Cytokinin Production By Fermentation Of Bacillus Licheniformis

Cytokinin is known as one of the six natural plant hormones, including IAA,GAs CTK, ABA, Eth and BR. Its physiological role is as following: 1) have good stimulative effect on cell fission and cytodifferentiation; 2) accelerate cell growth; 3) promote ateral buds; 4)break dormancy of some seeds and buds; 5) reduce the hydrolysis of chloroplast and postpone blade aging. It can be applied extensively in crops such as vegetable,fruits and other economic crops. This substannce ,for increasing the output and improving quality notably, exist generally in the body of plant. Though little in content, is it indispensable.A Bacillus licheniformis, isolated from soil in Beijing, was identified by Chinese Academy of Sciences and was found to produce cytokinin by fermention. namely, if Amaranthus caudatus was provided with cytokinin and tyrosine, the cotyledone of etiolation seedling could synthesize betacyanin, All betacyanins, soaked by reiteration forst, could be measured with colorimetry, so its activity was tracked and rationed at the same time. Studies on four elements such as soy bean meal, maize powder, yeast extract and CaCO3 were conducted in three levels using the orthogonal form of L9(34) to investigate the effects on biosynthesis. The result demonstrated the optimized fermentation medium was: 0.8% of soybean meal, 1% of maize powder, 0.2% of yeast extract.Solvent-extraction and chromatography were used to preliminarily extract and separate cytokinin in fermentation culture broth. The structure was analysized by comparing the Rf value of the thin layer chromatogram (TLC) , the UV-spectrum, the retention time of RP-HPLC and the spectrum of NMR. TLC was carried out on plates precoated with silical gel F254, for better separating methods of column chromatography. Powder of spraying drying was extracted with 80% of methanol, followed by extraction with n-butanol at room temperature. The combined extracts were concentrated in vacuo to give a crude extract, which was partitioned between H2O and n-butanol to provide n-butanol extract and water soluble fraction. The n-butanol extract was concentrated and subjected to column chromatography eluted with solvent mixture of methanol/chloroform (92:8-9:1, by vol) to give three fractions. Fraction B, which had effective activity ,was concentrated and subjected to column chromatography (methanol /chloroform 9:1, by vol) again, get cytokinin, which showed a high specific activity.the cucumber, The bioactive component obtained could promote the growth of the cucumber and enhance the disease resistance of cucumber. Compared with their control, the output was increased by 34.8%, 25.3%, 19.8%, respectively, reaching notable effect on increasing cucumber yield. Moreover, efficiency of inhibition to cucumber downy mildew and anthracnos could be up to 36% and 61.5%, respectively.