In vitro Culture of Embryos of Cucumis zeyheri Sond. (2n=24)

Cucurbit Genetics Cooperative Report 5:54-56 (article 26) 1982

J.B.M. Custers
Institute for Horticultural Plant Breeding, P.O., Box 15, Wageningen, The Netherlands

A. Khatun
Bangladesh Jute Research Institute, Dacca-17, Bangladesh

It has been shown previously that embryos from the cross Cucumis zeyheri* (Gene Bank no. 0181) x C. Metuliferus (Gbn 1734) stopped growth during cotyledon development and seeds with such embryos did not germinate. Embryo culture did not result in the development of viable plants from these embryos (2, 3), possibly because the medium was unsuitable. Trying various modifications of the medium might be a next approach in the culture of these hybrid embryos. However, we decided first to culture embryos of the maternal parent, because this might reveal special culture requirements for the species and likely also for the hybrid. Moreover, more general questions about the culture of non-hybrid embryos in the genus Cucumis still require answers (1).

Embryos of various developmental stages from selfed C. zeyheri (2n=24) (Gbn 0181) were incubated in 6 cm Petri dishes, each containing 3.5 ml of MS-medium supplemented with casein hydrolysate (1 g/l), Difco Bacto agar (7.5 g/l), IAA (0.01 mg/1), and various concentrations of sucrose (5, 20, 35, and 50 g/l) and kinetin (0, 0.1, 1, and 10 mg/l), selected on the basis of previous experiments (1). The cultures were kept under 16h light (Philips TL 34, approx. 750 lux) at a temperature of 24.5±1.5°C.

Table 1 gives data on embryo development after 2 1/2 weeks of culture. At that time, the variation in development was maximal. Two main types of development were present, viz. continuation of normal embryonic development (no chlorophyll development, organ proportions similar to those of in situ embryos) and precocious germination (cotyledon expansion, chlorophyll development, root development and, ultimately, development of a growing point). The tendency of the embryos to continue embryonic development gradually increased to a maximum in the late-intermediate-stage and then decreased quickly during the mature-stage. In addition, this tendency increased with higher sucrose concentration, whereas a high kinetin concentration counteracted it. Opposite to this, when precocious germination occurred, the highest kinetin concentration of 10 mg/l inhibited the development of a growing point.

A longer period of culture gradually changed the reaction pattern. Most embryos which started to develop embryonically switched from embryogenesis to germination. The weaker the embryonic tendency, the earlier was this transition. All the mature-stage embryos ultimately germinated and developed growing points. The same held true for the mid- and late-intermediate-stage embryos, except on 50 g/l sucrose, where no growing points appeared.

Most early-intermediate-stage embryos on 20 and 35 g/l sucrose also developed complete plants ultimately, but 10 mg/l kinetin diminished their frequency; Sucrose at 50 g/l kept these embryos in the embryonic phase continuously. On 5 g/l sucrose these embryos grew weakly and showed starvation, probably because of shortage of carbon.

The frequency of immature-stage embryos which developed a growing point did not increase after 2 1/2 weeks of culture. Reasons for this were the increasing tendency for embryonic development on 50 g/l sucrose and the low survival and weak growth on 5 and 20 g/l sucrose. at 35 g/l, the embryos seemed to need a low to moderate content of cytokinin for the completion of the last steps in embryo morphogenesis.

The results of the present experiments show that it is possible to get 100 percent plant formation with C. zeyheri (2n=24) embryos from the beginning of the intermediate-stage onwards. As far as nutrient components of the medium are concerned, we expect that the present procedure will be suitable for the culture of the hybrid embryos of C. zeyheri (2n-24) x C. metuliferus, which should reach at least the intermediate-stage, as judged from their ultimate size in situ of 1 – 1.5 mm (2).

*Formerly C. zeyheri (2n=24) was incorrectly named C. africanus L.f.(4).

Table 1. In vitro development of embryos of Cucumis zeyheri (2n-24) after excision at various stages and culture for 2 1/2 weeks with different sucrose and kinetin concentrations.

Developmental Stage and Size of the Embryos at Excision
		Immature		Intermediate					Mature
Sucrose (g/l)	Kinetin (mg/l)	early (globular)	late (heart)	early		mid	late		early	late
		0.07-0.1mm	0.3-0.4mm	0.8-1.2mm	1.2-1.8mm	2-2.5mm	3.2-3.5mm	4.6-5.0mm	5.5-6.0mm	5.6-6.0mm
5	0.0	d	d	d-g	g+	g+	e	e-g	g+	g+++
	0.1	d	d	d-g+	g+	g+++	e	e-g+	g+	g+++
	1.0	d	d	g	g++	g++	e-g	g++	g++	g+++
	10.0	d	d	g	g	g	e-g	g	g	g+++
20	0.1	d	d	g+	g++	g++	e	e	e	g++
	0.1	d	d-g+	g+	g++	g+++	e	e	e	g+++
	1.0	d	g+	g+++	g++	g+++	e	e-g	e-g+	g+++
	10.0	d	g	g	g	g+	e-g	e-g++	g+++	g++
35	0.1	g	g	e-g+	e-g	e	e	e	e	e-g+
	0.1	g++	g+	g++	e-g	e	e	e	e	g++
	1.0	g+	g++	g+++	g+	e-g++	e	e	e	g++
	10.1	g	g	g	g	g+	e	e	e-g+	g
*50	*0.0	e	e	e	e	e	e	e	e	e-g
	0.1	e-g+	e-g	e	e	e	e	e	e	e-g
	1.0	e-g+	e-g+	e	e	e	e	e	e	e-g+
	10.0	g	g	e	e	e	e	e	e	e-g

d = death or low rate of survival; e = continuation of normal embryonic development; g = precocious germination; +, ++, and +++ = 0=33, 33-67, and 67-100 percent of growing point development, respectively. The number of embryos per treatment was 6, 12 or 18.

Literature Cited

1. Custers, J.B.M. 1981. Heart shape stage embryos of Cucumis species more successful in embryo culture than advanced stage embryos. Cucurbit Genetics Coop. Rpt. 4:48-49.
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.
3. Custers, J.B.M., A.P.M. den Nijs and R.W. Riepma. 1981/ Reciprocal crosses between Cucumis africanus L.f. and C. metuliferus Naud. III. Effects of pollination aids, physiological condition and genetic constitution of the maternal parent on crossability. Cucurbit Genetics Coop. Rpt. 4:50-53.
4. Varekamp, H.Q., D.L. Visser and A.P.M. den Nijs. 1982. Rectification of the names of certain accessions of the IVT-Cucumis collection. Cucurbit Genetics Coop. Rpt. 5:58-59.