DOI: 10.5176/2251-2489_BioTech15.11
Authors: Anshika and Lata I Shukla
Abstract: Rice is a popular and important crop estimated to provide more than one fifth of the calorie consumed by the world population. The increasing number of indica rice which were earlier considered to be recalcitrant and difficult to regenerate are being addressed due to regional and taste preferences for people of India. Rice varieties ASD16 and CR1009 are recalcitrant to known synthetic media. This work is first report for development of embryogenic calli for recalcitrant rice varieties ASD16 and CR1009. ASD16 and CR1009 are the indica rice varieties, grown in Pondicherry and Tamil Nadu. CR1009 is popular idli rice, while ASD16 has a distinct taste, texture and longer shelf life for cooked rice. The E3 media containing MS basal, 30g/l sucrose, 2.5mg/l 2-4-D, 0.2mg/l kinetin, 2.5mg/l proline, 300mg/l casein hydrolysate, 20mg/l glutamine and 4g/l phytagel. The calli raised in white light gave better results than the one grown in dark. Among the two embryogenic calli ASD16, gave regeneration with development of plantlets and rooting was also observed. The procedures followed include E1-E4 media for generation of embryogenic calli and media R1-R4 were used for regeneration of calli. The calli raised on media showed different growth conditions. The MS media supplemented with proline, casein hydrolysate and glutamine enabling cell division, cell expansion and cell differentiation in CR1009 and ASD16. Also, the calli raised on E3 media were incubated in 16 hour white light. The calli of CR1009 showed relatively higher cellular expansion when compared with ASD16. The development of healthy embryogenic calli requires optimal concentrations of essential nutrients, which facilitates the processing of signals generated by phytohormones that are present in the media. The generation of embryogenic calli required 2.5mg/l 2,4-D in E3 media. In absence of essential amino acid the cells which are
undergoing mitosis under the influence of phytohormones can result in browning, development of necrotic areas leading to death of calli. Some additives like casein hydrolysate present in the media which is a mixture of 18 amino acids provide calcium, phosphate, several micronutrients and vitamins. This in turn could have a role to play in reducing the browning of calli thereby maintaining the viable and actively dividing cells in the calli. The embryogenic and regenerating calli were generated in sterile conditions and these were exposed to 16hr light and 8hr dark conditions at 298K and relative humidity of 80{6e6090cdd558c53a8bc18225ef4499fead9160abd3419ad4f137e902b483c465}. The results obtained suggested E3media (MS, 30g/l sucrose, 2.5mg/l 2-4-D, 0.2mg/l kinetin, 2.5mg/l Proline, 300mg/l casein hydrolysate, 20mg/l glutamine and 4g/l Phytagel) are suitable as embryogenic media for ASD16 and CR1009.
R3 (MS, 30g/l maltose, 1mg/l NAA, 3mg/l BAP, 2.5mg/l Proline, 300mg/l casein hydrolysate, 4g/l Phytagel) gave regeneration efficiency of 91.6{6e6090cdd558c53a8bc18225ef4499fead9160abd3419ad4f137e902b483c465}, 10 plantlets /callus were obtained at for ASD16 rice variety.
Further work is in progress to develop the regenerating media by modifying the vitamin constituents and evaluation of molecular mechanisms underlying the phytohormones uptake in development of procedures for generation of regeneration for rice variety CR1009.
Keywords: callus, recalcitrant, regeneration, CR1009, ASD16
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