Abstract
The efficient transformation of plants with large DNA molecules containing a set of useful genes would provide vast possibilities for the genetic improvement of agricultural as well as nonagricultural plants. The development of the bioactive beads (BABs) transformation method has proven useful for introduction of large DNA molecules into plant cells. In this chapter, the BABs transformation method used for the transformation of a 100-kb BAC DNA construct containing wheat genes into rice will be presented. Furthermore, the improved production method for BABs will be described. With the conventional method for producing BABs, the bead size varies, and the larger beads tend to carry fewer DNA molecules than the smaller beads. Thus, in order to facilitate the preparation of BABs with more uniform sizes, a simple set-up composed of a sine wave sound generator and microsyringe pump was fabricated. Using this bead-maker set-up, uniform and smaller beads could be produced which enhance the transformation efficiency.
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Acknowledgments
The authors would like to thank Prof. Takiko Shimada (Ishikawa Prefectural University, Japan) and Prof. Yasuhiko Mukai (Osaka Kyouiku University, Japan) for giving suggestions on the tissue culture of rice and providing the BAC DNA. We also would like to thank Prof. Motoyasu Otani (Ishikawa Prefectural University, Japan), Prof. Go Suzuki (Osaka Kyouiku University, Japan), and Prof. Shin’ichiro Kajiyama (Kinki University, Japan) for their advice and technical assistance throughout the study. This work is supported by a fund from the Handai Frontier Research Center’s Strategic Research Base supported by the Special Coordination Fund for Promoting Science and Technology of the Government of Japan to K. F. This research was partially supported by a Grant-in-Aid for Scientific Research (B) (No. 19380194) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We would also like to express special thanks to the Global COE (Center of Excellence) Program, “Global Education and Research Center for Bio-Environmental Chemistry,” of Osaka University. N.W. was supported by a Research Fellowship from the Japanese Society for the Promotion of Science for Young Scientists. N.K. would like to thank the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) and the International Graduate Program for Frontier Biotechnology, Graduate School of Engineering, Osaka University for fellowship support.
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Wada, N., Cartagena, J.A., Khemkladngoen, N., Fukui, K. (2012). Bioactive Bead-Mediated Transformation of Plants with Large DNA Fragments. 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_9
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DOI: https://doi.org/10.1007/978-1-61779-558-9_9
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