Plant Cell Reports

, Volume 23, Issue 9, pp 606–616 | Cite as

Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures

  • J. A. Charity
  • L. Holland
  • L. J. Grace
  • C. Walter
Genetic Transformation and Hybridization

Abstract

An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.

Keywords

Genetic engineering Conifers Stable transformation Nurse cultures Plant regeneration 

Abbreviations

bar

Gene coding for phosphinothricin acetyl transferase

EDM

Embryo development medium

ELISA

Enzyme linked immuno sorbent assay

GMO

Genetically modified organism

MUG

4-Methylumbelliferyl β-d-glucuronide

nptII/NPTII

Neomycin phosphotransferase gene or protein

PGR

Plant growth regulator

PVPP

Polyvinylpolypyrolidone

uidA/GUS

β-Glucuronidase gene or protein

SEM

Standard error of the mean

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • J. A. Charity
    • 1
  • L. Holland
    • 1
  • L. J. Grace
    • 1
  • C. Walter
    • 1
  1. 1.Cellwall Biotechnology CentreForest ResearchRotoruaNew Zealand

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