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

  • Genetic Transformation and Hybridization
  • Published:
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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.

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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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Acknowledgements

We would like to acknowledge funding for research from the New Zealand Forestry Consortiums, Geenz Ltd and LAG and the Foundation for Research, Science and Technology of New Zealand. Technical assistance was provided by Mrs Barbara Geddes, Ms Karen Hignett, Mr Kevin Steele, Mrs Cathie Reeves and Mrs Susan van der Maas. We thank North Carolina State University for the permission to use EHA105 pTOK47. The contribution of Crop and Food Research in finalising the construction of pGUL and pKEA is acknowledged, as is the permission to use the vectors in this research programme. NPTII ELISA and GUS fluorometric analysis was assisted by Mrs Tomoko Pearson, Mrs Judy Moody, Miss Nicola Moore and Mrs Megan Skiffington. We are grateful to Miss Lorelle Phillips for assistance with extraction of DNA, Dr Karen Nielsen and Miss Carmella Lee for assistance with probing the Southern blots. We would also like to thank Dr Phillip Wilcox for statistical analysis, Prof. Richard Gardner for his advice and guidance, as well as Dr Krystyna Klimaszewska and the three anonymous reviewers for critical review of the manuscript. Mrs Bev Harniss and Mrs Tania Elder are gratefully acknowledged for preparing the manuscript for publication.

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  1. Cellwall Biotechnology Centre, Forest Research, Private Bag 3020, Rotorua, New Zealand

    J. A. Charity, L. Holland, L. J. Grace & C. Walter

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  1. J. A. Charity
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  2. L. Holland
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  3. L. J. Grace
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  4. C. Walter
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Correspondence to J. A. Charity.

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Communicated by S.A. Merkle

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Charity, J.A., Holland, L., Grace, L.J. et al. Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures. Plant Cell Rep 23, 606–616 (2005). https://doi.org/10.1007/s00299-004-0851-6

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  • DOI: https://doi.org/10.1007/s00299-004-0851-6

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