Abstract
Weed management is a critical issue in the production of narrow-leaf lupin (NLL) (Lupinus angustifolius L.). The gene pool has limited herbicide tolerance, early vigour or short season productivity, hampering conventional breeding approaches. Previous transformation of NLL used the bar gene encoding tolerance to the herbicide glufosinate, for selection. However that methodology achieved <1 % success at T1, with one confounding factor being chimerism of the T0 shoots. Therefore glyphosate tolerance was examined as a potential alternative selectable marker with possible commercial application. Agrobacterium-mediated gene transfer using four vectors with two promoter and two transit peptide (tp) sequences in factorial combination was followed by three methods of selection post-transformation, following failure to achieve transformation by the published glufosinate selection method. An overall heritable transformation efficiency of 0.14 % was achieved from a total of 36,607 inoculated explants. Transformation with constructs containing the ubiquitin 3 promoter had a higher success rate (0.20 %) than with the CaMV promoter (0.07 %) however no difference between the two tp sequences was observed. The highest rate of transformation (0.45 %) was obtained by glyphosate selection during early phase of growth on regeneration media. This regeneration selection method more frequently produced highly tolerant events, with more heritable expression stability in T2 generation than alternative methods. Single insertion, single copy events were obtained. However these were recovered with significantly lower frequency than previously observed with the glufosinate selection method. Nonetheless such events have potential to contribute weed management options for broad-acre NLL production.
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Acknowledgments
The authors gratefully acknowledge the financial support of the Grain Research and Development Corporation (GRDC) through research grant UWA00129. The authors also thank Dr. Linda Tabe (CSIRO) for the vector pTAB 10; Professor Craig Atkins and Dr. Penny Smith for use of the transit peptide sequences; Dr. Jorge Mayer; Professors Craig Atkins and Kadambot Siddique for useful discussion and contributions to this study.
Author contribution
Project design SJB and WE; construct development KM, SA and MFH; transformation, shoot culture YK, NK, PK, LH and COL; molecular testing MFH, SA, KM, COL, DD, LH, and PK; plant growth and herbicide bioassay JJ, YK, LH and PS; communication and team integration TF and LH; data analysis SJB, PS, WE, LH, DD, YK, PK; plant images YK; manuscript preparation SJB, WE, MFH, LH and PS.
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Barker, S.J., Si, P., Hodgson, L. et al. Regeneration selection improves transformation efficiency in narrow-leaf lupin. Plant Cell Tiss Organ Cult 126, 219–228 (2016). https://doi.org/10.1007/s11240-016-0992-7
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DOI: https://doi.org/10.1007/s11240-016-0992-7