Cucurbit Genetics Cooperative Report 19:42-46 (article 16) 1996
S.H.T. Raharjo and Z.K. Punja
Dept. Biological Sciences, Simon Fraser University, Burbnaby, British Columbia V5A 1S6, Canada
Introduction. A few methods for initiating cell suspension culture, development of somatic embryos, and regeneration of plantlets in several fresh market cultivars of cucumber have been described (e.g. 2,3). A procedure for initiation and maintenance of suspension cultures of a pickling cucumber cultivar, Endeavor, for high-frequency regeneration of normal-appearing plantlets has recently been developed in our laboratory (9). This objective of this paper is to describe the incorporation of cell suspension culture and Agrobacterium-mediated transformation procedures to select and multiply transformed embryogenic aggregates in order to recover a high frequency of transformed plantlets.
Materials & Methods. The cucumber cultivar used throughout this study was Endeavor, a pickling type, which is an F1 hybrid (seed provided by Campbell Soup Co., Davis, CA). The decoated seeds were surface-sterilized by dipping in 70% ethanol for 30 sec, followed by soaking in a 10% solution of commercial bleach (Javex, 6.25% sodium hypochlorite) for 5 min, and rinsing three times in sterile distilled water. The seeds were germinated and seedlings maintained in Magenta culture vessels (Magenta Corp., Chicago, IL) containing 50 ml of half-strength Murashige and Skoog (MS) basal medium (5).
Agrobacterium tumefaciens strain EHA 105, a supervirulent leucinopine type, with a binary vector pMOG196 (provided by Dr. L, Melchers, MOGEN Int. nv, The Netherlands) was used. The vector contained a petunia acid chitinase gene (Linthorst et al., 1990), in addition to the neomycin phosphotransferase (NPT II) gene as a selectable marker and the cauliflower mosaic virus (CaMV) 35S promoter for constitutive expression. The Agrobacterium was maintained on Laura-Bertaini (LB) medium with 100 mg/l kanamycin, pH 5.4. The bacterial density was adjusted to 108 cells/ml and acetosyringone was added to a final concentration of 100 μM, 1h prior to infection of explants.
The explants used for infection were petiole segments (4 to 5 mm long) taken from the first and second true leaves of 10 to 21 day-old seedlings. Inoculation was done by dipping the explants into the bacterial suspension for 5 min, followed by blotting with sterile paper towels. The explants were transferred onto cocultivation medium, which was MS medium with 2,4D/BA (5.0/5.0 μM), pH 5.4, and cocultivated at 27 C in the dark for 2 to 4 days.
Following cocultivation, the explants were rinsed with MS medium and transferred onto selective medium, i.e. MS basal medium with 2,4 D/BA (5.0/5.0 μM), kanamycin (50 mg/l) and carbenicillin (500 mg/l). Calli which developed from the explants were subcultured onto fresh medium of the same composition 4 to 5 weeks after infection. Following one to two subcultures, embryogenic calli formed were used to initiate a suspension culture to further screen and multiply putative kanamycin-resistant cell aggregates. The procedures for the initiation and maintenance of the suspension cultures of pickling cucumber have been described (6) (Fig. 1).
Genomic DNAs from putatively transformed plants and from non-transformed (negative control) plants were extracted using previously described procedures (4). PCR amplification for the identification of the transgene in the genomic DNAs was conducted using two specific primer sequences (courtesy of Dr. M.M. Moloney, University of Calgary) of the NPT II coding region. This PCR was run for 30 cycles. The PCR products were analyzed by electrophoresis on 2.0% agarose gels. The oligomers were first tested by amplifying the characteristic 800 bp region of the NPT II gene by using 10 mg of pMOG196 as positive control template.
Results. Cocultivated petiole explants swelled and began to developed callused areas after 3-4 weeks on selective callus initiation medium. The calli surviving this selection step were characterized by an increase in size and development of pale yellow color and no browning. Explants which did not increase in size or did not form calli on the surface were considered to be nonviable. Surviving calli were subcultured onto fresh medium of the same composition where they would develop embryogenic (yellow and friable) sectors. In comparison, most non-cocultivated explants were bleached and did not develop further. The frequency of embryogenic calli which developed further and grew on kanamycin-containing medium, recorded 8 weeks after cocultivation, was approximately 12%.
Small portions of the calli growing on kanamycin-containing medium that appeared embryogenic (yellow in color and granular or friable in appearance) were dissected and transferred into liquid MS medium containing 2,4 D/BA (1.0/1.0 μM)and 50 mg/l kanamycin to initiate the suspension culture. After 2 to 3 weeks of shaking on a gyratory shaker, the calli started to break apart, forming a suspension of cells and aggregates. In the suspension culture, the kanamycin-resistant embryogenic aggregates grew faster on solid selective medium.
Following plating of kanamycin-resistant embryogenic aggregates onto solid medium containing NAAA/BA (1.0/1.0 μM) and 50 mg/l kanamycin, shoots were obtained within 3 to 4 weeks (Fig. 2a). When the shoots were excised and transferred onto MS medium without growth regulators and with 50 mg/l kanamycin, they elongated to form plantlets (Fig. 2b) and developed roots after 2-3 subcultures (2-3 weeks) onto the same medium. When the rooted shoots were 5 cm or higher, they were transferred to pots where they developed into plants.
Proof of transformation was confirmed by PCR amplification of the NPT II gene using two specific primer sequences of the NPT II coding region. Six randomly selected plants from three separate flasks of suspension cultures produced a band of the expected size of 800 bp for the NPT II fragment at the same position as those of positive control, amplified DNA from the binary vector pMOG196 (Fig. 3).
Discussion. This report describes the incorporation of a liquid suspension culture with genetic transformation in cucumber. In previous reports on cucumber transformation, regeneration of plantlets was either not achieved or not reported. Direct shoot regeneration from Kanamycin-resistant cucumber calli following Agrobacterium– mediated transformation has been reported to occur at a low frequency (10). Medium with optimal combinations and concentrations of plant growth regulators for direct plantlet regeneration of cv. Endeavor, i.e. 2,4D/BA or NAA/BA (5.0/5.0 μM) (8) failed to induce regeneration from cocultivated explants. Repetitive divisions of embryogenic aggregates in suspension, culture, however, has been shown to be a useful means to multiply the embryogenic tissues and to achieve an increased regeneration rate (1,2,7).
Suspension culture proved to be useful for multiplying embryogenic aggregates and for production of cell clumps which were capable of shoot formation upon transfer onto MS medium devoid of plant growth regulators. This procedures may be useful in transformation studies of pickling cucumber conducted in other laboratories.
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