Cucurbit Genetics Cooperative Report 4:48-49 (article 26) 1981
J. B. M. Custers
Institute for Horticultural Plant Breeding, P. O. Box 16, Wageningen, The Netherlands
Experience with general prerequisites of the artificial culture of vital embryos from self pollinated plants of Cucumis species is likely to be helpful to the culture of abortive embryos from interspecific crosses in this genus. Our attempts to get such experience by using embryos from various selfed cultivars of Cucumis sativus were hampered by difficulties in isolating the very young embryos from their ovules. Last year the ovules of the cross C. metuliferus [ Gene bank no. (Gbn) 1734] x C. africanus (Gbn 0181) proved more amenable to excision of embryos, likely because the tissues at the ovule tip were less hardened. As the embryos of this cross grew almost normally in situ (1, 2), we selected them to study the effect of embryo size on development in tissue culture.
The embryos were isolated at various times after pollination and incubated on MS medium with the addition of casein hydrolysate 1 g/l, sucrose 35 or 50 g/l, Difco Bacto agar 7.5 g/l, kinetin 0, 0.1, 1, or 10 mg/l, and IAA 0.02 mg/l. The cultures were kept under 16 hr TL 34 light (1,000 lux) at 25°C and 8 hr darkness at 23°C per day.
The size of the embryos proved decisive for the success of in vitro culture. Approximately 15% of the late globular stage embryos 0.07-0.10 mm in diameter [13-17 days after pollination (d.a.p.)] developed into plants on the basal medium with kinetin 0.1 mg/l + sucrose 35 g/l. In about three weeks they reached a developmental stage suitable for transplanting to soil. The other combinations of the variables were unsuccessful. The low rate of success is possibly due to damage of the embryos by the isolation. The heart shape stage embryos 0.1-0.8 mm in length (17-22 d.a.p.) appeared rather successful in culture. The early heart shape stage embryos 0.1-0.3 mm in length produced plants on kinetin 0.1 mg/l + sucrose 35 g/l, but they needed 1 mg/l when the medium contained sucrose 50 g/l. The late heart shape embryos 0.3-0.8 mm in length developed well into plants on kinetin 1 mg/l + sucrose 35 g/l, but the less on sucrose 50 mg/l. The other combinations of the variables were unsuccessful. The three suitable combinations for the heart shape embryos resulted in a success of 32%. The period of culture till transplanting to soil was as long as for globular embryos. Further cotyledon extension growth in situ (22-33 d.a.p.) diminished the results of the embryo culture. Embryos 0.8-2.0 mm in length did start growing, greening and occasionally rooting, but a growing point never appeared regardless of the combination of kinetin and sucrose. Embryos 2-4 in length remained completely white and did not grow at all on kinetin 0 and 0.1 mg/l, irrespective of the sucrose concentration. Increasing the kinetin concentration sometimes resulted in partially increasing or entire greening of the cotyledons, but a growing point did not develop. Embryos 4-5 mm in length reacted similarly, but later a few did form a growing point on kinetin 10 mg/l + sucrose 35 g/l. Maturation of the embryos (33-50 d.a.p.) improved their suitability for tissue culture. Almost all embryos 5-6 mm in length rapidly developed into complete plants on all the kinetin concentration studied + sucrose 35 g/l, whereas sucrose 50 g/l retarded this development.
Small batches of embryos obtained from selfed plants of C. africanus, C. metuliferus and C. sativus were also tried on the above media. Their age and size affected the success of in vitro culture similarly as found with the hybrid embryos. Thus, the results obtained with the hybrid embryos may be representative of embryos from selfing.
On the whole, the results indicate the occurrence of a temporary germination inhibitor complex in the cotyledons. We could not overcome this inhibitor complex by adding kinetin. GA3, which was tried later on, appeared also not capable of overcoming it. This postulated inhibitor complex was found only during the stage of cotyledon extension. It is, therefore, not similar to the germination inhibitor factor, which normally occurs in the seed coats of mature seeds. We conclude, that in the case of embryo abortion during advance embryonic stages, it is best to start the artificial culture from the early heart stage embryos rather than to wait until just before degeneration.
Literature Cited
- 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.
- Custers, J. B. M., A. P. M. den Nijs and A. W. Riepma. 1981. Reciprocal crosses between Cucumis africanus L. f. and C. metuliferus Naud. III. The crossability as affected by pollination aids and by the physiological condition and the genetic constitution of the mother plants. Cucurbit Genetics Coop. Rpt. 4:50-53.