Establishment Of High Frequency Of Regeneration System And Research Of Transformation System Of Mulberry

Morus alba Linn. is a perennial dicotyledonous woody plant ,its leaves are natural diets for Bombyx mori L..so getting leaves is the main aim to plant the trees.Meanwhile it is also traditional medicine plants in our country.So Morus alba Linn. is important economic trees and also important potential basis for sustainable development of sericultural industry.The transmit of woody plant trailed behind crop and other herbage plant,one important reason is that tissue culture and regeneration of woody plant is very difficult,lack appropriate recipient material,difficult to establish of efficient regeneration system,the other reason is that it has gene-dependent and low transmit efficiency.So using winter mulberry buds of Nong 14,Yu 71-1,S305,Da 10 as materials,this study founded high efficiency regeneration and optimized the transmit condition,by transmiting GUS and checking transformation efficiency in mulberry,provided reference for gene transmit in mulberry.The mainly results are:1 In this test we studied the infection of auxin 6-BA,NAA,2,4-D,lanthanon lanthanum and breed to mulberry bud polarization,screened out the proper polarization culture medium MS+6-BA1.2mg/L+2,4-D0.2mg/L+LaCl3 40mg/L. There is no significant influence between different breed.2 Researched the factors of regeneration of mulberry: TDZ,NAA,6-BA,IAA, breed,age and leaf position by orthogonal analysis,the results showed that the best embryogenic callus induction culture medium is MS+TDZ 1.0 mg/L+NAA 0.2 mg/L;The adventitious shoot polarization medium is MS+6-BA 3.0 mg/L+IAA 0.1 mg/L;The callus initiation rate of using 15d test-tube of S305 plantlet is 93.9% and adventitious bud differentiation rate is 98.33%,so we established the high frequency regeneration system of mulberry.TDZ can depress lipid peroxidization of leaves, protect the integrality of cell membrane structure,prevent degradation of chlorophyll,control the activity of POD delaying leaf-senescence and established the frequency efficiency of lesf regeneration.3 On the efficient regeneration system base, Observing of paraffin section showed that callus epidermis includes a flat of tidiness and lesser cells,cortex includes various and disorder arrangement parenchyma cells.The regenerate of adventitious bud come from the epidermis and its under regeneration system of callus,has big nucleolus.This result illuminate that the adventitious bud derivated from mulberry leaves can be transmitted.4 Because of the growing characteristics of woody plant that made genetic transformation become difficult and particular. We used GUS-staining optimized the transformation system by setting up various processing:bacterial suspension OD600,infection time,co-culture time,Kan selecting concentration etc then we obtained the optimal transmit system of S305: bacterium OD600 0.2-0.4,infected 15 min and Kan 30-40 mg/L,500 mg/L Cef.5 Using GUS to check the transmit rate and GUS-staining indicated that the expressed rate is 10.5%.The PCR results showed that GUS has conformity into the mulberry plant.