Cucurbit Genetics Cooperative Report 3:50-51 (article 30) 1980

Reciprocal Crosses Between Cucumis africanus L. F. and C. metuliferus Naud. II. Embryo Development In Vivo and In Vitro

J. B. M. Custers and G. van Ee

Institute for Horticultural Plant Breeding, Wageningen, the Netherlands

Using mentor pollen and amino-ethoxy-vinyl-glycine (AVG), we were able to obtain a number of fruits with embryos in crosses between Cucumis africanus and C. metuliferus (Nijs et al. 1980). The temperature during fruit development was set at 23°C D/18°C N, but on hot days it rose above 30°C. The fruits were opened at various times after pollination and all full-sized and almost full-sized ovules were examined. The size and stage of embryo development were determined, after which all successfully isolated embryos were incubated in vitro. MS medium was used with the addition of casein hydrolysate (1 g/l), sucrose (20, 35 and 50 g/l), Difes Bacto agar (7.5 g/l), kinetin (0.1, 1 and 10 mg/l), and IAA (0.02 mg/l). The cultures were kept at 25±0.8°C in a 16 hr photo-period (Philips TL 33; approximate intensity 1,500 lux).

In vivo results. Table 1 gives data on embryos excised from the fruit at different times after pollination. Different pollination aids as well as different accessions of the species did not affect embryo size, so all data were pooled per cross. The rate of embryo extension growth was almost similar in the reciprocal crosses for about four weeks. Thereafter, growth slowed down in C. africanus x C. metuliferus, but continued more rapidly in the reciprocal cross. In the former combination there was large variation in the size of the embryos, which appeared to increase with time. In both crosses the transition from globular to heart shape stage occurred when the embryos were 0.10 to 0.12 mm in diameter. When they were 1.2 mm long, the distance from radicle tip to the apex measured about 0.8 mm, whereas in the reciprocal cross, both length and width of the cotyledons increases considerably.

In vitro results. Only a limited number of the possible combinations of the variables was tested. Almost all small embryos (0.09 to 0.13 mm) started growing and greening on medium containing 0.1 mg/l kinetin and 5% sucrose; within two weeks the cotyledons were about 0.6 mm long. Although 5% sucrose was beneficial initially, subculturing on 2% sucrose prolonged embryo life. Development of complete plants, however, did never occur. When incubated on 10 mg/l kinetin, these small embryos died immediately.

Early heart shape stage embryos (0.14 to 0.40 mm) reacted rather like the globular ones, except that the cotyledon length reached up to 1 to 2 mm. Late heart shape stage embryos (0.5 to 0.8 mm) showed the same development, but sometimes exhibited a reaction pattern as found in the advanced stage embryos.

Advanced stage embryos (0.9 to 1.2 mm) remained fully white when incubated on 0.1 mg/l kinetin and 5% sucrose and did not grow. Some growth and marginal greening of cotyledons occurred on lower sucrose concentrations. The combination 1 mg/l kinetin + 3.5% sucrose brought about light green, glassy cotyledons, and sometimes new formation of leaf-like structures, but a growing point did not appear. The combination 10 mg/l kinetin + 5% sucrose induced the formation of small, thick, dark green cotyledons and sometimes hypocotyl extension growth and main root development. Promising embryoids developed as well, but organized growing points did not.

The embryos, 3.0 to 4.2 mm long, of C. metuliferus x C. africanus remained completely white on medium containing 0.1 mg/l kinetin and 5% sucrose. However, 10 mg/l kinetin induced development of normal plantlets, a number of which could be transferred into soil. These plants appeared to be real hybrids.

The embryos, 1.3 to 2.1 mm long, of C. africanus x C. metuliferus dissected 100 days after pollination looked weakened and shriveled. They retained vitality on medium containing 10 mg/l kinetin and hypocotyl and roots developed. Some embryos formed weak true leaves two months after incubation, but they are not yet ready to be transferred into soil.

Table 1. Size of embryos in ten fruits of Cucumis africanus x C. metuliferus and in five fruits of the reciprocal cross at different times after pollination.

	C. africanus x C. metuliferus			C. metuliferus x C. africanus
Days after pollination	n	Mean size (mm)	CV (%)	n	Mean size (mm)	CV (%)
15-17	14	0.15	22	12	0.12	17
"	5	0.14	17	-	-	-
18-20	10	0.39	24	2	0.24	6
"	6	0.34	21	-	-	-
"	9	0.30	14	-	-	-
21-23	41	0.79	18	-	-	-
24-26	-	-	-	9	0.87	40
27-29	30	1.05	43	12	3.44	15
"	84	1.06	30	-	-	-
38	-	-	-	21	4.06	7
100	27	1.70	28	-	-	-
"	38	1.61	30	-	-	-

n: number of measured embryos per fruit.
Mean size: mean diameter of globular or mean total length of heart shape and more advanced stage embryos (mm).
CV: coefficient of variation (variance/mean).

Literature Cited

1. Nijs, A. P. M. den, J. B. M. Custers and A. J. Kooistra, 1980. Reciprocal Crosses Between Cucumis africanus L. f. and C. metuliferus Naud. I. Overcoming Barriers to Fertilization by Mentor Pollen and AVG. Cucurbit Genetics Coop. Rpt. 3: 60-62.