Received: 16-10-2014
Accepted: 22-07-2015
DOI:
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Optimization of Transformation Protocol for Japonica Rice cv.Taichung 65 through Agrobacterium tumefaciens
,
Mai Duc Chung
1
,
Nguyen Thi Hue
1
,
Jeremy Lavarenne
2
,
Mathieu Gonin
2
,
Nguyen Thanh Hai
3
,
Do Nang Vinh
1
,
Pascal Gantet
2
Keywords
Agrobacterium, callus. Rice, embryogenic. japonica, Taichung 65, transformation
Abstract
In the present study, we have successfully developed a transformation protocol for a rice variety Taichung 65 mediated by Agrobacterium tumefaciens with high efficiency, easy manipulation and reduction in labour work. In the protocol, we have optimized all steps of transformation, standardized components of culture media and presented several manipulation experiments. Experimental results have shown that using petri dishes with high edge (15 mm) and exposing explants not only increased the rate of callus formation, but also enhanced the size and quality of calluses. Using bacterial suspension at density OD600nm = 0.1 was optimal with high GUS expression in callus (81.25%) and low contamination. During the selection phase, transformed calluses grew better in petri dishes with high edge (15 mm) and exposing explants. Checking for the presence of R4 promoter at the molecular level showed high frequency of regenerated plantlets carrying the transgenes (up to 90.24%). GUS expression in different tissues of transgenic plants indicated the high activity of promoter R4, leading to initiate transcription and synthesis of GUS enzyme. Analysis of T1 plants confirmed the stable inheritance of transgene to the next generation.
References
Chan M.T., Lee T.M., Chang H.H. (1992). Transformation of indica rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens. Plant Cell Physiol., 33: 577-583.
Chopita Sakulsingharoj, Poonsri Inta, Roypim Sukkasem, Saengtong Pongjaroenkit, Srimek Chowpongpang, Varaporn Sangtong (2014). Overexpression of OSB2 gene in transgenic rice up-regulated expression of structural genes in anthocyanin biosynthesis pathway. Thai J. Genet., 7(3): 173-182.
Enrico Scarpella, Saskia Rueband Annemarie H. Meijer(2003). The RADICLELESS1 gene is required for vascular pattern formation in rice. Development, 130: 645-658.
Gould J., Devey M., Hasegawa O., Ulian E.C., Peterson G., Smith R.H. (1991). Transformation of Zea may L. using Agrobacterium tumefaciens and the shoot apex. Plant Physiol., 95: 426-434.
Hiei Y., Ohta S., Komari T., Kumashiro T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T- DNA. Plant J., 6: 271-282.
Hiei Y., Komari T. (2008). Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protocols, 3: 824-834.
Hong S K., Kitano H., Satoh H, Nagato Y (1996). How is embryo sizegenetically regulated in rice? Development, 122: 2051-2058.
Inukai Y., Sakamoto T., Ueguchi-Tanaka M., Shibata Y., Gomi K., Umemura, I., Hasegawa Y., Ashikari M., Kitano H., and Matsuoka M. (2005). Crown rootless1, which is essential for crown root formation in rice, is a target of an AUXIN RESPONSE FACTOR in auxin signaling. Plant Cell, 17: 1387-1396.
Peng J. Kononowicz H.. Hodges T. K. (1992). Transgenic indica rice plants Theor. Appl. Genet., 83: 855-863.
Raineri D.M., Bottino P., Gordon M.P., Nester, E.W. (1990). Agrobacterium mediated transformation of rice (Oryza sativa L.). Biotech.,8: 33-38.
Satoh H,.Hong S.K., Nishimura A., Matsuoka M., Kitano H., Nagato Y. (1999). Initiation of shoot apical meristem in rice: characterization of four SHOOTLESS genes. Development, 126: 3629- 3636.
Xin X., Shinji K, Tatsuhito F., Chuntai W. (2005). A protocol for high-throughput extraction of DNA from rice leaves. Plant Mol. Biol. Rep., 23: 291-295.
Yara Asanori, Otani Motoyasu, Kusumi Kensuke, Matsuda Osamu, Shimada Takiko, Iba Koh (2001). Production of transgenic japonica rice (Oryza satica) cultivar, Taichung 65, by the Agrobacterium- mediated method. Plant Biotechnology, 18(4): 305-310.