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.
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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
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Tör, M., Ainsworth, C. & Mantell, S.H. Stable transformation of the food yam Dioscorea alata L. by particle bombardment. Plant Cell Reports 12, 468–473 (1993). https://doi.org/10.1007/BF00234714
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DOI: https://doi.org/10.1007/BF00234714