Tissue Culture Of Platanus Occidentalis L. And Salt Tolerant Gene Transformation With Safe Selection System

Soil salinity is a serious problem worldwide,and to plant trees in salinity soil could not only improve entironment but also bring with economic benefits.So it is a hotpot for breeders worldwide to breed new varieties with high salt tolerance.More and more people pay attention to Platanus occidentalis because of it's valuable for timber and it is also widely planted as a shade tree.In this paper,the regeneration system in vitro of P occidentalis and then the transformation of salt tolerant gene AtNHX1 was studied,the results were as below:1)A high-efficient tissue culture system was established from leaf discs.The leaves of P.occidentalis were selected and a high-efficient regeration system was established.The best induced medium we selected was WPM+6-BA(5.0 mg l^-1)+IBA(0.1 mg l^-1)+ sucrose(3%),and the highest frequence and shoots per explantis recheated 90%and 13.72,respectively.The suitable strong medium and root medium were MS+6-BA(0.3 mg l^-1)+NAA(0.03 mg l^-1)+Ad(2%)+ sucrose(3%)and 1/2MS+IBA(0.1 mg l^-1)+ sucrose(2%),respectively.The genetic stability of regenerated plants was identified using RAPD markers and the results showed that the somaclonal polymorphisms were lower than 0.0104%.2)A mannose seletion system for P.occidentalis transformation was developed.The result of explant sensitivity to mannose showed that regeneration rates and number of shoots per explant decreased with increasing mannose and decreasing sucrose concentration.It means that leaves of P.occidentalis can not use mannose as carbon source to induce shoots and we can use mannose as selective agent for P.occidentalis transformation.After transformation,P.occidentalis leaves could be induced different number of positive shoots strained by different mannose and sucrose concentration.The results of PCR displayed that the high trangenic rate were recorded as 12.76%with 10 g/l mannose and 15 g/l sucrose in the medium.3)Transgenic plants were obtained by using mannose selection system.Genetic transformation was confirmed by PCR and CPR(chlorophenol red assay),and transgenic plants were obtained.