Abstract
Rice shoot apices have been selected as the potential explant for genetic transformation since lesser time for regeneration and lower incidence of somaclonal variants were reported as compared to embryogenic callus. Utilizing this explant, efficient regeneration and Agrobacterium transformation protocol have been developed for the recalcitrant Malaysian indica rice, MR219. In this study, 4 mg/L of KIN was selected as the suitable cytokinin for multiple shoot formation as compared to TDZ and BAP. KIN was able to produce relatively high number of shoots as well as producing good quality shoots with the highest total chlorophyll content, and moderately high total soluble sugar content. In-vitro shoots were successfully acclimatized in glasshouse without signs of infertility. The optimization of the Agrobacterium-mediated protocols revealed that Agrobacterium supervirulent strain EHA101, shoot apices derived from 4 days old seedlings, excised wounded explants, 30 minutes of bacterial immersion time and 72 hours (3 days) of co-cultivation gave the highest percentage of plant transiently expressing two reporter genes – GFP and GUS. After 30 days of hygromycin selection and PCR verification of the putative transformants, the transformation efficiency was reported to be 5.83%. The established regeneration system and transformation protocol for MR219 rice shoot apices will serve as an essential fundamental platform for the production of transgenic rice with superior traits.
Highlights
90% of world’s rice was produced and consumed in Asia (Ziegler and Barclay, 2008)
In-vitro multiple shoot induction Determination of a suitable cytokinin and its optimal concentration are crucial as rice shoot apices responded differently towards different cytokinins
Three different cytokinins – KIN, benzyladenine aminopurine (BAP) and TDZ were evaluated for their potential of inducing multiple shoots from shoot apices
Summary
90% of world’s rice was produced and consumed in Asia (Ziegler and Barclay, 2008). 85% of the world population preferred the long-grained indica varieties and securing 80% of the world rice trade (Dawe, 2008). With the global population estimated to reach 9.5 billion by 2050, cereal grain production has to increase by 70% in order to meet the demands and to ensure food security is maintained (Bakshi and Dewan, 2013). Severe yield loss (30 to 60%) due to biotic stresses in cereal production (fungal, bacterial, viral, insects and weeds) and abiotic stresses (environmental changes and soil infertility) could outrun the global demand causing food supply shortage (Ji et al, 2013). New varieties that carry high yield and stress resistant traits are continuously being developed in order to pave towards a more sustainable rice production. MR219 was still known to be susceptible to fungal diseases (Liew et al, 2012), insects (Ahmed et al, 2011) and abiotic stress such as salinity (Hwte et al, 2011), drought (Saragih et al, 2013) and aluminium/acidity (Shamsuddin et al, 2013)
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