The Optimization Of Genetic Transformition System And The Transgenic Research Of AtPAP2 In Populous Tomentosa

With tall and graceful form,Populus tomentosa Carriere is one of unique fast-growing species in China.As a kind of garden greenery or street tree generally planted in the north,it is the excellent species for ornamental and timber use.PAP2(R2R3-MYB)gene can promote the synthesis of plant anthocyanin.Based on the establishment of leaf regeneration and stem propagation system,PAP2 from Arabidopsis was transferred into Populus tomentosa by Agrobacterium mediated method.The main contents and results in the research are as follows:1.Applying direct differentiation in leaf regeneration simplified steps and saved time.According to the differentiation of adventitious buds,the most suitable differential medium was selected:MS+0.4 mg · L-16-BA+ 0.01 mg · L-1TDZ+0.1 mg · L-1IBA,the mixed hormone of 6-BA and TDZ promoted leaf differentiation,and leaf differentiation rate can reach 100%.The step of shoot elongation lifted the inhibition of TDZ on bud growth,and largely improved the propagation coefficient of single leaf.Using gelrite as coagulants was better than agar in rooting medium.Shoot could root after 8 days without adding hormone,which shortened rooting time,increased the number and the length of the lateral roots,generated two lateral roots.The root had fine growth status and grew better.2.The access methods and procedures for genetic transformation of Populus tomentosa and the research of factors affecting the transformation efficiency were built through using the leaf disc transformation.The primary culture leaf was selected as explants without pre-culture.When the concentration of bacteria liquid was OD600=0.3,added AS 200 ?mol · L-1 and suspended 1 h.Explants were infected about 10 min,co-cultured 3 days.Explants were screened once by 40 mg ·L-1 Kan with 150 mg ·L-1 Ti for inhibiting Agrobacterium.After the culture of callus and differentiation,the rate of callus formation reached 83.5%,and the rate of positive plants was 100%,the transformation efficiency reached above 55%.The method reported in related articles varied a lot in the infected bacteria OD value and transformation efficiency.The experiment showed that leaf explants in different subculture of stem had varied optimal infection OD value and transformation efficiency.Leaf explants after many subcultures were more adaptable to bacterium solution but the transformation efficiency was greatly reduced.It was possible that the plant material was a significant factor and the explants from the primary culture were more suitable for genetic transformation of Populus tomentosa.The appropriate bacterial concentration and infection time made the repeated washing steps needless.3.GUS staining showed that the target gene had been integrated into the genome of Populus tomentos.Comparing to non-transgenic plants,the phenotype of PAP2 transgenic plants had no significant difference.Transplant the transgenic and non-transgenic poplar plantlet seedlings.The gene expression of PAP2 transgenic plants is much higher than that of non-transgenic plants by real time fluorescence quantitative PCR.Further study on the content of leaf anthocyanins showed that they had no significant difference under low light.Under bright light,the content of anthocyanin in transgenic plant was slightly higher than that in non-transgenic plants.This illustrated that transgenic plants showed more sensitive to light,more likely to be induced by light.