An investigation of an Agrobacterium-mediated transformation protocol for L. sativa L., yielded some insights into both methods of evaluation, and to the recalcitrance of a head lettuce cultivar. The methodology employed a direct count of the numbers of transformed cells or cell clusters as visualised by the generation, in histo, of the blue precipitate resulting from the activity of T-DNA derived β-Glucuronidase on 5- bromo-4-chloro-3-indolyl-glucuronide. This method yielded insights into the rise and decline of the numbers of transgenic foci over the first fourteen days following co cultivation. A time window from day eight to day ten post inoculation was found when the numbers of blue spots (representing metabolically viable, transgene-expressing cells or cell clusters) appeared to reach a maximum. This allowed the evaluation of gene transfer frequencies before the effects of selection, and the differential capacities of the explants' cells became influential factors.
An experimental design which accounts for the grouping of the explants into pottles was investigated for its potential to represent the patterns of blue spots observed, with a view to comparing the modifications to a transformation protocol in terms of the numbers of blue spots, observed after staining at ten days post inoculation.
Despite some technical complications, the experimental design showed some promise towards the possibility of evaluating the frequencies of Agrobacterium-mediated gene transfer before the confounding effects of the differential regeneration capacities of the explants' cells in a selective medium became an influential factor.
The effects of acetosyringone supplementation of the Agrobacterium overnight culture or the cocultivation medium yielded no appreciable increase in the numbers of blue spots observed at ten days post inoculation. Neither did the effects of mild UV irradiation of the explants during co cultivation appear to be augmentative. It was found that the recalcitrance of the head lettuce cultivar, Prizehead, is probably not due to an inability to accept and express T-DNA.[Show full abstract]
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