Development of Callus and Somatic Embryos from Zygotic Embryos of Cucumber (Cucumis sativus L.)

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Cucurbit Genetics Cooperative Report 11:1-2 (article 1) 1988

J.B.M. Custers, J.E.M. van Deelen and J.H.W. Bergervoet
Institute for Horticultural Plant Breeding (OVT), P.O. Box 16, 6700 AA Wageningen, The Netherlands

In the cross Cucumis sativus x C, melo seeded fruits often develop, but the embryos cease growth at the globular-0shaped stage (2). Embryo rescue procedures, suitable for embryos from self-pollinations, failed for these hybrid embryos (3)/ This may in part be caused by the difference in the base chromosome number, i.e. x=7 in C. sativus and x=12 in C. melo. To overcome this barrier, we are planning to induce callus formation from the hybrid embryos in order to induce chromosome elimination and rearrangements. This might result in cells with an adapted karyotype, from which plants might be regenerated. The present study was undertaken to establish procedures of callus formation and plant regeneration especially from young embryos. Embryos from selfed C. sativus were used as a model system.

For supply of the embryos, we used C. sativus var. hardwickiiIVT Gene bank no. (Gbn) 1811A, which was grown in an insect-proof glasshouse with temperature set at 25˚C day / 18˚C night. Immature seeds were taken out of fruits 7 to 24 days after pollination, and embryos 0.07 to 4.8 mm long from globlar-shaped cotyledonary stages (Table 1) were isolated and incubated on nutrient medium. For each embryo stage at least four different fruits were used. We cultured the globular and heart-shaped embryos enveloped in the embryo sac. A modified Murashige-Skoog medium was used containing as macro salts (in mg/1) NJ4NO3 330, KNO3, Ca(NO3) 2 ∙ 4H20 350, MgSO4 ∙ 7H20 165, KH2PO4 85, normal micro salts and organic components, 250 mg/l Edamin, 3% (w/v) sucrose, and 0.55% (w/v) Oxoid agarose. For growth regulators, 4μM 2,4-D were added, since that combination was reported to induce callus formation and embryogenesis on cucumber leaf explants (1). The medium was filter-sterilized, except for the agarose which was autoclaved. The pH was adjusted to 5.8. Six to nine embryos were incubated per 60 mm petri plate. The cultures were kept in the dark at 27˚C. Calli obtained from the embryos were transferred to fresh medium after 6 weeks and thereafter subcultured every 4 weeks. Somatic embryos were transferred to a plant development medium, Murashige-Skoog with 0.5μM K and 0.7% (w/v) Difco Bacto agar.

Table 1 presents the results after three weeks of culture. More than 90% of the globular-shaped embryos did not respond at all (NR) during culture. With older embryos four types of development have been identified: secretion of an exudate (EX), regeneration of somatic embryos directly from the cotyledons (SE), formation of beige callus (BC), and normal growth of the embryos (NG). A drop of a kind of translucent exudate, 5 to 8 mm in diameter, was generally secreted by the heart-shaped embryos, if they were responsive. It contained numerous single cells, which divided and regularly produced cell aggregates and embryo-like structures, 1 to 4 per drop. Direct somatic embryo formation, 1 to 3 embryos per cotyledon, occurred in the heart-shaped and early cotyledonary stage, but the frequencies were rather low. More commonly, in these stages, a watery, beige callus was formed, which mostly was accompanied by a spongy, white calls. The two types of calli markedly differed in regeneration capacity. The white callus failed to grow when subcultured, whereas the beige callus, after several subcultures, occasionally formed protuberances of a bright-yellow callus which was embryogenic. Normal development of the embryos in vitro was observed in cotyledonary stage embryos. With prolonged culture, however, these embryos also started to form callus, but this callus was grayer and hardly formed an embryogenic, yellow callus upon subculture.

Results of plant development from the somatic embryos obtained, were rather disappointing so far. Frequency of shoot growth was only 3% and most plants grew abnormally. Since only a few plants were successfully transplanted in soil, more research is required to improve the frequency of recovery of whole plants from somatic embryos.

Notwithstanding he low frequency of plant development from the somatic embryos, the study showed the ability of cucumber zygotic embryos to form undifferentiated tissue and to regenerate plantlets from it. The procedure established might be useful for callus formation and regeneration from hybrid embryos of the cross C. sativus x C. melo. These embryos will be cultured in further experiments.

Table 1. Response of cucunber embryos from self-ppollinated C. sativus var. hardwickii Gbn 1811A of various stages on Murashige-Skoog medium with 4μM BA and 4μM 2,4-Dz .

Embryos

Response in culture (%)
Stagey
Size (mm)
No.
NR
EX
SE
BC
NG
Globular 0.07 – 0.15 76 91 8 0 1 0
Early heart-shaped 0.15 – 0.3 102 25 53 9 13 0
Late heart-shaped 0.3 – 0.6 63 2 21 8 63 6
Early cotyledonary 0.6 – 1.2 84 5 7 15 43 30
Mid cotyledonary 1.2 – 2.4 77 0 0 0 18 82
Late cotyledonary 2.4 – 4.8 42 0 0 0 7 93
z Data were taken after 3 weeks of culture. NR: no response, EX: production of a drop of exudate with embryogenic cells, SE: regeneration of somatic embryos directly from the cotyledons, BC: production of beige callus, NG: normal embryo growth.
y Globular and heart-shaped embryos were incubated along with the surrounding embryo sac.

Literature Cited

  1. Malepszy, S. and A. Nadolska-Orczyk. 1983. In vitro culture of Cucumis sativus. I. Regeneration of plantlets from callus formed by leaf explants. Z Pflanzenphysiol. 111:273-276.
  2. Niemirowicz-Szczytt, K. and B. Kubicki. 1979. Cross fertilization between cultivated species of genera Cucumis L. and Cucurbita L. Genetica Polonica 20: 117-24.
  3. Nijs, A.P.M. den and J.B.M. Custers. 1988. Introducing resistances into the cucumber by interspecific hybridization. In: Bates, D.M. and R.W. Robinson (eds.), Biology and Chemistry of the Cucurbitaceae (In Press).