Cucurbit Genetics Cooperative Report 7:69-70 (article 30) 1984
Malter, A. B., R. J. Lebowitz, and J. A. Juvik
Department of Horticulture, University of Illinois, Urbana, IL 61801
Tissue culture may offer novel means of studying and improving Cucurbitaceae species. Cultures have already been used to analyze secondary metabolites (Yanagawa, et al., 1971), propagate gynoecious inbreds (Handley and Chambliss, 1979), regenerate plantlets from cotyledon-derived callus (Ding-Tai, et al., 1980; Jaleska, 1972 and 1974), and to study morphogenesis effects (HaIder and Gadgil, 1981). The following text describes the preliminary results from embryo culture experiments using two species; Cucurbita andreana and Cucurbita martinezii.
Embryo culture of C. andreana and C. martinezii was achieved on a medium consisting of half strength Murashige and Skoog (1962) salts, 0.1 mg/liter thiamine HCl, 2.5 mg/liter niacin, 2.0 mg/liter pyridoxine HCl, 25 mg/liter ascorbic acid, 500 mg/liter malt extract, 40 mg/liter coconut milk, 300 mg/liter myo- inositol, 25 g/liter glucose, 0.1 mg/liter NAA, and 0.1 mg/liter kinetin. The medium was solidified by adding Bactoagar (8 gm/liter) after it was adjusted to pH = 5.65 using KOH. Eight ml aliquots of medium were dispensed into 25 x 150 mm glass culture tubes. The tubes, capped with Bellco Kaputs, were then sterilized by autoclaving for 15 minutes at 1.06 kg/cm2 at approximately 115 C.
Seeds from each species were surface disinfested by placing them in a solution consisting of 10% Chlorox and 0.1% Triton X-100 surfactant for 15 minutes. The disinfested seeds were then rinsed twice for 5 minutes in sterile distilled water and allowed to dry under a sterile transfer hood. The coats of each individual seed were removed and their embryos cultured on the medium described above. Cultured embryos were maintained at 23% under 2.2 Klux intensity light.
Radicals emerged from most of the C. andreana embryos within 9-12 days. Their cotyledons developed chlorophyll during this same period. After 12 days, 40% of the developing seedlings had produced a shoot and 4-17 lateral roots. After 16 days, 30% of the developing seedlings had produced one or more true leaves.
Embryo cultures of C. martinezii exhibited slightly less vigorous development than those of C. andreana. Radicals from most of these embryos required 12-16 days to emerge, however 56% of these seedlings had developed single leaves and lateral roots by the end of this 16 day period.
Seedlings from both species were subcultured at least 3-4 weeks after initial culturing when their shoots had developed at least two nodes and two or more true leaves. The seedlings were divided into segments consisting of individual shoot nodes, cotyledons, leaf blades with their petioles, leaves with basal buds, and short stem segments with the cotyledons still attached (see Table 1). These 5 types of tissue explants were placed on fresh medium and maintained under the same environmental conditions described earlier. Shoots and roots only differentiated from two of the different types of explants; leaves with basal buds, and short stem segments with the cotyledons still attached (see Table 1). Roots alone developed on some of the cotyledon explants.
Table 1. Differentiation seen in Cucurbita tissue explants.
| Explant Type | No. of Cultures | Differentiation | ||
|---|---|---|---|---|
| Shoots | Roots | |||
| C. andreana | ||||
| Shoot node | 15 | – | – | |
| Cotyledon | 12 | + | – | |
| Leaf blade w/petiole | 15 | – | – | |
| Leaf w/basal bud | 13 | + | + | |
| Stem segment w/cotyledon | 10 | + | + | |
| C. martinezii | ||||
| Shoot node | 12 | – | – | |
| Cotyledon | 10 | + | – | |
| Leaf blade w/petiole | 12 | – | – | |
| Leaf w/basal bud | 13 | + | + | |
| Stem segment w/cotyledon | 10 | + | + | |
| + = Present, – = Absent | ||||
These results indicate that in vitro differentiation is controlled by endogenous factors as well as exogenous nutrients and hormones. Ding-Tai et al. (1980) and Jaleska (1972, 1974) reported similiar endogenous differentiation factors affecting Cucumis melo and Cucurbita pepo tissue cultures.
Literature Cited
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- Handley, L.W. and O.L. Chambliss. 1979. In vitro propagation of Cucumis sativus L. HortScience 14:22-23.
- Halder, T. and V.N. Gadgil. 1981. Morphogenesis in some plant species of the family Cucurbitaceae. pp. 98-103. In: Proceedings of the COSTED symposium on tissue culture of economically important plants. A.N. Rao (ed.), Singapore.
- Jaleska, S. 1972. Embryoid formation by fragments of cotyledons and hypocotyls in Cucurbita pepo. Planta 103:278-280.
- Jaleska, S. 1974. Embryogenesis and organogenesis in pumpkin explants. Physiol. Plant. 31:257-261.
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