Establishment of Withania somnifera hairy root cultures for the production of withanolide A.
Withania sominifera (Indian ginseng) was transformed by Agrobacterium rhizogenes. Explants from seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain R1601. Hairy (transformed) roots were induced from cotyledons and leaf explants. The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptII and rolB specific primers and, subsequently, by Southern analysis for the presence of nptII and rolB genes in the genomes of transformed roots. Four clones of hairy roots were established; these differed in their morphology. The doubling time of faster growing cultures was 8-14 d with a fivefold increase in biomass after 28 d compared with cultured, non-transformed seedling roots. MS-based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated (SH, LS and N6), with MS-based medium supplemented with 40 g/L sucrose being optimal for biomass production. Cultured hairy roots synthesized withanolide A, a steroidal lactone of medicinal and therapeutic value. The concentration of withanolide A in transformed roots (157.4 microg/g dry weight) was 2.7-fold more than in non-transformed cultured roots (57.9 microg/g dry weight).
Murthy
Hosakatte N
HN
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK. nmurthy60@yahoo.co.in
Dijkstra
Camelia
C
Anthony
Paul
P
White
Daniel A
DA
Davey
Mike R
MR
Power
J Brian
JB
Hahn
Eun J
EJ
Paek
Kee Y
KY
D015139:Blotting, Southern; D018929:Cell Culture Techniques / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis; D005798:Genes, Bacterial; D018515:Plant Leaves / Q000382:microbiology; D018517:Plant Roots / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic; D032341:Withania / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D054358:Withanolides
C433551:3-rhamnopyranosyl(1-4)-glucopyranosyl-12-diacetoxy-20-hydroxywitha-5,24-dienolide; D003470:Culture Media; D004269:DNA, Bacterial; D054358:Withanolides; D004875:Ergosterol