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Plant transformation is a technique that allows us to transfer genes from one species to another in order to introduce new characteristics into the recipient. The plant transformation technique has become widely adopted as a method both to understand plant physiology and to improve plant characteristics. There are now many established gene-transfer methods, both direct and indirect, for the stable introduction of novel genes into plant species. Examples include Agrobacterium-mediated transformation, particle bombardment (biolistic), and protoplast electroporation (Klein and Fitzpatrick-McElligott, 1993; Tzfira and Citovsky, 2006). Recently, a direct gene-transfer method called bioactive beads (BABs)mediated transformation has been developed (Sone et al., 2002; Wada et al., 2011a, 2011b). This method involves immobilization of DNA molecules on alginate beads and their transfer to plant cells with the assistance of a polyethylene glycol (PEG) solution. Alginate, a hydrophilic polysaccharide that solidifies in the presence of Ca2+ ions, is utilized as a barrier membrane to produce calcium-alginate beads for immobilizing high-density DNA molecules. The original procedure for bead production was described in Sone et al. (2002) (Fig. 1). Firstly, a plasmid DNA solution was mixed with a CaCl2 solution. Isoamyl alcohol was then added to a 1.5 ml micro-tube containing an aqueous phase 1% sodium alginate solution to form a water/oil mixture that was emulsified by sonication using an ultrasonic disrupter (UR-20P; Tomy Seiko, Tokyo, Japan) for 10 s. Bioactive beads (BABs) encapsulating DNA molecules were generated after immediately adding a CaCl2 solution containing plasmid DNA into the emulsified solution. The DNA-immobilizing BABs were utilized in combination with PEG solution for protoplast transformation.

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Bioactive Beads-Mediated Transformation of Rice with Large DNA Fragments Containing Aegilops tauschii Genes, with Special Reference to Bead-Production Methodology