Plant Cell Reports
Plant Cell Reports (1987) 6:470-472
© Springer-Verlag1987
Cell regeneration and sustained division of protoplasts from cotton ( Gossypium hirsutum L.) * Kamel Saka, Frank R. Katterman, and John C. Thomas
**
Forbes Building No. 36, Room 201, University of Arizona, Tucson, AZ 85721, USA Received May 5, 1987 / Revised version received September 25, 1987- Communicated by J. M. Widholm
ABSTRACT Protoplasts were isolated from 12 day old subcultured phytohormone habituated callus tissue of Gossypium hirsutum L. (0.5% cellulysin-Calbioehem, 0.6% macerase-Calbiochem, 0.7M mannitol, and pH 5.0). After separation and purification (0.35M sucrose floatation medium), the protoplasts were cultured (K3 media of Kao et al., 1974 with 0.9 ~M BAP, 5 BM IAA and 0.35M sucrose) in both liquid and solid medium at a density of 5XI05 protoplasts/ml. Four weeks after isolation, cell regeneration and callus formation was observed. Abbreviations: IAA, indoleacetic acid; BAP, 6-benzyl-adenine. INTRODUCTION Recently, several workers have reported on the isolation and culture of Gossypium (cotton) protoplasts through cell wall regeneration and subsequent cell division of 50 to i00 cell clusters (Bhojwani et al., 1977; Finer & Smith, 1982; Ei-Shihy & Evans, 1983; Thomas & Katterman, 1984; Firoozabady and DeBoer, 1986). Without the ability to continue division to plant regeneration, the use of protoplasts in cotton improvement is not feasible. This communication describes a method for the isolation and culture of Gossypium hirsutum L. stem callus protoplasts which leads to normally growing callus tissue that can be subcultured. MATERIALS AND METHODS Callus Cultures. Phytohormone habituated stem callus of Gossypium hirsutum L. cv. Deltapine 16, (Sandstedt, 1975) was used for protoplast isolation and culture. The calli were cultured on Murashige and Skoog (1962) salts and vitamins with 3% (w/v) sucrose and 1% w/v Bactoagar at pH 5.6 in 125 ml Erlenmeyer flasks with foam plugs. These cultures were subcultured bi-monthly and grown under (50 uEm-2s -I) light at 30C with a 16 hr/day photoperiod. iArizona Experimental Station Publication No. 4373. 2person to whom reprint requests are directed. 3Current address: Dept. of Biology, Texas A&M University, College Station, TX 77843.
Protoplast Isolation. Investigations were performed on the types (cellulysin-Calbiochem, celluloseWorthington diagnostics, macerase-Calbiochem and pectinase-Sigma) and concentrations of cellulase (0.5-4% w/v) and pectinase (0.2-1.2% w/v) enzymes, osmoticum (mannitol 0.2-1.0M), pH (4.2-8.6), incubation time (2-14 hrs.) and the subculture age of callus (4-24 days) on the release of viable protoplasts from calli. The following isolation mixture was derived from the optimized procedure. Peripheral tissue (one gram fresh weight) of 12 day old subeultured callus was digested in 5 ml of filter sterilized enzyme mixture consisting of 0.5% (w/v) cellulase (Behring Diagnostics), 0.6% (w/v) macerase (Calbiochem), 0.7M mannitol, pH 5.0. Digestions were carried out in 14 cm plastic petri dishes. The dishes were incubated in the dark at 25C on a gyratory shaker at 40 rpm for a period of 6 hrs. Protoplasts were purified by means of sucrose floatation medium. After passage through a 95 um mesh filter the filtrate obtained was transferred with a wide bore pipette into a 500 ml separatory funnel to which were added 3 volumes of the culture medium containing 0.35M sucrose. The mixture was allowed to stand 4 hrs. The floating protoplast band was washed twice with 3 volumes of culture media by centrifugation at 121xg for 5 min. Protoplast viability in the protoplast band was determined by means of Evans blue (Kanai & Edwards, 1973). Culture of Protoplasts. Media of Bhojwani et al., (1977), Finer & Smith (1982) K 3 (Kao et al., 1974) and Thomas & Katterman (1984) were tested. The K 3 was modified to contain 0.9 ~M BAP, 5.5 ~M IAA and 0.35M sucrose. After purification by floatation the protoplasts were cultured in each medium at a density of 5XI05 protoplasts/ml. Protoplasts were cultured in three ways: liquid medium, solid medium, and microdroplets. Due to the phytohormone habituated nature of the original callus, the plating efficiency of the successful media, both with and without phytohormones, was determined. Liquid Culture. After floatation, the protoplasts were placed in a 14 cm plastic petri dish containing i0 ml of culture medium to have a final plating density of 5XI05 protoplasts/ml. The osmotic potential of the medium was lowered by substituting 4 ml of fresh medium that contained a lower osmotic level for each i0 day period (0.25M, 0.15M, and
* Arizona Experimental Station Publication No. 4373 ** Current address: Department of Biology, Texas A & W University, College Station, TX 77843, USA Offprint requests to: F. R. Katterman
471
Fig. i. Development of G. hirsutum protoplasts in culture from cell regeneration through callus formation. Protoplasts were prepared under optimal conditions and plated on Gelrite as explained in Materials and Methods. a. Visible colonies formed from micro calli after plates were inverted as described in Results (2-3 weeks). b. Further proliferation of callus colonies (4-5 weeks), c. Callus tissue ready for subculturing (6-7 weeks). O.08M sucrose). Plating Culture. Protoplast suspension in 1 ml of liquid medium was layered on top of the solid 0.16% w/v Gelrite (Kelco) medium to a density of 5XI05 protoplasts per plate. Droplet Culture. A series of ten ul droplets of protoplasts was arranged in the petri dish. For each droplet, 40 ul of medium was added to a final density of 5XI03 protoplasts. Every 4 days, 20 ul of fresh medium with a lower osmoticum was added to each droplet. All dishes for each technique were sealed with parafilm, incubated in the dark for 4 days and then transferred to light as described for callus culture. RESULTS Protoplast Isolation. The optimal isolation mixture described in the Methods section was utilized for protoplast isolation from callus tissue.
Culture of Protoplasts. Of the various media tested, K 3 was most effective in supporting division of protoplasts and colony formation. Cell colonies could be seen after 8 days in all of the liquid cultures. Large colonies of 50 to i00 cells occurred only when protoplasts were cultured in K 3 medium. Protoplast regeneration and sustained division during culture in K 3 medium, resulted from a simple manipulation. When the culture plates were inverted, calli formed from individual protoplasts that adhered to the Gelrite surface after 6 weeks (Fig. i) and from the microdroplets after 4 weeks. When the microcolonies in the liquid culture were transferred individually to the Gelrite plates as 50 ul droplets and the plates inverted after 1 week, calli were also obtained 3 weeks later. If the liquid cultures themselves were gently inverted after a number of microcolonies had attached to the bottom of the dish, colonies grew and formed callus. These plates were turned right side up every 2 days to insure constant moisture and nutrient uptake.
The separation of the protoplasts from cells and debris was evident by the formation of a floating band of protoplasts at the surface of the floatation medium. The results of this study (Table i) showed that yields of more than 70% viable protoplasts were obtained after 4 hrs. standing.
In general, the phytohormones in the modified K 3 as opposed to the hormone-free medium, enhanced callus formation in all culture methods employed. Both types of m~dia (hormone & hormone free) exhibited a plating efficiency of 4% and 2%, respectively. The calli obtained were transferred to the callus maintenance medium and incubated under the same conditions as described for callus growth.
Table i. Effects of the floatation purification protoplast yield. Data are means of duplicate experiments with three replicates.
DISCUSSION
Viable Protoplast Yield (% total protoplasts in floating band)
Standing Time (hrs)
25.00 31.65 46.70 71.33
1
2 3 4 aStd deviation
on
of the mean
+ 3.80 a
$ 5.89 $ 3.11 ~ 2.55
The friable nature and vigorous growth of G. hirsutum habituated callus made it a very suitable source for protoplast isolation, as .shown by a 65% conversion rate of the original cells into protoplasts when taken 12 days after subculture. These observations are consistent with those of Finer and Smith (1982) who obtained the highest yield of 20% with a 14 day old callus of G__t. klotzschianum, while Thomas and Katterman (1984) reported an optimum release at 3 to 5 days after subculture of G.
472 hirsutum anther callus. These variations may be attributed to differences in growth rates and tissue origins of the callus. For protoplast purification, a 4 hour floatation method in the culture media with 0.35M sucrose gave preparations free of cell debris and whole cells thus eliminating the possibility of callus formation from the separate whole cells that retained their cell walls after enzyme digestion. The media developed by Bhojwani et al. (1977), Finer and Smith (1982) and Thomas and Katterman (1984) were ineffective for the promotion of protoplast growth from cotton habituated callus. Only the modified Kao medium was capable of supporting protoplast survival up to 8 weeks. Kao's basal medium has also been used successfully for several other species; Nicotiana sylvestris (Nagy and Maliga, 1976), Trigonella foenum-graecum (Shekhawat and Galston, 1983), and Panicum miliaceum L. (Heyser, 1984). In this investigation, IAA (5.5 ~M) and BAP (0.9 ~M) as well as other trial hormone combinations, stimulated cell wall regeneration, but these compounds were not indispensable, demonstrating that habituated callus protoplasts from cotton are self-sufficient in this regard. Protoplasts from some hormoneindependent tissues, however, do show variation in their response to growth regulators. In Parthenocissus tricuspidata for example, hormones were essential to initiate cell wall formation and division only during the early stages of culture (Scowcroft, et al., 1973). It is well known that plant cell divisions are dependent upon culture medium, the osmoticum, the cell protoplast plating density, and the culture conditions i.e. light and temperature. Results obtained from the inverted plate technique suggest that in the case of cotton protoplasts, two additional factors are critical for successful callus formation; adequate gas supply and humidity. For example, when the
droplet cultures were inverted the protoplasts shifted to the boundary of a liquid and gaseous phase which permitted adequate humidity and improved gas exchange. A similar mechanism may also apply to the solid Gelrite and the liquid culture techniques. Although the agarose bead culture method (Shillito et al., 1983) also increases the surface area for gas exchange, it did not improve further development of G. hirsutum cells (Firoozabady and DeBoer, 1986). In conclusion, a simple reproducible protocol for isolating and culturing protoplasts of cotton (G. hirsutum) to callus tissue has been developed.
REFERENCES Bhojwani SS, Power JB, Cocking EC (1977) PI. Sci. Lett. 8:85-89. Ei-Shihy OM, Evans PK (1983) In: Proc. 6th Int'l Protopl. Symp., Basel, Switzerland, pp. 24-25. Finer JJ, Smith RH (1982) PI. Sci. Lett. 26:147-151. Firoozabady E, DeBoer D (1986) Plant Cell Reports 5:127-131. Heyser JW (1984) Z. Pflanzenphysiol. 113:293-299. Kao KN, Constabel F, Michayluk MR, Gamborg OL (1974) Planta. 120:215-227. Kanai R, Edwards GE (1973) Plant Physiol. 52:484-490. Murashige T, Skoog F (1962) Physiol. Plant 15:473479. Nagy, JI, Maliga P (1976) Z. Pflanzenphysiol. 78:453455. Sandstedt R (1975) Beltwide Cotton Prod. Res. Conf. pp. 52-53. National Cotton Council, Memphis, Tenn. USA. Scowcroft WR, Davey MR, Power JB (1973) PI. Sci. Lett. 1:451-456. Shekhawat NS, Galston AW (1983) Plant Cell Reports 2:119-121. Shillito RD, Paszkowski J, Potrykus I (1983) PI. Cell Reports 2:244-247. Thomas JC, Katterman FRH (1984) PI. Sci. Lett. 36:149-154.
Plant Cell Reports (1987) 6:470-472
© Springer-Verlag1987
Cell regeneration and sustained division of protoplasts from cotton ( Gossypium hirsutum L.) * Kamel Saka, Frank R. Katterman, and John C. Thomas
**
Forbes Building No. 36, Room 201, University of Arizona, Tucson, AZ 85721, USA Received May 5, 1987 / Revised version received September 25, 1987- Communicated by J. M. Widholm
ABSTRACT Protoplasts were isolated from 12 day old subcultured phytohormone habituated callus tissue of Gossypium hirsutum L. (0.5% cellulysin-Calbioehem, 0.6% macerase-Calbiochem, 0.7M mannitol, and pH 5.0). After separation and purification (0.35M sucrose floatation medium), the protoplasts were cultured (K3 media of Kao et al., 1974 with 0.9 ~M BAP, 5 BM IAA and 0.35M sucrose) in both liquid and solid medium at a density of 5XI05 protoplasts/ml. Four weeks after isolation, cell regeneration and callus formation was observed. Abbreviations: IAA, indoleacetic acid; BAP, 6-benzyl-adenine. INTRODUCTION Recently, several workers have reported on the isolation and culture of Gossypium (cotton) protoplasts through cell wall regeneration and subsequent cell division of 50 to i00 cell clusters (Bhojwani et al., 1977; Finer & Smith, 1982; Ei-Shihy & Evans, 1983; Thomas & Katterman, 1984; Firoozabady and DeBoer, 1986). Without the ability to continue division to plant regeneration, the use of protoplasts in cotton improvement is not feasible. This communication describes a method for the isolation and culture of Gossypium hirsutum L. stem callus protoplasts which leads to normally growing callus tissue that can be subcultured. MATERIALS AND METHODS Callus Cultures. Phytohormone habituated stem callus of Gossypium hirsutum L. cv. Deltapine 16, (Sandstedt, 1975) was used for protoplast isolation and culture. The calli were cultured on Murashige and Skoog (1962) salts and vitamins with 3% (w/v) sucrose and 1% w/v Bactoagar at pH 5.6 in 125 ml Erlenmeyer flasks with foam plugs. These cultures were subcultured bi-monthly and grown under (50 uEm-2s -I) light at 30C with a 16 hr/day photoperiod. iArizona Experimental Station Publication No. 4373. 2person to whom reprint requests are directed. 3Current address: Dept. of Biology, Texas A&M University, College Station, TX 77843.
Protoplast Isolation. Investigations were performed on the types (cellulysin-Calbiochem, celluloseWorthington diagnostics, macerase-Calbiochem and pectinase-Sigma) and concentrations of cellulase (0.5-4% w/v) and pectinase (0.2-1.2% w/v) enzymes, osmoticum (mannitol 0.2-1.0M), pH (4.2-8.6), incubation time (2-14 hrs.) and the subculture age of callus (4-24 days) on the release of viable protoplasts from calli. The following isolation mixture was derived from the optimized procedure. Peripheral tissue (one gram fresh weight) of 12 day old subeultured callus was digested in 5 ml of filter sterilized enzyme mixture consisting of 0.5% (w/v) cellulase (Behring Diagnostics), 0.6% (w/v) macerase (Calbiochem), 0.7M mannitol, pH 5.0. Digestions were carried out in 14 cm plastic petri dishes. The dishes were incubated in the dark at 25C on a gyratory shaker at 40 rpm for a period of 6 hrs. Protoplasts were purified by means of sucrose floatation medium. After passage through a 95 um mesh filter the filtrate obtained was transferred with a wide bore pipette into a 500 ml separatory funnel to which were added 3 volumes of the culture medium containing 0.35M sucrose. The mixture was allowed to stand 4 hrs. The floating protoplast band was washed twice with 3 volumes of culture media by centrifugation at 121xg for 5 min. Protoplast viability in the protoplast band was determined by means of Evans blue (Kanai & Edwards, 1973). Culture of Protoplasts. Media of Bhojwani et al., (1977), Finer & Smith (1982) K 3 (Kao et al., 1974) and Thomas & Katterman (1984) were tested. The K 3 was modified to contain 0.9 ~M BAP, 5.5 ~M IAA and 0.35M sucrose. After purification by floatation the protoplasts were cultured in each medium at a density of 5XI05 protoplasts/ml. Protoplasts were cultured in three ways: liquid medium, solid medium, and microdroplets. Due to the phytohormone habituated nature of the original callus, the plating efficiency of the successful media, both with and without phytohormones, was determined. Liquid Culture. After floatation, the protoplasts were placed in a 14 cm plastic petri dish containing i0 ml of culture medium to have a final plating density of 5XI05 protoplasts/ml. The osmotic potential of the medium was lowered by substituting 4 ml of fresh medium that contained a lower osmotic level for each i0 day period (0.25M, 0.15M, and
* Arizona Experimental Station Publication No. 4373 ** Current address: Department of Biology, Texas A & W University, College Station, TX 77843, USA Offprint requests to: F. R. Katterman
471
Fig. i. Development of G. hirsutum protoplasts in culture from cell regeneration through callus formation. Protoplasts were prepared under optimal conditions and plated on Gelrite as explained in Materials and Methods. a. Visible colonies formed from micro calli after plates were inverted as described in Results (2-3 weeks). b. Further proliferation of callus colonies (4-5 weeks), c. Callus tissue ready for subculturing (6-7 weeks). O.08M sucrose). Plating Culture. Protoplast suspension in 1 ml of liquid medium was layered on top of the solid 0.16% w/v Gelrite (Kelco) medium to a density of 5XI05 protoplasts per plate. Droplet Culture. A series of ten ul droplets of protoplasts was arranged in the petri dish. For each droplet, 40 ul of medium was added to a final density of 5XI03 protoplasts. Every 4 days, 20 ul of fresh medium with a lower osmoticum was added to each droplet. All dishes for each technique were sealed with parafilm, incubated in the dark for 4 days and then transferred to light as described for callus culture. RESULTS Protoplast Isolation. The optimal isolation mixture described in the Methods section was utilized for protoplast isolation from callus tissue.
Culture of Protoplasts. Of the various media tested, K 3 was most effective in supporting division of protoplasts and colony formation. Cell colonies could be seen after 8 days in all of the liquid cultures. Large colonies of 50 to i00 cells occurred only when protoplasts were cultured in K 3 medium. Protoplast regeneration and sustained division during culture in K 3 medium, resulted from a simple manipulation. When the culture plates were inverted, calli formed from individual protoplasts that adhered to the Gelrite surface after 6 weeks (Fig. i) and from the microdroplets after 4 weeks. When the microcolonies in the liquid culture were transferred individually to the Gelrite plates as 50 ul droplets and the plates inverted after 1 week, calli were also obtained 3 weeks later. If the liquid cultures themselves were gently inverted after a number of microcolonies had attached to the bottom of the dish, colonies grew and formed callus. These plates were turned right side up every 2 days to insure constant moisture and nutrient uptake.
The separation of the protoplasts from cells and debris was evident by the formation of a floating band of protoplasts at the surface of the floatation medium. The results of this study (Table i) showed that yields of more than 70% viable protoplasts were obtained after 4 hrs. standing.
In general, the phytohormones in the modified K 3 as opposed to the hormone-free medium, enhanced callus formation in all culture methods employed. Both types of m~dia (hormone & hormone free) exhibited a plating efficiency of 4% and 2%, respectively. The calli obtained were transferred to the callus maintenance medium and incubated under the same conditions as described for callus growth.
Table i. Effects of the floatation purification protoplast yield. Data are means of duplicate experiments with three replicates.
DISCUSSION
Viable Protoplast Yield (% total protoplasts in floating band)
Standing Time (hrs)
25.00 31.65 46.70 71.33
1
2 3 4 aStd deviation
on
of the mean
+ 3.80 a
$ 5.89 $ 3.11 ~ 2.55
The friable nature and vigorous growth of G. hirsutum habituated callus made it a very suitable source for protoplast isolation, as .shown by a 65% conversion rate of the original cells into protoplasts when taken 12 days after subculture. These observations are consistent with those of Finer and Smith (1982) who obtained the highest yield of 20% with a 14 day old callus of G__t. klotzschianum, while Thomas and Katterman (1984) reported an optimum release at 3 to 5 days after subculture of G.
472 hirsutum anther callus. These variations may be attributed to differences in growth rates and tissue origins of the callus. For protoplast purification, a 4 hour floatation method in the culture media with 0.35M sucrose gave preparations free of cell debris and whole cells thus eliminating the possibility of callus formation from the separate whole cells that retained their cell walls after enzyme digestion. The media developed by Bhojwani et al. (1977), Finer and Smith (1982) and Thomas and Katterman (1984) were ineffective for the promotion of protoplast growth from cotton habituated callus. Only the modified Kao medium was capable of supporting protoplast survival up to 8 weeks. Kao's basal medium has also been used successfully for several other species; Nicotiana sylvestris (Nagy and Maliga, 1976), Trigonella foenum-graecum (Shekhawat and Galston, 1983), and Panicum miliaceum L. (Heyser, 1984). In this investigation, IAA (5.5 ~M) and BAP (0.9 ~M) as well as other trial hormone combinations, stimulated cell wall regeneration, but these compounds were not indispensable, demonstrating that habituated callus protoplasts from cotton are self-sufficient in this regard. Protoplasts from some hormoneindependent tissues, however, do show variation in their response to growth regulators. In Parthenocissus tricuspidata for example, hormones were essential to initiate cell wall formation and division only during the early stages of culture (Scowcroft, et al., 1973). It is well known that plant cell divisions are dependent upon culture medium, the osmoticum, the cell protoplast plating density, and the culture conditions i.e. light and temperature. Results obtained from the inverted plate technique suggest that in the case of cotton protoplasts, two additional factors are critical for successful callus formation; adequate gas supply and humidity. For example, when the
droplet cultures were inverted the protoplasts shifted to the boundary of a liquid and gaseous phase which permitted adequate humidity and improved gas exchange. A similar mechanism may also apply to the solid Gelrite and the liquid culture techniques. Although the agarose bead culture method (Shillito et al., 1983) also increases the surface area for gas exchange, it did not improve further development of G. hirsutum cells (Firoozabady and DeBoer, 1986). In conclusion, a simple reproducible protocol for isolating and culturing protoplasts of cotton (G. hirsutum) to callus tissue has been developed.
REFERENCES Bhojwani SS, Power JB, Cocking EC (1977) PI. Sci. Lett. 8:85-89. Ei-Shihy OM, Evans PK (1983) In: Proc. 6th Int'l Protopl. Symp., Basel, Switzerland, pp. 24-25. Finer JJ, Smith RH (1982) PI. Sci. Lett. 26:147-151. Firoozabady E, DeBoer D (1986) Plant Cell Reports 5:127-131. Heyser JW (1984) Z. Pflanzenphysiol. 113:293-299. Kao KN, Constabel F, Michayluk MR, Gamborg OL (1974) Planta. 120:215-227. Kanai R, Edwards GE (1973) Plant Physiol. 52:484-490. Murashige T, Skoog F (1962) Physiol. Plant 15:473479. Nagy, JI, Maliga P (1976) Z. Pflanzenphysiol. 78:453455. Sandstedt R (1975) Beltwide Cotton Prod. Res. Conf. pp. 52-53. National Cotton Council, Memphis, Tenn. USA. Scowcroft WR, Davey MR, Power JB (1973) PI. Sci. Lett. 1:451-456. Shekhawat NS, Galston AW (1983) Plant Cell Reports 2:119-121. Shillito RD, Paszkowski J, Potrykus I (1983) PI. Cell Reports 2:244-247. Thomas JC, Katterman FRH (1984) PI. Sci. Lett. 36:149-154.
