Isolation and Culture of Cucumis metuliferus Protoplasts

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Cucurbit Genetics Cooperative Report 12:26-29 (article 12) 1989

William H. McCarthy, Todd C. Wehner and Margaret E. Daub
Department of Horticultural Science, Box 7609 (1st and 2nd authors) and Department of Plant Pathology, Box 7616, North Carolina State University, Raleigh, NC 27695-7609 (3rd author)

Research supported in part by a grant from Pickle Packers International. The authors wish to thank Mr. D.F. Moxely for technical assistance.

In the southeastern United States, approximately 12% of the potential cucumber yield is lost to root knot nematodes (Meloidogyne spp.). Screening of the Cucumis sativus germplasm revealed no resistant accessions (4). Within Cucumis, the species C. metuliferus has shown medium- to high-level resistance to root knot nematode (3). Traditional sexual hybridization techniques have been unsuccessful in producing hybrids between C. sativus and C. metuliferus (1). Protoplast fusion is one possible method of overcoming the barriers which exist between these two species. Before fusion work can take place, techniques for protoplast isolation and culture of C. metuliferus need to be established. The objective of this study was to develop a procedures for protoplast isolation and culture of C. metuliferus protoplasts.

Methods. Cucumis metuliferus PI 482454 seeds were sterilized using the industrial disinfectant LD (Alcide Corporation, Norwalk, Conn. USA 06851) for 30 minutes at the suggested rate of 1:1:10 for base, activator, and double glass-distilled water, respectively. Seeds were rinsed 5 times with sterilized water, and placed onto C1 medium (Table 1) and incubated in the dark at 30°C. After 84 hours, seedlings were placed in a growth room held at 22°C and 16 hours of light (8,000 lux). Twenty four hours before protoplast isolation, seedlings were transferred back to 30°C in darkness.

An enzyme solution was prepared consisting of 0.7 mM KH2PO4, 7 mM CaCl2 * 2H20, 0.5 M mannitol, 3 mM MES [2-(N-morpholino)ethanesulfonic acid], 2% cellulysin(Cal. BioChem.), and 0.5% macerase (Cal. BioChem.). This solution was then mixed at a 1:1 ratio with C2 medium (Table 1) as described by Durand et al. (2), modified by adding an additional 230 mg/l CaCl2 * 2H20 (5). Ten ml of enzyme-C2 solution were added to 0.5 grams of cotyledons (5 to 7 days old), which were then vacuum infiltrated at 9.33 kPa for 20 seconds. The infiltrated tissue was put into sterile 50 ml flasks on a gyrator run at 60 rpm at 25°C in the dark. After 6 hours of digestion, the protoplasts were separated by gently swirling the 50 ml flasks. Protoplasts were isolated from cell walls and other debris by filtering through sterilized miracloth (Cal. BioChem.).

Protoplasts were washed 3 times with C2 medium by centrifuging at 100 g for 3 minutes. Viability was determined using a fluroscein diacetate stain (7). Protoplasts were cultured in 5 ml of C2 medium at a density of 1×105 protoplast/ml, in 10 x 60 mm petri plates, and incubated in the dark at 25°. Five days after protoplast isolation, half of the plates were moved to a 30°C chamber in the dark. Seven days after protoplast release, 1 ml of C3 (Table 1) medium was added to each plate. Fourteen and 21 days after isolation, 1 ml of C4 (Table 1) medium was added to each plate. Protoplast culture plates were briefly swirled daily to increase aeration.

Estimates of plating efficiency (percentage of protoplast which had undergone cell division) were made 8 to 10 days after isolation. Plating efficiency was estimated by visual observation of 5 samples per plate, at 320X magnification. Plating efficiency was calculated by counting the number of cells with clearly defined (1 or more) cell divisions. Using the sample results, total number of divided cells per plate was calculated. This number was then compared to the total number of protoplasts in the plate (5×105) to produce an estimate of plating efficiency. Approximately 3 weeks after isolation, the number of microcalli per plate (clumps of 8 to 64 cells which appeared to have originated from 1 cell) were estimated. The number of microcalli per plate was estimated by counting the number of microcalli in 5 samples per plate (100X magnification), and calculating an approximate number per plate from the random visual counting. Experiment 1 was a randomized complete block with 4 replications.

In experiment 2, protoplasts were isolated and cultured using the methods described above. After 3 weeks, microcalli suspensions were pipetted onto C5 medium (Table 1) containing different amounts of 2,4 dichlorophenoxyacetic acid (2,4-D), indoleacetic acid (IAA), kinetin (kin), and benzylaminopurine (BA) (Table 2). The callus cultures were maintained at 22°C in the dark for 3 weeks before being rated for percentage of the petri plate covered with callus. Callus color was rated 1 to9 (1-3 = white, 4-6 = yellow, 7-9 = brown). For both experiments, protoplast viability and number of protoplasts isolated per gram of tissue were determined. Experiment 2 was a randomized complete block with 4 replications.

Results. Protoplast viability (as determined by fluroscein diacetate staining) was consistently between 80 and 100%, and the number of viable protoplasts isolated per gram of tissue was 8.2±2.5 X 106. In both experiments, protoplasts rapidly regenerated cell walls and underwent cell division. Cell wall regeneration was determined by observed changes in protoplast shape, and actual cell division. In experiment 1, protoplasts cultured at 25°C had a plating efficiency PE) of 4%. Protoplasts cultured at 30°C had a PE of 7%. Analysis indicated there was a significant difference between the 2 temperatures for plating efficiency. After 3 weeks of culture at 25°C, each plate had an average of 3970 microcalli, while culture of protoplasts at 30°C produced an average of 5025 microcalli per plate.

In experiment 2, medium A3 (Table 2) was best for producing a large amount of yellow, friable calls. the color ratings showed no significant differences among media, but protoplasts cultured at 30°C were significantly whiter. Callus color appeared to indicate potential for continued proliferation because callus with ratings above 5 usually had little or no continued growth, even when transferred to fresh media. Although no plant regeneration recurred from any of the 4 media, medium A3 provided the means for producing large amounts of callus which could subsequently be transferred to a embryo inducing medium.

From these two experiments, successful isolation of a large number of viable protoplasts, and regeneration of cell walls of C. metuliferus protoplasts was achieved. A rapid method of producing protoplast-derived callus, suitable for possible plant regeneration was also found. In future experiments, we will attempt to increase the plating efficiency of isolated C. metuliferus protoplasts, and regenerate plants from culture.

Table 1. Components used for culture media for C. metuliferus protoplasts.z

Medium

Component
C1
C2
C3
C4
C5
Mannitol – 0.3 M 0.15 M – –
2,4-D – 0.5 mg/l 0.5 mg/l 0.5 mg/l –
Kinetin – 1.0 mg/l 1.0 mg/l 1.0 mg/l –
Agar (W/v) 0.8% – – – 0.8
Salts and vitamins 1/2 MSy Mod. DPx Mod. DPD Mod. DPD Mod. DPD
Sucrose (g/l) 15.0 17.1 17.1 17.1 17.1

zAll media were adjusted to a pH of 5.8.
y Murashige and Skoog salts (6).
x Durand, Potrykus and Donn medium (2) modified by Jia et al. (5).

Table 2. Results of callus production from C. metuliferus protoplasts.z

Protoplast culture temperature

25°C

30°C

Code no.

Media Componentsy
% plate covered
Color rating
% plate covered
Color rating
A1 0.01 mg 2,4-D 1.0 mg BA 3.0 6.1 5.0 5.0
A2 0.20 mg IAA 0.5 mg BA 0.0 – 3.0 5.5
A3 0.25 mg 2,4-D 0.5 mg KIN 10.0* 5.3 8.0 4.4
A4 0.50 mg 2,4-D 1.0 mg KIN 2.0 5.3 5.0 3.7

z Data are means of 4 replications.
y Base medium was C5 (Table 1).
*Significant at 5% level.

Literature Cited

  1. Deakin, J.R., G.W. Bohn and T.W. Whitaker. 1971. Interspecific hybridization in cucumis. Econ. Bot. 25: 195-211.
  2. Durand, J. I. Potrykus and G. Donn. 1973. Plantes issues de protoplatstes de Petunia. Z. Pflanzenphysiol. 69:26-34.
  3. Fassuliotis, G. 1970. Species of Cucumis resistant to the root-knot nematode, Meloidogyne incognita acrita. J. Nematol. 2:174-178.
  4. Fassuliotis, G. and G.J. Rau. 1963. Evaluation of Cucumis spp. for resistance to the cotton root-knot nematode, Meloidogyne incognita acrita. Plant Dis. Reptr. 47:809.
  5. Jia, Shi-rong, You-ying Fu and Yun Lin. 1986. Embryogenesis and plant regeneration from cotyledon protoplast culture of cucumber (Cucumis sativus L.) J. Plant Physiol. 124:393-398.
  6. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
  7. Widholm, J.M. 1972. The use of fluroscein diacetate and phenosafranine for determining viability of cultured plant cells. Stain. Tech. 47:189-194.