A simple procedure for regeneration of cucumber plants (Cucumis sativus L. cv. Poinsett 76) from cotyledon and hypocotyl explants has been developed. Somatic embryogenesis was induced on Murashige and Skoog (MS) salts and vitamins medium supplemented with 2,4-D at 2.0 mg·liter-1 and kinetin at 0.5 mg·liter-1. Development of embryos was accomplished on MS medium with NAA at 1.0 mg·liter-1 and kinetin at 0.5 mg·liter-1. Eighty-five percent of the mature somatic embryos formed showed a typical bipolar structure. All developed into morphologically normal plantlets when transferred to MS medium containing no growth regulators. Chemical name used: 2,4-dichlorophenoxyacetic acid (2,4-D).
Paula P. Chee
An embryo culture method overcomes the lengthy dormancy requirement of Taxus L. spp. (yew) seeds. When zygotic embryos excised from mature T. brevifolia L. seeds were cultured in darkness for 4 weeks on one of three basal salt media (B5, Litvay, and Murashige and Skoog), radicle emergence and seedling development was highest on B5 basal salt medium. After 1 to 2 weeks on B5 basal salt medium, seedling development of T. brevifolia, T. cuspidata L., T. baccata L., and T. baccata stricta L. ranged from 2% to 36%. BA at 2.25 μm had no effect on radicle emergence; 22.5 μm prevented it. Embryos excised from mature or nearly mature seeds had the highest frequency of radicle emergence and seedling development. Cultured embryos developed seedlings in only 8 to 10 weeks. Chemical name used: N 6-benzyladenine (BA).
Paula P. Chee
A procedure for the regeneration of muskmelon (Cucumis melo L.) cv. Topmark via shoot organogenesis from cotyledon explants is described. The best induction medium for a morphogenic response was MS salts and vitamins medium with BA at 1.0 mg·liter-1. Further vegetative bud development was completed by transferring organogenic tissue to MS medium containing BA at 0.05 mg·liter-1 . The shoots were rooted in MS medium containing NAA at 0.01 mg·liter-1. Morphologically normal plantlets were obtained. Chemical abbreviations used: 6-benzylaminopurine (BA); indoleacetic acid (IAA); naphthaleneacetic acid (NAA).
Paula P. Chee
Plant regeneration from tissue cultures of summer squash (Cucurbita pepo L. ev. YC60) has been observed. Embryogenic callus tissues were initiated when cotyledons of mature seeds were excised and cultured on Murashige and Skoog (MS) medium supplemented with either 22.7 μm 2,4-D or a combination of 4.7 μm 2,4,5-T, 4 μm BA, and 0.5 μm kinetin. Clusters of somatic embryos were found in callus tissue. Maturation of these somatic embryos was effected by transfer of embryogenic callus tissues to MS supplemented with 0.5 μm NAA and 0.25 μm kinetin. Regenerated mature plants were morphologically normal and set fruits containing seeds that germinated normally. Chemical names used: 6-benzylaminopurine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); α - napthaleneacetic acid (NAA); 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).
Paula P. Chee and Jerry L. Slightom
Cotyledon explants of cucumber (Cucumis sativus L. cv. Poinsett 76) seedlings were cocultivated with disarmed Agrobacterium strain C58Z707 that contained the binary vector plasmid pGA482GG/cpCMV19. The T-DNA region of this binary vector contains plant-expressible genes for neomycin phosphotransferase II (NPT II), β -glucuronidase (GUS), and the coat protein of cucumber mosaic virus strain C (CMV-C). After infection, the cotyledons were placed on Murashige and Skoog medium containing 100 mg kanamycidliter. Putative transformed embryogenic calli were obtained, followed by the development of mature embryos and their germination to plants. All transformed RO cucumber plants appeared morphologically normal and tested positive for NPT IL Southern blot analysis of selected cucumber DNAs indicated that NPT II, GUS, and CMV-C coat protein genes were integrated into the genomes. Enzyme-linked immunosorbent assay and Western blot analysis indicated that the CMV-C coat protein is present in the protein extracts of progeny plants. These results show that the Agrobacterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for producing transgenic plants in Cucurbitaceae.
Carol Gonsalves, Baodi Xue, Marcela Yepes, Marc Fuchs, Kaishu Ling, Shigetou Namba, Paula Chee, Jerry L. Slightom, and Dennis Gonsalves
A single regeneration procedure using cotyledon explants effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens (using `Burpee Hybrid' and `Hales Best Jumbo') or microprojectile bombardment (using `Topmark') methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), β-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 μm 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg·liter-1 and carbenicillin (Cb) at 500 mg·liter-1. Our comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R0 plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes. This analysis showed that plants transformed by A. tumefaciens contained all three genes, although co-transferring the genes into bombarded plants was not always successful. R1 plants were challenge inoculated with CMV-FNY, a destructive strain of CMV found in New York. Resistance levels varied according to the different transformed genotypes. Somaclonal variation was observed in a significant number of R0 transgenic plants. Flow cytometry analysis of leaf tissue revealed that a significant number of transgenic plants were tetraploid or mixoploid, whereas the commercial nontransformed cultivars were diploid. In a study of young, germinated cotyledons, however, a mixture of diploid, tetraploid, and octoploid cells were found at the shoot regeneration sites.