| China is one of the main production areas of Oriental pear varieties,and the cultivated area and annual yield of pear varieties rank first in the world.Pear is a woody plant,and its propagation methods are mainly seedling cultivation and graft propagation.Due to the long childhood period of pear varieties,high gene heterozygosity,long growth cycle,self-incompatibility and other problems,resulting in its low reproduction efficiency,and the development of plant tissue culture technology and transgenic technology has broken the disadvantages of traditional breeding technology,greatly promoted the improvement of pear variety regeneration efficiency and the selection and breeding of fine varieties.Plant tissue culture technology is beneficial to shorten the breeding cycle of pear,improve the regeneration efficiency,and carry on the efficient and rapid propagation of pear varieties.The development of transgenic technology is beneficial to improve the characteristics of pear varieties,improve the resistance of pear varieties,and promote the breeding of superior pear varieties.This experiment by using tissue culture technology of pear with cytokinin 6-BA and auxin IBA ratio of different concentrations of hormones for variables,to explore ’pear no.1in pear varieties transgenerational and regeneration of the best culture conditions,in order to optimize’ pear no.1 in pear varieties of regeneration technology,explore ’pear no.1 in pear varieties of optimum transgenerational regeneration system,form the system complete regeneration system.In this study,the genetic transformation technology of wild Qiuzi pear(Pyrus ussuriensis M.)was optimized,and the concentration of hygromycin that could survive in its leaves was screened,which laid a foundation for the growth of positive seedlings in the genetic transformation test.Specific research results are as follows:1.Establishment of regeneration system of ’Zhongli No.1’.After original culture of more than 30 varieties of pears,the survival rate for each are not identical,this study selected the highest survival rate ’pear no.1 in cultured the transgenerational regeneration,in the light,temperature and humidity and the status of the explant grow consistent case,select the best successive transfer culture medium for MS + 6-BA(1.5 mg/L)+ I-BA(0.3mg/L)+ sugar 30 g/L + AGAR 7.5 g/L,p H = 5.8,transgenerational effect best,plant tiller up to six,proliferation rate is 4.25,2.14 cm plant height,pollution rate was 9.8%.When the medium was MS+6-BA(0.5mg/L)+I-BA(0.2mg/L)+ sucrose 30g/L+ AGAR 7.5g/L,the water impregnation was serious,and when the concentration of 6-BA was higher than2.0mg/L,the glass impregnation was serious.Under the same experimental method,different dark culture time was set,and the optimal regeneration conditions of ’Zhongli No.1’ leaves were obtained as follows: NN69+6-BA(1.0mg/L)+NAA(0.5mg/L)+TDZ(1.0mg/L)+ sucrose 30g/L+ AGAR 7.5g/L,p H=5.8,dark culture for 50 days,leaf regeneration rate was 100%,and the number of regenerated buds was 3.24.The optimal rooting culture conditions for ’Zhongli No.1’ were selected by 1/2MS medium as basic medium and auxin IBA as plant hormone.The optimal rooting effect was obtained by 1/2MS medium+I-BA(1.5mg/L)+ sucrose 30g/L+ AGAR 7.5g/L,p H=5.8,and the number and length of roots were large and long,5.12 and 6.1cm,respectively.2.Screening of hygromycin concentration.The concentration of hygromycin that can survive in pear leaves has not been reported.According to relevant studies,the current minimum concentration of 4.0mg/L would still make leaves black and die.Therefore,nine concentration gradients were set in this experiment,from the highest concentration of4.0mg/L to the lowest concentration of 0.002mg/L.60 leaves were inoculated in each concentration gradient,and then dark culture was conducted for a month.When the concentration of hygromycin was 4.0mg/L and 3.0mg/L,the leaves turned black and died.When the concentration of hygromycin was 2.0mg/L,the veins turned white,and the leaves under 1.0mg/L survived normally with occasional blackening.When the concentration was below 0.5mg/L,the leaves grew well and were green in color.3.Exploration on genetic transformation system of ’Qiuzili ’.In this experiment,by setting different concentrations of Agrobacterium bacterium,different types of leaves used for transgenic and different transgenic genes,it was concluded that when OD value of fluorescent protein gene 843 was 0.43,transgenic leaves had the best growth state and higher germination rate.The optimal OD values of ATG-6 and ATG-1 resistant genes were0.28 and 0.67,respectively.The Agrobacterium tumefaciens activity of ATG-6 gene was better,and the leaves spillover occurred when OD value was higher than 0.28.In the course of leaf infection,the method of cutting up the leaf and cutting off the main vein on the leaf were adopted.Compared with the two methods,the leaf regeneration rate under the method of leaf shredding was very low and almost no regeneration phenomenon,while the transgenic leaves grew normally and the regeneration rate was higher when the main vein was only cut. |
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