Researches On The Genetic Transformation Of BADH Gene To Watermelon(Citrullus Lanatus)

Citrullus lanatus is a popular cucurbitaceous fruit and come from Africa.The narrower genetic background,so traditional breeding is difficult of improving same characters.Genetic engineering technology offers a new way for improve new oriented varieties.Establishment of high efficient regeneration system is the basis of improve germplasm resources using Genetic engineering technology.At present,there is no a systematic deep-going theoretic study of genetic transformation system.The results of references are different from each other even just opposite and short of repeated manipulation because of different varieties,different methods,cultural conditions and different personal experiences.Since the situation,it is necessary to do basic research to the watermelon regeneration system.95%of the watermelon component is water.It needs millions of stere water in Beijing to plant watermelon per year.The conflict between short of water and high requirement of it becomes more and more sharp and obvious.Since then,it is urgent to transform the drought-tolerant gene.Betaine is an important osmoregulation substance,which can increase plant stress resistance.Since the expression of betaine is very easy and convenient,betaine synthetase gene is considered to be one of the most important and the most effective resistant gene of drought stress.This experiment tries to select high efficient regeneration genotype to optimize regeneration system.Optimize watermelon genetic transformation system on this basis and transfer BADH gene into the plant to get new varieties of drought-resistant.The results are as follows:1.Get 5 high efficient regeneration genotypes and optimize the in vitro regeneration system of watermelon cotyledonThrough the comparative analysis of 23 pairs of genotypes,choose five high efficient regeneration genotypes and the highest induction rate is 90%.Cut out for peak green watermelon cotyledons,put them on the culture medium MS+3.0mg/LBA to induce buds,and then transfer the buds to the culture medium MS+0.2mg/LKT.When the buds are about 2cm,transfer them to the culture medium MS+0.5mg/LIAA to induce roots.After roots being strong the young seeding is cultivated on the sterilized soil that is soaped with 1/2MS firstly and then plant the seeding after new roots appearing.2.The agrobacterium tumifaciens transformation system has been establishedThrough the comparative test of the same factors that influence conversion,the optimum transformation conditions are cutting out for transformation materials and cultivating on MS+3.0mg/LBA for 1～2 days' pre-culture.Centrifugation of bacterial liquid with OD₆₀₀=0.4 which get the second activation,then adding the same volume of MS with 200μmol/L acetosyringone and pre-culture leaves.After ten minutes the leaves which remove liquid cultivate in MS+3.0mg/LBA+200μmol/L acetosyringone for 3～4 days.After co-culture,making leaves oscillate 30 minutes on 1/2MS+300mg/LCef.Then transfer leaves into differentiation culture which is MS+3.0mg/LBA+100mg/LKm+300mg/LCb.And take the induced buds into multiplication culture,MS+0.2mg/LKT+75mg/LKm+100mg/LCb.Rooting medium is 1/2MS+0.5mg/LIAA +30mg/LKm+50mg/Lcef.3.The identification of watermelon transferred with betaine aldehyde dehydrogenase geneOn the transformation systems of optimization watermelon's cotyledon,transferred with betaine aldehyde dehydrogenase gene,after this disposal we get 0.8%induction rate of positive-resistant bud, and get 15 elongate buds and four buds are positive by PCR.