Planta Med 2003; 69(2): 143-147
DOI: 10.1055/s-2003-37709
Original Paper
Physiology
© Georg Thieme Verlag Stuttgart · New York

Agrobacterium tumefaciens-Mediated Genetic Transformation of the Cardenolide-Producing Plant Digitalis minor L.

Ester Sales1 , Juan Segura1 , Isabel Arrillaga1
  • 1Departamento de Biología Vegetal, Facultad de Farmacia, Universidad de Valencia, Burjassot, Valencia, Spain
Further Information

Publication History

Received: May 13, 2002

Accepted: October 3, 2002

Publication Date:
07 March 2003 (online)

Abstract

A repeatable transformation system has been established for Digitalis minor using Agrobacterium tumefaciens. Leaf explants from 30-day-old seedlings were inoculated with either EHA105 (carrying the nptII and gusA genes) or AGL1 (with the bar and gusA genes) strains. Among the tested factors influencing T-DNA transfer to plants, the EHA105 strain and the addition of acetosyringone to the co-culture medium increased transformation. The highest transformation efficiency (8.4 %) was obtained when freshly isolated explants, soaked in a bacterial suspension with an OD550 of 0.9, were subcultured on selection medium after a 4-day co-culture with the bacteria. Evidence of stable transgene integration was obtained by PCR, growth on media selective for nptII or bar genes, and expression of the gusA gene. Southern hybridisation, performed in six plants, provided information about the number of inserts. More than 200 transgenic plants were recovered from 65 independent explants. Thirty of these plants were successfully established in soil. This is the first report on transgenic Digitalis spp plants using an A. tumefaciens-mediated leaf disc transformation procedure.

Abbreviations

AS:acetosyringone

BA:benzyladenine

CEF:cefotaxime

GEN:geneticin

IAA:indoleacetic acid

KAN:kanamycin

MS:Murashige & Skoog medium (1)

PPT:phosphinothricin

References

  • 1 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.  Physiol Plant. 1962;  15 473-97
  • 2 Verpoorte R, Alfermann A W. Metabolic engineering of plant secondary metabolism. Kluwer Academic Publishers Dordrecht; 2000
  • 3 Kreis W, Reinhard E. The production of secondary metabolites by plant cells cultivated in bioreactors.  Planta Med. 1989;  55 409-16
  • 4 Stuhlemmer U, Kreis W, Eisenbeiss M, Reinhard E. Cardiac glycosides in partly submerged shoots of Digitalis lanata .  Planta Med. 1993;  59 539-45
  • 5 Haussmann W, Kreis W, Stuhlemmer U, Reinhard E. Effects of various pregnanes and two 23-nor-5-cholenic acids on cardenolide accumulation in cell and organ cultures of Digitalis lanata .  Planta Med. 1997;  63 446-53
  • 6 Pradel H, Lehmann U, Diettrich B, Luckner M. Hairy root cultures of Digitalis lanata. Secondary metabolism and plant regeneration.  J Plant Physiol. 1997;  151 209-15
  • 7 Lehmann U, Moldenhauer D, Thomar S, Diettrich B, Luckner M. Regeneration of plants from Digitalis lanata cells transformed with Agrobacterium tumefaciens carrying bacterial genes encoding neomycin phosphotransferase II and β-glucuronidase.  J Plant Physiol. 1995;  147 53-7
  • 8 Sales E, Nebauer S G, Arrillaga I, Segura J. Plant hormones and Agrobacterium tumefaciens strain 82.139 induce efficient plant regeneration in the cardenolide-producing plant Digitalis minor .  J Plant Physiol. 2002;  159 9-16
  • 9 Hellens R, Mullineaux P. A guide to Agrobacterium binary Ti vectors.  Trends Plant Sci. 2000;  5 446-51
  • 10 Jefferson R A, Kavanagh T A, Bevan M V. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.  EMBO J. 1987;  6 3901-7
  • 11 Toki S, Takamatsu S, Noriri C, Ooba S, Anzai H, Iwata M, Christensen A H, Quail P H, Uchimiya H. Expression of a maize ubiquitin gene promoter-bar chimeric gene in transgenic rice plants.  Plant Physiol. 1992;  100 1503-7
  • 12 Manders G, Otoni W C, d’Utra F B, Blackhall N W, Oower J B, Davey M R. Transformation of passionfruit (Passiflora edulis fv flavicarpa Degener.) using Agrobacterium tumefaciens .  Plant Cell Rep. 1994;  13 697-702
  • 13 Stummer B E, Smith S E, Landgridge P. Genetic transformation of Verticordia grandis (Myrtaceae) using wild-type Agrobacterium rhizogenes and binary Agrobacterium vectors.  Plant Sci. 1995;  111 51-62
  • 14 Sawada H, Ieki H, Matsuda I. PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains.  App Environ Microbiol. 1995;  6 828-31
  • 15 Schöpke C, Taylor N, Cárcamo R, Konan N DK, Marmey P, Henshaw G G, Beachy R N, Fauquet C. Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures.  Nat Biotechnol. 1996;  14 731-5
  • 16 Diemer F, Caissard J C, Moja S, Jullien F. Agrobacterium tumefaciens-mediated transformation of Mentha spicata and Mentha arvensis .  Plant Cell Tiss Org Cult. 1999;  57 75-8
  • 17 Cervera M, Pina J A, Juárez J, Navarro L, Peña L. Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration.  Plant Cell Rep. 1998;  18 271-8
  • 18 Gershenzon J, Kreis W. Biochemistry of terpenoids: monoterpenes, sesquiterpenes, diterpenes, sterols, cardiac glycosides and steroid saponins. In: Wink M, editor Biochemistry of plant secondary metabolism, Annual Plant Reviews 2. Sheffield Academic Press Sheffield; 1999: pp 222-99

Isabel Arrillaga

Departamento de Biología Vegetal

Facultad de Farmacia

Universidad de Valencia

Avda. Vicent A. Estelles s/n

46100 Burjassot

Valencia

Spain

Email: isabel.arrillaga@uv.es

Phone: +34-96-386-4922

Fax: +34-96-386-4926