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Table 3. Callus growth from protoplasts of C. metuliferus after 6 weeks on 4 different media, at 22°C in the
dark. z
Medium Plate coverage(%) Color rating
no. Media components y 25°C 30°C Mean 25°C 30°C Mean
B1 0.01 mg 2,4-D + 1.0 mg BA 3.0 6.9 5.9 3.0 6.0 5.0
B2 0.20 mg IAA + 0.5 mg BA 1.5 16.1 11.2 5.0 5.4 5.3
B3 0.25 mg 2,4-D + 0.5 mg kin. 0.0 5.0 2.5 - 4.0 4.0
B4 0.50 mg 2,4-D + 1.0 mg kin. 0.0 9.3 6.2 - 6.6 6.6
Mean (LSD 5% x ) 1.0 10.3 (3.2) 4.0 5.6 (0.4)
z Data are means of 5 replications; protoplasts were initially cultured for 3 weeks at 25 or 30°C; initial medium was C2
(Table 1).
y Media B1 to B3 consist of C3 medium; medium B4 consists of C4 medium; all growth regulators expressed in mg " L -1
.
x For comparison of column means.
In experiment 3, growth regulator concentrations
remained the same, but the basal medium was
changed from C4 to C3. In contrast to experiment 2,
no significant differences were observed for media
types affecting amount of callus. However, media
type B3 produced whiter callus than other media
(Table 3). There was significantly more growth at 30
than at 25°C, repeating the trend seen in experiments
1 and 2. Effects of temperature on color, however,
were the opposite of results of experiment 2, with
protoplasts cultured at 25°C being significantly
whiter (Table 3). After 6 weeks on medium B3 or
B4, more than 10 roots per plate were obtained. As
with experiment 2, no embryogenesis was observed.
Results of all 3 experiments suggest that a culture
temperature at or near 30°C produces more microcalli
and/or callus. In experiment 3, callus from
protoplasts cultured at 30°C was browner, perhaps
reflecting a depletion of nutrients in the medium.
This effect was probably due to increased growth at
the higher temperature compared to 25°C, and can
probably be corrected by more frequent subculturing.
In summary, a method has been developed which
consistently provides large numbers of viable
protoplasts from cotyledon tissue of C. metuliferus.
The technique allows development of callus and roots
from the protoplast cultures. Using our procedure,
30°C was better than 25°C for producing microcalli,
and large amounts of callus on the callus induction
media. Although there was a large amount of
variability associated with liquid culture, agar
solidified MS medium containing 0.25 mg " L -1 2,4-D
and 0.5 mg " L -1 kinetin was best suited for producing
large amounts of friable, light yellow callus.
Literature Cited
1. Deakin, J. R., G. W. Bohn and T. W. Whitaker.
1971. Interspecific hybridization in Cucumis.
Economic Bot. 25: 195-211.
2. Durand, J., I. Potrykus and G. Donn. 1973.
Plantes issues de protoplastes de Pétunia. Z.
Pflanzenphysiol. 69: 26-34.
3. Fassuliotis, G. 1967. Species of Cucumis
resistant to the root-knot nematode, Meloidogyne
incognita acrita. Plant Dis. Rptr. 51: 720-723.
4. Gamburg, O. L., R. A. Miller and K. Ojima.
1968. Nutrient requirements of suspension
cultures of soybean root cells. Exp. Cell Res. 50:
151-158.
5. Jarl, C. I., G. S. Bokelmann and J. M. De Haas.
1995. Protoplast regeneration and fusion in
Cucumis: melon ! cucumber. Plant Cell Tiss.
Org. Cult. 43: 259-265.
Cucurbit Genetics Cooperative Report 24:102-106 (2001) 105