| Rapeseed (Brassica napus) is a major important source of vegetable oil and protein. It is also the most important oil crop in China. About 7,400,000 hectares of rapeseed have been annually grown in China since 2000. Stem rot (Sclerotinia sclerotiorum) is a major disease on rapeseed, and has become a major limiting factor on rapeseed production. The genetic engineering of rapeseed for disease resistance becomes an important issue. Synergistic action of plant defense genes appears to be an effective avenue in anti-fungal genetic engineering of plant.The studies were carried out on the methods of Agrobacterium-mediated transformation in Brassica napus by using isolated microspores and cotyledonary petioles of po/-CMS hybrid parents (restorers and maintainers) as receptor. Resistance to Sclerotinia sclerotiorum was studied. The main results are as follows:1. Factors that affect the embryogenesis were studied using isolated microspore of po/-CMS hybrid parents as receptor by Agrobacferium-mediated method. The Agrobacterium-mediated transformation system of isolated microspores in Brassica napus with binary genes was established. Ten transgenic haploid plants of the restorer were obtained. Nine plants of them were successfully doubled by colchicine treatment and T0 self-pollinated seeds were collected.2. By screening and optimizing such factors that may affect transformation efficiency as Agrobacterium suspension medium composition, concentration of bacterium used for infection, duration of infection and cocultivation, temperature of cocultivation with Agrobacterium, acetosyringone, medium pH and so on, a high-efficient Agrobacferium-mediated genetic transformation system of po/-CMS hybrid parents in Brassica napus with binary genes was established. The transformation efficiency of restorers and maintainers reached 8.8%, 5.3%, respectively.3. The transformation method was improved with 5-7 d delay in kanamycin selection, exposure of regenerated green shoots to a higher kanamycin concentration in rooting medium containing low sucrose so as to eliminate non-transformants. The transformation efficiency was raised significantly.4. A total of 109 independent transgenic plants were obtained. Among them, 66 plants were regenerated from restorers, i.e. 6, 41, 9, 10 plants from peng 34-1, peng40-1, 93275, 14786, respectively; 43 plants were regenerated from maintainers, i.e. 9, 23, 11 plants from 95B, 96B,98B, respectively.5. Integration of the introduced transgene was examed by PCR analysis, PCR-Southern blot and segregation of the kanamycin-resistance trait in the progeny.6. Disease resistance test and changes in activities of chitinase and β -1, 3-glucanase of the T, transgenic lines indicated that most of the foreign genes expressed. In some plants the resistance was significantly improved, while in a few plants the resistance was similar to CK. In field identification of resistance to S. Sclerotiorum, disease incidence and disease index of transgenic line Peng 40-12 were on average 3.38% and 1.34, respectively, which were 78.7%, 85.2% and77.4%, 83.5%, respectively, lower than those of the control non-transgenic line Peng40 and Zhongyou 821.7. The Kanamycin spraying method can be used for large-scale screening of positive plants, segregation investigation and acquirement of homozygous transgenic line in open field.
|
PDF Full Text Request