Study On Transferring Insecticide Protein Gene From Bacillus Thuringensis Into Maize Callus Via Agrobacterium-mediation

Maize(Zea mays L.) is the important source of both grain and forage crop and morden food and chemical industry. It has also many kinds of hereditary characteristics. Reseach fellows pay close attention on to the study on genetic transformation of maize.Now ,A number of techniques are currently in use for genetic modification of plants. These include Agrobacterium-mediated transformation, polyethyleneglycol-mediated transformation, pollen tube pathway method, microinjection, protoplast and intact cell technology, and micro particle bombardment technology. Plant transformation mediated by Agrobacterium tumefaciens has become the most common method for the indtroduction of forfeign DNA into plant cells. Agrobacterium tumefaciens also has been used extensively for gentic engineering in maize, but to date, genetic transformation of maize is still seriously limited by genotypes, only a few of maize inbred line were reported, example All8 and B73; and tranformation frequency of maize is still low relative to that obtained in other plant. A successful transformation of maize using Agrobacterium relies on several factors including the frequency of regeneration in transgenic maize cells, maize genotypes, Agrobacterium strain, bacterium infection and transformation competency of the target tissue. At an attempt to improve transformation efficiency, present experiment was conducted to compare the effect of several factors on transformation of maize callus via Agrobacterium-mediation.In experiment, factors influencing the callus induction of immature embryos in maize inbred line 7922, H99, P2, Mol7, 吉 846, 吉 63 , etc. such as different media, different concentration of phytohormone, different carbon source and their concentration, were studied. The result shows that NB is optimal media of callus induction on immature embryos in different maize inbred line; concentration of 2,4-D 2mg/l can obviously enhance inducing frequency; concentration of sucrose 20g/l and 20g/l glucose or 30g/l sucrose and 10g/1 glucose is the better conditions of callus induction. Since the T-DNA transfer process is mediated by the cooperative action of proteins encoded by genes in the Ti plasmid Vir genes and the bacterial chromosome. Vir genes inducer such as AS, AS-OH, etc. can enhance Vir genes expression and increase the transformation rate, experiment result indicated that 200ug/l AS has the optimalIVeffect on maize transformation, but result also indicated that AgNO3 can not clearlyincrease induction frequency.The GUS gene has a higher expression rate when maize callus were pre-cultivated 3days before infected by Agrobacterium. It shows 3 days per-cultivation time issuitable for maize callus transformation.During different Agrobacterium growth phase, its infecting ablity is different. WhenAgrobacterium grow reach log phase (a density of OD600nm-0.5), it has stronginfecting ablity; we recommend infecting time for 25-30 min is better in maize callustransformation.In experiment, the embrogenic callus of inbred line 7922, P2, H99 were selected onMs medium contraining different concentration of PPT ,the result shows that 6mg/lPPT is the most suitable selected concentration; and we found that maize callus weremore suitable to regeneration plant on Ms medium containing 6-BA lmg/1 and KT2mg/l.It was proved by PCR analysis that the target Bt. gene had been integrated into thegenome of regenerated plants.Rate of PCR transformation was 5.9%.