An efficient regeneration system and Agrobacterium-mediated transformation of Chinese upland rice cultivar Handao297

Abstract

A highly efficient tissue culture system and Agrobacterium-mediated transformation protocol for Chinese upland rice cultivar Handao297 has been established with mature embryos as explants. Up to 81.2% of mature embryos were induced to regenerate good-quality calli on NB medium (a medium combining N6 macronutrient components and B5 micronutrient and organic components) containing 3 mg/l 2,4-dichlorophenoxyacetic acid in 10 days. More than 80% of the calli were morphogenic within 1 week and regenerated green plantlets within 1 month on Murashige and Skoog medium supplemented with 0.5 mg/l 6-benzyladenine, 0.5 mg/l kinetin, 1 mg/l zeatin, 0.5 mg/l thidizazuron (TDZ), 0.5 mg/l naphthaleneacetic acid, 0.15 mg/l indoleacetic acid, and 0.15 mg/l indolebutyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of upland rice Handao297. Furthermore, some important factors affecting transformation frequency were investigated with Agrobacterium strain AGL1 containing the plasmid pCAM-BIA1381. The addition of 30 mg/l hygromycin B followed by 60 mg/l hygromycin B to the selection induction medium facilitated the revival of calli from selection and reduced false positive calli. Hygromycin B at 10 mg/l was most effective in suppressing non-transgenic callus growth in the differentiation medium. The addition of TDZ to the differentiation medium promoted the morphogenesis of calli and facilitated the generation of adventitious shoots by five to tenfold in comparison to medium without TDZ.

Efficient Agrobacterium-mediated transformation of Pennisetum glaucum (L.) R. Br. using shoot apices as explant source

“…Zhao et al (2000); Howe et al (2006) in sorghum and Ishida et al (2007) in maize, reported 5 min of inoculation period as optimal, while Sarker and Biswas (2002) reported 50 min of inoculation period as optimal along with 5 min vacuum treatment in rice. Our results are in accord with Lee et al (2006) in orchardgrass and Zhao et al (2011) in chinese upland rice, where they recommended an inoculation period of 30 min.…”

Osmotic stress stimulates shoot organogenesis in callus of rice (Oryza sativa L.) via auxin signaling and carbohydrate metabolism regulation

“…Many factors affect shoot regeneration in plant tissue culture: such as genotype Glowacha et al 2010;Park et al 2011), exogenous and endogenous hormones (Jiménez 2005;Barreto et al 2010;Sun and Hong 2010;Huang et al 2012), carbon sources (Huang and Liu 1998;Iraqi et al 2005;Huang et al 2006;Silva 2010;Feng et al 2010), and osmotic requirements (Geng et al 2008;Pan et al 2010;Huang et al 2012). Despite many shoot regeneration and transformation protocols developed in rice culture, the regeneration frequency is low and varies highly among cultivars (Al-Khayri et al 1996;Hoque and Mansfied 2004;Khaleda and Al-Forkan 2006;Zhao et al 2011). The regeneration ability of non-regenerable rice callus could be promoted by treatment with an osmotic agent such as sorbitol or mannitol Geng et al 2008;Feng et al 2011).…”

Agrobacterium‐mediated transformation of reed (Phragmites communisTrinius) using mature seed‐derived calli

“…Incorporation of a single or very few copy of transgenes is one of the advantages of Agrobacterium ‐mediated transformation. Similar results were also obtained in other grass species (Lee et al ., , ; Kim et al ., ; Zhao et al ., ). RT‐PCR analysis also revealed high‐level expression of GUS mRNA as the gene is driven by a constitutively expressing CaMV 35S promoter (Fig.…”

“…Some evidence also suggests that acetosyringone may suppress virulence in some strain/plant species interactions at a high concentration of acetosyringone (Godwin et al, 1991). By contrast, positive effects of acetosyringone on transformation efficiency have been demonstrated in other monocot species, such as rice (Zhao et al, 2011), maize (Ishida et al, 1996), barley (Trifonova et al, 2001), and wheat (He et al, 2010). Effect of cocultivation period.…”