Inducement Of Callus And Establishing Cell Suspension Culture System Of Pomegrante

In this paper,the different organs tissues of pomegranate(Punica granatum L) plant are selected as the material, plant tissue and cell culture technology were used to induce callus and establish cell suspension culture system. The callus inducement and regulation , and the characteristics and growth regularity of differentiated loose callus suspension particles in cultural system, were studied systematically. The amount of flavonoids from different sources of pomegranate tissues was measured by spectrophotometric method, to provide the experimental and theoretical basis for the industrial production of flavonoids.In the Callus inducement period, the effects of different explants types,different sterilization conditions, different hormones and hormones levels on pomegranate callus induction rate, the raritas rate and the browning rate were studied.The results showed as follow: raritas callus were more easily induced from the mature leaves near the top, the combination of alcohol with a volume fraction of 75% 4 s and mercuric chloride with a mass fraction of 0.1% 4 min has a better effect for disinfection. The best callus inducement medium hormone prescription of pomegranate callus was: MS + 1.0 mg / L 2,4-D + 1.0mg / L 6-BA +1.0 mg / LNAA.In the continuing generation process of Pomegranate callus, as it appeared that the callus grew excessively dense and it was not conducive to achieve suspension culture, so the premise of successfully accomplishing pomegranate cell suspension culture was to obtain loose, rapidly growing callus. The effects of raritas callus by different types and different levels of hormone were studied. The results showed that The best secondary culture prescription for Pomegranate was:MS + 1.0mg/L2 ,4-D +0.1 mg / LKT. At the same time, developmental growth curve in 36 days of the pomegranate callus was determined. In this cultivating stage, injured tissues showed a "S"-type growth cycle, entered to logarithmic phase in the 12th day, and in the 30th day the cell biomass reached its maximum of 6.48g, which has increased 5.34 times.The cell suspension culture system of pomegranate was established, the effects of cell suspension culture systems by the inoculation capacity, shaker speed, sucrose concentration, liquid volume, hormone prescriptionss, such factors as pH change in the culture process were studied. At the same time the determination of developmental growth curve in the cell suspension culture and the accumulation of flavonoids were partly researched.In this paper, the optimum conditions of pomegranate derived cell suspension culture are as follows: with the composition and content of standards of MS basic medium, liquid medium was prepared , the sucrose concentration was regulated to 3%, that is, 30g / L, the liquid volume of the culture bottles is 25mL/150mL, 3g calli were inoculated in every bottle. The shaker speed was 110rpm, the best prescription for hormones was:MS +1.0 mg/L2 ,4-D +0.1 mg / L KT. Under the culturing conditions above-mentioned, suspended cells went into logarithmic phase in 6th day, the cell biomass reached its maximum in the 21th day,the dry weight reached 21.68g / L. After 21 th day the cell biomass began to decline into the growth of quiescent phase. At this stage Flavonoids content produced by secondary biological cell metabolites reached to the peak value of 69.05mg / g. pH value changed in the cultivating process, first decrease, then the trend of rising became apparent.The amount of flavonoids was measured by spectrophotometric method, content of the pomegranate callus and the cells in liquid culture in regular collection of pomegranate leaves, pomegranate leaves and solid cultivation, and analyzed of trends at different times. The results showed that: the content of total flavonoids megranate skin over time and showed a gradual downward trend, by the end of June the flavonoid content in pomegranate skin reached a peak value of 124.7mg / g, When the fruits mature from the end of September to early October, the Flavonoids content in pomegranate skin stabilized basically to 64.8mg / g. The flavonoids content in Pomegranate leaves changed little, there is a peak period in the late September, the concentration of 30.40mg / g. Pomegranate leaves were 73.13mg / g and 69.05mg / g, induced through tissue culture of callus and cell suspension cultures .Their total flavonoids was equal to the average level of megranate skin, but was higher than the pomegranate leaves.