Studies On The Regeneration System From Leaves Of Pear Cultivars And Rootstock In Vitro

This article was mainly involved in the cultivars of Pear named "Xishui" and "Qinfeng" and rootstock of Western Pear named "BA29".Regeneration system was studied using the leaves of plants in vitro.The adventitious bud regeneration system had been modified by studying the effect factors on regeneration such as the basic medium,the types and concentration of plant growth regulators,cultivate condition etc.The main results were as followed:1.Internal influence factors of regeneration from leaves of Pear Cultivars and Rootstock:(1)Genotype plays an important role in the efficiency of regeneration.(2)The regeneration ability in vitro of leaf was higher than stem segment's and leaf stalk's.Regeneration ability of whole leaf was greater than part of leaf.Young leaf at the tip of plantlet was suitable material part for regeneration of Xishui and BA29.Leaf from the tip or middle part of plantlet could be used to regeneration of Qinfeng.(3)Subculture generation plays another important role in leaf regeneration.Leaf from plantlet in the subculture of 15th time was suitable for leaf regeneration of Xishui and Qinfeng,while 8th to 15th was suitable for BA29.2.External influence factors of regeneration from leaves of Pear Cultivars and Rootstock:(1)NN₆₉Was confirmed as the best suitable medium of the leaf regeneration in vitro of Xishui.Qinfeng was less sensitive to the different medium.BA29 needed MS or NN₆₉ medium to induce leaf regeneration in vitro.(2)The best combination of plant growth regulators was confirmed on NN₆₉medium. TDZ1.5mg/L+IAA(0.1～0.5)mg/L or IBA(0.1～0.5)mg/L was suitable for Xishui,6-BA 5.0mg/L+ IBA 0.3mg/L was best for Qinfeng,and TDZ1.5mg/L+NAA0.3mg/L was optimum to BA29.(3)The leaf should be cut by 3 times vertical to main vein before inoculated.The leaf of Qinfeng and BA29 should be inoculated with the method of abaxial side in contact with medium,while Xishui was less sensitive to the different inoculated method.(4)For Qinfeng and BA29,2～3 weeks of dark culture was suitable.As to Xishui,it made no difference between 1～3 weeks of dark culture and light culture.What's more, 4～5 weeks of dark reduced the regeneration efficiency.(5)It is favorable for leaf regeneration of BA29 to properly increase the ratio of NO₃^--N. The most ratio of NH₄⁺-N/NO₃^--N was 1:5,and the leaf regeneration frequency could be increased to 100%.(6)AgNO₃ and Tryptone could improve leaf regeneration of BA29.AgNO₃ could also advance the peak period of regeneration,the best concentration was 1～2mg/L.The best concentration of Tryptone was 800～1200mg/L.However,Peptone could not improve leaf regeneration,and 800mg/L of Peptone might reduce the regeneration efficiency.3.Transferring the part leaf with adventitious buds into new subculture medium was suitable for elongation of adventitious shoots.MS+6-BA 0.5mg/L+IBA0.1mg/L was the best proliferation medium for Xixhui and BA29,and AS+6-BA0.8mg/L+IBA0.1mg/L is suitable for proliferation of Qinfeng.Kraft paper+seal membrane was the best seal material for multiplication of plantlets from regeneration,which was economy and convenient.It could make the plantlets grow up well,and could avoid mould contamination which from moisture of cotton plug.4.The plantlets from regeneration of Xixhui should be cultured on MS+IBA 1.0mg/L+NAA0.5mg/L+AC0.5mg/L for 5days,and then be transferred to medium without Auxin.The plantlets from regeneration of Qinfeng should be cultured on MS+IBA0.5mg/L+NAA1.0mg/L for 5～10 days,and then be transferred to medium without Auxin.5.The optimum concentration of Cefotaxime was 300 mg/L during the gene transformation to inhibit bacterial.25mg/L kanamyein could be used as a selecting pressure for the regeneration of adventitious buds with antibiotic-resistant.