| Maize is the second most important crop of China.. According to the statistic, there is 6,589,800 acres of corn attacked by Chilo partellus, which leads to loss of 7-8%,even 15-20%,of the yield per year. My study aims to gain transgenic maize plants that can resist Chilo partellus. As a result, I got a lot of regenerate plants. Results of PCR,PCR-Southern, Southern blotting and Dot blotting assays of these plants proved that some of them carried such foreign insecticidal genes as modified cowpea trypsin inhibitor(SCK) gene and modified Bacillus thuringiensis(Bt) gene. We had gained Tl seeds of these transgenic plants.Microprojectile bombardment is usually the preferable method in transformation of maize, for it is often referred to as an efficient method. Though, it is expensive and can only transform a little material once. Though relatively inefficient on maize, Agrobacterium tumefaciens will make up for its shortcomings. According to many studies in this field, Agrobacterium tumefaciens has great potentialities. In my study, to establish an efficient experimental system, I transformed maize by Agrobacterium tumefaciens, which paved the way of applying this method to maize transformation successfully.In my experiment, I studied the technological details concerning embryonic callus inducement, transformation operation, selection, regeneration and transplanting.1. Embryonic callus inducementThe rate and quality of embryonic callus varied with genotype. Generally speaking, 152X141 was better than 18(Red). By increasing the osmotic pressure of medium, mannitol prevented the callus cells from absorbing too much water so as to improve its quality. To reduce the browning rate of embryos, it was viable to better the culture conditions and increase the 2,4-D concentration. In callus subculture, increase in 2,4-D content kept the callus from differentiation, though, high concentration of 2,4-D did harm to the callus.2. TransformationSuch factors affected the transformation efficiency as Agrobacterium tumefaciens culture conditions, manners of subculture, involvement of As and antibiotics. The first period of Agrobacterium tumefaciens culture should be no1 longer than 8-10 hours unless medium and oxygen were ample. As for subculture manners, centrifuging to collect the culture and resuspend it in fresh medium was better than transferring it to fresh medium directly because the former avoided pollution of antibiotics to fresh medium. During the first period of Agrobacterium tumefaciens culture, antibiotics should be added to the medium after 2 hours of culture while As should be added to the medium immediately before immersion of callus during the second period and coculture medium also contained As. 7-8 days of preculture of callus before transformation activated the callus cells, which was good for infection. And treating the material on high osmotic pressure medium for several hours also increased its sensitivity. Selecting the transformed calli after a certain period of recovery on antibiotic-free medium produced more resistant calli than selecting immediately after transformation.3. Selection and regenerationIn selection, the concentration of HygB should be kept under the deadly level lest that it had damaged the differentiation potentialities of transformed cells. And the HygBconcentration should be low at the beginning so that it didn' kill the non-transformed cells which could nurse the then weak transformed ones. In differentiation medium, high concentration of HygB aggravated the browning of callus. The reaction to Kinetin of different genotypes varied. To 18(Red) ,the proper concentration was 0.5mg/mL while it was 1mg/mL as far as 152X141 was concerned. High concentration of Kinetin led to deformity and weakness of regenerate plants. Transplanting of young plants was critical to the whole experiment. It was proved that, to transplanted individuals, natural condition was better than greenhouse. Though, to protect the plants from heavy rain, high temperature, intense sunlight, co...
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