Callus Formation from Cotyledon and Hypocotyl of Cucumis melo L. and Cucumis metuliferus

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Cucurbit Genetics Cooperative Report 17:88-89 (article 26) 1994

A. Beharav and Y. Cohen
Bar-Ilan University, Dept. of LIfe Sciences, Ramat Gan 52900, ISRAEL

For many years, viral disease in muskmelon (Cucumis melo) (2n = 24) has been of great concern for seedsmen and growers (5). The development of resistant cultivars can eliminate the disease.

Various accessions of Cucumis metuliferus (African horned cucumber) (2n=24) contain genes for some desirable characters that should be valuable additions to the gene pool of the muskmelon if they could be transferred (2, 6 and literature cited therein).

Our efforts to achieve the interspecific hybrid between C. melo and C. metuliferus through conventional breeding procedure between a few reciprocal combinations (in summer 1992) were unsuccessful because of the existence of a prefertilization barrier (1).

Tissue culture is a promising tool for the recovery of important traits such as disease resistance. Selection in vitro would allow for rapid screening of large populations of cells if suitable selection agents are available and if cell culture techniques exist for the regeneration of plants from single cells or clumps of cells. We report here on the callus formation from explant of C. melo and C. metuliferus on various nutrient media.

Plant material consisted of cotyledon and hypocotyl of axenic cultures of Cucumis melo cultivar ‘Charentais’ and Cucumis metuliferus line ‘Italia’. After seed coat removal they were sterilized in 0.75% solution of sodium hypochlorite for 30 and 20 min for C. melo and C. metuliferus seeds, respectively, followed by 3 rinses in sterile bidistilled water. This procedure was found to be suitable in preliminary study. The seeds were transferred to medium consisting of 1.6 g/liter Murashige and Skoog’s (MS) (4) inorganic salts and vitamins, 10 g/liter sucrose and 1% agar (Sigma). Cotyledons and hypocotyl from 8-day-old plants were cut to 3 mm wide strips and 8 mm length strips, respectively, and placed on the culture media.

The basal nutrient medium consisted of MS, 0.1 g/liter m-inositol, 1 mg/liter thiamine HCl, 40 g/liter sucrose, and various concentrations of BAP and NAA. The pH was adjusted to 5.75 prior to addition of 0.8% purified agar (Sigma). The media were autoclaved at 121 ˚ C for 20 min and about 20 ml were dispensed into petri dishes. the cultures were kept under 16 h of low light (1000 lux) at 25 + 1 ˚ C.

Table 1 gives data on callus development after 25 days of culture. The best developments were observed on medium consisting of 1 mg/liter NAA for C. metuliferus. The results demonstrate the possibility of applying them: 1) to develop cell suspension culture from the callus culture and apply the suspension in fusion and transformation experiments; 2) to develop shoot regeneration from the callus culture; 3) the differences found between C. melo and C. metuliferus to the selection of hybrid cells after fusion.

Table 1. Callus development of Cucumis melo (‘Charentais’) and Cucumis metuliferus (‘Italia’) after 25 days of culture with different BAP and NAA concentrations.

Callus Developmentz

BAP

NAA
C. melo
C. metuliferus
(mg/liter)
(mg/liter)
Cotyledon
Hypocotyl
Cotyledon
Hypocotyl
0.5 0y 1 2 0 b
1 0x 1 0 0 2
1x 3 3 b w
2x 3 3 2 2

z Green callus rated from 0, none; 1, little; 2, moderate; 3, much.
b = brown callus; w = white callus
y according to Jain and More (3).
x according to personal knowledge.

Literature Cited

  1. Beharav, A., and Y. Cohen. 1994. The crossability of Cucumis melo and C. metuliferus, an investigation of in vivo pollen tube growth. Cucurbit Genet. Coop. Rpt. 17:97-100.
  2. Fassuliotis, G. 1977. Self-fertilization of Cucumis metuliferus Naud. and its cross-compatibility with C. melo L. J. Amer. Soc. Hort. Sci. 102:336-339.
  3. Jain, J. and T.A. More. 1992. In vitro regeneration in Cucumis melo cv. Pus madhuras. Cucurbit Genet. Coop. Rpt. 15:62-64.
  4. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-479.
  5. Perring, T.M., C.A. Ferrar, K. Mayberry, and M.J. Blua. 1992. Research reveals pattern of cucurbit virus spread. Calif. Agr. 46:35-40.
  6. Punja, Z.K., F.A. Tang, and L.H. Watkins. 1988. Identification of resistance to root knot nematodes and virus diseases in Cucumis metuliferus and approaches to hybridization with Cucumis sativus by protoplast fusion. Phytopathol. 78 (Abstr.).