Studies On In Vitro Culture Of Cucumber Hairy Roots And Its Transformation With LAE Gene From Digitalis Lanata Ehrh

An efficient system for induction and culture of Cucumis sativus L hairy roots transformed by Agrobacterium rhizogenes ATCC15834, effects of 6-BA on the growth and morphology , activities of SOD and POD and production of endogenous ethylene of cucumber hairy roots were investigated as well as genetic transformation of cucumber with LAE gene from Digitalis lanata Ehrh.. The main results were as follows:Hairy roots could be induced 10 days after infection 10-day-old cucumber cotyledon explants of C. sativus L "Jinyan 4" with A.rhizogenes ATCC15834 harbouring wild agropine-type Ri plasmid, pRiA4b. The obtained hairy roots could be divided into three phenotypes and could have a rapid growth on exogenous growth regulator-free MS medium. Phenotype I cucumber hairy roots were similer to non-transformed roots (control) but had more numerous lateral roots; Phenotype III hairy roots were much shorter and thicker, enlongated very slowly, more lateral roots and had no root hairs. Phenotype II hairy roots were intermediate in the number of lateral roots and root hairs. The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes and virC gene of Ri plasmid from A.rhizogenesATCC15S434. Among the three kinds of phenotypic hairy roots tested, phenotype I hairy roots grew most rapidly. Its multiplication coefficient of dry weight reached 17.83 after in vitro culture for 15 days and was 1.2 times and 1.6 times as much as that of phenotype II and phenotype III roots, respectively.Addition of various concentrations of 6-BA in the medium could not only influence the morphology, but also enhance the growth and delay the senescence of cucumber hairy roots. Among the 6-BA concentrations tested, 6-BA at the concentration of 3.0 mg/L in the medium was the most effective, where the main roots of hairy rootsbecame shorter and thicker and formed almost no lateral roots; When hairy roots were cultured in vitro for 20 days, the highest multiplication coefficient of biomass(fresh weight), ca 17.52 was gained. Compare to the control (hairy roots cultured on exogenous growth regulator-free MS medium) , cytokinin 6-BA in the medium could decrease the decline of soluble protein content, significantly enhance the activities of SOD and POD in hairy roots, decrease the production rate of ethylene in cucumber hairy roots and delayed the appearance of growth-peak by 5 days.The expression vector of LAE (lanatoside 15'-O-acetylesterase gene) gene from D. lanata Ehrh. was constructed by PCR amplification and then transferred into A.rhizogenes ATCC15834 with Ri (root inducing) plasmid through using freeze-thaw method. An efficient transformation system was also developed by genetic transformation of 10 day-old cotyledon explants of C. sativus L."Jinyan 4 " with A.rhizogenes ATCC15834 containing LAE gene. The hairy roots transformed with LAE gene could also have an autonomous growth on exogenous growth regulator-free MS medium. Transformation of hairy roots confirmed by PCR amplification of virC and LAE genes showed that LAE gene had already integrated into the genome of cucumber hairy roots.This has laid a foundation for investigation active expression of LAE gene of Digitalis lanata in hairy roots and also provided the possibility of utilization cucumber hairy roots with LAE gene to perform biotransformation of cardenolides from Digitalis plants.