Abstract
A mutated rice acetolactate synthase (mALS) gene expressed under the control of the rice callus-specific promoter (CSP) (CSP:mALS) becomes a useful selectable marker for producing transgenic rice seed with higher accumulation of recombinant protein. When amounts of transgene products in mature seeds are compared between transgenic rice lines containing the CSP:mALS selection gene cassette and those with the hygromycin phosphotransferase (HPT) gene under the control of the CaMV 35S promoter (35S:HPT), the former transgenic rice seeds usually resulted in 1.2- to 2-fold higher accumulation of transgene products than in the latter. It is considered that specific expression of a selection marker gene at the selection stage may allow enhanced transgene products in seeds.
This chapter represents a highly efficient Agrobacterium-mediated rice transformation system using the CSP:mALS gene cassette in place of the conventional constitutive selection using bacterial antibiotics. This selection stage specific expression using a rice-derived selection marker mALS will be especially beneficial for developing transgenic rice seeds accumulating bioactive proteins or peptides contributing to human health promotion; pharmaceutical products such as vaccine, antibodies, and biopharmaceuticals; and industrial enzymes.
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Notes
- 1.
mL AA-1 (1 g MnSO4∙4H2O, 0.3 g H3BO4, 0.2 g ZnSO4∙7H2O, 25 mg Na2MoO4∙2H2O, 2.5 mg CuSO4∙5H2O, 2.5 mg CoCl2∙6H2O, 75 mg KI), 100 mL AA-2 (15 g CaCl2∙2H2O), 100 mL AA-3 (25 g MgSO4∙7H2O), 100 mL AA-4 (4 g Fe-EDTA), 100 mL AA-5 (15 g NaH2PO4∙2H2O), 100 mL AA-6 (20 mg nicotinic acid, 200 mg thiamine hydrochloride, 20 mg pyridoxine monohydrochloride, 2 g myo-inositol), 100 mL AA-7 (1.77 g l-arginine, 95 mg glycine).
- 2.
L DKN-macro (20× stock) (5.46 g NaH2PO4, 16 g KNO3, 1.34 g (NH4)2SO4, 5 g MgSO4, 3 g CaCl2∙2H2O), 100 mL DKN-micro (1,000× stock) (0.16 g MnSO4∙4H2O, 0.22 g ZnSO4∙7H2O, 0.3 g H3BO3, 12.5 mg CuSO4∙5H2O, 12.5 mg Na2MoO4∙2H2O), 100 mL modified B5-vitamins (1,000× stock) (100 mg nicotinic acid, 1 g thiamine hydrochloride, 100 mg pyridoxine monohydrochloride, 10 g myo-inositol, 200 mg glycine), 100 mL R2-iron (1,000× stock) (750 mg Na2∙EDTA, 550 mg FeSO4∙7H2O).
References
Takaiwa, F., Takagi, H., Hirose, S., and Wakasa, Y. (2007) Endosperm tissue is good production platform for artificial recombinant proteins in transgenic rice. Plant Biotechnol. J. 5, 84–92.
Wakasa, Y., Yang, L., and Takaiwa, F. (2009) Production of bioactive peptide in transgenic rice seed, in Modification of Seed Composition to Promote Health and Nutrition, Agronomy Monograph Series 51, ASA and SSSA, USA. pp.101–120.
Chaleff, R. S. and Mauvais, C. J. (1984) Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants. Science 224, 1443–1445.
Shimizu, T., Nakayama, I., Nagayama, K., Miyazawa, T., and Nezu, Y. (2002) Acetolactate synthase inhibitors, in Herbicide Classes in Development. Vol. 1 (Boger, P., Wakabayashi, K., and Hirai, K. eds.), Springer, Berlin, pp.1–41.
Charest, P. J., Hattori, J., DeMoor, J., Iyer, V. N., and Miki, B. L (1990) In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes. Plant Cell Rep. 8, 643–646.
Gabard, J. M., Charest, P. J., Iyer, V. N., and Miki BL (1989) Cross-resistance to short residual sulfonylurea herbicides in transgenic tobacco plants. Plant Physiol. 91, 574–580.
Li, Z., Hayashimoto, A., and Murai, N. (1992) A sulfonylurea herbicide resistance gene from Arabidopsis thaliana as a new selectable marker for production of fertile transgenic rice plants. Plant Physiol. 100, 662–668.
Ray, K., Jagannath, A., Gangwani, S. A., Burma, P. K., and Pental, D. (2004) Mutant acetolactate synthase gene is an efficient in vitro selectable marker for the genetic transformation of Brassica juncea (oilseed mustard). J. Plant Physiol. 161, 1079–1083.
Okuzaki, A., Shimizu, T., Kaku, K., Kawai, K., and Toriyama, K. (2007) A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice. Plant Mol Biol. 64, 219–224.
Wakasa, Y., Ozawa, K., and Takaiwa, F. (2007) Agrobacterium-mediated transformation of a low glutelin mutant of ‘Koshihikari’ rice variety using the mutated-acetolactate synthase gene derived from rice genome as a selectable marker. Plant Cell Rep. 26, 1567–1573.
Wakasa, Y., Ozawa, K., and Takaiwa, F. (2009) Higher-level accumulation of foreign gene products in transgenic rice seeds by the callus-specific selection system. J. Biosci. Bioeng. 107, 78–83.
Chu, C. C., Wang, C. C., Sun, C. S., Hsu, C., Yin, K. C., Chu, C. Y., and Bi, F. Y. (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Scientia Sinica 18, 659–668.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–493.
Ogawa, T., Fukuoka, H., Yano, H., and Ohkawa, Y. (1999) Relationships between nitrite reductase activity and genotype-dependent callus growth in rice cell cultures. Plant Cell Rep. 18, 576–581.
Daigen, M., Kawakami, O., and Nagasawa, Y. (2000) Efficient anther culture method of the japonica rice cultivar Koshihikari. Breed Sci. 50, 197–202.
Ozawa, K. and Kawahigashi, H. (2006) Positional cloning of the nitrite reductase gene associated with good growth and regeneration ability of calli and establishment of a new selection system for Agrobacterium-mediated transformation in rice (Oryza sativa L.). Plant Sci. 170, 384–393.
Parkhi, V., Ray, M., Tan, J., Oliva, N., Rehana, S., Bandyopadhyay, A., Torrizo, L., Ghole, V., Datta, K., and Datta, S. K. (2005) Molecular characterization of marker-free transgenic lines of indica rice that accumulate carotenoids in seed endosperm. Mol. Gen. Genomics 274, 325–336.
Saika, H. and Toki, S. (2010) Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacterium-mediated transformation. Plant Cell Rep. 29, 1351–1364.
Ozawa, K. (2009) Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.). Plant Sci. 176, 522–527.
Acknowledgments
This work was partly supported by research grants from the Ministry of Agriculture, Forestry, and Fishery of Japan (Genomics and Agricultural Innovation: GMC0003) to F. T. and Grant-in-Aid for JSPS fellows to Y. W. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Wakasa, Y., Takaiwa, F. (2012). Use of a Callus-Specific Selection System to Develop Transgenic Rice Seed Accumulating a High Level of Recombinant Protein. In: Dunwell, J., Wetten, A. (eds) Transgenic Plants. Methods in Molecular Biology, vol 847. Humana Press. https://doi.org/10.1007/978-1-61779-558-9_36
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DOI: https://doi.org/10.1007/978-1-61779-558-9_36
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