In planta transformation of narrow-leafed lupin (Lupinus angustifolius) seedlings
Ratanasanobon, K.N., Wylie, S. and Jones, M.G.K. (2004) In planta transformation of narrow-leafed lupin (Lupinus angustifolius) seedlings. In: ComBio 2004, 26 - 30 September 2004, Burswood Convention Centre, Perth.
In planta transformation of narrow-leafed lupin (Lupinus angustifolius) by vacuum infiltration with Agrobacterium was investigated. The apical area of young shoots was wounded by sonication prior to infiltration with Agrobacterium cells containing a reporter gene (35S gus) and a selectable marker gene (35S bar) for herbicide tolerance. Sonication time and infiltration time were optimized. Thirteen minutes of sonication with 10 minutes of infiltration with Agrobacterium tumefaciens AGL0 were the best conditions for delivery of A. tumefaciens cells. Longer times increased the number of fatalities of seedlings. Other important parameters, namely bacterial growth phase and the composition of infiltration solution, were investigated to increase stable transformation as determined by the number of blue spots in the tissue. Expression of the reporter gene was not stable over time. Seeds from 1720 treated plants were screened for expression of the bar gene by the application of 50 mg/l phosphinothicin (PPT). All the seedlings died in a manner similar to the controls. We then looked at the viability of Agrobacterium cells over time on the seedlings following infiltration to determine if the cells were dying before they could transfer the T-DNA to the seedlings. Firstly, we determined by gus gene expression on explants in vitro that T-DNA transfer began at Day 3 of the co-cultivation period, then increased rapidly to a maximum by Day 5. The viability of A. tumefaciens cells in planta was examined by counting the numbers of viable cells on the treated seedlings over time post-inoculation. Cells were isolated from seedlings and plated on medium containing lactose as a carbon source. The presence of 3-ketolactose around colonies indicated that they were A. tumefaciens. The assay showed that the cells had a very low rate of survival on the seedlings, and that by Day 4, when T-DNA transfer in vitro was approaching maximum efficiency, survival of A. tumefaciens cells on the plant was very low, about 103 times less than required for successful transformation of lupins via the routine in vitro route (CLIMA lupin transformation protocol). These findings may explain why in planta transformation has not yet been possible in lupin.
|Publication Type:||Conference Item|
|Murdoch Affiliation:||Western Australian State Agricultural Biotechnology Centre|
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