An Improved Agrobacterium-Mediated Transformation Protocol for Recalcitrant Elite Indica Rice Cultivars K.K. KUMAR, S. MARUTHASALAM, M. LOGANATHAN, D. SUDHAKAR and P. BALASUBRAMANIAN* Rice Transformation Laboratory, Department of Plant Molecular Biology and Biotechnology, Center for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore-641003, Tamil Nadu, India Abstract. We report here a high-efficiency transformation protocol for recalcitrant indica rice cultivars IR64 and IR72 with the selectable marker gene hph and the gusA reporter gene. Factors that favor high-efficiency transformation were found to be use of 2-month-old mature seed-derived embryogenic calli, maltose as a source of carbon, a higher concentration of 2,4-dichlorophenoxyacetic acid, and both phytagel and agar as gelling agents. The putative transgenic (T0) plants were analyzed for integration of the transgene through polymerase chain reaction and Southern blotting analyses. Various factors thought to be responsible for increased transformation efficiency are discussed. Key words: Agrobacterium, indica rice, mature seed-derived calli, rice transformation, transgenic rice Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BAP, 6-benzylaminopurine; MS, Murashige and Skoog; NAA, α-naphthaleneacetic acid; OD, optical density; PCR, polymerase chain reaction. Agrobacterium-mediated transformation protocol for indica Introduction rice cultivars Kumar et al.
Agrobacterium-mediated transformation is routinely used for the transformation of many monocots, including rice. Several reports are available on the Agrobacterium-mediated transformation of japonica and indica rice cultivars. However, the efficiency with which the indica rice cultivars are transformed is very low. Different explant types are used in the Agrobacterium-mediated transformation of rice, including mature seed-derived calli, immature embryo-derived calli, immature embryos, and shoot apex (Hiei et al., 1994; Aldemita and Hodges, 1996; Park et al., 1996). Although Aldemita and Hodges (1996) obtained relatively high transformation efficiency for indica rice by using immature embryos for cocultivation, this technique has many limitations, such as nonavailability throughout the year and laboriousness. Hiei et al. (1994) and Rashid et al. (1996) *
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Kumar et al.
reported successful transformation of japonica and easily amenable indica rice genotypes by using mature seed-derived calli as explants and found that 3-week-old mature seed-derived calli are the most suitable explants for Agrobacterium-mediated transformation. There are several reports on transformation of recalcitrant indica rice cultivars, for example, IR64 and IR72, through Agrobacterium (Datta et al., 1999, 2000, 2001; Khanna and Raina, 1999, 2002; Tu et al., 1998; Zhang et al., 1998), with a relatively low frequency of transformation. We report here an improved transformation protocol for 2 elite indica rice cultivars, IR64 and IR72, by use of mature seed-derived calli as explants. Materials and Methods Plant materials used Mature seeds of rice cultivars IR64 and IR72 (obtained from Paddy Breeding Station, Tamil Nadu Agricultural University, Coimbatore) were manually dehusked, after which they were sterilized with 70% ethanol for 3 min and then with 0.1% mercuric chloride for 5 min, followed by 3 washes in sterile distilled water. Sterilized seeds were cultured on callus induction medium (MS salts [Murashige and Skoog, 1962] containing B5 vitamins, 30 g/L maltose, 3.0 mg/L 2,4-dichlorophenoxyacetic acid [2,4-D], 0.3 mg/L kinetin, 4.0 g/L phytagel, and 2.0 g/L) agar at 25°C in the dark. After 3-4 wk, the proliferating calli were subcultured onto the same...