The effect of cocultivation treatments on transformation efficiency in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) |
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Authors: | Lenka ?vábová Miroslav Griga |
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Institution: | (1) Plant Biotechnology Department, AGRITEC Ltd., Zemědělská 2520/16, CZ 787 01 Šumperk, Czech Republic |
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Abstract: | The effect of chemical additives (acetosyringone, AS; L-cysteine, CYS; dithiothreitol, DTT; glutathione, GSH; cellulase, CEL;
pectinase, PEC) and light regimes (16/8 light/dark photoperiod, 16L/8D; continuous light, 24L; continuous dark, 24D) applied
during cocultivation procedure of pea explants with Agrobacterium tumefaciens on transformation efficiency was studied. A hypervirulent strain of A. tumefaciens EHA 105 with two plasmids, namely pGT89 and pBIN19, both carrying reporter gus-int gene, and bar or nptII selectable marker gene, respectively, was used for genetic transformation of cotyledonary node explants of three dry seed
pea cultivars Adept, Komet and Menhir. The focus was laid on cocultivation step (48 h) of transformation protocol. After chemical
or physical treatments, transient GUS expression was recorded 20 days after cocultivation as a measure of successful transformation,
using a four category scale (0 – without GUS expression, 1 – weak, 2 – medium and 3 – strong GUS expression) for calculation
of IGE (Intensity of GUS Expression). Of the tested chemical cocultivation additives, 100 μM AS and 50 mg CYS significantly
improved GUS expression (IGE value), while DTT, GSH and both macerating enzymes (CEL, PEC used either separately or in combination)
either had no positive effect or were even negative. There were no statistically significant differences between the light
regimes tested. Nevertheless, cocultivation in 24L, without chemical additives, reproducibly resulted in the highest frequency
of explants scored in category 3 of GUS expression (followed by 24D and 16L/8D treatment). However, application of 100 μM
AS reverted this trend. Cv. Adept yielded higher transformation frequencies than cvs. Menhir and Komet. Plasmid pGT89 produced
a higher IGE value than pBIN19. Based on our results, the improved cocultivation step for pea consists of 48 h cocultivation
at 20 ± 2°C, with 50 mg l−1 CYS and 100 μM AS, 16L/8D photoperiod (or without AS in continuous light). |
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Keywords: | Acetosyringone Agrobacterium-mediated transformation Cellulase Cocultivation additives Dithiothreitol Glutathione L-cysteine Light regime Pectinase |
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