Transform Gene PuNHA Into Malus Robusta Rehd. And Pyrus Betulaefolia Bge.

There is widespread of saline soil in China,which owned total area for 991.3 billion hm² at present.There is large population in China,while whatever per capita of the land or the water resource is lower than the average level of world.Therefore,the development of large waste land and the prevention and cure of irrigated area salinization will be the key for the food problem resolution of 21^th.The theory and practice significance will be profound.Apple is one of the four greatest fruits in the world,which is the head regardless of the cultivation area,the yield or the consumption in China.The pear,alike the apple,is one of the longest cultivated fruiter.It has lots of traits,including long lifetime,high yield,abound nutrition. With grafting method for the reproduction,the resistance to salt of apple and pear are moderate of deciduous trees,which are mainly up to the stock.Stocks determine the survival of fruits and behavior of over ground breeds towards the adaptability of local soil environment to the large extents.To choose the stock for salty resistance is first for the fruit trees cultivation on the alkaline soil.Or else,growth of stocks and grafting trees are restrained.Stocks and breeds with high resistance are eager to need on the production.Developed inherited transform skill in the 1980s offers a new technical way to species breed. To transmit the salt-resistant gene to the fruit trees with inherited transform skill can cultivate better stock breeds.That is significant for fully use the alkaline soil,develop the agriculture,improve the environment and help the farmers earn more money.Currently,reports on the research of fruits salt-resistant gene transforming are just few,while that of apples,pears and stocks at present are no found.Take apple stock Malus robusta Rehd.of the northern alkaline soil in the common use and more salt-resistant plant Pyrus betulaefolia Beg.as materials,the research made use of the high-effect carrier and transgenic engineering technology to not just transform the PuNHA gene into the materials,but detect parts of genetic mutants with PCR,Southern and Northern method.The results were as follows:Firstly transform the PuNHA gene into the apple stock M.robusta Rehd.of the northern alkaline soil that is in the common use,we got numerous transgenic Malus robusta mutants.The 4mg/L herbicide concentration selected in the experiment that the growing point destroyed as explants,the percent transformation was the best.The high quality system was that:the regenerated media(MS+4.0 mg/LBA+0.2 mg/LNAA+4mg/L herbicides+500 mg/L Carbenicillin);the elongate media(MS+1.5 mg/LBA+0.3 mg/LNAA+4mg/L herbicides+500 mg/L Carbenicillin);the rooting media(1/2MS+0.5 mg/LIBA+4mg/L herbicides+500 mg/L Carbenicillin).Detected with PCR,we found one different strip in the electrophoresis.The result showed that exogenous gene was already integrated into the genome of M.robusta Rehd.Examined the mutants with Southern,both 35S promotor DNA and transforming plants DNA had cross-breeding signal,but untransforming ones had no signal.The results showed that PuNHA gene was correctly plugged into the apple nucleus genome.Checked out the mutants with Northern,the mRNA expression quantity of the transformed was increased,which was more than the wild type.The results showed that PuNHA gene was validly expressed in the transgenic plants.Treated M.robusta Rehd.under the concentration of salt as 0‰,2‰,3‰,4‰,5‰for 30 days.The salt-resistant ability of non-transformed plants was low after 30 days 3‰salty stress treated.Acted as slowly growing,short and small plants,blades turning yellow,they would not grow normally on the culture.However,the transgenic plants still grew normally with green color leaves under the condition of 3‰salty stress.The advantage is obviously higher than non-transformed plants that proved the salt-resistant ability of transgenic plants apparently rose up from 2‰to 3‰.Proliferated media of P.betulaefolia Beg.was MS+2.0 mg/LBA+0.4 mg/LIAA,with the P. betulaefolia Beg.of materials.Regenerated media was that MS+3.0 mg/LKT+0.3 mg/LNAA+1.0 mg/LAgNO₃.To take them under the dark cultivation for 21 d could apparently increase the regenerated rate of P.betulaefolia stem.Taking the growing point organization destroyed as explants to induce the bud regeneration,regenerated media was MS+3.0 mg/LKT+0.3 mg/LNAA.The rooting media was 1/2MS+0.5 mg/LIBA.The herbicides concentration selected with transgenic P. betulaefolia Beg.was 3mg/L.Firstly transformed the PuNHA gene into P.betulaefolia Beg.and parts of transgenic mutants were got.The high quality system was that taking the callus of light stem section and the growing point organization destroyed as explants after 21 d dark cultivation in the regenerated media that infected by Agrobacterium with OD550 of 0.4,co-cultivated for 2 d in the regenerated media without herbicides pressure,and then turned into the selected media(MS+3.0 mg/LKT+0.3 mg/LNAA+3mg/L herbicides+500 mg/L Carbenicillin).Resistant buds were transgenic mutants under the pressure selection.