Cell Suspension Culture And Plant Regeneration Of Triploid Dioscorea Zingiberensis Plantlet

Dioscorea zingiberensis C.H.Wright, known as yellow ginger, rotor root. Its roots rich in the diosgenin(commonly known as saponin) are the main raw materials of synthetic steroid hormone and steroid pill, andbeacause of it difficult to synthetesize, called "medicinal gold" by people. Its wild resources are largelyplanted in Hubei, Shanxi, Henan and other regions. But due to excessive excavation, the wild resourcesbecame extinct. As the development of steroid hormone drugs and the expansion of application scope, thedemand of diosgenin is increasing at a rate of10%, the wild resources of D. zingiberensis will becomeincreasingly shortage. In our previous study, triploid plantlets were obtained by using endospermcultivation technology, rapid propagation, dedifferention, diosgenin content and resistance to diseases underdifferent light intensity were compared between diploid and triploid plantlets. In this study, in order toobtain more diosgenin production and lay the foundation of cell engineering and gene engineering, triploidcell suspension culture system and its plant regeneration process have been established. The main resultswere as follows:1. Callus induction and subculture. The suitable was MS+2,4-D1.5mg·L-1+6-BA1.0mg·L-1, theinduction rate was80.56%. The callus were classified into four types after subcultured, type Ⅰwhich waslight yellow, small, loos and rapid growth was best for establishing the suspension culture system. Thesuspension culture cell growth curve was like “S”, there were3phases: delay period (0~3d), index growthperiod (3~12d) and rest period (12~15d).2. Establishment of the triploid suspension cell and the study of physiological and biochemicalcharacteristics of growth cycle. The suspension cell vitality and soluble protein contents of triploid D. Zingiberensis both increased at the early stages of the training, reach maximum at6d ahead the timeof itsgrowth curve peak; The pH of culture medium was raised firstly, then declined maintain between4.8~7.0;The medium conductivity fell to the lowest point at12d, rebound after rest period; In early culture stagephosphate, nitrate, ammonium salt concentrations were118.75mg·L-1、2.44×103mg·L-1、3.71×102mg·L-1,at the end of the cell cycle, there were9.26%,9.26%and6.13%of phosphate, nitrate, ammonium saltconsumed respectively; Diosgenin of the suspension cell compound slower at delay period, compoundquickly after3d, and reached the highest yield(0.58%) at9d and then decreased, remain0.41%after12d.3. The optimization of triploid cell suspension system. The suit able medium for triploid cellsuspension culture was3/4MS+NAA1.0mg·L-1+6-BA0.2mg·L-1; with4.5g·L-1inoculationconccentrantion,12d subculture cycle,30g·L-1sucrose, pH value6.0. The effect of differentconcentrations of yeast inducer, Cu2+and sodium acetate on the cell growth and content of saponins werestudied. When the concentration of yeast inducer was2.5%(v/v), cell dry weight and diosgenin reach thehighest level,18.77g·L-1and0.81%respectively; When the concentration of sodium acetate was0.5mg·L-1, cell dry weight and diosgenin content were the highest,15.0g·L-1and0.79%respectively.Compared with the control, adding different concentration of Cu2+has no influnce.4. Plant regeneration of triploid suspension culture cell. The differentiation rate of mediumMS+6-BA2.0mg·L-1+NAA0.4mg·L-1was highest,51.39%. Take2~3cm bud stems to rooting medium,root number increased after15d, The rooting transplanting survival rate was80%after one-monthacclimatization management.