Hairy Root Induction And Plant Regeneration Of Datura Arborea L.

Alkaloids, which has the second to the fourth atom of N in its molecular structure, belongs to one kind of plant secondary products. It provides many pharmacologically active compounds. D.arborea L.belongs to Datura, its leaf and flower can product Hyoscyamine and Scopolamine, it is one of the resource plants of Tropane alkaloids. Scopolamine is basal important medicine, and widely used in clinic. It has function to ease pain and hocus so as to be used to cure palsy,faint,angina of stomache,ache of trigeminal. There is a large demand of scopolamine in market.This study established invitro culture,genetic transformation and plants regeneration system based on hairy roots. It settles a firmly foundation for metabolic engineering of TA and molecular breeding of D.arborea L. to be realized using plant secondary metabolic engineering .An efficient in vitro plants regeneration system and clonal propagation via leaf of medical plant D.arborea L. was established. The results showed that: The highest shoots induction (91.4％) was achieved from leaves cultured on MS medium containing 3.0 mg/L 6-BA and 0.2mg/L NAA, with an average of 13.5 shoots developed per explant; The developed shoots were rooted on 1/2MS medium supplemented with 1.0mg/L IBA, the rooting rate is 94.5％.This study took the method of "leaf tray" to transform asepsis leaf by "disarmed" C58C1 strain which also carried Ri plasmid pRiA4 . We established the effective genetic transformation system of D.arborea L. and the conditions of transformation were optimized. The induction rate of hairy roots is more than 80.0％. The strain has strong ability to induce hairy roots when its OD is about 0.6. When explants was pre-cultured for 2 days and co-cultured with Agrobacterium rhizogenes for 3 days, we get the highest induction rate of hairy roots. Adding 100μmol/L AS in the medium also promoted the transferring rate(83.3％).PCR analysis for rolB and rolC confirmed that plasmid pRiA4 was integrated into genome of hairy roots of D.arborea L.The contents of alkaloids in the samples were analyzed by HPLC. It showed that the amount of Hyoscamine and Scopolamine in the hairy roots had much more times than that in the wild plant. So the result effectively showed that we can get the optimal hairy root clones which product large amount of Hyoscyamine and Scopolamine by genetic transformation . This study has important business values. It offers a good way to produce TA.Transfer the examined hairy roots to the buds induction medium to induce buds, then analyze the content of TA by HPLC. The system of plants regeneration of hairy roots is established. The result showed that the best medium for buds induction by "one-step" method is MS+6-BA2.0 mg/L +NAA0.2 mg/L. We take the "two-step" method to induce buds,the result showed that:the optical medium for callus induction is MS+6-BA4.0mg/L+KT0.5mg/L+2,4-D0.5mg/L; MS+6-BA4.0mg/L +NAA0.2mg/L is used to induct buds. The optical rooting medium is 1/2MS+IAA 0.1mg/L.The result of HPLC proved that the content of TA in regenerating plants is much more than that in the wild plant.Transplant the regenerated plants of hairy roots to flowerpots, most of the plants grow healthy and keep strong in a good condition.