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Using green fluorescent protein sheds light on Lupinus angustifolius L. transgenic shoot development

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Abstract

Narrow-leaf lupin (NLL) is the main legume crop grown in rotation with wheat and other cereals in Western Australia. Efforts to improve NLL germplasm by use of genetic technologies have been hampered by the lack of an efficient genetic transformation method, an issue that is in common with dominant crop legumes globally. Prior research has primarily used the bar gene for phosphinothricin (PPT) resistance. The aim of recent research has been to investigate alternative selection methodologies, in order to determine whether the limitations of low frequency of transgenic shoots, combined with chimerism at T0 could be overcome. Investigation of hygromycin resistance as a selectable marker compared to PPT is reported here. The results suggested that hygromycin resistance was a more suitable selectable marker for NLL transformation than PPT. Surprisingly, from investigation of transformation using the GUS reporter gene, it was also observed that transformation frequency was greater when selection treatment was reduced or delayed, compared to the existing protocols. To further investigate this observation, an eGFP expressing construct was prepared. Observations within the first week after Agrobacterium exposure of lupin explants demonstrated that transformation of NLL explant cells was not a rate-limiting step. Instead, the results indicated that the current selection methodology was killing the cells that were competent to regenerate into transgenic shoots. It was concluded that further research on the development of the treated explants should focus on delayed selection and exposure to Agrobacterium of cells below the apical meristem.

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Abbreviations

eGFP:

Enhanced green fluorescent protein

GUS:

Beta-glucuronidase

NLL:

Narrow-leaf lupin

MPH:

Micropropagation medium with hygromycin

PCR:

Polymerase chain reaction

PPT:

Phosphinothricin

Rg:

Regeneration medium

T0:

Initial generation of transgenic shoot

T1:

Progeny of T0 generation

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Acknowledgments

The first author acknowledges with deep gratitude the Australian Award Scholarship funded by the Australian Government. The authors sincerely thank Dr Mark Waters (School of Chemistry and Biochemistry, The University of Western Australia) for the Gateway vectors pHGWFS7,0, pB7WG2 and pDONR221. The authors acknowledge the facilities of CELLCentral, School of Anatomy Physiology & Human Biology. The University of Western Australia and the facilities, scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis (CMCA), The University of Western Australia, a facility funded by the University, State and Commonwealth Governments.

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Authors and Affiliations

  1. Centre for Plant Genetics and Breeding (PGB), School of Plant Biology M080, The University of Western Australia, Crawley, WA, 6009, Australia

    An H. Nguyen & William Erskine

  2. School of Plant Biology M090, The University of Western Australia, Crawley, WA, 6009, Australia

    An H. Nguyen, Teguh Wijayanto & Susan J. Barker

  3. Faculty of Biology, Hanoi University of Science, Hanoi, Vietnam

    An H. Nguyen

  4. Fakultas Pertanian, Universitas Halu Oleo, Kendari, Sulawesi Tenggara, Indonesia

    Teguh Wijayanto

  5. Institute of Agriculture M082, The University of Western Australia, Crawley, WA, 6009, Australia

    William Erskine & Susan J. Barker

Authors
  1. An H. Nguyen
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  2. Teguh Wijayanto
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  3. William Erskine
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  4. Susan J. Barker
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Correspondence to Susan J. Barker.

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Nguyen, A.H., Wijayanto, T., Erskine, W. et al. Using green fluorescent protein sheds light on Lupinus angustifolius L. transgenic shoot development. Plant Cell Tiss Organ Cult 127, 665–674 (2016). https://doi.org/10.1007/s11240-016-1079-1

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  • DOI: https://doi.org/10.1007/s11240-016-1079-1

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