Optimizaton The Technology Of Clean DNA Transformation To Cultivate Safety Transgenic Rice Plant

Whole plasmids are used in traditional genetic transformation. However, a general problem arises subsequently that vector backbone sequences are undesirably integrated into the plant genome along with transgene. This tends to promote transgene re-arrangement within the host genome, resulting in the silence and incomplete expression of transgene in progenies.The undesirable vector backbone sequences may not only promote transgene rearrangements and affect transgene or endogenous gene expression negatively, but have disadvantage on the safe assessment of the transformants as"desert DNA". The direct DNA transforming systems can transfer minimal gene expression cassettes (promoter,open reading frame,terminator) into plant genome and generate"safer" transgenic plants.The bar gene expression cassettes without plasmid vector backbone sequences was introduced to the Japonica rice variety "Nipponbare" and the Indica rice variety "Minghui63". Using the marker-free transformation system to study some factors that affecting the transforming efficiency of liner gene expression cassettes to rice varieties by particle bombardment.The results show:(1) The effect on clean DNA transformation frequency of rice calli derived from different inducing conditions on28℃in light,28℃in dark,32℃in light and32℃in dark. The results show that32℃light-induced calli as a transformation of receptor material, to get the highest conversion efficiency, up9.32%; To the first culture, the first subculture, the second subculture, as the receptor material, to syudy the effect on clean DNA transformation frequency of rice calli from different stages.The results showe that after one more subculture, the calli used as the injured tissue for bombardment, it can be gained the highest conversion rate; In the same culture conditions, the rice "Nipponbare" and rice "Minghui63" for calli induction, calli derived from a subculture of for biolistic transformation of the receptor material, the results show that, during Clean DNA transformation, the conversion rate of rice was significantly higher than rice.(2) To select spermidine and protamine as the DNA precipitant, to study the effect on clean DNA transformation frequency of different DNA precipitant.The results show that when the DNA concentration is2ug per six shots, compared with spermidine, protamine group last rate of positive plants and conversion rate are increasing very significantly. In the case of the same concentration, protamine as a precipitant, the conversion rates is higher than spermine as the precipitant.To select2.5ng per shot,25ng per shot, and2ug per six shots as the different DNA concentration to study the effect on clean DNA transformation frequency of different DNA concentration. The results show that when spermidine as the precipitating agent,2.5ng per shot as the DNA concentration, rate of positive plants and conversion rate than2ug per six shots group are significantly reduced. Protamine as a precipitant,2.5ng per shot as the DNA concentration, the final rate of differentiation, rate of positive plants and conversion rate than plants2ug per six shots group are very significantly reduced.25ng per shot as the DNA concentration, the final rate of differentiation than plants2ug per six shots group is significantly reduced. These show that when the protamine as precipitant, on different concentrations, the differentiation rate, conversion rate and the rate of positive plants all have a certain impact.Use the same DNA precipitation agent, in the concentration range of2.5ng per shot,25ng per shot and2ug per six shots,the2ug per six shots can be resulted the highest conversion rate, reaching9.64%, and in the three concentration range, the conversion rate as the DNA concentration increases. In addition, Southern bloting results show that different concentrations of DNA used for clean DNA transformation, and finally obtained the copy number of transgenic plants vary. Also, the use of low concentrations of DNA for bombardment, are all given a simple low-copy integrated transgenic plants.