Plant Regeneration from Cotyledon Protoplasts of Cucumis melo L. cv. Cantaloup Charentais.

Cucurbit Genetics Cooperative Report 9:74-77 (article 23) 1986

Roig¹, L. Zubeldia², M.C. Orts¹, M.V. Roche¹, and V. Moreno²
Departments of Microbiology¹ and Genetics², E.T.S. Ingenieros Agronomos, Universidad Politecnica de Valencia, 46022 Valencia, Spain.

In a previous work (1) we described suitable methods for obtaining callus from mesophyll protoplasts of axenic plants of three melon cultivars. Recently we have reported the morphogenic response of those kind of calli, both via embryogenesis and organogenesis (2). Whole plants cold be regenerated from protoplast-derived calli growing on a definite sequence of media. In this paper we show the achievement of plant regeneration from protoplasts originating from another source, namely cotyledons from seedlings of Cucumis melo L. ‘Cantaloup Charentais’.

Methods. Seeds of melon were surface sterilized by immersion in 12.5% commercial bleach (equivalent to 6.25 g/l of active chlorine) for 20 min. After three rinses with sterile distilled water, they were aseptically sown on agarified MG medium (1) in 20X195 mm test tubes. Cotyledons from 11-13 days old seedlings were used for protoplast isolation, either directly or after preculturing them in C medium (1) for two days. Strips of 1-2 mm width from these explants were placed in 100 ml Erlenmeyer flasks containing 8 ml of enzyme solution at the rate of 0.15 g of tissue/ml of enzyme, following the procedure previously described (1). After removing the enzyme solution, protoplasts were purified by flotation over 20% sucrose, picking up the ring which appeared in the interphase formed between the mannitol solution containing the protoplasts and the pad of sucrose. The protoplast yield was estimated by four cell counts in a5 Howard mould-counting chamber. Protoplasts, at a initial density of 1.5-2 c105 cells/ml, we2re cultured on 50 mm Petri dishes containing 4 ml of ZEPC 0.6M-mannitol liquid medium (1), at 27 ˚ C under darkness, for 20 days. Cells and small cell aggregates originating from protoplasts were transferred to the same liquid medium but at 0.3M-mannitol, at the rate of 2-4 x104 units/ml. After 20 days more in culture, the cells and small cell clusters were subcultured in a modified CEN medium (2) consisting of Murashige and Skoog (4) salt solutions, 1 mg/1 thiamine-HCl, 100 mg/l myo-inositol, 1 g/l casaminoacids, 30 g/l sucrose, 0.1 mg/1 indole-3acetic acid and 10 mg/1 kinetin, at the rate of 0.5-1.0 x104 units/ml, in 100 ml flasks containing 20 ml of liquid medium and incubated at 25.0+0.5 ˚ C and constant illumination (400 lux) on an orbital shaker at 95 r.p.m. In order to obtain organogenesis, the requiting microcalli were successfully transferred every 15 days to the sequence of solid media IK 0169 (3), IK 0060 (3) and a modified NB 00101s (2) to content 10 g/l sucrose. All seedlings and callus cultures were incubated in a growth chamber at 27+2 ˚ C during the 16 h light period (1,500 lux, cool fluorescent tubes Gro-lux, Sylvania) and at 24+1 ˚ C during the dark.

Results. Table 1 shows the protoplast yields obtained from cotyledons under the two conditions assayed and their behaviour when they were cultivated in EPC 0.6M-mannitolliquid medium. Contrarily to what happened with mesophyll protoplasts (1) we do not observe here a decrease of yield due to the preculture of the source of the protoplasts; in fact, the number of protoplasts per gram of tissue obtained from precultured as well as from non-precultured cotyledons was similar to the one produced by precultured ;eaves of axenic plants (1).

Morphologically, cotyledon protoplasts are very close to mesophyll ones, presenting a great quantity of big chloroplasts distributed in the cytoplasm and a notable vacuolization, but being clearly superior in size and containing a higher number of other intracellular organelles (mainly amyloplasts) which makes it necessary their purification by floating them over a pad of sucrose.

The behaviour of the protoplasts in the ZEPC liquid medium was absolutely dependent on the preculture of the cotyledons. So, precultured cotyledons produced viable protoplasts with 75-80% of cell wall regeneration within 2-3 days of culture and sustained divisions in the 45% of the regenerated cells after 20 days in culture. On the contrary, the non-precultured cotyledons gave rise to protoplasts which experimented cell wall regeneration at very low rates and only exceptionally initiated cell wall regeneration at very low rates and only exceptionally initiated first divisions; sustained divisions were never observed. Previously (1) we already pointed out the advantageous effect of preculturing the source of protoplasts in the case of varieties with low response and/or when mother plants were grown under suboptimal conditions; the present results using cotyledons as source of protoplasts are a new evidence in this direction.

The percentage of cells undergoing sustained divisions in the ZEPC 0/6M as well as their further growth after being subcultured in the ZEPC 0.3M liquid medium did not differ from the data reported for mesophyll protoplasts (1), reaching in both cases a high level of mitotic activity.

When cells and small cell clusters grown on ZEPC 0.3M were transferred to the CEN agitated liquid medium, whitish or light0green microcalli were formed after 20days in culture. These could be collected by being filtered through a 100 μm nylon mesh. Batches of around 250 mg (fresh weight) of microcalli were placed on IK 0160 solid medium and grown for 15 days, giving rise to big whitish and friable calli carrying several green zones of apparently organized growth and some peripheral true buds. The transfer of these organized tissues to auxin-free IK0060 solid medium encouraged their growth, though the amount of unorganized friable callus formed was still high. The quantity of unorganized friable callus formed was still high. The quantity of unorganized tissue decreased gradually after two passages of the green zones through NB 00101S medium, where compact and green calli were developing greatly, giving rise to the formation of a great number of buds and shoot-buds. Clearly distinguishable shoots could be observed in some calli after their third passage through the NB 00101S medium. The number of calli presenting developed shoots and the number of shoots per callus depended on two factors: the exclusive transfer of organized zones of calli in each subculture and the short interval elapsed among them (10-15 days every time). Table 2 summarizes the results obtained after each step of this procedure.

In order to obtain whole plants, the clearly developed shoots were individually transferred to a modified NB NB 00101S medium (without 6-benzylaminopurine or casaminoacids). Most of the shoots displayed clear abnormalities due, perhaps, to changes in their chromosome number and were unable to root. However, some of them became whole plants (usually rosette-shaped plants and occasionally normal plants) which could be propagated in vitro by subculturing their apices and axillary buds in the abovementioned medium.

In short, we can now avail ourselves of two different protoplast systems which, under the proper cultural conditions, allow a moderate rate of plant regeneration, via organogenesis, in melon.

Nowadays, in our laboratories, we are using the protoplast fusion technique in order to obtain interspecific somatic hybrids between Cucumis melo L. and several wild species of genera Cucumis and Cucurbita, in which resistances to plagues and diseases have been described. The obtention of such a kind of hybrid plants should represent the first step in our final goal, i.e., the transfer of resistance genes from the wild species to the cultivated one. So the attainment of plant regeneration from protoplasts of melon is an important step towards the stated end.

This research was supported by Grant No. 3021/83 from CAICYT Fund, M.C. Orts and M.V., Roche are grateful for a Grant from the Autonomous Government of the Valencian Community (Spain).

Table 1. Isolation and culture of protoplasts from non-precultured (NP) cotyledons and from precultured (P) ones in C mediun for 20 days.

	Isolation	Culture
	Protoplast yield (x106) /g of tissue	% of cells undergoing divisions
NP	0.82 + 0.53	0.0
P	1.37 + 0.77	45.30 + 9.57

Table 2. Morphogenic response of protoplast-derived calli after subculturing for 15 days in each of the successive solid culture media.

			% of calli with budsz
Sequence of media		No. of calli	++	+	–	% of calli with shoot-buds	% of calli with shoots
1) IK 0160		177	77.4	22.6	0	1.7	0
2) Ik 0060		138	97.8	2.2	0	31.2	0
3) NB 00101S		93	77.4	22.6	0	35.5	0
4) NB 00101S		59	94.9	0	5.1	59.4	0
5) NB 00101S		41	100.0	0	0	100.0	9.7
6) NB 00101S		56	100.0	0	0	100.0	10.7
7) NB 00101S		64	100.0	0	0	100.0	20.0
Z++	:	calli with numerous buds peripherally distributed
+	:	calli with a few buds appeared in discrete zones.
–	:	calli without buds

Literature cited

1. Moreno, V., L. Zubeldia, and L.A. Roig. 1984. A method for obtaining callus cultures from mesophyll protoplasts of melon (Cucumis melo L.). Plant Sci. Let. 34:195-201.
2. Moreno, V., L. Zubeldia, B. Garcia-Sogo, F. Nuez, and L.A. Roig. 1985. Somatic embryogenesis in protoplast-derived cells of Cucumis melo L. In: Proc. Intnl. Eucarpia Symp. on “Genetic Manipulation in Plant Breeding”. Berlin (West) (in press).
3. Moreno, V., M. Garcia-Sogo, I. Granell, B. Garcia-Sogo, and L.A. Roig. 1985 Plant regeneration from calli of melon (Cucumis melo L.) cv. Amarillo Oro. Plant Cell, Tissue and Organ Culture. (in press).
4. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.