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A protocol for high-frequency somatic embryogenesis in Cucumis melo L. was developed using `Male Sterile A147 as a model cultivar. Basal halves of quiescent seed cotyledons were cultured on embryo induction (EI) medium containing concentration ranges of the auxin 2,4-D and the cytokinins BA, Bin, TDZ, or 2iP before transfer to embryo development (ED) medium. Medium with 2,4-D at 5 mg·liter-1 and TDZ at 0.1 mg·liter-1 was superior, with 49% of explants responding and an average of 3.3 somatic embryos per explant (6.8 somatic embryos per responding explant). More explants produced embryos when incubated on EI medium for 1 or 2 weeks (30% and 33%) than for 3 or 4 weeks or with no induction. However, 2 weeks was 2.9 times better than 1 week in terms of number of embryos per explant. One week of initial culture in darkness, followed by a 16 hour light/8 hour dark photoperiod, produced more responding explants (26%) than two or more weeks in darkness or no dark period at all; but 1 and 2 weeks of darkness resulted in a similar number of embryos per explant (2.1 and 2.8). Sucrose concentration in EI and ED media had a highly significant effect on embryo induction and development. EI medium with 3% sucrose resulted in more embryogenic explants than EI medium with 1.5% or 6% sucrose. However, treatments with 3% sucrose in EI medium and 3% or 6% sucrose in ED medium produced significantly more embryos per explant (8.5 and 11.9) than other treatments. Treatments did not affect embryo induction directly and regeneration per se but, instead, frequency and efficiency of somatic embryo development. The optimal treatments were tested with 51 other commercial varieties. All varieties underwent somatic embryogenesis, exhibiting a response of 5% to 100% explant response and 0.1-20.2 embryos per explant. Chemical names used: N-(phenylmethyl)-lH-purin-6-amine (benzyladenine or BA); N-(2-furanylmethyl)-lH-purin-6-amine (kinetin or BIN); N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ); N-(3-methyl-2-butenyl)-lH-purin-6-amine (2iP); (2,4-dichlorophenoxy) acetic acid (2,4-D).
Design modification of a particle inflow-type gun for particle bombardment significantly simplifies construction and reduces fabrication time. The gun consists of a high-speed electric solenoid valve mounted on and through a vacuum jar. DNA-coated tungsten particles are placed on the support grid of a filter housing and accellerated by a burst of pressurized helium, which is controlled by a timer. Specimens are held between plastic screens and their distance from the particle support grid is adjusted with a miniature laboratory apparatus positioner. Transient expression of GUS in cantaloupe cotyledons and grape somatic embryos was equivalent to that obtained with a conventional particle inflow gun. The device was constructed with locally-available hardware in 40 minutes using a hand drill, some thread taps and a thread die.