Cucurbit Genetics Cooperative Report 4:48-49 (article 26) 1981
Heart Shape Stage Embryos of Cucumis Species
More Successful in Embryo Culture than Advanced Stage Embryos
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.
- 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.