Induction And Cryopreservation Of Eruca Sativa Mill Hairy Roots And Analysis Of Its Secondary Metabolites

Eruca Sativa Mill, an oil crop of the Cruciferae family, is one of the Cruciferae plants most resistant to drought and barren. In addition, like other species of Cruciferae family, it contained a secondary metabolite, named glucosinolate. This metabolite would be converted to isothiocyanates (ITCs) by the myrosinase, which benefits to the therapy of cancer. So it is considered to be a promising species as a potential genetic resource of glucosinolates and genetic transformation for other crucifers, and also can be employed as an experimental system for studying drought, salt and disease resistance. Furthermore, hairy roots culture is considered a effective way for produce useful plant secondary metabolites in now days due to its faster growth speed and capacity of growing well on medium without hormone. So the genetic transformation of E. sativa Mill mediated by Agrobacterium rhizogenes R1000 was carried out. This work laid a foundation for mass production of hairy roots and then useful secondary metabolites. The main results of this research were summarized as follows:1). The most vigorous strain for transforming E. sativa Mill was A. rhizogenes R1000, A₄ and LBA9402 were the second and third, respectively;Among the three types of explants (Cotyledon, Embryoid, Hypocotyls) tested, leaf explants was the most ideal explants for the transformation;Before infection, 2-days pre-culture of explants on MS medium without hormone could increase the transformation rates, amounted to 29.5%;Co-cultivation of explants with bacteria in MS medium was found that 20-minutes incubation was efficient for higher transformation frequency.2). PCR analysis revealed that rol B gene of T-DNA on Ri plasmid had been integrated into the genome of hairy roots of E. sativa Mill.3). The most effective media for induction of both calluses andregeneration plantlets from hairy roots of E. sativa Mill was MS medium + 2.0mg/L NAA+O.lmg/L BA and MS medium +1.5mg/L BA+0.5mg/L IAA, respectively.4). The best condition for the expanded reproduction of the hairy roots was to use fresh hairy roots, three-litre Erlenmeyer flasks containing one litre 1/2MS liquid medium, room temperature and a rotatory shaker at 110 rpm.5). SDS-PAGE of soluble proteins of common roots and hairy roots of E. sativa Mill showed that there is remarkable difference in band patterns between both.6). The total content of glucosinolates in hairy roots of E. sativa Mill was about 62.5 % times of that in its natural roots, determined by Bac^. HPLC analysis showed that four kinds of glucosinolates existed in the studied hairyroots of E. sativa Mill.7). The obtained hairy roots were successfully conserved usingcryopreservation of encapsulation—vitrifiation, with survival rates of 75%.