Cucurbit Genetics Cooperative Report 16:81-83 (article 31) 1993
Caiping Ma, X.P. Zhang, B.B. Rhodes and J.W. Adelberg
Horticulture Dept., Box 340375, Clemson University, Clemson, SC 29634
Protoplast culture and subsequent regeneration could lead to more rapid genetic progress with watermelon. Experiments were conducted to define conditions for protoplast culture of cotyledons.
Effects of Mannitol, Cellulase and Pectinase on Protoplast Isolation from Watermelon Cotyledons.
A factorial experiment was performed to determine effects of mannitol cellulase and pectinase on protoplast isolation. Three levels of mannitol (0.3, 0.4 and 0.5 M), three levels (0.5, 1.0, 1.5, and 2.0%) of cellulase (E.C. 3.2.1.4, C-9422, Sigma) and two levels (0 and 0.4%) of pectinase (E.C. 3.2.1.15, P-4625, Sigma) were dissolved in the following buffer: 0.1 M glycine, 10 mM CaCl2* 2H20, 0.7 mM KH2P04 at pH 5.7. Enzyme solutions were sterilized by filtration. Cotyledons of 1 week old seedlings grown in the greenhouse were used as the explant source. Cotyledons were first washed with tap water one hour, then surface-disinfected with 10% Clorox for 10 min., then rinsed four times in sterile distilled waer. Lower epidermis of the cotyledon was peeled off with fine curved forceps, cut into 0.4 x 0.4 mm segments and put into a sterile enzyme solution. The tissue was incubated in darkness at 28 ˚ C for 4-6 hr. At the end of digestion, the cotyledon tissue and enzyme solution was shaken gently by hand to release the protoplasts. Enzyme solution and protoplasts were filtered through a nylon mesh (45 μ M pore size), and filtrate was transferred to a centrifuge tube and spun at 200 RCF (Microcentrifuge Model 5-9A, Fisher Scientific) for 3-5 min. Protoplasts were resuspended in 0.5 ml of buffer solution with different concentrations of mannitol but without cellulase and pectinase. Density of protoplasts was determined with a hemocytometer, and viability of the protoplasts was examined after staining with 0.1% phenosafranin.
The optimal concentration of enzyme solution for isolating watermelon cotyledon protoplasts was 0.4 M mannitol, 1.5% cellulase and 0.4% pectinase (Table 1). Large standard errors indicate that lal three factors are important for watermelon protoplast isolation, Sorbitol at 0.4 M was an equally effective osmoticum for isolation of osmoticum in trials not presented here.
Effect of Temperature and Time during the Enzyme Digestion on Protoplast Denisty and Viability.
Materials and procedures were the same as in the first trial. Enzyme solution consisted of 0.4 M mannitol, 1% cellulase, 0.4% pectinase, 0.1 M glycine, 10 mM CaCl2 * H20 and 0.7 mM K2H2P04 at pH 5.7. Three temperatures and digestion times were tested. Every treatment had three replications. In order to obtain a high intact protoplast density in a short time, we selected a suitable digestion period of 4-6 h at 29 ˚C (Table 2).
Effect of Calcium Concentration in Enzyme Solution
It is known that Ca+2 can stabilize the protoplast membrane and enhance the survival of protoplasts. Four levels of Ca+2 in enzyme solution were evaluated: 0, 10, 15 and 20 mM. Enzyme solution consisted of 1% cellulase, 0.4% pectinase and 0.4 M mannitol at pH 5.7. In this experiment, immature cotyledons were used to isolate protoplasts. First, proximal portion of cotyledons were dissected from the embryo, cut in half lengthwise and transferred to MS medium with 10 mM BA, 3% sucrose, and 0.7% agar at pH 5.7. Tjese were maintained at 25+2 ˚ C, with a 16 h light period produced by cool white fluorescent lamps at 30 μ mol m-1 sec-2 . After 10-14 days of culture, green, expanding cotyledons were cut into 0.1 cm strips and put into sterile enzyme solution kept in darkness at 26 ˚ C for 4h. Every treatment had three replicates. The results are shown in Table 3.
Protoplast viability was highest at 20 mM Ca+2 although the protoplast density was lower at 15 mM Ca+2 . Unless a high density is desired, 20 mM Ca+2 is suitable for protoplast isolation (Table 3).7
Effect of Cotyledon Source on Protoplast Isolation
Because harvested watermelon seeds are often difficult to decontaminate prior to tissue culture, protoplast isolation for cotyledons of harvested (dried) seed and cotyledons of seed from the initial fruit were compared.
- Fresh seed from mature fruit. Mature fruit harvested in the grenhouse were surface disinfected with 95% ethanol, and seeds were removed asepticaly. Embryos were removed from seed coats. Cotyledon explants were dissected from embryos and transferred tto MS salts medium with 0.7% agar, 3% sucrose, 10 mM BA, 100 mg 1-1 myo-inositol, 2 mg 1-1glycine , 0.2 mg 1-1nicotinic acid at pH 5.7. Tissue was maintained at 25+ 2 ˚ C under light. After 8u-15 days the cotyledons were cut into 0.1 cm strips and put into the enzyme solution.
- Aseptic seed from mature fruit were dried in the laminar flow hood and kept in sterile bottles. Seeds were soaked in sterile distilled water before use. After 24-48 h. embryos were removed from seed coats and cultured as above.
In a second test with dried seeds, aseptic mature seeds were soaked in sterile distilled water and shaken on a platform shaker at 150 rpm for 24 hours. Seeds were transferred to a Magenta GA7 vessel with a layer of wet filter paper and kept in the dark at 30 ˚ C. After 2 days, embryos with ca, 1 cm radicle were removed from seed coats and transferred to the same medium used in test 1, but with out BA. Cotyledons 8-14 days old were cut into 0.1 cm strips and put into the enzyme solution.
Cotyledons of fresh seeds from mature fruit were the best explant source for isolation and culture of protoplasts. The density of the protoplasts was 1.0-1.3 x 105 ml-1 and protoplast viability was 68-74%.
Protoplasts were cultured on liquid and agar gelled B5 medium (Gambourg et al. 1968) with different levels of growth regulators. Protoplasts became more oval after 2-3 days, indicating the synthesis cell wall. The protoplasts survived 15 days. However, protoplasts from dried seeds died in 2-3 days although the density and viability was not significantly different from protoplasts of fresh seeds. Results indicate that cotyledons from fresh seeds of mature fruit should be explants for protoplast isolation.
Table 1. Effectof 3 levels of mannitol, 2 levels of pectinase and 4 levels of cellulase on watermelon cotyledon protoplast density and variability.
Ingredient |
Density (x 104) |
Variability (%) |
|
| Mannitol | |||
| 0.3 M | 14.5 +12.4a | 46.9+28.3 | |
| 0.4.M | 14.7+13.8 | 58.6+15.6 | |
| 0.5 M | 13.7+18.7 | 51.6+22.3 | |
| Pectinaseb | |||
| none | 2.6+2.2 | 55.2+15.2 | |
| 0.4 | 16.0+8.7 | 47.0+25.6 | |
| Cellulasec | |||
| 0.5% | 15.2+14.90 | 48.5+16.0 | |
| 1.0% | 14.5+15.3 | 47.1+21.0 | |
| 1.5% | 16.6+14.1 | 60.2+10.4 | |
| 2.0% | 10.9+6.2 | 48.7+30.4 | |
aStandard errors.
b,cPectinase was E.C. 3.2.1.15,P4625; Cellulase, E.C.3.2.1.4,C-9422, both Sigma.
Table 2. Effect of temperature and digestion time on watermelon cotyledon protoplast density and variability.
20 |
25.5 |
29 |
||||
D(x 104)a |
V |
D(x104) |
V |
D(x104) |
V |
|
digestion (h) |
||||||
| 4 | 8.2 | 85.4 | 12.3 | 92.4 | 19.4 | 88.2 |
| 6 | 12.0 | 87.8 | 16.0 | 83.8 | 20.0 | 82.2 |
| 8 | 12.8 | 78.9 | 22.3 | 75.8 | 27.4 | 72.5 |
| 10 | 19.0 | 69.0 | 26.2 | 77.9 | 23.8 | 61.0 |
aD = density; V = viability
Table 3. Effect of Ca+2 in enzyme solution on density and viability of protoplasts from immature watermelon cotyledons.
Ca+2(mM)a |
||||
| 0 | 10 | 15 | 20 | |
| Density (x104) | 1.9 | 3.9 | 2.5 | 2.2 |
| Viability (5) | 64.7 | 80.8 | 88.7 | 94.1 |
aCaCl2* 2H20
Literature Cited
- Gambourg,O.L., r.A. Miller, and K. Ojima. 1968. Plant cell cultures. I. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 50:151-158.