The Preliminary Study Of Kenaf Efficient Regeneration System Optimization And Double Insectresistant Genes Transformation

Kenaf (Hibiscus Cannabinus L.) of Malvaceae family is annual bast fiber crop which is an important raw material in linen and paper industry. Kenaf of herbicide-resistant and insect-resistance can not only reduce the production cost,but also improve the output and quality.This test studied the effect factors of callus induction and adventitious buds differentiation of kenaf, optimized the efficient regeneration systemof kenaf.Mature efficientregeneration system can shorten the breeding time of Kenaf quality variety,and laid thefoundation for transgenosis molecular breeding of kenaf. In the test of genetic transformation, through the research of double insect-resistant genes transformed kenaf cotyledons, the exogenous insect-resistant genes were integrated in the genomic DNA of kenaf, and laid the foundation for the future study of kenaf transgenic breeding. The results are as follows.The results of the kenaf optimization of efficient regeneration system:(1) In the callus induced process, anthers can induce the best callus,hypocotyls were worse, cotyledons were the worst.The best induction medium of anther was:MS+2,4-D (2.0mg/L)+KT(1.0mg/L)+NAA(1.0mg/L);(2) The best induction medium for callus of otyledons and hypocotyls was MS+6-BA (3mg/L)+NAA(1mg/L); the best differentiation mediumwas MS+6-BA(lmg/L)+2,4-d (1mg/L)+GA3(1mg/L);(3) Added 0.3% activated carbon in the callus differentiation medium, can induce a few adventitious buds; but at the same time produced a large amounts of roots;(4) In the process of directly induce the adventitious buds, hypocotyl almost can’t induction adventitious buds, cotyledon was better than hypocotyl; added AgNO3 in the induction medium could improve the induction of adventitious buds. The best induction medium for adventitious buds was MS+TDZ(0.1mg/L)+NAA(0.1mg/L)+AgNO3(1mg/L);(5) Among the three Kenaf varieties of Fu Hung992, Fu Hung952and the Fu Hung hangl, the highst induction rate was Fu Hung 992, the second was Fu hong hang 1 and the worst was Fu Hung 952;(6) A lot of mutants existed in regeneration plants of the tissue culture of for there are Mutant stems and the anthocyanins and crown etc changed, but these mutations is beneficial mutations is worth doing further research.Tth results of agrobacterium mediated double insect-resistant genes transformed Fu hong 992:(1)The original saved plasmid transformed the competence EcoliDH5a, then extr acted plasmid to enzyme cut confirmation, the result showed that the original saved plasmid carrying the double insects-resistance gene Bt and pta;(2) Extract plasmid transformed agrobacterium competence EHA105,extracted plasmid then through specific primers PCR confirmated that the plasmid in the EHA105 carrying the double insects-resistance gene Bt and pta;(3) Through the critical concentration screening of herbicide(Basta) resistance to kenaf,got the lethal concentration of Basta to kenaf varieties Fu Hung 992 was 7mg/ L, this was the filtering concentration of Basta used in the filter culture;(4) Resistant adventitious buds were got through cotyledons selecttion culture of Fu Hung 992 after agrobacterium infection;Extracted the genome DNA of resistant adventitious buds, and used the double insect-resistant genes to specific primer PCR, got 680 bp of Bt and 804 bp fragment of pta;(5)Transformed 98 cotyledons of Fu Hung 992, got 12 resistance buds; lof the 12 resistance buds was positive plant, the transformtion rate of cotyledons was 1.02%.