Cucurbit Genetics Cooperative Report 3:2-4 (article 1) 1980

In vitro adventitious bud formation on seedling and embryo explants of Cucumis sativus L.

J.B.M. Custers and J.H.W. Bergervoet

Institute for Horticultural Plant Breeding, Wageningen, The Netherlands

We have previously reported on the effects of illumination, explant position, and explant polarity on adventitious bud formation of seedling explants of Cucumis sativus cv. 'Hokus' (1). This paper reports on experiments on explant length, seedling age, light intensity during seedling growth, and concentration of growth regulators. Also, a preliminary test of the developed system and its suitability for the ultimate goal of regeneration of adventitious buds from very young embryos was conducted.

In most experiments a set of standard conditions was maintained and only one factor was varied in any experiment. Experimental material, aseptic germination, time of explant excision, nutrient medium, growth regulator concentrations (kinetin 10 mg/l and IAA 0.1 mg/l), explant position on the medium, and assessment of the results were as previously described (1). Seedlings were grown under 16 hr/day Philips TL 33 light (approx. intensity 2,000 lux). Three explants, each 1 cm long, were excised from hypocotyls 5 cm in length. The explants originated from the upper, middle, and lower region of the hypocotyl. They were kept under 16 hr Philips TL 34 light (approx. intensity 1,700 lux) at 24.5 ± 0.8°C and 8 hr darkness at 23.0 ± 0.7°C per day.

The different treatments and results are sown in Table 1. The explant length proved to be very critical, 2.5 mm being too small. In the experiments on seedling age and light intensity during seedling growth, the hypocotyls reached different lengths; thus, some hypocotyls were divided into two explants, each about 5 mm long.

In all experiments bud formation frequency increased from the basal to the apical parts of the hypocotyl. The decrease in bud formation in the four-day treatment, as well as the 13,000 lux seedling treatment, was likely caused by the small explant length. Large explants from seedlings grown at 0 or 1,400 lux, however, show decreasing bud formation, especially when excised from the lower regions. The kinetin concentration proved to be very critical, 10 mg/l was the optimum concentration. IAA at 0.01 mg/l was the most beneficial for the regeneration of the buds and also promoted growing point extension and development into complete plants, which rarely occurred at he other IAA concentrations.

To determine if the above system would be applicable to ontogenetically younger cucumber tissue, explants from immature embryos were excised from cotyledons obtained by selfing plants of C. sativus var. hardwickii (Gbn. 0777 and 1811). explants were taken at different times after pollination and incubated on standard medium darkness or 16 hr/day light. The explants from 2.5-4.0 mm long cotyledons (55 in total out of four fruits) regularly regenerated buds, especially in continuous darkness, whereas those from 0.8-1.2 mm long cotyledons (18 in total out of two fruits) did not and did not grow. These results agree well with those obtained with in vitro culture of the embryos from the crosses between C. africanus and C. metuliferus (2).

Table 1. The effects of explant length, seedling age, light intensity during seedling growth, and kinetin and IAA concentration on the percentage of hypocotyl explants of Cucumis sativus cv. 'Hokus' with adventitious bud formation.^z

Explant position in the hypocotyl	Treatment variable
	Explant length (mm)
	2.5	5	10y	20
upper region	25	75	83	92
middle region	0	25	58	42
	Seedling age (days)/ hypocotyl length (mm)
	4/10	8/50y	12/70
upper region	58x	100	92
middle region	--w	67	42
	Light intensity during seedling growth (lux)/hypocotyl length (mm)
	0/110	1,400/60	4,300/35y	13,000/20
upper region	53	93	87	40
middle region	0	7	33	20
lower region	0	0	20	33
	Kinetin concentration (mg/l)
	0	5	10y	20
upper region	0	8	83	25
middle region	0	0	42	0
lower region	0	0	8	8
	IAA concentration (mg/l)
	0	0.01	0.1y	1
upper region	50	92	83	50
middle region	42	75	58	0
lower region	8	8	0	0

^z All data were taken after six weeks of culture. The number of explants per treatment was 12 or 15.
^y Standard treatment.
^x The upper half of the hypocotyl was incubated.
^w The lower hypocotyl half + a radicle part incubated; radicle extension growth caused explant reversal into its normal position and bud formation did not occur.

Literature Cited

1. Custers, J.B.M. and L.C. Buijs. 1979. The effects of illumination, explant position, and explant polarity on adventitious bud formation in vitro of seedling explants of Cucumis sativus L. cv. 'Hokus'. Cucurbit Genetics Coop. Rpt. 2:2-4.
2. Custers, J.B.M. and G. van Ee. 1980. Reciprocal crosses between Cucumis africanus L.f. and C. metuliferus Naud. II. Embryo development in vivo and in vitro. Cucurbit Genetics Coop. Rpt. 3:50-51.