We report here an efficient and high-frequency protocol for direct shoot bud differentiation from mature leaves and petioles of greenhouse-grown Begonia tuberhybrida plants. Shoot buds were induced directly on the adaxial surface of leaf tissues from not only at the cut ends, but also across the entire surface of both leaf and petiole segments. The highest frequency of shoot bud formation was 90%, and the maximum number of shoots (132) per leaf explant was achieved with modified Murashige and Skoog (MS) (Murashige and Skoog, 1962) media supplemented with 1.0 mg·L-1 α-naphthalene acetic acid (NAA) and 2.0 mg·L-1 thidiazuron (TDZ). In petioles, the highest frequency of shoot buds was 82%. A maximum number of 33 shoots per explant was achieved with 0.5 mg·L-1 NAA and 2.0 mg·L-1 TDZ. The number of shoots produced in both explants was drastically reduced in the treatment with benzyl-aminopurine (BAP) alone or in combination with NAA and/or TDZ. The regenerated shoots were rooted on MS medium supplemented with 0.5 mg·L-1 NAA. All the elongated shoots developed into complete, rooted plantlets within 3 months. All the plantlets were successfully transferred to soil in pots in the greenhouse and they produced morphologically normal flowers. Chemical names used: α-naphthalene acetic acid (NAA), N-phenyl-N'-1,2,3-thiadiazol-5ylurea (TDZ; thidiazuron), 6-benzyl aminopurine (BAP).
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