Improved Cucumber Transformation by a Modified Explant Dissection and Selection Protocol

Abstract

Agrobacterium-mediated transformation of Cucumis sativus L. cotyledons was investigated, to identify important factors that affect transformation efficiency. The factors evaluated were initial explant preparation, including preculture, pricking of the explant, inoculation time and co-cultivation regime. We also modified the selection regime, compared to previously published protocols. Our results show that dissecting the proximal half of the cotyledon by a V-shaped cut resulted in a higher transformation rate, compared to two other methods of dissection, as indicated by transient β-glucuronidase gene expression. Selection on 100 mg·L-1 kanamycin resulted in the early development of nontransformed shoots, whereas a gradual increase of kanamycin concentration up to 200 mg·L-1 resulted in the subsequent formation of transgenic shoots on the same medium. The overall transformation frequency in these experiments, expressed as the number of rooted, confirmed transgenic plants per initial number of explants, was 1.7%. The stable integration of T-DNA was confirmed in the primary transformants and their progeny. Abbreviations: ABA = abscisic acid; AdS = adenine sulfate; AS = acetosyringone; BA = 6-benzylaminopurine; GA3 = gibberellic acid; IBA = indolebutyric acid.