Study On Transfer Of Chitinase Gene Into Wheat

Plasmid DNA, containing bean chitinase gene, was delivered into spring wheat NE7742 via particle bombardment, ilgrobacteriurn tumefaciens, and pollen tube pathway. The expression and inheritance of transgenic plants were analyzed, and the disease resistance of the transformed progeny was tested. Viewpoint has been put forward on the factors influencing gene transfer; also the question occurred in the study of transgenic wheat. The result was summarized as follows:1.The Plasmid DNA carrying bean chitinase genes was transferred into 8139 germinating immature embryos by using PDS1000/He high velocity micro projectile bombardment. Seven transgenie plants derived from NE7742 T0have been obtained and confirmed by PCR and PCR桽outhern hybridization. The transgenic plants cane from 4 groups in different bombard parameter combination. The transfer frequency was 0. 56 %, 0. 36%, 0. 20%, and 0. 35% respectively. One thousand and five hundred forty six NE7742 immature embryo calli were treated with .4grobacterium tuz,efaciensediate method and method of Biolistic working in combination with ,4grobacterium tumefaciens. The transferring frequency was 0. 46% and 0. 32%, respectively, but no regenerated plant was obtained from transgenic calli. Two transgenic plants were obtained from 385 NE7742 florets by pollen tube pathway operation. The transferring frequency was 0. 52%. The author suggests that different way should be taken from each of the three transferring methods used in the studies according to the potential of plant regeneration in genotypes used as acceptors. Biolistic and ilgrobacterium turaefaciens is suitable for genotypes with high frequency potential of plant regeneration cultured in vitro, and pollen tube pathway for those in low frequency.2.Immature embryo used as acceptor in gene transfer is better than that of young spike because of quickly callus formation and good callus quality. The frequency of plant regeneration in calli from immature embryo of the three genotypes used in the study was quite different. It is 7. 84% in Lio條0, 13. 68% in 918569, and 54. 88% in NE7742. The study indicated that geneticin (G418) showed significant selection effect because of the natural resistance to kanamycin inwheat. The sensitivity of three genotypes to geneticin showed different. Liao?0 ranked the first, 91B569 next, and NE7742 the last. The optimum concentration of G4l8 used in resistant calli selection was 25 mg/L, 3Omg/L, and 35mg/L for Liao條O, 91B569, and NE7742, respectively. Besides, the effect of bombard factor on the distribution and density of gold particle was found, and distance effect is more significant than that of pressure. Under the condition both of 6 cm and 9cm in distance of bombardment, the inside circle gained more gold particle than outside, and the different was markedly. However, well distribution of gold particle was found in 12cm distance.3.Analysis of T1 from transgenic plants T0 98?, 98?, and 98? self梒rossing progenies showed the segregation ratio between the transformed and non?transformed plants were 1:2.3, 1:4, and 1:4, respectively. This non桵endelian inheritance segregation phenomenon may be caused by non梥ingle transformed cell or other reason unknown yet. Aminoglucose method was used in the study to determine the chitinase activity in leaf of transgenic plants 98T~ and CK. The result showed that the endo梒hitinase activity of 98?, 98?, and 98? was 29. 78, 34.69, and 30.09 activity units, respectively, and 14.81 in CK. It meant the endo梒hitinase activity in transgenic plant was about two times that of CK. Conclusion could be reached that bean chit inase gene under the control of promotor 35S has been expressed in transgenic plants obtained in the study, but in lower level of expression. The test of transgenie and non梩ransgenic plants to powder mildew and scab infection was carried out in green house to evaluate the diseases resistance. No significant difference in powder mildew resistance between transgenic plants and CK was found. As to wheat scab, the infect ion...