Genetic Transformation Of Populus Deltoides (I-63×I-69) Transferred By BtCry1A

Bacillus thuringiensis (Bt) is a very important entomopathogen which can produce specifically toxic crystal proteins against many agriculturally and biomedically detrimental pests. Bt(or endotoxin Bt-toxin) gene is a gene that is wildly used for controlling the pests in the world. In 2005, the areas of transgenic crops have increased to 2630 ha. Poplars are seriously attacked by insects in many areas and insect-resistant poplars are expected in forest production. Bt gene has been successfully transformed into Populus alba, Populus nigra, Populus tomentosa, POpulus simonii, Populus deltoides. However, it is little known about the plantation of transgenic insect-resistant poplars to date. Further researching of transgenic poplars with Bt gene is fundamental for poplars insect-resistant breeding. In the present studies, the modified BtCrylA gene was transformed to the explants of Populus deltoides (I-63×I-69) mediated by Agrobacterium tumefaciens. PCR amplification of the chromosomal DNA demonstrated that the BtCrylA gene was detected in the transformed plants. ELISA, SDS-PAGE and MTT analysis indicated that the insecticidal protein was efficiently expressed in P. deltoides (I-63×I-69). The anti-insect experiments showed that the transformed poplar plants obtained evidently the resistance to the larvae of Clostera anachoreta (Fabricius). These results suggested that transgenic plants would offer a new way for protecting the poplars against attacking of insect pests. The following are results:Water-cultured method was used in this experiment, the optimal induction medium is MS + 1.0 mg/L BA + 0.1 mg/L 2,4-D + 30g/L sucrose + 6.5g/L agar. Among the callus culture of bud differentiation, the differentiation rate can reach 71.1% when cultured on the medium of MS + 0.5mg/L BA + 0.1mg/L 2,4-D + 30g/L sucrose + 6.5g/L agar. The best medium for buds differentiation of the leave directly is (1/2N)MS + 0.2mg/L BA + 0.02mg/L NAA + 25g/L sucrose +7g/L agar, with the bud differentiation rate of 85%. KT has the stimulation to the plants rooting of P. deltoides (I-63×I-69), the better rooting medium is MS + 0.05mg/L KT + 0.02 mg/L NAA + 6-7 g/L agar + 30g/L sucrose, with the rooting rate of 82.2%.Several antibiotics were used to eliminate A .tumefaciens LBA4404 during plant transformation. Their effects on inhibition of A. tumefaciens LBA4404 and bud differentiation of leaf from P. deltoides (I-63×I-69) were analyzed. Proliferation of A. tumefaciens LBA4404 was completely suppressed in the medium containing 150-200mg/L cefotaxime with the buds differentiation rate of 73.3%. Analyzing from many angles, the lowest limit concentration of kanamycin used for selection of transformation seedings was 30mg/L when taking buds as acceptor; at rooting stage, lower limit concentration was 15mg/L. The experiment also proved that streptomycin(str) has the same effect as NAA on the bud regeneration. The combination of 10 mg/L str and 0.2 mg/L BA with the differentiation rate of 71.1%, and 20mg/L str+0.02 mg/L NAA+0.2 mg/L BA with the differentiation rate of 100%.Several factors affecting BtCry1A gene transformation of P. deltoides (â… -63×Ⅰ-69) mediated by A. tumefaciens were studied, and a simple and effective protocol with optimized condition for transformation of P. deltoides (â… -63×Ⅰ-69) was established. The results demonstrated that the highest frequency of the transformation was 28.9% under these conditions: the 12 h of pre-eulture time, explants infected by A. tumefaciens (OD₆₀₀=0.3～0.4), co-culture time(4d), acetosyringone (150μmol/L) was added during co-culture and selection was delayed after 10 days of co-culture. Through successive selection in buds growth and root induction stage at high concentration of kanamycin presence we obtained 17 kanamycin-msistance rooted plants.PCR analysis showed that 6 of these 17 kanamycin-resistant rooted plants were BtCry1A gene positive. These 6 PCR positive plants were further confirmed by ELISA and SDS-PAGE analysis. Our results indicated that BtCry1A gene was efficiently expressed and the quantity of Bt insecticidal protein is about 3.88～2.36ppb, MTr analysis showed that the protein of PCR positive plant named X1 was strikingly insecticidal to cultured Trichoplusia ni Hubner cells in vitro. Bioassay results that X1 was significantly resistant to feeding by first larvae of Clostera anachoreta(Fabricins), compared with the untranaformed control plant. The significant decrease of leaf consumption by larvae, a slow weight gain of larvae and a higher larvae mortality rate of Clostera anachoreta (Fabricius) were observed.