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 Cooperative Report 2:2-4 (article 1) 1979

J. B. M. Custers and L. C. Buijs
Institute for Horticultural Plant Breeding, Wageningen, The Netherlands

In certain interspecific crosses in Cucumis, the embryos abort at the early embryonic stages. Artificial culture of very young embryos proved to be unsuccessful in many plants. Surprisingly, research on somatic embryo culture sometimes seems more successful. Therefore, it may be valuable to induce callus formation from the aborting embryos and to attempt subsequent regeneration of adventitious buds or embryoids. We have investigated adventitious bud formation on seedling explants of cucumber. On the medium used by Jelaska (1), our explants gave rise to adventitious roots only. High cytokinin concentrations were necessary to induce bud differentiation. The degree of the bud formation, however, was limited. Therefore, attention was paid to illumination, to the place in the seedling where the explant was excised, and to the position of the explant on the culture medium. This report deals with preliminary results of experiments on these factors.

The experiments were performed under different culture conditions:

• L¹: per day, 16 hr Philips TL 33 light (approx. intensity 4,000 lux) and 8 hr darkness at 25.0 1 0.5°C;
• D¹: continuous darkness at 25.0 1 0.5°C;
• L¹¹: per day 16 hr Philips TL 34 light (approx. intensity 1,700 lux) at 24.5 1 O.8°C and 8 hr darkness at 23.0 1 0.7°C;
• D11: continuous darkness at the same temperature regime as under L¹¹

Seeds of Cucumis sativus cv. ‘Hokus’ were surface sterilized and sown aseptically on MS medium (2) without growth substances. They were either kept in L1or D1. After eight days when seedlings were obtained, two explants were excised from a cotyledon and four from the hypocotyl. Explant lengths were proportional to the lengths of the original organs. The position in the seedling of each explant was marked. The explants were implanted vertically in the medium, the cotyledon explants either with the proximal or the distal wound down, and the hypocotyl explants always upside down. The MS medium was used with addition of casein hydrolysate 1 g/l, sucrose 4% (w/v), oxoid agar 0.7% (w/v), kinetin 10 mg/l, and the potassium salt of IAA 0.1 mg/l. The cultures were kept in L11 or D11. Examination of the results was done 2.5 and 6 weeks after explant incubation. Both percentage of explants which showed organ formation and the mean number of buds produced were calculated on the basis of the number of uncontaminated explants. The significance at P = 0.05 of differences between the means was assessed by Student’s t test.

Table 1. Organogenesis on Cucumis sativus L. cv. ‘Hokus’ seedling explants as influenced by light condition, position of the explant in the seedling, and its position on the medium. The number of explants per treatment was at least 15. Mean values designated by the same letter are not significantly different from each other at P = 0.05.

			Bud formation(%) after
Light condition	Explant position in the seedling	Explant wound placed in the medium	2.5 wks	6 wks	Root formation (%) after 6 wks	Mean number of buds after 6 wks
Seedlings in L1 and explants in L11	Cotyledon
	distal half	proximal	0	0	0	0	a
	proximal half	proximal	6	13	0	0.6	a
	distal half	distal	0	0	13	0	a
	proximal half	distal	0	0	6	0	a
	Hypocotyl
	apical quarter	apical	88	88	0	10.6	e
	upper median quarter	apical	88	94	0	6.4	d
	lower median quarter	apical	69	69	0	3.6	bd
	basal quarter	apical	63	69	0	3.6	bc
Seedlings in D1 and explants in D11	Cotyledon
	distal half	proximal	0	25	0	1.3	ab
	proximal half	proximal	12	77	0	6.5	d
	distal half	distal	0	0	0	0	a
	proximal half	distal	0	50	0	5.4	bcd
	Hypocotyl
	apical quarter	apical	0	41	50	2.8	b
	upper median quarter	apical	0	0	25	0	a
	lower median quarter	apical	0	6	41	0.3	a
	basal quarter	apical	0	0	58	0	a

The experimental design and the results of a first experiment are shown in Table 1. The L1L11 treatments as well as the D1D11 treatments show gradients of organogenesis. Bud formation increased from the basal to the apical parts of the hypocotyl and from the distal to the proximal part of the cotyledon. The L¹L¹¹ treatment as compared with the D¹D¹¹ treatment increased bud formation of the hypocotyl parts and decreased that of the cotyledon parts. Bud formation of the cotyledon explants was promoted by insertion of their proximal ends in the medium. Since bud formation on these explants, however, preferably occurred in the medium, this phenomenon may also be caused by the budding-gradient.

Although root formation was rather poor (only one of two very small roots on a rooted explant), the studied factors seemed to influence it opposite to bud form.

In a second experiment, the effects of L¹ L¹¹, D¹ D¹¹, L¹ D¹¹, and D¹ L¹¹ treatments were studied. The number of explants per treatment was 8 or 12. The effects of L¹L¹¹ and D1D¹¹ were almost identical with those of the first experiment. In the L¹D11 treatments, only two hypocotyl explants regenerated buds and in the D¹L¹¹ treatments, only one cotyledon explant did so.

The results of the two experiments indicate that 16 hr/day illumination and continuous darkness give rise to different budding stimuli and that the stimulus built up during the seedling phase is still too weak to give realization of bud formation. It may also be possible that the light-stimulus and the dark-stimulus antagonize each other.

Literature Cited

1. Jelaska, S. 1974. Embryogenesis and organogenesis in pumpkin explants. Physiol. Plant. 31:257-261.
2. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.