Autoteterploid Induction And Transformation Of Antisense-LFY Of Platanus Orientalis L

Platanus orientalis L. is a tree of strong stem, good effects of green and its leaves have great capability to resist poisonous gas and insects and become a famous street tree of the world, so that it be planted in many cities of our country. But its seed's fur not only pollute surroundings but also provoke disease of breathing system and skins and bring very inconvenience to citizen's living. So scholar of home and abroad proceed a great research to solve the problem of pollution of seed's fur. With the boom of modern science and technology, gene and cell engineering provide new methods to solve these problems.Factors affecting the transformation of P. orientalis by Agrobacterium tumefaciens are studied and the optimum transformation conditions are defined. The genetic transformation system of P. orientalis is originally established. Method of extraction DNA from leaves of P. orientalis is selected. The transformation of P. orientalis is verified by PCR and GUS histochemistry test. The results of experiments are as followes:1 Autotetraploids from diploidy leaves of Plantanus orientalis on the solid-liquid MS media supplemented with 0.1mg/L NAA and 15mg/L BA (optimal organogen- etic medium to the leaves of diploidy seedlings) with different colchicines concentrateion- s were induced,chromosome numbering of the plant root tip cells and DNA content ana- lysis of the leaf single cell were used to analyses the autotetraploids.The results indicated that the highest tetraploid induction rate could reach 23.4%when the leaves precultured on the optimal organogenetic MS medium for 12d and treated with 10 mg/L colchi- one on the double layer media for 24h in vitro.Compared with diploidy seedlings of Plantan us orientalis L.,leaf length and stomata size of tetraploid becomes bigger,the leaf length/width ratio and stomata density become s smaller.2 The Agrobacterium tumefaciens is made of the engineering Agrobacterium for transformation of P. orientalis. The expression vector pBI121 contains anti-LEAFY gene, and there are a position, which can be defied by HindIII. We obtain the anticipative DNA segement from plasmid is digestioned by HindIII and EcoRI. The anti-LEAFY gene can be examined by PCR as it is transferred into Agrobacterium tumefaciens.3 In this paper dealt with effects of different antibiotics on in vitro plantlet regeneration of P. orientalis. Induction frequencies of the calli, shoots and rootings with the leaves were reduced with Ka and Cef in the media. The rootings of their explants and calli, shoots were inhibited with 15 mg·L-1 and 40 mg·L-1 of Ka concentrations in proliferation and rooting media respectively; So were that with Cef concentrations of 500 mg·L-1 and 800 mg·L-1 in those media. There existed no leaf callus and shoot formations on the proliferation media with 30 mg·L-1 and 400 mg·L-1 of Ka and Cef. This would be referenced to the manipulation of gene engineering of P. orientalis.4 Several factors affecting the efficiency of genetic transformation mediated by Agrobacterium tumefaciens in P. orientalis. The results showed that the transformation efficiency of the leaves precultured for 9 d on preculture medium were optimal, which were infested in OD600 value is equal to 0.7 bacterium medium for 10 min , and then co-cultured in dark conditions and co-cultured medium with 100μmol ? L-1 acetosyringone for 3 d respectively.5 In this study dealt with effects of the calli browing of P. orientalis through different concentrations of antioxidant DTT and adsorbing substancecan PVP,AC in the media. The results indicated that DTT,PVP and AC in which DTT concentrations of 0.08 mg·L-1 could inhibit the browing in the best and the rate of browing is 19%;The rate of browing of 0.1% AC is 27% and PVP concentrations of 2 000 mg·L-1 is 48%. In other factors the rate of browing in 10℃,2 500 lx is12% and the result of inhibiting the browing are the best.6 This paper dealt with extraction of DNA from P. orientalis leaves with SDS-Ⅰ,SDS-Ⅱ,CTAB-Ⅰand CTAB-Ⅱmethods. CTAB-Ⅱwas considered as one of the best methods for extraction of DNA from P. orientalis.7 Through stringent selection of the infected explants on the medium added with Ka, climinated some explants which resistance to Ka. Then the expression of GUS of the plants, regenerated from the Ka resistant explants, were detedted by histochemical method. Blue was found in organs of transformed plants. The results of molecular biological assays with PCR confirmed that the anti-LEAFY gene was integrated into genome of P. orientalis. All assays proved that the transgenic P. orientalis plants were gained in this experiment, which provide the stable base to subsequent work of selective breeding on production-lever of transgenic P. orientalis without seeds.