Study On The Tissue Culture And Genetic Transformation Of Populus Alba×P.berolinensis

Populus alba×P.berolinensis is one of excellent tree species for fast-growing and high-yield forest and is cultivated large area in north China.Moreover,it is good for paper pulp.However, The get rid of lignin will improve paper cost and bring great air pollution.The conventional breeding program can not cultivate new Populus alba×P.berolinensis species which has low lignin content in short time because of long tree growth cycle.Recently,genetic transformation by Agrobacterium-mediated was widely used for cultivating new species.The establishment of high efficiency adventitious shoot regeneration system was the key step for genetic transformation by Agrobacterium-mediated.So in the present study,we first established high efficiency tissue culture system for Populus alba×P.berolinensis,Then anti-sense 4CL gene from Amorpha fruticosa was transferred into Populus alba×P.berolinensis to decrease lignin content in its body.The main result were as follows:1.MS medium supplemented with 2%(w/v) sucrose,8%agar,0.1mg/lTDZ and 0.02mg/l NAA produced the highest shoot regenerating rate,100%from leaf explants.The average number of regenerated shoots was 12 per explant.2.The optimum medium shoot regeneration from stem explants was MS medium supplemented with 0.1mg/l TDZ,2%(w/v) sucrose and agar 8g/l.Shoot regeneration rate was 70.3%with 4.6 shoots per explant.3.The optimum medium for rooting of the regenerated plantlets was 1/4MS medium supplemented with 0.5mg/l IBA,2%(w/v) sucrose and agar 8.0g/l.Root frequencies were 100%with root number,4 to 5.4.Among different carob source,sucrose was good for adventitious shoot regeneration. Light produced good effects for shoot regeneration.Among different light quality,normal light and red light was good for shoot regeneration.5.During adventitious shoot regeneration from leaf explants,The content of SOD,POD, soluble protein and soluble sugar decreased from 0 to 15 days then decreased.6.Among different carob source for root induction,from root production and growth during acclimatization,20g/l sucrose produced the best results.7.The efficient system of genetic transformation was established.1.5mg/l Hygromycin was used in the shoot regeneration medium to select the resistant shoots.500mg/l cefotaxine was used to kill the Agrobacteria after infection.Explants were pre-cultured for 3 days before infection.The pre-cultured explants were incubated for 5-10min in the bacterial suspension which concentration(OD₆₀₀) was between 0.1 and 0.5.Length of co-cultivation period was 3 days.The target genes integrated into the genomic DNA of C.acuminata were detected and confirmed by PCR and PCR Southern analysis.