Plant Cell Reports
Plant Cell Reports (1996) 15:859-864
9 Springer-Verlag1996
Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.) Kanniah Rajasekaran J. G. Boswell Company, 850 Plymouth Avenue, P.O. Box 787, Corcoran, CA 93212-0787, USA Received 6 December t995/Revised version received 24 January 1996 - Communicated by I. K. Vasil
Abstract.
Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0~ A coohng rate of 0.5~ provided a satisfactory recovery rate ( 3 0 % ) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of l~ Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soft.
Abbreviations: DMSO, dimethylsulfoxide; MS, Murashige and Skoog (1962); MMS, modified MS; NAA, a-naphthaleneacetic acid.
Introduction Embryogenic callus and cell suspension cultures are routinely used in genetic transformation studies and crop improvement. They are also convenient for long term preservation of germplasm and experimental materials (Kartha 1985). Plant cell cultures are prone to chromosomal, genetic and molecular changes. Both epigenetic as well as heritable changes have been described (Larkin and Scowcroft 1981, Lee and Phillips 1988). Such variations pose a serious problem for the maintenance of genetic uniformity in plants regenerated in vitro. In biotechnology research, where plants are often transformed with a single
gene, it is very important to retain the agronomic performance of the variety that is being transformed. True-to-type plants can be regenerated from embryogenic callus a n d cell cultures only when the regeneration system is rapid and efficient (Vasil 1986, Rajasekaran et al. 1986). Long term maintenance of callus and cell cultures often results in plants that are morphologically abnormal, and functionally sterile, thus making them unsuitable for breeding. Several previously published reports (Bajaj and Gill 1985, Trolinder and Goodin 1987, Stelly et al. 1989, Li et al. 1989) indicate that tissue culturerelated genetic variation and sterility among regenerated plants can be a crucial factor in cotton (Gossypium hirsutum L.) transformation programs. Somatic variations, however, can be controlled to a large extent if embryogenic source cultures are new and freshly established or ff obtained from new cultures which have been cryopreserved. Here I report the successful regeneration of plants from cryopreserved embryogenic callus and cell suspension cultures of several elite cotton varieties. These cultures have been successfully used in genetic engineering of cotton for herbicide tolerance and insect resistance (Rajasekaran et al. in preparation).
M a t e r i a l s and m e t h o d s Initiation of embryogenic callus and cell suspension cultures Embryogenic callus cultures were established in the followingcotton (Gossypium hirsutum L.) varieties accordingto the procedures of Rangan (1993) and Rangan et al. (in preparation): SJ2, SJ5, SJC1, GC510, Royale (4226), B1644, B1654, B3991, CSC28, Stoneville 506, Siokra, Coker 315, Chembred B2 and Chembred C4. Briefly, cotyledon and hypocotylexplants from7 to 10 d old seedlingswereplacedon a
860 callus induction medium (Murashige and Skoog 1962; MS) supplemented with 0.4 mg/1 thianaine HC1, 30 g]l glucose, 2.0 mg/l NAA, 1.0 mg/l kinetin, 100 mg/l myo-inositol and 0.8% agar. The cultures were incubated at 27 + 2~ u n d e r conditions of 16 h light and 8 h dark, light intensity at 60 btE m-2 s-~, in a n environmentally controlled incubator (Percival, Boone, tA). Callus formed on these explants within 3 to 4 weeks was selectively subcultured to enrich for friable, yellowish green callus every 3 to 4 weeks on the same medium except t h a t the carbon source was sucrose (20 g/l) instead of glucose. Embryogenic callus capable of forming small globular somatic embryos appeared two to four subcultures after initiation. Embryogenic callus was m a i n t a i n e d and multiplied by routine subculture every 3 to 4 weeks on MS medium containing 100 mg]l myo-inositol, 20 g/1 sucrose, 2.0 rag/1 NAA and 0.8% agar (maintenance medium). Cell suspension cultures were initiated from finely dispersed embryogenic callus cultures in liquid maintenance medium s h a k e n (120 rpm, 27 + 2~ on a gyratory shaker (New Brunswick G-10, Edison, NJ). The suspension cultures were enriched for small, isodiametric, densely cytoplasmic and highly embryogenic cells by discarding free floating cells and large aggregates (>840 ~tm) every week. Rapid multiplication of these cultures was done by subculturing every 4 to 6 days in the same medium (100 ml) in 1000 ml Erlenmeyer flasks for two to three weeks prior to cryopreservation. Callus and suspension cultures of Acala cotton variety B1654 were used in all the initial studies to develop tlie cryopreservation protocol for cotton. All the chemicals including the cryoprotectants used in this study were purchased from Sigma Chelrfical company (St. Louis, MO).
the microcomputer. The cooling rate and the phase change were monitored by placing a t e m p e r a t u r e probe (Thermocouple 'T' type) in one of the ampoules containing the same cryoprotectant mixture, which can be displayed on t h e microcomputer and]or on an attached recorder. Two cooling r a t e s (0.5 a n d 1.0~ per rain) were used to freeze callus and cell cultures. F u r t h e r details on the procedure are given in the Results and Discussion. At the end of the programmed freezing, frozen ampoules were quickly moved to a liquid nitrogen storage t a n k (CMR 2800, CryoMed) for long t e r m storage.
Thawing Frozen ampoules were removed from the storage container and thawed inunediately by continuously shaking in a water b a t h at 37 to 40~ for 2 to 3 min. The cryoprotectants were removed in two different ways, either by washing two times in liquid MMS medium and plating on solidified maintenance medium or by gradually removing the cryoprotectants by plating the thawed cells onto a filter paper (Whatman #1) positioned on top of solid maintenance medium. The solid medium was changed a t least t h r e e times a t 2 to 3 h intervals prior to plating the cells directly on solid maintenance medium for regrowth.
Regeneration of plantlets Embryogenic callus cultures obtained from thawed cultures were induced to form somatic embryos on solid maintenance medium. Plantlets were t h e n r e g e n e r a t e d from these cultures and were successfully transferred to soil using t h e procedures of Rangan et al. (in preparation).
R e s u l t s and D i s c u s s i o n
Viability assay Viability of cells after pretreatment with the cryoprotectants or after freezing and thawing was determined using fluorescein diacetate (for viable cells) and phenosafranine (for dead cells) according to the procedures of Widholm (1972).
Treatment of cells with clyoprotectants Suspension cells were filtered to get a homogeneous 4 years). 1. Collect the cells in a sterile 50 ml centrifuge tube. Remove the culture medium and resuspend the cells in a known volume of cold (4~ MMS medium with 5% sucrose. Cells should be kept in an ice bucket from this point onwards.
2. Prepare cryoprotectant mixture (2x the final concentration) as follows: MMS + 10% (v/v) glycerol + 10% (v/v) fresh DMSO (all sterile). 3. Add the cryoprotectant mixture to the cells over a period of one hour with intermittent shaking. The f i n a l concentration of both glycerol and DMSO should be 5% (v/v). Agitate the cells every 5 minutes. 4. Dispense the cold suspension into 3.6 ml cryovials such that the unpacked cell volume in each vial is approximately 70 to 80% of the total volume. Place these vials in a precooled (4~ freezer chamber attached to CryoMed Microcomputer Model 1010. 5. Start the preprogrammed freezing cycle: 9 Starting temperature 4~ 9 Slow cooling @ 0.5 or 1.0~ per min to the freezing point -4~ for embryogenic callus cultures or cell suspension cultures comprising
Plant Cell Reports (1996) 15:859-864
9 Springer-Verlag1996
Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.) Kanniah Rajasekaran J. G. Boswell Company, 850 Plymouth Avenue, P.O. Box 787, Corcoran, CA 93212-0787, USA Received 6 December t995/Revised version received 24 January 1996 - Communicated by I. K. Vasil
Abstract.
Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0~ A coohng rate of 0.5~ provided a satisfactory recovery rate ( 3 0 % ) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of l~ Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soft.
Abbreviations: DMSO, dimethylsulfoxide; MS, Murashige and Skoog (1962); MMS, modified MS; NAA, a-naphthaleneacetic acid.
Introduction Embryogenic callus and cell suspension cultures are routinely used in genetic transformation studies and crop improvement. They are also convenient for long term preservation of germplasm and experimental materials (Kartha 1985). Plant cell cultures are prone to chromosomal, genetic and molecular changes. Both epigenetic as well as heritable changes have been described (Larkin and Scowcroft 1981, Lee and Phillips 1988). Such variations pose a serious problem for the maintenance of genetic uniformity in plants regenerated in vitro. In biotechnology research, where plants are often transformed with a single
gene, it is very important to retain the agronomic performance of the variety that is being transformed. True-to-type plants can be regenerated from embryogenic callus a n d cell cultures only when the regeneration system is rapid and efficient (Vasil 1986, Rajasekaran et al. 1986). Long term maintenance of callus and cell cultures often results in plants that are morphologically abnormal, and functionally sterile, thus making them unsuitable for breeding. Several previously published reports (Bajaj and Gill 1985, Trolinder and Goodin 1987, Stelly et al. 1989, Li et al. 1989) indicate that tissue culturerelated genetic variation and sterility among regenerated plants can be a crucial factor in cotton (Gossypium hirsutum L.) transformation programs. Somatic variations, however, can be controlled to a large extent if embryogenic source cultures are new and freshly established or ff obtained from new cultures which have been cryopreserved. Here I report the successful regeneration of plants from cryopreserved embryogenic callus and cell suspension cultures of several elite cotton varieties. These cultures have been successfully used in genetic engineering of cotton for herbicide tolerance and insect resistance (Rajasekaran et al. in preparation).
M a t e r i a l s and m e t h o d s Initiation of embryogenic callus and cell suspension cultures Embryogenic callus cultures were established in the followingcotton (Gossypium hirsutum L.) varieties accordingto the procedures of Rangan (1993) and Rangan et al. (in preparation): SJ2, SJ5, SJC1, GC510, Royale (4226), B1644, B1654, B3991, CSC28, Stoneville 506, Siokra, Coker 315, Chembred B2 and Chembred C4. Briefly, cotyledon and hypocotylexplants from7 to 10 d old seedlingswereplacedon a
860 callus induction medium (Murashige and Skoog 1962; MS) supplemented with 0.4 mg/1 thianaine HC1, 30 g]l glucose, 2.0 mg/l NAA, 1.0 mg/l kinetin, 100 mg/l myo-inositol and 0.8% agar. The cultures were incubated at 27 + 2~ u n d e r conditions of 16 h light and 8 h dark, light intensity at 60 btE m-2 s-~, in a n environmentally controlled incubator (Percival, Boone, tA). Callus formed on these explants within 3 to 4 weeks was selectively subcultured to enrich for friable, yellowish green callus every 3 to 4 weeks on the same medium except t h a t the carbon source was sucrose (20 g/l) instead of glucose. Embryogenic callus capable of forming small globular somatic embryos appeared two to four subcultures after initiation. Embryogenic callus was m a i n t a i n e d and multiplied by routine subculture every 3 to 4 weeks on MS medium containing 100 mg]l myo-inositol, 20 g/1 sucrose, 2.0 rag/1 NAA and 0.8% agar (maintenance medium). Cell suspension cultures were initiated from finely dispersed embryogenic callus cultures in liquid maintenance medium s h a k e n (120 rpm, 27 + 2~ on a gyratory shaker (New Brunswick G-10, Edison, NJ). The suspension cultures were enriched for small, isodiametric, densely cytoplasmic and highly embryogenic cells by discarding free floating cells and large aggregates (>840 ~tm) every week. Rapid multiplication of these cultures was done by subculturing every 4 to 6 days in the same medium (100 ml) in 1000 ml Erlenmeyer flasks for two to three weeks prior to cryopreservation. Callus and suspension cultures of Acala cotton variety B1654 were used in all the initial studies to develop tlie cryopreservation protocol for cotton. All the chemicals including the cryoprotectants used in this study were purchased from Sigma Chelrfical company (St. Louis, MO).
the microcomputer. The cooling rate and the phase change were monitored by placing a t e m p e r a t u r e probe (Thermocouple 'T' type) in one of the ampoules containing the same cryoprotectant mixture, which can be displayed on t h e microcomputer and]or on an attached recorder. Two cooling r a t e s (0.5 a n d 1.0~ per rain) were used to freeze callus and cell cultures. F u r t h e r details on the procedure are given in the Results and Discussion. At the end of the programmed freezing, frozen ampoules were quickly moved to a liquid nitrogen storage t a n k (CMR 2800, CryoMed) for long t e r m storage.
Thawing Frozen ampoules were removed from the storage container and thawed inunediately by continuously shaking in a water b a t h at 37 to 40~ for 2 to 3 min. The cryoprotectants were removed in two different ways, either by washing two times in liquid MMS medium and plating on solidified maintenance medium or by gradually removing the cryoprotectants by plating the thawed cells onto a filter paper (Whatman #1) positioned on top of solid maintenance medium. The solid medium was changed a t least t h r e e times a t 2 to 3 h intervals prior to plating the cells directly on solid maintenance medium for regrowth.
Regeneration of plantlets Embryogenic callus cultures obtained from thawed cultures were induced to form somatic embryos on solid maintenance medium. Plantlets were t h e n r e g e n e r a t e d from these cultures and were successfully transferred to soil using t h e procedures of Rangan et al. (in preparation).
R e s u l t s and D i s c u s s i o n
Viability assay Viability of cells after pretreatment with the cryoprotectants or after freezing and thawing was determined using fluorescein diacetate (for viable cells) and phenosafranine (for dead cells) according to the procedures of Widholm (1972).
Treatment of cells with clyoprotectants Suspension cells were filtered to get a homogeneous 4 years). 1. Collect the cells in a sterile 50 ml centrifuge tube. Remove the culture medium and resuspend the cells in a known volume of cold (4~ MMS medium with 5% sucrose. Cells should be kept in an ice bucket from this point onwards.
2. Prepare cryoprotectant mixture (2x the final concentration) as follows: MMS + 10% (v/v) glycerol + 10% (v/v) fresh DMSO (all sterile). 3. Add the cryoprotectant mixture to the cells over a period of one hour with intermittent shaking. The f i n a l concentration of both glycerol and DMSO should be 5% (v/v). Agitate the cells every 5 minutes. 4. Dispense the cold suspension into 3.6 ml cryovials such that the unpacked cell volume in each vial is approximately 70 to 80% of the total volume. Place these vials in a precooled (4~ freezer chamber attached to CryoMed Microcomputer Model 1010. 5. Start the preprogrammed freezing cycle: 9 Starting temperature 4~ 9 Slow cooling @ 0.5 or 1.0~ per min to the freezing point -4~ for embryogenic callus cultures or cell suspension cultures comprising
