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 lM 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 lM 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 lM AS, 16L/8D photoperiod (or without AS in continuous light).

The effect of different antibiotics on the elimination of Agrobacterium and high frequency Agrobacterium-mediated transformation of indica rice (Oryza sativa L.)

“…Transient GUS expression was frequently used to assess stable transformation efficiency either by evaluating the frequency of GUS explants or by evaluating the quality/quantity of GUS expression. The latter demonstrated a better correlation between transient and stable transformation (Švábová & Griga 2008). Similarly, the transient GUS expression was determined by GUS + explants and their intensity of blue colour.…”

Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation

“…Pre-treatment of A. rhizogenes R1000 with 30 lM acetosyringone was optimum for genetic transformation of Torenia fournieri (Tao and Li 2006). Svábová and Griga (2008) reported that application of 100 lM acetosyringone in combination with 50 mg Lcysteine l -1 during co-cultivation of cotyledonary node explants of three dry seed pea cultivars with Agrobacterium tumefaciens EHA 105 carrying pGT89/pBin19 significantly improved intensity of transient GUS expression. Agrobacterium tumefaciens precultured with acetosyringone has also been shown to be endowed with an enhanced efficiency of transforming several plants including cabbage (Brassica oleracea var.…”

“…In Arachis hypogaea, the most suitable co-cultivation period was 48 h (Karthikeyan et al 2007), whereas in Centella asiatica cocultivation for 7 days was the most effective (Kim et al 2007). Co-cultivation for 48 h with 16 h photoperiod (or continuous illumination without acetosyringone) was most suitable for pea transformation using A. tumefaciens EHA105 (Svábová and Griga 2008).…”

Resolving browning during the establishment of explant cultures in Vicia faba L. for genetic transformation

“…Although, tested cultivars of broad bean were very susceptible to AMT, the regeneration of transgenic broad bean plants still remains the main problem of the practical use of Agrobacterium-mediated gene delivery in the broad bean (Jelenić et al, 2000). A range of chemical substances with a diff erent nature and action were used for cocultivation during AMT with the aim of enhancing the fi nal effi ciency of the transformation protocol (Švábová and Griga, 2008). Generally, these substances work through induction of virulence -acetosyringone (AS), reduction of resistance of the recipient cell (thiol compounds, antioxidants), or a facilitation to penetrate through the cell wall (mechanically or chemically) (Dandekar and Fisk, 2005;Opabode, 2006;Vrbová et al, 2013).…”