Plant regeneration and Agrobacterium-mediated transformation of cotyledon explants of Citrullus colocynthis (L.) Schrad.

Abstract

The effect of 6-benzylaminopurine (6-BA) alone or in combination with naphthaleneacetic acid or indoleacetic acid on the morphogenetic response of cotyledon explants of Citrullus colocynthis (L.) Schrad. was tested. The best results were obtained with a medium containing 25 µm 6-BA, which yielded organogenic calli at a frequency of 81.8%. When these organogenic calli were transferred to elongation medium (basal medium supplemented with 0.5 µm 6-BA), 80% produced well-developed shoots. These shoots rooted normally when cultured on rooting medium containing indolebutyric acid at 2.5 or 5.0 µm. Plants grew to maturity under greenhouse conditions and gave normal fruits. Cotyledon explants were transformed by cocultivation with Agrobacterium tumefaciens LBA4404 carrying the binary vector pBI121 which bears the reporter gene β-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII). Transformants were selected for growth capacity on medium with 100 mgl^–1 of kanamycin. On the basis of β-glucuronidase expression, the transformation frequency was 14.2%. Molecular characterization by polymerase chain reaction confirmed the presence of the two genes transferred (gus, nptII) in the transgenic plants. Sexual transmission of both genes was also confirmed by studying their expression in progenies from several transgenic plants.