Isolation and Fusion of Cotton Protoplasts

Protoplasts were isolated from five species of Gossypium. · Protoplast yield and viability were affected by incubation conditions, osmolarity, purification procedures, and cell source. Using an optimized procedure, highly viable protoplasts were isolated from cell suspensions, callus cultures, and leaf tissue of G. hirsutum, G. arboreum, G. k/otzschianum, G. harlmessii, and G. herbaceum. Protoplasts of G. harknessii were enucleated and successfully fused with protoplasts of G. hirsutum. IN1RODUCTION Modification of plants through tissue culture methods, such as somatic cell fusion has the potential of providing immediate benefits to agriculture (Evans and Sharp, 1986), because genetic characteristics can be transferred by somatic cell fusion without the necessity of isolation and identification of the genes responsible for the trait. Many aspects of plant improvement through somatic cell hybridization have been reviewed previously (Evans and Flick, 1983; Schieder, 1982). An important plant breeding tool is male sterility. Through sexual crosses, Meyer (1975) demonstrated that Gossypium harlmessii Brandegee cytoplasm in the mclear background of G. hirsutum L. resulted in plants with CMS. Production of these cotton plants required extensive backcrossing, and the seed set was limited Although the genetic basis for cytoplasmic male sterility (CMS) is not understood (Boeshore et al., 1985; Jigeng and Yi-mong, 1983; Levings and Pring, 1979), this trait has been transferred to a male fertile plant through protoplastfusion (Zelceret al., 1978) and subsequent hybrid regeneration. 1k fusion of G. harlmessii cytoplasts with G. hirsutum protoplasts should also produce G. hirsutum plants exhibiting CMS. As a first step in this process, a technique to rapidly obtain good yields of highly-viable protoplasts suitable for protoplast fusion was developed. Protoplast yields and viability exceeded other published accounts (Bhojwani et al., 1977; El-Shihy and Evans, 1983; Finer and Smith, 1982; Firoozabady and DeBoer, 1986; Khasanov and Butenko, 1979). Additionally, cytoplasts were prepared and fused with these protoplasts. MATERIALS AND ME1HODS Plant Material Cotton plants G. hirsutum L. var. Coker 310, Stoneville 213, and Paymaster 145, were grown in potting soil in an environmentally-controlled grvwth chamber which 1 Address all correspondence to:Dr. Michael H. Renfroe, Department of Biology MSC 7801, James Madison University, Harrisonburg. VA 22807 58 VIRGINIA JOURNAL OF SCIENCE was progranuned for 12 h of light with a temperature of 30 C and 12 h of darkness at 20 C. Plants were watered as necessaiy and fertilized with Osmocote slow-release fertilizer. Young, rapidly-expanding leaves were selected as source leaves for protoplast isolation Callus cultures of G. hirsutum L., G. harknessii Brandg., G. arboreum L., G. herbaceum L., and G. klotzschianum Anderss. were maintained on a medium consisting of: rnacrormtrients and micronutrients of Murashige and Skoog ( 1962) (MS salts); 2 mg/L NAA; 1 mg!L 2-iP; 30 g/L glucose; a vitamin mixture consisting of 1 mg!L thiarnine-HCl; 0.5 mg/L pyridoxine-HCl; 0.5 mg/L nicotinic acid; and 100 mg!L myo-inositol (Smith et al., 1977). Media were sterilized by autoclaving for 15 min at 121° C. Prior to autoclaving and addition ofagar, the pH was adjusted to 5.7-5.8 and medium was solidified with 0.8% Difeo Bacto agar. Cultures were subcultured at monthly inteivals. Suspension cultures of G. hirsutum and G. harknessii were maintained in a medium described above with the following modifications: NAA reduced to 1 mg/L; BAP, 0.1 mg/L substituted for 2-iP; agar was omitted. Suspension cultures were maintained at a 16:8 h photoperiod (601pm). Cultures were supplied with fresh medium weekly and serially subcultured eve:ry 3 weeks. · Protoplast isolation The general isolation procedure was developed using cotton cell suspension cultures of G. hirsutum. Basal isolation medium consisted of rnacronutrients of MS salt (Murashige and Skoog, 1962), 5 rnM MES (2-(N-morpholino)ethanesulfonic acid), 0. 7 M rnannitol, 5% (w/v) Cellulysin cellulose and 1 % (w/v) Macerase pectinase at a pH of 5.7. Effect of osmotic potential on protoplast isolation and viability was detennined by reduction of mannitol to 0.5 Mand 0.3 M. Effect of rnacronutrient composition on protoplast isolation and viability was detennined by testing full and half strength MS rnacronutrients and full strength macronutrients from Garnborg's ( 197 5) medium. Effects of enzyme concentration and length of incubation were tested by reduction of the enzyme concentration to 1/2 and by increasing the incubation period from 5 h to 24 h. Isolated pro top lasts were purified either by filtration through a nylon mesh with 100 nun pores, by centrifugation at 125 RCF for 6 min layered over a 20% (w/v) sucrose solution, or by a combination of filtration and centrifugation treatments. Prior to incubation in the isolation medium, cells from the suspension culture were plasmolyzed ina solution identical to the respective isolation medium without enzymes for approximately 30 min. Plasrnolyzed tissue was incubated in protoplast isolation medium for 5 hat 28° C in a water bath reciprocal shaker at 40 CPM. Protoplasts used for enucleation experiments were also isolated using this procedure. Cell counts were made using a haemocytometer. Cell viability was measured using the Evans' blue dye exclusion techniqu~ (Onyia et al., 1984). Protoplasts of the other species were obtained from callus cultures and young plant leaves using the procedure developed for cell suspension cultures. Cytoplast Preparation and Protoplast Fusion G. harknessii protoplasts were enucleated by centrifugation at 31,000 RCF for 1 h on an iso-osmotic step gradient (Lon and Potrykus, 1980). Enucleation of protoplasts to form cytoplasts was verified using epi-fluorescence microscopy (Zeiss) following ISOLATION OF COTION PROTOPLASTS 59 TABLE 1. Yield and viability of protopl$ts isolated in media of various osmotic strength after S hours incubation in protoplast isolation mediwn. Mannitol Water Potential of Yield Viability (M) Medium (MPa) (No/ml PCv8) (%) 0.3 -0.97 3.2X 10 s 96.7 0.5 -1.48 2.7 X 10 s 95.7 0.7 -2.07 1.9 X 10 s 91.9 • PCV =packed cell volwne incubation of protoplasts and cytoplasts for at least 1 h in DAPI (4,6-diamidino-2phenylindole) at 1 mg,'mL. Protoplasts of G. hirsutum were mixed with an excess of G. harknessii cytoplasts (approximately 2: 1 cytoplasts:protoplasts). A red pigmented cell line of G. hirsutum was used in some experiments to provide a visual marlcer for interspecific fusion. Protoplasts and cytoplasts were fused using the procedure by Evans (1983), modified by substitution of 0.5 M glucose for sorbitol in the enzyme wash solution. Fusion was promoted by a 50%, (w/v) PEG (mw 6000) solution (Evans, 1983). The PEG fusing solution was eluted with either a glycine buffer (50 mM glycine, 50 mM CaC12·2~0, 0.3 M glucose, pH 10.5) followed by a wash with culture media, or by a Tris buffer (5 mM Tris, 50 mM CaC~·2H20, 0.3 M glucose, pH 7 .0) followed by a wash with culture media, or eluted by washing with culture media alone (pH 5. 7-5.8). Pro top lasts and fusion products were cultured in various media based on Murashige and Skoog's (1962), Gamborg's (1975) or Kao and Michayluk's (1975) fonnulations. Liquid culture, agar or agarose-solidified media and nurse cultures were all used in an attempt to promote growth of protoplasts and fusion products. RESULTS Protoplasts of high viability were obtained from all.the Gossypium species examined and from leaf, as well as callus and suspension cultures, using this procedure. The highest yield and viability were obtained using the isolation medium with 0.3 M mannitol (Table 1). ~ the medium osmotic strength was increased, protoplast yield and viability decreased. Isolation medium containing 0.3 M mannitol was therefore selected for subsequent trials. Isolation medium macronutrient content had no effect on yield (Table 2). Similar results were obtained when macro-salts of MS were at fullor half-strength. Yield and viability were only slightly decreased by substitution of Gamborg's macronutrients. MS macronutrients at full-strength were chosen for routine use. Reduction of enzyme concentration by half had no effect on viability over a 5 h period but did decrease protoplast yield (Table 3). An increase in incubation period from 5 h to 24 h resulted in decreased total yield and decreased viability. Loss of viability was slightly greater at the lower concentration of enzymes (fable 3). A 5 h incubation period using 5% Cellulysin and 1 % Macerase was selected as the standard procedure. · Several purification procedures were compared for their effect on protoplast yield and viability (fable 4). Filtration of the protoplast suspension through a nylon mesh 60 VIRGINIA JOURNAL OF SCIENCE TABLE 2. Effect on protoplast yield of various macronutrient formulations in the protoplast isolation medium Protoplasts were isolated from suspension cultures of G. hirsutum •. Macro nutrient Yield Viability Formulation Strength (No/mlPCV) (%) Murashige & Skoog IX 4.4 X 10 5 96.2 Murashige & Skoog 0.5X 4.5 X 10 5 94.0 Gamborg IX 4.3 X 10 5 91.7 TABLE 3. Effect of enzyme concentration and incubation period on yield and viability of protoplasts. Cellulysin Mace race Incubation Yield Viability (%wlv) (%w/v) (hrs) (No/mlPCV) (%) 5.0 1.0 5 3.1X10 5 100 2.5 0.5 5 5.2 X 10 4 100 5.0 1.0 24 1.0 X 10 5 93.2 2.5 0.5 24 1.4 X 10 4 89.8 TABLE 4. Effect of protoplast yield and viability of several purification procedures by filtration, centrifugation, or their combination. Recovery% Nwnberof Initial Viability After of Pro top lasts Protoplasts/ Viability Purification Purification Method (%) mLPCVYield (%) (%) Filtration, 100 mM Mesh 100 4.0X 10 6 92.3 82.1 Floatation over Sucrose 68 1.7X 10 6 88.4 77.8 Filtration and Floatation 26 9.0X 10 5 86.9 85.7 with 100 mm pores, allowed protoplasts, cell fragments and cells with partially-digested walls to pass through resulting in an impure population of protoplasts. In contrast, purification by centrifugation was superior. Cell clumps and cell fragments sedimented into the sucrose layer while intact protoplasts floated above the sucrose in the suspension medium. Both purification by filtration and by centrifugation led to about a I 0% decrease in viability. Purification by centrifugation gave approximately two-thirds the yield of filtration. When filtration and centrifugation procedures were combined, recovery was reduced more than would be expected by a simple additive effect of the two procedures. The optimized procedure was used to isolate proto~lasts from five different species of Gossypium (Table 5). Yields ranged from I 0 -10 protoplasts per g dw of tissue. ISOLATION OF COTTON PROTOPLASTS 61 TABLE 5. Yield and viability ofprotoplasts isolatcJ from various cotton apecies 1nd tissue sourca. Species Material Yield Viability (No/gdw) {°lo) G. hirsutum leaf 1.1X10 7 93.1 callus 6.6X 10 6 67.9 suspension l.4X 10 8 89.5 G. harknessii callus 4.8X 10 7 85.1 suspension 1.1X10 8 81.8 G. arboreum callus 3.3 X 10 5 76.0 G. herbaceum callus 8.6X 10 7 85.3 G. klotzschianum callus 5.4X 10 7 84.8 Yields were higher for suspension cultured cells of G. hirsutum and G. harknessii than from callus of the respective species. Leaves of G. hirsutum yielded more protoplasts than did callus, but less protoplasts than did suspension cultures. All isolated protoplasts had good viability (Table 5). Protoplast yield from callus varied with the friability of the callus tissue. Callus of G. arboreum and G. hirsutum was very oodular and compact which resulted in lowered protoplast yields. However, callus of G. herbaceum, G. klotzschianum and G. harknessii was friable and soft and yields were accordingly greater. Protoplast to cytoplast fusion was initiated using a 50% (w/v) PEG fusion solution The PEG solution caused protoplast adhesion to occur, but there was virtually no fusion until the PEG solution was eluted with high pH glycine buffer. Following elution, 50% of all visible objects were still single protoplasts that had not fused. Additionally, 38% of the viewed objects were multicellular fusion products. Only 20% of the objects were fonned by adhesion of only two protoplasts. An additional 2% were bicellular fusion products in which the fusion event had clearly occurred at the time of evaluation However, when PEG solution was eluted with Tris buffer (pH 7 .0) or with culture media (pH 5.8) there was virtually no fusion of adhering protoplasts. Clearly the higher pH of the glycine buffer was effective in stimulating fusion of protoplasts and cytoplasts. Furthermore, by increasing the ratio of cytoplasts to protoplasts from 1: 1 to 2:1, the excess of cytoplasts increased the number of protoplast-cytoplast fusion products (4-24%). DISCUSSION Genetic modification of cotton through protoplast methods requires a procedure for isolating adequate nwnbers of viable protoplasts. We systematically evaluated isolation parameters and developed a system with widespread applicability to cotton species for the production of protoplasts suitable for fusion experiments. 62 VIRGINIA JOURNAL OF SCIENCE In past studies of cotton protoplast isolation (El-Shihy and Evam, 1983; Finer and Smith, 1982; Firoombady and DeBoer, 1986), enzyme concentration and incubation periods were evaluated, but concentrations were varied for a single, fixed time inteival, or incubation period was varied, for one concentration of the enzymes. Results of these earlier studies indicate that low enzyme concentration for prolonged periods give best protoplast yield and viability (PotJykus and Shillito, 1986). Our results indicated that a higher concentration of enzyme used for a shorter period of time produced a greater yield of protoplasts with better viability. No benefits were derived from prolonging incubation periods, instead, viability and yield decreased. Protoplast purification by filtration is the most commonly used technique (Evans, 1983). In our experiments, cotton protoplasts were isolated in greater numbers with greater viability with the flotation method. However, filtration did not remove ruptured cells, cell fragments, or cells with incompletely digested cell walls. The protoplast population that was recovered from flotation was extremely pure. Changes in macronutrient composition in the isolation medium had almost no effect on yield, but had a slight effect on viability. This may have reflected differences in osmolality of the solutions. During purification, protoplasts should be maintained in solutions of similar osmotic pressure (PotJykus and Shillito, 1986). Therefore, it may be best to select macronutrients for isolation media with the final culture medium in mind Macronutrient composition is probably of little overall consequence if the incubation and purification time is sufficiently short. The largest component of osmotic pressure in our solution was mannitol. Mannitol is a commonly employed osmoticum and has been used in cotton protoplast isolation in the range of 0.4 M (El-Shihy and Evans, 1983; Firooz.abady and DeBoer, 1986) to 0.7 M (Finer and Smith, 1982). Khasanov and Butenko (1979) tested mannitol concentrations over the range of0.3-0.9 Mand concluded that 0.5 M was optimum for yield. However, they did not evaluate protoplast viability. We obseived a reduction in viability after only 5 h. It is likely that these differences would be more pronounced after an extended incubation period when the cells would have been exposed to the water stress of the high concentration mannitol solution for a longer period of time. The effects of water stress should be more widely considered, especially in procedures with extended incubation periods. Our method of protoplast isolation and purification has wide applicability with cotton tissue sources and species. We isolated highly-viable protoplasts from five species of cotton and from leaf tissues as well as callus and suspension cultures. Suspension cultures and young, rapidly expending leaves from mature plants are good sources for the isolation of plant protoplasts. Khasanov and Butenko (1979) were unable to isolate protoplasts from cotton leaves, but could isolate protoplasts from cotyledons. Others have isolated protoplasts from cotton cotyledons (El-Shihy and Evans, 1983; Firoombady and DeBoer, 1986), young leaves (Firooz.abady and DeBoer, 1986) and callus cultures (Bhojwani, et al., 1977; Finer and Smith, 1982). In addition to evaluating leaves and callus, we extended the trials to include suspension cultures and found that cell suspensions invariably produced the highest yields of protoplasts. Our overall procedure results in a high yield of protoplasts with good viability. Furthermore, the procedure is relatively quick compared to other published procedures and is advantageous for use in fusion experiments. Chemical fusion procedures are ISOLATION OF COTTON PROTOPLASTS 63 harsh. Successful fusion and subsequent hybrid cell growth will be favored if the protoplasts are initially viable. We were able to demonstrate fusion not only ofprotoplasts, but also ofprotoplasts with cytoplasts using a standard fusion procedure (Evans 1983). Protoplast-cytoplast fusion products were obtained in 4-24% of all fusion events. Although numerous methods have been used for indirect selection of fusion products such as complementation (Carlson et al., 1972; Glimelius et al., 1978; Melchers and Labib, 1974) or inactivation (Medgyesy et al., 1980; Zelcer et al., 1978). We used a pigmented cell line to allow immediate visual scoring of fusion events. Cytoplasmically-detennined traits have been transferred when organelles were left in their native milieu inside an enucleated protoplast (Maliga et al., 1982) or in a nuclear-inactivated protoplast (Zelcer et al., 1978). To demonstrate the potential for such a system in cotton, we enucleated protoplasts using a published procedure (Lorz and Potrykus, 1980) to fonn cytoplasts. Protoplast fusion was not promoted by PEG alone, as reported by Kao and Michayluk (1974), but required a high pH treatment as descnbed by Keller and Melchers (1973). Elution of the PEG with a neutral buffer or with slightly acid culture medium did not promote fusion. For cotton protoplasts, it seems that a high pH elution step is essential for good fusion Regeneration of cotton plants from protoplasts has seemed intractable in the past (Bhojwani etal., 1977; El-Shihy and Evans, 1983; Finer and Smith, 1982; Firoozabady and DeBoer, 1986; Khasanov and Butenko, 1979) with protoplast cultures not growing well despite numerous approaches. However, forone cotton cultivar, plants have been regenerated from callus that developed from protoplasts (Peeters et al., 1994 ). We have taken the next step in demonstrating the potential for development of new cotton lines through protoplast-cytoplast fusions. Genetic modification by protoplast-protoplast or protoplast-cytoplast fusion may lead to agronomically-useful cotton hybrids. ACKNOWLEDGEMENTS · RHS initiated and supervised the project and provided the cotton cell lines. 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