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The effect of cocultivation treatments on transformation efficiency in pea (Pisum sativum L.)

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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Abbreviations

AS:

Acetosyringone

CCM:

Cocultivation medium

CEL:

Cellulose

CYS:

L-Cysteine

d/n:

Day/night

DTT:

Dithiothreitol

GSH:

Glutathione

IGE:

Intensity of GUS Expression

PEC:

Pectinase

24L:

Continuous light regime

24D:

Continuous dark regime

16L/8D:

16 h light/8 h dark photoperiod

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Acknowledgements

The authors wish to thank Ms. J. Vychodilová for excellent technical assistance and Dr. Sergio Ochatt for critical reading of manuscript and helpfull comments. This research was financially supported by the National Agency for Agricultural Research (grant QF 3072) and Ministry of Education of Czech Republic (grants MSM 2678424601 and 1M06030).

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Correspondence to Miroslav Griga.

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Švábová, L., Griga, M. The effect of cocultivation treatments on transformation efficiency in pea (Pisum sativum L.). Plant Cell Tiss Organ Cult 95, 293–304 (2008). https://doi.org/10.1007/s11240-008-9443-4

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Keywords

  • Acetosyringone
  • Agrobacterium-mediated transformation
  • Cellulase
  • Cocultivation additives
  • Dithiothreitol
  • Glutathione
  • L-cysteine
  • Light regime
  • Pectinase