Development of a genotype independent and transformation amenable regeneration system from shoot apex in rice (Oryza sativa spp. indica) using TDZ

Abstract

Agrobacterium-mediated transformation of indica rice has been established in only a limited number of cultivars because the regeneration of plants from transformed embryogenic calli is highly cultivar-specific. Establishment of a highly efficient plant regeneration system from shoot apex explants applicable to many cultivars of indica rice will accelerate the application of transformation technology in breeding programs and functional genomics study. We established an efficient shoot multiplication and plant regeneration system from shoot apices of indica rice using thidiazuron (TDZ) as a plant growth regulator. Shoot apices cultured on MS basal medium devoid of plant growth regulators formed solitary shoots in 90% of cultures. Addition of TDZ or benzylaminopurine to regeneration medium significantly influenced formation of multiple shoots directly from shoot apex explants without an intervening callus stage. Best shoot proliferation response (10.3 shoots per explant) was recorded when shoot apices were cultured on media supplemented with 4 mg/l TDZ. No synergistic effect on shoot proliferation was observed when indole-3-acetic acid and indole-3-butyric acid were supplemented to media containing 4 mg/l TDZ. The regeneration system was efficient in evoking multiple shoot proliferation in eight different cultivars of indica rice. Shoots were rooted in MS basal medium and plantlets were acclimatized with 100% survival rate. The shoot apex explants of all the eight cultivars of indica rice were found competent to Agrobacterium-mediated transformation while explants from IR-64 showed highest transient GUS expression. This variety-independent transformation amenable regeneration system from shoot apices may widely be applicable for genetic transformation of indica varieties.

Efficient regeneration and Agrobacterium-mediated transformation protocol for recalcitrant indica rice (Oryza sativa L.).

“…Moreover, TDZ has also been known to be (Lincy and Sasikumar, 2010). A concentration of 4 mg/L of TDZ was used effectively in producing the most number of shoots from shoot apices of two indica cultivars -KDML105 (Yookongkaew et al, 2007) and IR64 (Dey et al, 2012). Both studies were in agreement with this study in which the optimal TDZ concentration was 4 mg/L and an overall shorter shoot height was observed in indica cultivar MR219.…”

“…Besides that, genotypic variations was thought to influence the immersion time needed to infect indica rice shoot apices of different cultivars. A 30 minutes of immersion period was needed for indica IR64 which was in accordance with this current study (Dey et al, 2012). However, a relatively brief immersion time was only needed for indica varieties white ponni (10 minutes), pusa basmati 1 (10 minutes) and KDML105 (1 minute) (Arockiasamy and Ignacimuthu, 2007;Yookongkaew et al, 2007).…”

“…Both studies were in agreement with this study in which the optimal TDZ concentration was 4 mg/L and an overall shorter shoot height was observed in indica cultivar MR219. In other words, TDZ was suggested to be a genotype-independent cytokinin since Dey et al (2012) reported that they were no significant differences on the mean shoot numbers in eight other indica rice varieties. Nonetheless, increased TDZ concentration has greatly reduced the number of axillary rice shoots, fresh and dry weight of the in-vitro rice shoots (Dey et al, 2012).…”

“…In other words, TDZ was suggested to be a genotype-independent cytokinin since Dey et al (2012) reported that they were no significant differences on the mean shoot numbers in eight other indica rice varieties. Nonetheless, increased TDZ concentration has greatly reduced the number of axillary rice shoots, fresh and dry weight of the in-vitro rice shoots (Dey et al, 2012). Similar findings also explained that high concentrations of TDZ could be deleterious and the shoots produced were weak, fasciated and morphologically distorted (Subotić et al, 2008;Guo et al, 2011).…”

Efficient plant regeneration protocol for finger millet [Eleusine coracana (L.) Gaertn.] via somatic embryogenesis

“…The use of shoot apex in regeneration is critical since it can divide to produce viable new organs such as leaves, stems and adventitious roots (Itoh et al, 2006). Shoot apex is also most beneficial for its quick development therefore it can allow rapid development of plants (Ceasar and Ignacimuthu, 2008;Dey et al, 2012). Auxins have been shown to play an important role in inducing callus (Anjaneyulu et al, 2011).…”