Cucurbit Genetics Cooperative Report 19:63-65 (article 23) 1996
N. Spetsidis, G. Sapountzakis and A.S. Tsaftaris
Department of Genetics and Plant Breeding, Aristotelian University of Thessaloniki, Greece
‘Galia’ is one of the most important melon genotypes cultivated in greenhouses in the Mediterranean basin. In vitro techniques for clonal micropropagation have been used with good results in species of the Cucurbitaceae such as cucumber (Cucumis sativus; 2,5), pumpkin (Cucurbita pepo; 4) and watermelon (Citrullus lanatus; 1). The objective of this work was to test different growth regulator combinations for micropropagating the melon (Cucumis melo) hybrid ‘Galia’.
Seeds of ‘Galia’ (Hazera, 1939, L.T.D., Israel) were soaked in a 75% v/v ethanol solution for 1 minute, subsequently in a Triton x 100 (Merk) 0.01% v/v solution for 15 minutes, and finally surface sterilized in a Milton (Proctor & Gamble L.T.D.) solution (1% w/v Na0Cl and 16.5% w/v NaCl) for 25 minutes. The seeds were then rinsed three times with sterile water and placed under aseptic conditions containing Murashige and Skoog (MS) medium (3) solidified with 0.8% w/v agar (Sigma). Cultures were placed in a growth chamber to allow germination and shoot growth until the 6-8 leaf stage. Axillary buds were used as the explant source.
All the experiments were conducted in a growth chamber maintained at 25 C provided with 16 hours photoperiod by cool white fluorescent light at 1500 lux. Media were adjusted to a pH of 5.7, autoclaved at 121 for 20 minutes, and solidified with 0.8% w/v agar. Three experiments were conducted to estimate the optimum level of growth regulators and to quantify the rate of propagation. In every experiment eight replications were used. Four weeks after the establishment of each experiment the number of the shoots at least 0.5 cm long were measured.
Experiment 1 – Estimating the optimum level for cytokinin: Axillary buds derived from in vitro grown shoots were established in vessels containing MS medium supplied with 0.0, 0.5, 1.0 or 2.0 mg/16-benzlaminopurine (BA).
The means of the shoots produced per axillary bud are presented in Table 1. It is obvious that BA level affects the number of shoots produced, and the optimum level is 2.0 mg/l (2.37 shoots per explant).
Experiment 2 – Estimating the optimum level for auxin: Axillary buds derived from in vitro grown shoots were transferred in vessels containing MS medium supplied with the optimum BA level found in the previous experiment (2.0 mg/l) and 0.0, 0.1 or -/5 mg/l ind9ole-3-acetic acid ((AA).
The means of the shoots produced per axillary bud are presented in Table 2. Best results were obtained with 0.1 mg/l IAA, yielding 2.50 shoots per explant. A cluster of shoots produced from a developed axillary bud, which was cultivated four weeks in the above medium, is shown in Fig. 1.
Experiment 3 – The effect of callus removal from the explants: Axillary buds produced in vitro were cultivated on MS medium supplemented with the optimum levels of BA and IAA (2.0 mg/l BA, 1/0 mg/l IAA). Two weeks after culture establishment, the formed callus was removed from the explants base, and he explants were transferred to fresh media of the same composition.
In the previous experiments, a significant mass of callus formed at the explants base, which possibly depressed growth. This experiment was aimed to study this problem. The explants which had callus removed two weeks after the initial establishment showed significantly more shoots per explant (4.75 vs. 3.62, LSD:0.89, at P=0.05).
Elongation and rooting of the produced shoots, after their excision from the clusters, was carried out on MS medium with no growth regulators within 4 weeks (Fig. 2).
In conclusion, the data indicated that:
a) The presence of cytokinin (BA) in the culture media increased the shoot propagation rate, and the optimum level was 2.0 mg/l. This level is higher than ones reported for other cucurbits. BA levels of 0.5 and 1.0 mg/l have been proposed for two cucumber genotypes (5),and 1 mg/l for pumpkin (4). Optimum levels of kinetin ranged from 0.1 mg/l in cucumber (2) to 1.0 mg/l for watermelon (1).
b) The presence of auxin (IAA) in the media affected the number of shoots produced, with the best results AT 1.0 IAA. This auxin level was similar to those proposed for one cucumber genotype (0.1 mg/l IAA) (2) and for watermelon (0.04 mg/l IAA) (1). For two other cucumber genotypes (5) and for pumpkin (4), best results were obtained in media lacking auxin.
Table 1. The mean number of shoots produced, per in vitro grown axillary bud, of the melon hybrid ‘Galia’, four weeks after cultivation on MS media supplemented with different levels of BA.
BA (mg/l) |
0.0 |
0.5 |
1.0 |
2.0 |
| Shoots | 1.00 | 1.87 | 1.79 | 2.37 |
Sample size: 8 explants per treatment]
LSD: 0.95 (at P=0.05)
Table 2. the mean number of shoots produced, per in vitro grown axillary bud, of the melon hybrid ‘Galia’, four weeks after cultivation on MS media supplemented with 2 mg/l BA and different levels of IAA.
IAA (mg/l) |
0.0 |
0.1 |
0.5 |
| Shoots | 1.87 | 2.50 | 2.25 |
Sample size: 8 explants per treatment.
LSD: 1.29 (at P=0.05).
Literature cited
- Barnes, L.R. 1979. In vitro propagation of watermelon. HortScience 11:223-227.
- Handley, L.W. and O.L. Chambliss. 1979. In vitro propagation of Cucumis sativus L. HortScience 14:22-23.
- Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:47-497.
- Pink, D.A.C. and D.G.A. Walkey. 1984. Rapid propagation of Cucurbita pepo L. by culture of meristem tips. HortScience 24:107-114.
- Sapountzakis, G. and A.S. Tsaftaris. 1994. Micropropagation of the cucumber hybrids ‘Brunex’ and ‘Bambin’. cucurbit genetics Coop. Rep. 17:50-53.