Cucurbit Genetics Cooperative Report 12:20-22 (article 9) 1989
J.B.M. Custers and E.C.P. Verstappen
Institute for Horticultural Plant Breeding (IVT), P.O. Box 16, 6700 AA Wageningen, The Netherlands
In general, in vitro culture of cucumber (Cucumis sativus L.) meets with several problems. In regeneration studies, adventitious buds as well as somatic embryos develop poorly into plants, which show structural abnormalities and absence of apex formation (1,3). The in vitro culture of complete plants is hampered by vitrification, precocious flower formation, and cessation of growth (2). In order to improve this, in vitro growth of small shoot tips and axillary buds was studied. Attention was paid to the effect of better aeration during the continuous culture of cucumber plants.
Seeds of C. sativus cultivar Hokus (Rijk Zwaan, De Lier) and C. sativus var. hardwickii (IVT Gene bank number 0777) were aseptically germinated in honey jars on 40 ml Murashige-Skoog medium with 3% (w/v) sucrose and 0.6% (w/v) agar (Oxoid Bacteriological). The jars were closed with white, partially transparent plastic lids. Three seeds were incubated per jar. The cultures were grown at a 16 hour photoperiod ( Philips TL 84, 1 Klux in the jar) at 25°C day / 23°C night. Shoot cuttings, including the cotyledons, were excised from the seedlings 8 days after germination and subcultured on fresh media. After 2 weeks, shoot tips and nodal cuttings were collected from the plants obtained.
Three experiments were designed. In experiment 1, shoot tips of different sizes (2, 3 and 5 mm in length) were compared for their growth capacity. In experiment 2, nodal cuttings with internodes of different length (2, 10 and 20 mm) were examined for the ability of the axillary buds to develop into complete plants. In both experiments, the period of culture was 4 weeks, under the same conditions as described for the seedlings.
Experiment 3 was designed to study continuous in vitro culture of cucumber plants by successive subculturing. Each subculture was started from nodal cuttings with a 15 mm internode. Effects of growth conditions were studied. Aeration was changed using 3 methods of closing the jars: a plastic lid, one layer of vitafilm (Good Year) and 3 layers of vitafilm. Light was reduced by covering the jars with layers of cheese cloth.
Shoot tips. As a consequence of choosing main axes of different length from ‘Hokus’ plants, the size of the basal leaf of the cuttings differed considerably (Table 1). All cuttings survived incubation, but the amounts of growth and plant formation were different. The large cuttings generally developed into normal plants with 4 full-grown leaves having blade lengths up to 40 mm. In contrast, most cuttings of 2 and 3 mm in length initially showed arrest of growth. After growth had started, very compact plantlets developed with 2 to 4 small leaves with blades about 10 to 20 mm in length. Upon subculture, these plants did not regain normal growth, but instead formed numerous flower buds in the axils.
Nodal cuttings. As consequence of the varying length of the internodes attached, the distance rom axil to medium was different (Table 2). Normal plants developed from the long cuttings of 10 and 20 mm length. The short cuttings, however, formed compact plants with small leaves. In general, the features of these plants were similar to those grown from shoot tips.
Table 1. Capacity of plant formation in vitro of Cucumis sativus cv. Hokus shoot tip cuttings of different sizes. The plants obtained after 4 weeks are classified by length.z
Cutting size (lengths in mm) |
Classes of plant length (% ) |
|||
Main Axis y |
Blade of basal leaf |
50-10 mm |
10-50 mm |
50-75 mm |
| 2 | 0 | 90 | 5 | 5 |
| 3 | 3-7 | 80 | 10 | 10 |
| 5 | 10-16 | 0 | 14 | 86 |
z Each treatment comprised 21 shoot tips.
y Length measured from apex to base.
Table 2. Capacity of plant formation in vitro of Cucumis sativus cv. Hokus nodal cuttings with internodes of different lengths. The plants obtained after 4 weeks are classified by length.z
Internode length (mm) |
Distance from axil to medium (mm) |
Classes of plant length (%) |
||
5-10 mm |
10-50 mm |
50-75 mm |
||
| 2 | 0 | 90 | 10 | 0 |
| 10 | 5-7 | 0 | 10 | 90 |
| 20 | 15-17 | 0 | 5 | 95 |
z Each treatment comprised 21 shoot tips.
Continuous in vitro culture. In the initial culture, the nodal cuttings yielded plants that grew well, but after 2 and 3 subcultures, plants developed which showed several irregularities such as abundant flower formation, vitrification and stunted growth. The leaf color became light green. These problems were more obvious in ‘Hokus’ than in the C. sativus var. hardwickii accession. Sealing the culture jars with vitafilm instead of using the plastic lids considerably improved the condition of the plants. One layer of the film proved to be better than 3 layers. In that treatment, plants were produced having vital, dark green leaves and without flower bud formation, but plant extension growth as well as leaf size were reduced. Moreover, the culture medium desiccated rapidly. These problems could be overcome by covering the jars sealed with one payer vitafilm with one layer of cheese cloth, which reduced the light to approximately 1 klux, and by application of 5 ml sterilized water on top of the solid medium. Under these conditions continuous culture of cucumber plants was successful.
From the results in this study, we concluded that cucumber cultures that grow well can be obtained by starting from relatively large cuttings (shoot tip or nodal.) Cultures started from small cuttings proved to be less successful. As can be deduced from the experiment with the nodal cuttings, the distance from the culture medium rather than the size of the cuttings appeared to be important. Apparently, close contact of the apex or axillary bud with the culture medium prevents their normal development into plants, possibly because of a disturbance in functioning of the endogenous hormones. This might also be an explanation for the poor development into plants of adventitious buds and somatic embryos of cucumber under normal tissue culture conditions. The continuous culture of cucumber plants is improved considerably under special conditions, viz. culturing in jars sealed with a thin vitafilm instead of closing them with an air-tight lid. This suggests that the culture needs aeration. Cucumber plants in culture apparently produce certain harmful gases, such as ethylene, which can diffuse through the thin vitafilm.
Literature Cited
- Kim, S.G., J.R. Cjamg. H.C. Cha and K.W. Lee. 1988. Callus growth and plant regeneration in diverse cultivars of cucumber (Cucumis sativus L.). Plant Cell, Tissue Organ Cult. 12:67-74.
- Rute, T.N., R.G. Butenko. and K.A. Maurinya. 1978. Influence of cultivation conditions on morphogenesis of the apical meristems of cucumber plants in cultures in vitro. Soviet Plant Physiol. 25:432-438.
- Ziv, M. and G. Gadasi. 1986. Enhanced embryogenesis and plant regeneration from cucumber (Cucumis sativus L.) callus by activated charcoal in solid/liquid double-layer cultures. Plant Sci. 47:115-122.