Cucurbit Genetics Cooperative Report 14:81-84 (article 30) 1991
Selective Methods for the REcovery of Somatic Hybrids of Cucumis melo x metuliferus and C. sativus x C. metuliferus
M. Dabauza, L.A. Roig and V. Moreno
Plant Cell & Tissue Culture Laboratory, Biotechnology Department, Universidad Politecnica de Valencia, Cno. de Vera, 14, 46020- Valencia (Spain)
Introduction. Methods for the isolation and culture of Cucumis metuliferus protoplasts have been previously reported by our group (18) and by other authors (8, 17, 20). These studies have given of valid information about the cultural response of protoplasts from this cucurbitaceous wild species and they are relevant works since, in accessions of this species, resistances to root-knot nematode (2), SqMV (16), WMV-1 (16), ZYMV (17) powdery mildew (PM1) and aphids (1) have been described. In spite of the sexual cross between a feral Cucumis melo (PI 140471) and a line of Cucumis metuliferus (PI 292190) published in 1969 (14), other authors (3, 6, 7, 15) reported the impossibility of reproducing this particular cross, even using the same parental lines. As a consequence, efforts for introgressing those resistance carrying genes into the genetic context of cultivated cucurbitaceous species by conventional sexual crosses have been severely impeded by the existence of incompatibility battiers (1, 7). Somatic hybridization by protoplast fusion should allow the attainment of genetic bridges through which the transfer of interesting traits, from the wild species to the cultivated ones like melon or cucumber, should be feasible.
Materials and Methods. Seeds of Cucumis metuliferus (kindly supplied by Dr. Jacos, Stellenbosch University, South Africa) were surface sterilized and germinated on MG medium as previously described (9,12). Cotyledons from 5 day-old seedlings were used for protoplast isolation after pre-culturing them in C medium (12) during three days. Strips of 1-2 m width from these explants were placed in 100 ml Erlenmeyer flasks containing 8 ml of a filter sterilized enzyme solution consisting in LG medium (M & S macroelements at 1/4 strength, 0.3 M mannitol., 0.1 M glycine, 1 mM CaCl2:2H20, 0,51 mM MES) and 1.5% cellulase Onozuka R-10 (Yakult Pharmaceutical Ind. Ltd.) at pH 5.7. The incubation was performed under darkness at 28
˚ C for 12 h in a reciprocal shaker at 100 strokes/min (amplitude 20 mm). After incubation, the protoplasts were filtered through an 85
μ m nylon mesh, purified by flotation over F6 medium (0.6 M sucrose, 0.05 M glycine, 0.01M CAC12:2H20 at pH 5.7) and rinsed twice in LG medium. After protoplast counting, they were cultured in 35 mm diameter glass Petri dishes containing 2.5 ml of the culture liquid medium at a final concentration of 1 x 105 protoplast/ml.
Two different media were used for protoplasts culture: DNB 10/05/05 and DIB 10/05/05 (henceforth denominated DNV and DIV). Both of them contained B5 mineral solution (4) 0.058 M sucrose, 0.055 M glucose, 0.55 mMmyo-inositol nd 0.51 mM MES. Additionally, DNB medium contained 0.03 mM thiamine-HCL, 8.12 5Mnicotinic acid, 4,86 5M pyroxodine-HCl, 0.44 mannitol, 4.52 5M 2,4-D, 2.68 5M NAA and 2,22 5M 6-BA. DIB medium contained Shahin's vitamins (19), 100 mg/l casein hydrolysate, 50 ml/l coconut milk, 0.27 M adenine sulfate, 0.02 M xylitol, 0.36 M mannitol, 4.52 5M 2,4-D, 2,85 5M IAA and 2.22 5M 6-BA. The pH was adjusted to 5.7 in both media before autoclaving. Incubation took place under darkness at 28
˚ C. Four consecutive reductions in the osmolarity (0.50-0.42-0.32-0.25-0.21M) of the culture media were effected by the addition of mannitol-free fesh medium every 7 days.
After 30 days in culture, the microcalli grown in each of two media were subcultured into different media following two different ways: those grown on DNB followed the embryogenic pathway designed for cucumber protoplasts (in preparation) and the ones grown on DIB followed the organogenic pathway established for melon protoplasts (10). Thus, microcalli grown in DNB medium were subcultured in two media consisting in MB basal medium (M&S salts + 0.09M sucrose + 0.55 mM myo-inositol + 1.71 mM glutamine + 0.27M adenine sulfate + Sh-vitamins) supplemented with 4.52 5M 2,4D + 4.44 5M 6-BA (DB 10/10 medium) or 13.43 5M NAA = 4.92 5M 2-iP (NP 25/10 medium) and incubation was made under darkness at 28
˚ C, while those proceeding from DIB medium were subcultured onto IB 25/10 medium (5) and further transferred to solid IK 01/60 Cul medium and incubated under photoperiod as described for melon protoplasts (10).
A double-layer ("soft-hard") technique was utilized for subculturing the microcalli. It consists of removing the worn-out protoplast culture medium and replacing it with 3 ml of the new liquid medium to be tested. The microcalli are resuspended in this liquid medium and mixed with 3 ml of the same, but agarified (1%), liquified medium. The resulting mixture (final agar concentration 0.5%) is agitated and rapidly distriibuted ("soft layer") over the 'hard layer' consisting of 20 ml of the same medium soidified with 0.8% agar and previously distributed in 90 mm diameter plastic Petri dishes. Final cell count concentration is 1-2 x 104 minicalli/ml in each replicate. Fifteen replicates were used per treatment.
1. - Protoplasts isolation and culture. Protoplast yield was 1-2 x 106 protoplasts/g of tissue. After 2 days, cell wall regeneration took place in 80% of the protoplasts in both DNV and DIB culture media. First divisions began on the third day and cell aggregates could be observed on the 7th day in culture in DNB medium, while in DIB medium first divisions started a little later (6 days). Nevertheless in both media the growth is similar and considerable after 30 days in culture: the liquid medium has disappeared and replaced by a continuous mass of microcalli.
2. - Cultural and embryogenic response of C. metuliferus p-calli. C. metuliferus protoplast-derived microcalli grew slowly in both media DB 01/01 and NP 25/10 and gave rise to small minicalli that originated only one embryogenic callus in all theDB medium replicates and three embryogenic calli when NP medium was used. These calli were subcultured to hormone-free medium to promote the development of proembryos, but they did not grow anymore and became necrosed in 10 days.
On the contrary, media as well as cultural conditions utilized in this experiment allow an intense mitotic activity and the regeneraiton of plants through somatic embryogenesis in protoplast-derived calli of Cucumis sativus (in preparation). As a consequence, this completely different response in bioth cucurbitaceou species could be efficiently used to perform selective methods for the recovery of somatic hybrids after protoplast fusion between cucumber and C. metuliferus. The selective scheme would imply the use of protoplasts from a recessive mutant of cucumber (such as glabrous or yellow-green) and their fusion with protoplasts from a normal line of C. metuliferus. It would be based, therefore, on the complementation between the embryogenic capability of cucumber and the wild phenotype of the wild species. Only the hybrid cells would expect to be able to regenerate normal hairy or green plants, respectively.
3. - Cultural and organogenic response of p-calli. In this case the response of C. metuliferus protoplast-derived calli was not positive either. After 21 days in culture in the first sequential medium (IB 25/10), the microcalli became small minicalli and did not grow (or did so very slowly) even when transferred ti tge IK 01/60 solid medium, becoming necrotic, although occasionally some calli grew a little more and showed small cream-greenish globular structures that did not develop anymore.
This response is, again, completely different to that normally found when protoplasts of Cucumis melo are used following the same sequential media and incubation conditions designed by our group (10). Melon protoplasts cultivated in DIB liquid medium regenerate the cell wall and start cell division very quickly; afer cubculturing the microcalli into IB25/10 medium they row fast up to the point of 5-10 mm sized cream-greenish minicalli. The transfer of these minicalli to IK 01/60 solid medium causes the formation of big calli carryig organized growing zones with shoot-buds from which plants can be regenerated.
Again, thse results pointed out the possiility of applying the differences found between C. melo and C. metuliferus to the selection of hybrid cells after their protoplast fusion. The scheme should be the same as that mentioned above but taking advantage this time of the organogenic response of melon in the sequence of media described: only the hybrid cells would regenerate normal hairy or green plants through shoot formation.
Experiments aimed at the attainment of somatic hybrids between C. metuliferus and cucumber or melon lines are in progress.
Acknowledgements: The authors express their appreciation to CICYT (Comision Interministerial de Ciencia y Tecnologa, Ministry of Education and Science, Spanish Government) for financial support (Project BIO89-0446). M. Dabauza is grateful for her Grant from the Autonomous Government of the Valenvian Community (Spain).
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