In Vitro Culture And Gentic Transformation Of Different Genotypes Of Platanus Acerifolia

London Plane tree(Platanus acerifolia Willd.) is an important landscape tree used for improving city environment in China and has favourable heterosis.However,in April to May,pollen and seed hairs disperse widely,in addition to polluting the environment but also serious affect human health.To solve this problem,many researchers try to improve the measures of cultivation and management,or use chemical or physical mutagenesis, but have not yet resoved the problem completely.Tissue culture and genetic engineering for genetic improvement is a more effective means of breeding.The final objective of this study was to breeding sterile individuals of London Plane tree.This purpose can be achieved by genetic transformation of sterile genes.For the purpose,a stable in vitro propagation system and high frequency plant regeneration system should be developed first.In my study,different genotypes explants of Platanus acerifolia were cultured,compared with the sterile materials kept in our lab.A new propagation system and regeneration system from leaves for genetic transformation was established,and the improved Agrobacterium-mediated transformation system was developed.In addition,we conducted a PCR detection to detect the transgenic plantlets conserved in the laboratory.Then they were exercised and transplanted to different matrix. The main results as following:1.The buds enclosed in petiole bases of PE,P19 and the tetraplont of Platanus acerifolia were disinfected and cultured.The results showed that the buds in autumn or the four to six buds on the fresh branches in spring were the best explants.The disinfecting time of HgCl₂ was 10 minutes in November or 12 minutes in April.On the medium MS+6-BA0.3mg/L+NAA0.05mg/L,the sprouting rate of P19 was upmost. The sprouting rate of PE was 33.3%.The sprouting rate of the tetraplont was only 13.3%. The best medium for the buds of P19 sprouting was MS+6-BA 0.3 mg/L+NAA 0.05 mg/L.2.On the medium MS+6-BA0.3mg/L+NAA0.05mg/L,the proliferation coefficient of PH2 was 3.9,the next was P19 which was 2.5,PE was only 1.6.MS,WPM and 1/2 MS were unimportant for multiplication of PH2.The best medium for shoot multiplication of PH2 was MS+6-BA0.6mg/L,the best medium for shoot multiplication of P19 was MS+6-BA0.6mg/L+NAA0.05mg/L3.On the same medium MS+IBA 0.1 mg/L,the average NO.of roots of PH2 was 7.0,the taproot and the lateral roots was thick.The roots of PE were less and shorter,and the NO.of roots per shoot was 4.2.The P19 was 6.4.MS,WPM,1/4MS were not suitable for rooting of PH2,and the best basal medium was 1/2MS.On medium 1/2MS+0.1 mg/L IBA,the state of roots was best.4.On MS+BA6.0mg/L+IBA0.5mg/L,the frequency of shoot regeneration of PH2 was 44%,the P19 was only 12%.The best medium for adventitious shoot regeneration of PH2 was MS+BA 4.0 mg/L+IBA 1.0 mg/L,it was MS+BA 6.0 mg/L+IBA0.5 mg/Lfor P19.It was better for adventitious shoot regeneration to be cultured in dark for 7 days first.5.In the transformation of PtLFY,the improved Agrobacterium-mediated transformation system was developed.The results indicated that EHA105 Agrobacterium strain was better than C58;forty days to fifty days aged leaves were the best explant for infection;treated with 0.4mol/L mannitol for 5 hours could be better for infection.With the improved Agrobacterium-mediated transformation system,5 risistant buds were obtained,but they died in the proliferation.6.The transgenic plantlets in our lab was detected by PCR,the results showed that the interest genes had incorporated into the genome of Platanus acerifolia.And detected after one year the result was still the same.7.The plantlets which were grown for 40-50 days and with more roots can be transplanted successfully.Ten days was best for the plantlets exercising.The best transplanting medium was soil and sand and perlite.The proportion of them was 1:1:1. The survive rate of PH2 and P19 were higher evidently than the two kinds of transgenic plantlets.