Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Manuel Rey, María Victoria González, Ricardo J. Ordás, Raffaela Tavazza, Giorgio Ancora

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Transfer and expression of the β-glucuronidase gene (gusA) in cultured cotyledons of radiata pine (Pinus radiata D. Don) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pBI121, delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters; the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus-expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.
Original languageEnglish
Pages (from-to)630 - 636
Number of pages7
JournalPhysiologia Plantarum
Volume96
Issue number4
DOIs
Publication statusPublished - 1996
Externally publishedYes

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Physiology
  • Genetics
  • Plant Science
  • Cell Biology

Cite this