Cucurbit Genetics Cooperative Report 12:35-36 (article 14) 1989
F. van der Mark, J.H.W. Bergervoet and J.B.M. Custers
Institute for Horticultural Plant Breeding (IVT), P.O. Box 16, 6700 AA Wageningen, The Netherlands
Agrobacterium rhizogenes includes hairy root disease in many dicotyledonous plants. The root including ability is conferred to plant cells by bacterial genes (T-DNA) which are transferred from the root including (Ri) plasmid of the bacterium to the plant genome. The Ri plasmid of the often used agropine type strains of A. rhizogenes consists of two distinct transformation elements, designated TL- and TR-DNA. TL-DNA contains genes relevant for hairy root induction, whereas TR-DNA contains genes involved in the production of auxin. However, recent reports indicate that TR-DNA alone can cause production of hairy roots (1). Transformation of cucumber (Cucumis sativus L.) with A. rhizogenes has been reported only once (4). An agropine type strain was used. The results showed that the frequency of Ri T-DNA transfer into cucumber was rather low and that mostly only a small part of TL- or TR-DNA was integrated in the plant genome. This contrasts with A. rhizogenes transformation of other plant species, as most Ri-plants analyzed so far contain both TL- and TR-DNA, whereas sometimes the integration of TL-DNA alone is found (1). In this paper, we present preliminary results of A. rhizogenes transformation of cucumber inbreds Gy 3 and ‘Straight Eight’.
Methods Hypocotyl explants were inoculated on the basal wound with agropine type A. rhizogenes strain LBA-9402 and inserted upside down in a Murashige-Skoog solid medium supplemented with 3% (w/v) sucrose. Developing roots were excised from the hypocotyl explants and root cultures were made on the same medium for examining autonomous growth and expression of hairy root phenotype, i.e. excessive formation of lateral roots and partial nongeotropism. After 3 weeks of culture, growing roots were divided in 1 cm explants which were rechecked on a medium without hormones for another 3 weeks. Subsequently, root clones with the hairy root phenotype were tested for agropine and mannopine production. Opine positive clones were subcultured on media with hormones for induction of plant regeneration. Two embryo-inducing media were used; (M1) MS with 5 μM 2,4-D + 5 μM BA (4), and (M2) MS with 4 μM 2, 4-D + 4 μM BA.
Results. The 2 inbred lines reacted rather similarly. After 6 weeks of culture, 75% of the inoculated hypocotyl explants showed root formation from the treated wound surface. No rooting was ever observed from the control explants. A total of 174 roots was excised from the hypocotyl explants, and 58% showed rapid growth on a medium without hormones. Large clones could be obtained from these roots, which clearly expressed the hairy root symptoms. The test on opine synthesis was carried out for only 25 fast growing root clones. In 20% of these clones, opines could be detected. Upon subculture on hormone containing media, these roots formed gray, slowly-growing callus on M1, whereas on M2 a more vital callus was formed from which regular protuberances of an embryonic, yellow callus developed. We transferred the yellow callus into a liquid medium of the same hormone composition as M2. Several somatic embryos appeared in this medium, but until now it was impossible to regenerate plants.
In the root cultures, evident differences were found between the roots derived from the A. rhizogenes infected cucumber hypocotyl sections. Approximately 40% of the roots did not show the hairy root phenotype. These roots probably originated as an indirect result of the process of transformation. Approximately 60% of the roots exhibited autonomous growth, accompanied by expression of the hairy root phenotype. This suggests that only these roots contained genes of the Ri T-DNA responsible for hairy root formation, i.e. TL-DNA, TR-DNA, TR-DNA or both. Since only a low percentage of the fast growing roots actually produced opines, the integration of TR-DNA seems to be less common than that of TL-DNA. In agreement with the results obtained by Trulson et al. (4) it is concluded that the integration of T-DNA via A. rhizogenes transformation in cucumber is a rather complicated event resulting in roots with different parts of the T-DNA. A careful analysis of the integrated DNA by Southern hybridization is needed to evaluate this phenomenon.
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