Attempts to Overcome Barrier of Interspecific Hybridization between Cucumis melo and C. metuliferus

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Cucurbit Genetics Cooperative Report 17:90-93 (article 27) 1994

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

Our efforts to achieve the interspecific hybrid between Cucumis melo and Cucumis metuliferus through conventional breeding procedure between a few reciprocal combinations (in summer 1002) were unsuccessful because of the existence of a prefertilization barrier (1). The barrier was characterized by arrested pollen tube growth in the stigma or the upper style. In any event, viable embryos can be recovered 15 to 20 days after pollination, but not at fruit maturity, perhaps due to embryo abortion (8). To overcome the barrier, techniques like bud pollination (5), growth regulator application (5) and use of irradiated mentor pollen (5,6) – suggested in other crop plants and in Cucumis – combining with embryo culture, were employed to find out their potential to overcome the barrier of hybridization between the two species.

Plant material: The plants were grown in the field under insect-proof net and flowers were hand-pollinated during May-June 1993. We used 10 lines (Monoecious PI124111F and ‘Tam-Uvalde’; Andromonoecious ‘Venrantais’, ‘Chartenais-T’, ‘Mainstream’, ‘Gulfcoast’, ‘Ananas-Yokneam’ (Hazera Seed Co., Haifa, Israel); and Genoecious ‘Gylan’ E6/10 and ‘Gylan’ 5/5/5) as a female parent. Six accessions of C. metuliferus (PI 292 190 So/1, 2 ,3 and 4, ‘Italia’ and one from Tzofar, Israel) were used as a male parent. Using different cultivars of the same species in interspecific cross results in variable fruit set and embryo development (4. 10).

Irradiated mentor pollen: Anthers with pollen were collected in the morning from fresh male C. melo flowers and irradiated with 100, 150 and 200 Krad gamma rays. The flowers were kept overnight at 5˚ C. The next morning, anthers with the mentor pollen (MP) were mixed with fresh anthers of C. metuliferus in a volume ratio of about 2:1 (7) for use in crosses., We applied the MP first and the foreign pollen directly thereafter or in the reverse order. Standard pollination procedure for Cucumis were used with special precautions to avoid contamination by pollen from foreign sources.

Benzyladenine: 1% of the cytokinin N6 -benzyladenine (BA) in lanolin was applied to the base of ovaries immediately after pollination. This concentration of BA was suitable in promoting fruit set in hand pollinated C. melo (9).

Bud pollination: Hand-pollination of the flowers was attempted at its stage: 24, 48 or 72 hr prior to anthesis.

Embryo culture: Fruits that developed were checked for ovules with embryos 20-25 days after pollination. After seed coat removal, they were washed during 24 hr and then placed on 5 culture media, with or without previous sterilization, depending on the culture media, of which the composition is given in Table 1. Seeds placed on media containing sucrose were sterilized in 0.75% solution of sodium hypochlorite for 0 (control), 5, 10 or 15 min, followed by 3 rinses in sterile bidistilled water. The pH of the media was adjusted to 5.75 prior to addition of 0.75% purified Bacto agar (Difco). The media were autoclaved at 121 ˚ C for 20 min at 15 psi. The cultures were kept under 16 h of low fluorescent light (1000-1500 lux) at 25 +1 ˚ C .

Seeds that started to show signs of contamination were removed from the culture medium, immersed for 10 sec in 70% ethanol followed by 3 rinses in sterile bidistilled water, and then placed back on medium 4 (Table 1). Seedlings with fully expanded cotyledons were transplanted into pots with a sterilized mixture of peat substrate and sand, then irradiated with 1/10 MS as needed. Plantlets at the initiation of the 2nd leaf developed from those seedlings were transferred into a glasshouse, where temperature ranged from 21 ˚ C (night) to 30 ˚ C (day). Plants at the stage of 3 leaves were planted out into soil in the field with a thick net.

Using the techniques of BA and bud pollination was ineffective in overcoming the hybridization barrier between the two species. Out of 50 and 45 pollination in various combinations, using the BA and bud pollination, respectively, no fruit set was observed.

Fruit and seed set of 10 lines of C. melo after pollination with self-irradiated pollen mixed with fresh pollen of C. metuliferus are presented in Table 2. Twenty and 21 normal fruits were developed from about 60 pollinations after irradiating the mentor pollen with 100 and 150 Krad, respectively. Fruit set was intensely reduced by the higher radiation dose (200 Krad). From about 35 pollinated flowers, only 2 normal fruits were developed after irradiation of the mentor pollen with this dose. Those results could easily be explained with our results about the effect of the various doses on pollen viability in vitro (2).

Mentor pollen technique effectively induced fruit set on all of the C. melo genotypes examined, but the success of obtaining fruits containing embryos was dependent on genotype (Table 2). In those fruits, the number of embryos was generally low.

The embryos which were sterilized and initially placed on media containing sucrose appeared to be highly contaminated, and after their removal to medium 4 (1/3 MS), most of them did not germinate. Some of the embryos which were cultured on media without sucrose were also infected, but most of them germinated even after being removed twice with a short immersion in ethanol. Embryos that were initially placed on medium consisting of 1/3 MS were observed to provide normal development for most of the embryos. On the contrary, embryos initially placed on medium consisting of only H2O did not grow, and therefore, were transferred to medium 4 after a few days.

Nine embryos developed into plantlets and survived transplanting to soil. All 9 plants that developed from these embryos resembled the C. melo maternal parent. Fruits that developed after self-pollination of these plants were also similar to the maternal parent fruits and, therefore, the 9 plants were considered to be noninterspecific hybrid. Fruits of C. melo, which developed after self-pollination with irradiated pollen with at least 100 Krad gamma-rays never contained mature seeds (2). Since all fruits which contained embryos in this study were obtained on the andromonoecious genotypes and never on the monoecious or genoecious lines (Table 2), we assume that those embryos resulted from viable maternal pollen, although all the anthers were removed from the hermaphrodite flowers 24 h before flowering.

We reached the following conclusions:

  1. A significant factor in the embryo culture was the age of isolated embryos. The age of 20-25 days, counting from the date of pollination, was suitable to form plants.
  2. The seeds isolated at the above-mentioned age appeared to be highly contaminated, yet not sufficiently mature to survive after sterilization with sodium hypochlorite. Therefore, a method of disinfect ion must be worked out that will provide sterility and surviving embryos. One possibility is using a mixture of nonionic detergents, as described by Rakoczy-Trojanowska and Malepszy (12).
  3. Initially cultured embryos at the above-mentioned age on media container 1.3 MS are suitable to allow for normal plantlet development.

Table 1. General composition of the media used.

Designation

Composition of Medium
1 The content of mineral components and vitamins according to Murashige and Skoog (MS) (11), 1 g/l casein hydrolysate, 0.1 g/l m-inositol, 1 mg/1 thiamine-HCl, 1 mg/l puridoxin HCl, 1 mg/l nicotinic acid, 35 g/l sucrose, 0.02 mg/l IAA and 0.5 mg/l kinetin (according to Beharav and Cohen (3).
2 Same as 1, with 2 modifications: I. only 1/3 MS; II. only 10 g/l sucrose
3 1/3 MS and 10 g/l sucrose.
4 1/3 MS.
5 Only H2 O.

Table 2. Effect of Cucumis melo genotype treated witih various Gamma-irradiation doses of mentor pollenmixed with fresh pollen of C. metuliferus on fruit and seed sets.

Genotype

Irradiation dose (Krad) of mentor pollen

No. of fruit set

No. of fruit with embryos

No. of embryos in the fruits containing embryos

MONOECIOUS
PI 124111F 150 3 0 —
200 1 0 —
‘Tam-Uvalde’ 150 4 0 —

GENOECIOUS
‘Gylan’ E6/10 100 4 0 —
150 1 0 —
‘Gylan’ 5/5/5 100 7 0 —
150 7 0 —

ANDROMONOECIOUS
‘Mainstream’ 100 1 0 —
Gulfcoast 100 1 1 1
150 2 1 8
‘Ananas-Yokneam’ 100 2 1 1
‘Hemed’ 100 2 1 2
‘Vedrantais’ 100 2 2 1, 9
150 2 1 5
100 1 0 —
150 2 1 2
Charentais-‘T’ 200 1 * —

* This fruit harvested early due to harvesting of the field.

Literature Cited

  1. Beharav, A. and Y. Cohen. 1994a. The crossability of Cucumis melo and C. metuliferus, an investigation of in vivo pollen tube growth. Cucurbit Genet. Coop. Rpt. 17:97-100.
  2. Beharav, A. and Y. Cohen. 1994b. Eeffect of gamma-radiation on vitality and competitive ability of Cucumis melo and C. metuliferus pollen. Cucurbit Genet. Coop. Rpt. 17:94-96.
  3. Beharav, A. and Y. Cohen. 1994c. In vitro culture of embryos of Cucumis melo L. Plant Cell, Tissue and Organ Culture (submitted).
  4. Castetter, E.F. 1930. Species crosses inthe genus Cucurbita. Amer. J. Bot. 17:41-57.
  5. Chatterjee, M. and T.A. More. 1991. Techniques to overcome barrier of interspecific hybridization in Cucumis. Cucurbit Genet Coop. Rpt. 14:66-68.
  6. Den Nijs, A.P.M., J.B. M. Custers, and A.J. Kooistra. 1980. Reciprocal crosses between Cucumis africanus and C. metuliferus Naud. I. Overcoming barriers to fertilization by mentor pollen and AVG. Cucurbit Genet. Coop. Rpt. 3:60-62.
  7. Des, A.P.M. and E.H. Oost. 1980. Effect of mentor pollen on pistil-pollen incongruities among speies of Cucumis L. Euphytica 29:267-271.
  8. 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.
  9. Jones, C.M. 1965. Effect of benzyladenine on fruit set inmuskmelon. Proc. Amer. Soc. Hort. Sci. 87-335-340.
  10. Kwack, S. N. and K. Fujieda. 1985. Pollen tube growth and embryo development in interspecific crosses of Cucurbita. J. Faculty Agr. Kyushu University 30:1-8.
  11. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-479.
  12. Rakoczy-Trojanowska, M. and W. Melepzy. 1986. Obtaining of hybrids within the family Cucurbitaceae. I. Characteristics of hybrids from Cucurbita x C. pepo. Genet. Pollination 27:259-272.