Plant Cell Reports

, Volume 12, Issue 7, pp 468–473

Stable transformation of the food yam Dioscorea alata L. by particle bombardment

Authors

  • Mahmut Tör
    • Wye College, University of London
  • Charles Ainsworth
    • Wye College, University of London
  • Sinclair H. Mantell
    • Wye College, University of London
Article

DOI: 10.1007/BF00234714

Cite this article as:
Tör, M., Ainsworth, C. & Mantell, S.H. Plant Cell Reports (1993) 12: 468. doi:10.1007/BF00234714

Summary

A biolistic particle gun was used to deliver genetic material into intact yam cells. Cultured suspension cells of D. alata were bombarded with microprojectiles coated with pBI221.2 DNA and histochemical assays were carried out to show transient GUS expression in bombarded cells. Stably transformed D. alata cells were recovered from cultured cells after bombardment with microprojectiles coated with pRT99gus harbouring both the nptII and uidA genes. Bombarded cells were selected on a medium containing geneticin (G418). Two months after bombardment, calli resistant to G418 were assayed for GUS expression. There was a 100% correlation between resistance to G418 and GUS expression. From these calli, four cell lines were established and GUS activity in each line was determined fluorometrically. The use of a specific GUS inhibitor showed that the GUS activity was due to the introduced uidA gene rather than to any intrinsic GUS-like activity originating from the plant. Incorporation of the introduced DNA into the plant genomic DNA was confirmed by Southern analysis.

Key words

Dioscorea alataGUStransformationparticle gun

Abbreviations

GUS

β-glucuronidase

MU

4-Methylumbelliferone

MUG

4-Methylumbelliferyl-β-D-glucuronide

PVP

Polyvinylpyrrolidone

SDS

Sodium dodecyl sulphate

TAE

Tris-acetate-EDTA buffer

X-Gluc

5-Bromo-4-chloro-3-indolyl-β-D-glucuronide

Copyright information

© Springer-Verlag 1993