co-cultivated with Agmbacteriwn twnefaci ens. Resistant adventitious buds emerged after 4 weeks on selective medium. The buds were transferred onto elongation medium and regenerated shoots grew to 2.5-3 cm high after 4-6 weeks. The shoots were cut off and transferred onto rooting medium . After about 3 weeks, tie rooted plantletc were hardened off and transferred into pots. All the plants could flower and set seeds nomially. Among the 8 transplanted survival T0 plants, seven gave positive PCR reaction; and 4 of the 7 T1 plant lines had PCR positive plants. DNA was extracted from the leaves of positive plants in the 4 T1 plant lines, and southern blot was conducted with probe of .Bt gene. All the 4 lines are positive, indicating that the Bt gene has integrated into the receiver genome and can be passed to progenies. The leaves from transgenic plants could produce calhis on medium containing kanamycin; the leaves arid stern slices from transgenic plants could be dyed with X-Gluc; and two transgenic plant lines showed obvious resistance to flax budworms. These results indicate that the transgenes, iq* II 5gw and Bt all be expressed in the transgetuc plants.The factors influencing A~vbactei?an-mediated soybean tramfonnation were studied ~ith the sections of soybean hypocotyl from 5-day-old seedlings as explants and the resistant callus as talget The results show:1.The sensitivity of soybean cultivars to Agyt&&~ter1wn twnefaciens is commonly low and there is significant difference between genotypes. fl~ average reaction frequency is only 4.0%.2.There is certain difference between tie semitivity of soybean cultivara to selective agents. Among the 4 tested agents, the inhibiting effect of PPT is the best and stable between cukivws; and hygromycin is the secorxL Die effective concentration of kanamycin is the highest The suitable concentrations of the 4 agents we kanamycin 100?00 mgl? G418 50-75 mgl? hygromycin 20-30 mg1~ arid PFr 5 mgl?3.The duration of infection and co-culture with Agmbacteriwn influences soybean transformation evidently. The suitable infection duration is about 8 minutes; and the appropriate co-culture duration is 2-4 days. There s~n to be certain aws action between infection dur~ionand co-culture duration. The best combination is infection 8 minutes and co-culture 3 days under the experimental conditions.4. Both the concentration of acetosyringone and pH value at stages of infection and co-culture influence the transformation frequency of soybean. The suitable pH value is between 5.4 and 5.6. When it exceeds 5.8, the transformation frequency decreases obviously. Acetosyringone (50?200 jiM) can increase the transfonnation frequency of soybean. But it is inadvisable to use it with too high concentration. Overhigh concentration (400 jtM) decreases the transformation frequency instead. The optimum concentration is 1 0(4iM.5.Precuiture of explants on h~ertonic medium before infection of Agrobacteriwn can raisethe transformation frequency of the sections of soybean hypocotyl. The optimum duration of preculture is one day.With pollen tube pathway transformation system of soybean, about 5000 buds were treated and more than 4500 seeds were obtained. Four PCR positive T~ plants were screened out with the special primers of fit gene. PCR-Southem analysis was conducted with the 4 positive T1 plants. The PCR-pmducts of both the 4 plants and plasmid bad hybridization signals while the negative ck bad not This indicates that the PCR result is reliable. Two T2 plant lines had PCR positive plants which had week gus expression and no clear resistance to flax budwoims. One of the two lines segregated in 3/1 ratio and the other had 1/4 albinos, 2/4 positive and 1/4 negative plants.Compared with Agrobacteriwn-mediated transformation, the pollen tube pathway transfor...
|
PDF Full Text Request