Attempts at Colchicine Doubling of an Interspecific Hybrid of Cucumis sativus L. x C. hystrix Chakr.

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Cucurbit Genetics Cooperative Report 20:24-26 (article 12) 1997

J.F. Chen and J.E. Staub
Vegetable Crops Research Unit, USDA/ARS, Departments of Horticulture, University of Wisconsin-Madison, WI. 53706 U.S.A.

Introduction. Traditional approaches for interspecific hybridization have been used in Cucumis. These include growth regulator application (Custer and den Nijs, 1986), pollen irradiation (Beharav and Cohen, 1994), use of mentor pollen (Kho et al., 1980), and bud pollination (Chatterjee and More, 1991). We previously reported the successful hybridization and recovery of F1plants (2n=19) by embryo rescue from a cross between Cucumis sativus L. (2n=2x=14) and C. hystrix Chakr. (2n=2x=24) (Chen et al., 1996; Chen et al., 1997). Reciprocal crossing of F1plants to either parent and self-pollination indicated that the hybrids were male and female sterile.

Colchicine, an antimitotic substance has been used to double the chromosome number in several plant species in attempts to increase fertility (Tosca et al., 1995; Taira et al., 1991). Different ploidy levels exist in the genus Cucumis (Shifriss, 1942; Kubicki, 1962). Autotetraploid cucumbers have been produced by soaking seed in solutions of colchicine (Smith and Lower, 1973). However, the fertility of such tetraploid plants is about 1/5 that of the diploids from which they are derived. Since colchicine application has been used successfully for the induction of higher ploidy levels in cucumber a study was designed to double the chromosome number of interspecific hybrids of Cucumis sativus x C. hystrix. Doubled hybrids (2n=38) would then be evaluated for fertility restoration.

Materials and Methods. The initial parental germplasm used (C. sativus and C. hystrix), the method of interspecific hybridization, they embryo rescue procedures, and the culture of F1plants have been reported previously (Chen et al., 1996; Chen et al., 1997).

Embryo treatment. Rescued F1embryos were incubated on MS medium (Murashige and Skoog, 1962) containing 0.01-1.0% colchicine (Sigma) for 1-12 days at 25C. A factorial design was used in two experiments (each in different growing seasons; spring and fall 1996) to examine the effects of colchicine concentration (%) (Experiment 1: 0.0, 0.01, and 0.05%; Experiment 2: 0.0, 0.10, 0.50, and 1.00%) and treatment duration (Experiment 1: 0, 2, 4, 6 days in solution; Experiment 2: 0, 8, 10, 12 days in solution). A third experiment was conducted to determine the effect of preculture duration on the recovery rate of embryos from colchicine treatment. Embryos were precultured on MS medium for either 2, 4 and 6 days before transfer to colchicine treatment. In each there were two replications of each treatment.

After colchicine treatment, the embryos were rinsed thoroughly with liquid MS medium, and then cultivated on a colchicine-free MS medium to produce plantlets. The plantlets in the 2- to 4- leaf stage were transferred to a soil medium containing field soil and sand (3:1 v/v), and fertilized using a standard 20:20:20 N:P:K solution.

Shoot treatment. Several application techniques were employed. The apical shoots of small hybrid plants (4th to 5th leaf stage) were treated by immersion in aqueous colchicine solution (0.02%) three times in each of three immersion intervals (4 times, 3 hours each for two days, 5 times, 3 hours each for two and half days; 6 times, 3 hours each for three days). Twenty-one shoots in total were treated (seven in each treatment in interval).

A second experiment was conducted to test the effect of DMSO on the chromosome doubling activity of colchicine treatment. In this factorial experiment the colchicine concentration was 0.02 and 0.05%, each in 1% DMSO. Treatment intervals consisted of: 1) one time, for 8 hours for one day; 2) Two times, for 8 hours each, for two days; 3) three times, for 3 hours each, once every another day for six days; 4) six times, for 3 hours each, once every another day for twelve days, and; 5) nine times, for 3 hours each, once every another day for eighteen days. Thirty-five shoots in total were treated (seven in each treatment interval).

A third treatment consisted of the application of 0.5 and 1.0% (wt/wt) colchicine in a lanolin paste (Fougera) on the apical meristems of plants in the 4th to 5th leaf stage. Twenty-six shoots in total were treated.

Evaluation of ploidy level. The ploidy level of colchicine- treated plants were evaluated by chromosome analysis and by flow cytometry. Root tips of selected plants were examined by standard root tip squash preparations and actocarmine staining techniques.

Results and Discussion. Embryo culture. A total of 256 embryos (239 colchicine-treated and 17 control) were used in the experiments described above. Seventy-seven plantlets were recovered from the colchicine treated embryos (32.2% regeneration rate). The average regeneration rate was 41.4% (4 of 10 embryos and 3 of 7 embryos) for control embryos. Variation in the initial leaf morphology was observed, but no chromosome doubled plants were obtained.

Results of Experiment 1 suggest that a considerable number of plantlets could be obtained (32 to 47%) from treatment of embryos with 0.01 or 0.05% colchicine (table 1). Thus, the treatment concentrations used in Experiment 2 were increased. It appears that, given the doses used, a concentration of 0.01% colchicine provided for the highest number (42%) of regenerated plantlets. This regeneration rate was similar to control (40%). Apparent colchicine toxicity (13-23% regeneration rate) occurred at concentrations higher than 0.50% colchicine in this experiment. Nevertheless, plantlets with chromosome numbers higher than 2n=19 were not recovered.

Repeated application of colchicine at any concentration caused a reduction of the number of plantlets recovered (25-33%) when compared to the control (40-43%) treatment in Experiments 1 and 2 (Table 1). Repeated application of colchicine at or beyond 10 days resulted in no plantlets recovered.

Preculture of embryos for six days on MS media (Experiment 3) resulted in a higher recovery of plantlets (52%) when compared to controls (40%) (Table 2).

Apical shoot treatments. Because colchicine toxicity was observed in embryo Experiments 1-3 and no higher-ploidy-level plantlets were recovered from the higher colchicine treatments, an experiment (shoot experiment) was designed to determine whether addition of DMSO to the colchicine application solution would result in recovery of plants with higher ploidy levels.

All 21F1plants treated with 0.02% colchicine survived treatment. All news leaves of the plants after treatment were deformed. However, after continued growth (~10 nodes above treatment node) all plants appeared to revert back to normal morphology. Treated plants did not change ploidy level.

When 35 plants were treated with 0.02 and 0.05% colchicine and 1% DMSO, 20 plants died. The remaining plants showed distortions in leaf and shoo morphology typical of colchicine treatment alone. These plants eventually recovered and appeared normal, but the treatment did not change the ploidy level of any of the plants.

All of the 26 plants treated with colchicine (0.5 and 1.0%) in a lanolin paste died after treatment. Plant growth reduction was observed in control plants (lanolin paste smear without colchicine). Therefore, it appears that lanolin paste under our conditions is somewhat toxic to cucumber plants and that a combination of lanolin paste and colchicine at the doses used inhibits plant growth and development.

It is concluded that colchicine treatment of embryos or shoot tips with colchicine in the concentrations and treatment durations used in our studies does not result in the doubling of C. sativus x C. hystrix F1hybrid embryos or plants. The colchicine treatment and culture of single cells from callus should be evaluated for its potential efficacy.

Table 1. Regeneration rates of embryos rescued from a Cucumis sativus x C. hystrix mating after colchicine treatment.

Effect of concentration
Conc.(%)
Treated numbers
Regeneration numbersrate
Regeneration (%)
Experiment 1
0.00 10 4 40.0
0.01 47 22 46.8
0.05 56 18 32.1
Total 113 44
Experiment 2
0.00 7 3 42.8
0.10 57 24 42.1
0.50 40 5 12.5
1.00 39 8 22.8
Total 143 40

Effect of treatment duration
Days1
Treated numbers
Regeneration numbers
Regeneration rate (%)
Experiment 1
0 10 4 40
2 68 21 30.7
4 70 17 24.7
6 68 22 32.5
Total 216 64
Experiment 2
0 7 3 42.8
8 20 5 25.0
10 10 0 0
12 10 0 0
Total 47 8

1 Days in solution

Table 2. Results of preculture (before colchicine treatment) of rescued embryos on the regeneration rate of embryos from a Cucumis sativus x C. hystrix mating.

Days

Treated numbers

Regeneration numbers

Regeneration rate (%)
0 10 4 40.0
2 47 20 42.5
4 52 19 36.5
6 48 25 52.0
Total 157 68

Literature Cited

  1. Chen, J.F., J.E. Staub and Y. Tashiro. 1996. Regeneration of interspecific hybrids of Cucumis sativus L. x C. hystrix Char by direct embryo culture. Cucurbit Genet. Coop. Rpt. 19:34-35.
  2. Chen, J.F., J.E. Staub and Y. Tashiro, S. Isshiki and S. Miyazak. 1997. Successful interspecific hybridization between Cucumis sativus L. and C. hystrix Chakr. Euphytica (in press).
  3. Beharav, A. and Y. Cohen. 1994. Effect of gamma-radiation on vitality fertilization ability of Cucumis melo and C. metuliferus pollen. Cucurbit Genet. Coop. Rpt. 17:94-96.
  4. Custers, J.B.M. and A.M.P. den Nijs. 1986. Effects of aminoethoxyvinylglycine (AVG), environment and genotype in overcoming hybridization barriers between Cucumis species. Euphytica 35:639-647.
  5. Kho, Y.O., A.M.P. den Nijs and J. Franken. 1980. Interspecific hybridization in Cucumis L. II. The crossability of species, an investigation of in vitro pollen tube growth and seed set. Euphytica 29:661-672.
  6. Chatterjee, M. and T.A. More. 1991. Interspecific hybridization in Cucumis spp. Cucurbit Genet. Coop. Rpt. 14:69.
  7. Tosca, A., R. Pandolfi, S. Citterio, A. Fascoli, and S. Sgorbati. 1995. Determination by flow cytometry of the chromosome doubling capacity of colchicine and oryzalin in gynogenetic haploids of Gerbera. Plant Cell Rpts. 14:455-458.
  8. Taira, T., Z.Z. Shao, H. Hamawaki and E.N. Larter. 1991. The effect of colchicine as a chromosome doubling agent for wheat-rye hybrids as influenced by pH, method of application, and post-treatment environment. Plant Breeding 106:329-333.
  9. Smith, O.S. and R. L. Lower. 1973. Effects of induced polyploidy in cucumbers. J. Amer. Soc. Hort. Sci. 98:118-120.
  10. Shifriss, O. 1942. Polyploids in the genus Cucumis. J. Hered. 33: 144-152.
  11. Kubicki, B. 1962. Polyploids in muskmelons and cucumbers. Genetica Polonica 3:161-179.
  12. Murashige, T. and F.A. A revised medium for growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-497.