Abstract
For pulse legume research to fully capitalise on developments in plant molecular genetics, a high throughput genetic transformation methodology is required. In Western Australia the dominant grain legume is Lupinus angustifolius L. (narrow leafed lupin; NLL). Standard transformation methodology utilising Agrobacterium tumefaciens on wounded NLL seedling shoot apices, in combination with two different herbicide selections (phosphinothricin and glyphosate) is time consuming, inefficient, and produces chimeric shoots that often fail to yield transgenic progeny. Investigation of hygromycin as an alternative selection in combination with expression of green fluorescent protein indicated that transformation of NLL apical cells was not the rate limiting step to achieve transgenic shoot materials. In this research it was identified that despite ready transformation, apical cells were not competent to regenerate. However a deep and broad wounding procedure to expose underlying axillary shoot and vascular cells to Agrobacterium, in combination with delayed selection proved successful, increasing initial explants transformation efficiency up to 75 % and generating axillary shoots with significant transgenic content. Based on knowledge gained from studies of plant chimeras, further subculture of these initial axillary shoots will result in development of low chimeric transgenic materials with heritable content. Furthermore, the method was also tested successfully on other Lupinus species, faba bea and field pea. These results demonstrate that development of a high yielding transformation methodology for pulse legume crops is achievable.
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Abbreviations
- Cc:
-
Co-cultivation medium
- CZ:
-
Central zone
- eGFP:
-
Enhanced green fluorescent protein
- GM:
-
Genetic manipulation;
- MPH:
-
Micropropagation medium with hygromycin
- NLL:
-
Narrow-leaf lupin
- Rg:
-
Regeneration medium
- PPT:
-
Phosphinothricin
- PZ:
-
Peripheral zone
- RZ:
-
Rib zone
- SAM:
-
Shoot apical meristem
- 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 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.
Author contributions
Project design and data analysis AHN, SJB and WE, transformations AHN and LH, plant tissue culture AHN, microscopy AHN, figure preparation AHN and SJB, manuscript preparation AHN, SJB, WE and LH.
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Nguyen, A.H., Hodgson, L.M., Erskine, W. et al. An approach to overcoming regeneration recalcitrance in genetic transformation of lupins and other legumes. Plant Cell Tiss Organ Cult 127, 623–635 (2016). https://doi.org/10.1007/s11240-016-1087-1
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DOI: https://doi.org/10.1007/s11240-016-1087-1