Somatic Hybridization of Muskmelon (Cucumis melo L.) with Kiwano (Cucumis metuliferus Naud.) and Squash (Cucurbita pepo L.) by Protoplast Electrofusion

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Cucurbit Genetics Cooperative Report 13:36-39 (article 14) 1990

I. Debeaujon and M. Branchard
Laboratoire de Génétique Végétale – C.N.R.S. (U.R.A. 115). Bât. 360
Université de Paris Sud – 91405 Orsay cedex – France

The introduction of disease and pest resistances and the increase in cold tolerance are two important objectives of muskmelon (Cucumis melo L.) improvement programs.

Considerable progress has already been assessed in these areas through conventional breeding techniques. However the resistances to Squash Mosaic Virus (SqMV) and root-knot nematode (Meloidogyne incognita) still remain to be introgressed into the cultivated muskmelon. Cucumis metuliferus Naud. (African horned cucumber or kiwano) was found to contain genes that confer resistance or tolerance to these major plagues (4,12). But its cross-incompatibility with Cucumis melo L. is very severe, hampering the formation of hybrid progenies (5,10).

Grafting on Cucurbita sp. is generally used to improve muskmelon growth at low temperature; however, this method is costly and time-consuming. Sexual crossings would be another way to combine the two genera Cucumis and Cucurbita but all attempts failed, due to strong incompatibility barriers (9).

Consequently, somatic hybridization by protoplast fusion appears to be a judicious approach to overcome this sexual incompatibility. Several studies already reported somatic hybridization in Cucurbits at the interspecific (3,7,13,14,17) and intergeneric (1,3,13) levels.

In this report, we describe results concerning the isolation and culture of muskmelon protoplasts and their electrofusion with kiwano or squash protoplasts. Our aim is to obtain somatic hybrids with the agronomically valuable traits mentioned above.

Plant material: Cotyledons and leaves of axenic cultures of muskmelon, squash and kiwano (leaves only) were used in our experiments. Cucurbita pepo L. cv. Diamant F1 and Cucumis melo L. cv. Preco F1 were provided by Dr. Mounier Mirabel, Nunehms Zaden, Valence-France. Cucumis melo L. cv. Charentais T, Cucumis metuliferus (originally from Fassuliotis), and Cucurbita moschata L. were provided by Drs. Risser and Pitrat, INRA, Avignon-France.

Muskmelon seeds were sterilized in 2% calcium hypochlorite (70% active Chloride, Prolabo) for 3 min. followed by 3 rinses in sterile bidistilled water. Squash and Kiwano seeds were placed in 4% calcium hypochlorite for 15 min. and then soaked for 24 h in bidistilled water. After seed coat removal they were placed in 2% calcium hypochlorite for 3 min. followed by 3 rinses in sterile bidistilled water (7).

Fully expanded cotyledons were cut off as apical buds were aseptically planted into 250 ml bottles containing 50 ml of MEL (7) modified medium with 3% sucrose and 0.7% agar. Table 1 gives culture duration on MG and MEL media.

Table 1. Culture duration of mother plants on MG and MEL media for the obtention of cotyledon and leaf mesophylls as source of protoplasts.

Plant

Cotyledon (MG)

Mesophyll (MEL)
muskmelon 12 days 18-21 days
squash 7 days 11-13 days
kiwano – clonal propagation

The first to fourth expanded leaves were used as a source of mesophyll protoplasts. The incubation was carried out in a culture room at 27±1C (day) and 21±1C (night). The photoperiod was 16 h under 50 Em2.s1 provided by cool white fluorescent tubes GRO-LUX Sylvania.

Protoplast isolation: Leaves and cotyledons were cut in 1 mm wide strips and preplasmolysed during one hour in SB solution consisting of CPW salts (6), 0.1M glycine, 0.1M glucose and 3mM MES. The osmotic pressure was adjusted to 600 mosmol/kg with mannitol. The pH was adjusted to 5.7 with KOH. Enzymatic digestion was performed in SB completed with 1.5% cellulase onozuka R-10 (Yakult, Tokyo) and 0.3% macerozyme R-10 (Yakult, Tokyo); tissues were incubated overnight (15-16 h) in the dark at 27±1C. The enzyme-protoplast mixture was then filtered on a 63 m stainless steel mesh. Protoplasts were pelleted (100 g, 5 min.), resuspended in CPW solution with 21% w/v sucrose and centrifuged (120 g, 10 min.). Intact floating protoplasts were rinsed in SB solution (100 g, 5 min.) and finally transferred to culture medium.

Protoplast fusion: Electrofusion was carried out using the electric apparatus described by Sihachakr et al. (15). The movable multi-electrodes were connected to both a function generator (Enertec 4415) and a DC square pulse generator (self-constructed unit). They were placed in a Petri dish containing 0.6 ml of mixture (1:1) of protoplasts of both parents at a density of 3.105/ml of a 0.5 M mannitol solution. Following the application of 15 s AC field at 125 V/cm and 1 Mhz for aligning protoplasts, 6 square pulses developing 1150 V/cm for 60 s each were applied for protoplast fusion.

Protoplast culture: Immediately after the fusion process, 0.4 ml LCM culture medium (16) was progressively added to the fused protoplast mixture, completed with 1 ml, 1.2% agarose-containing medium 24 h later. This gave a final concentration of 105 protoplasts /ml. After 2 weeks culture in darkness, plating efficiency (number of dividing protoplasts/total number of protoplasts) was established and agarose blocks transferred on solid LCM medium with 0.75 mg/1 BAP. When microcalli have reached the size of about 1-2 mm they were isolated and cultured according to the protocol of Branchard and Chateau (2), for initiation and development of somatic embryos.

Protoplasts isolation: The same isolation procedure was applied to all our genotypes with satisfactory yield of intact protoplasts. That is 0.5-1.5.106 prot./g cotyledon tissue and 1.5-5.106 prot./g mesophyll tissue.

Protoplast fusion and culture: Without any fusion treatment, muskmelon protoplasts began to divide on the third day of culture; the plating efficiency determined 14 days after isolation was on average 25% in LCM medium, whatever genotype and organ source. But until now plant regeneration was obtained only from ‘Charentais T’ cotyledons, with 3 months being necessary from protoplast isolation to development of embryos into plantlets. Kiwano and squash protoplasts underwent divisions but a lower rate than muskmelon ones, with plating efficiencies being respectively 9% and 7%. Kiwano calli showed vigorous and sustained division on the contrary of squash calli whose growth was very slow and which tended to turn brown with time. None of these calli were able to differentiate embryogenic structures in our conditions.

The fusion treatment did not seem to significantly affect plating efficiency compared with control. The number of binary fused protoplasts varied between 18 and 28% according to the parental species and the origin of protoplasts (results assessed thanks to a DAPI (4′-6’diaminido-2-phenylindole) coloration of protoplast nuclei). No regeneration was observed after fusion experiments yet.

We do not have any early markers to select our heterocaryons or hybrid microcalli. So the fact that squash and kiwano protoplasts divide with low frequency and that they are unable to regenerate into plants appears to be a great advantage. Moreover, our fusion procedure is quite efficient and then increases the probability for heterofusions to occur. Both these facts will help considerably the recovery of hybrid products.

But the main problem encountered was the bad regeneration efficiency of muskmelon protoplast-derived calli. Some experiments are underway to improve the regeneration rate.

From now on, isozyme studies will be undertaken to confirm hybridity at the callus and plant levels; we focus our attention on malate dehydrogenase and acid phosphatase systems.

Literature Cited

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