Vitro Culture And UDP-glycosyltransferase76G1 Gene Cloning Of Stevia Rebaudiana Bertoni Strain "1096"

Stevia rebaudiana is introduced into China Since 1980's,for Stevia rebaudiana is a cross-pollination plant,steviol glycosides content in its leaves varies greatly after several years,key to up question is to reform growing method and to select new culturer,to get more and better leaves, we try to develop new method to select better strains.The tissue culture technology not only to be able to maintain the good characteristics of Stevia rebaudiana,but may also propagate quickly and breed large quantities of seedlings,In a short time,which are not required to consider environic condition.Using tissue culture technology will also promote genetic improvement and make up for the deficiency of traditional breeding method of Stevia rebaudiana.Steviol glycosides,which material containing not less than 95%,are approved to be used as addictive at the sixty-third meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA).The Committee established a temporary ADI of 0～2 mg/kg bw for steviol glycosides,so key to the question of stevia industry is to reform extract method,to improve Purity,to decrease production costs,to product high RebaudiosideA content steviol glycosides with good taste,or to culture stevia rebaudiana new variety with high RA content characteristic.In the present study,we established rapid propagation system of stevia rebaudiana strain "1096" which screened by improved DNS method in our laboratory.We discussed the influence of the different density sucrose and mannitol on "1096" strain in vitro conservation,colned the gene of UGT76G1 which Controls steviolbioside and stevioside to RebaudiosideA..The main experimental results were as follows:1.The effects of different combinations of growth substances and explants on callus induction, bud differentiation,root formation and transplant were investigated in Stevia rebaudiana strain "1096".The results showed that stem apex seemed to be the best explant for callus induction and bud differentiation,followed by stem segment with axillary bud and the third was leaf.Both the stem segment without axillary bud and root of plantlets could be induced to form calli but not adventitious buds.The optimal combinations of 6-BA for adventitious bud induction on stem apex was 1.0mg/L,on stem segment with axillary bud was 1.5mg/L on MS medium;The best adventitious buds induction medium from leaves was MS medium with 1.0mg/L 6-BA and 1.0mg/L IAA.The proliferation culture medium for bud differentiation was MS medium with 0.5 mg/L 6-BA and 0.05 mg/L NAA.Rooting medium is 1/4MS+IBA 0.1 mg/L+NAA 0.1mg/L +active carbon 1.0g/L,the rooting rate reached up to 100%,transplant survive was 95%.2.1t is feasible to conserve the stevia rebaudiana germplasm in vitro.The 1/4MS medium with 10g/L mannitol is better for "1096"strain vitro conservation than sucrose.After regeneration culture, the plantlet can restore the original shape,there is no difference in leaf shape and plant type between the plantlet of recovering growth and control group.3.Clone gene of UGT76G1 of "1096"strain by reverse transcription and touchdown PCR. Cloning product and its encoded protein was analyzed with bioinformatics software.Lay a foundation for improving taste of steviol glycosides,the further breeding of high RA content new stevia rebaudiana Variety by genetic engineering method.