The specificities of tissue culture of wheat greatly limit the use of chloroplast transformation technologies in this crop. One limitation in wheat tissue culture is that it is difficult to regenerate plantlets from leaf tissue explants of regenerated plantlets, resulting in difficulty in obtaining homoplastic plants via multiple rounds of antibiotic selection of chloroplast transformants. Thus, a repeated in vitro regeneration system from leaf tissues was studied in this research. Our results showed that 2 mm leaf basal segments of the 4 cm high leaves from regenerated plantlets can give the best callus induction at present study. The best callus induction medium was Murashige and Skoog (MS) basal medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid and 1 mg/L naphthalenacetic acid, which gave a callus induction rate of up to 87.2%. The optimal differentiation medium was MS basal medium supplemented with 10 mg/L silver nitrate and 1 mg/L 2,3,5-triiodobenzoic acid, which gave a regeneration rate up to 33.7% for the wheat lines tested. This is the first report showing that leaf basal segments of in vitro regenerated plantlets can be used for regeneration of wheat. The establishment of a repetitive regeneration system should pave the way for the development of chloroplast transformation and the plant regeneration systems starting from leaf material of in vitro regenerated wheat and other cereal crops.
Chugh, A. and Khurana, P. (2003) Regeneration via somatic embryogenesis from leaf basal segments and genetic transformation of bread and emmer wheat by particle bombardment. Plant Cell, Tissue and Organ Culture, 74, 151-161. doi:10.1023/A:1023945610740
Lee, S.M., Kang, K., Chung, H., Yoo, S.H., Xu, X.M., Lee, S.B., Cheong, J.J., Daniell, H. and Kim, M. (2006) Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny. Molecules and Cells, 21, 401-410.
Dolezelova, M., Dolezel, J. and Nesticky, M. (1992) Relationship of embryogenic competence in maize (Zea mays L.) leaves to mitotic activity, cell cycle and nuclear DNA content. Plant Cell, Tissue and Organ Culture, 31, 215-221.
Fei, S., Read, P.E. and Riordan, T.P. (2000) Improvement of embryogenic callus induction and shoot regeneration of buffalograss by silver nitrate. Plant Cell, Tissue and Organ Culture, 60, 197-200.
Mahalakshmi, A., Khurana, J.P and Khurana, P. (2003) Rapid induction of somatic embryogenesis by 2,4-D in leaf base cultures of wheat (Triticum aestivum L.). Plant Biotechnology, 20, 267-273.
Choi, Y.E, Kim, H.S., Soh, W.Y. and Yang, D.C. (1997) Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid. Plant Cell Report, 16, 738-744.