| Cell culture technology is an effective way in mordern biotechnology which no t only oriented train active ingredient or active substance, but also the basis for i ndustrial production target substance.Tobacco is an important experiment material i n plant tissue culture.In recently years, using tobacco suspension culture cells to c any out research on plant resistant, gene transfer and production of secondary met abolites have achieved some progression.Therefore, the establishment of a stable to bacco cell suspension culture system is of great significance. There’s many varietie s of tobacco, the current report on the establishment of clouds 21 tobacco cell sus pension system is little. In this paper, we use clouds 21 tobacco leaves as experi ment material. Using plant tissue culture technoiogy to obtain callus and chosse th e best culture conditions for brittle callus growing, then established tobacco suspen sion culture system.At last, elected suitable conditions for established clouds 21 to bacco cell suspension system. It’s not only provid a method for the cell culture gr owing, but also provide material for the study of metabolism in VB6.We choose MS+6-BA 3mg/L+NAA 0.2mg/L medium to induce callus, we plac ed the inoculated flasks in incubator with darkness culture.The results showed that: After 10 days growing, the tobacco leaves became large, many yellow granular cal lus formed around it.After 15 day, callus growing proliferated, this the first generat ion callus.The callus which growth quickly and loose friable is necessary, so we designe d a combination of different hormones in different levels and subculture cycle to o btain the callus.The results showed that MS+6-BA 0.5mg/L+NAA 1.0 mg/L for mula cubculture is the most appropriate formula, we can get 100% brittle callus by three generations. These callus growing exuberant and loose friable which can be u sed for liquid suspension culture.In the process of establishing tobacco cell suspension culture system, we study the effect on the initial inoculation and hormones. We found the 7.5% inoculation for cells growth is best. Using this inoculation brittle callus inoculated to the MS +2,4-D 1.0 mg/L liquid medium for suspension culture. The result showing tha t:cells growing slowly in the first six days, then growing rapidly. Till the nine d ay, the fresh weight of cells reached 6.73 times compared with the initial inoculu m. The fresh weight of cells reached 12.54 times in the twelve day, in fifteen da y the fresh weight has no significant change compared with the twelve day. It ca n determined that subculture cycle for each ten days is suitable for the clouds 21 tobacco suspension cells growing.Cells culture medium and suspension cells of clouds 21 tobacco content were 0.22ug/mL、3.28μg/g fresh weight. The constitute ratio of cells culture medium abo ut VB6 were as follows:PMP 7.58%, PM 20.40%, PL 1.96%, PN 70.06%. There salmost not have PLP in cells culture medium; The constitute ratio of VB6 in susp ension cells were as follows:PMP 0.76%, PM 50.86%, PLP 19.06%, PL 16.13%, PN13.19%. |
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