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Long term survival of embryoids of carrot (Daucus carota L.)
Plant Science Letters, 2 (1974) 221--224 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
LONG TERM SURVIVAL OF EMBRYOIDS OF CARROT (DA UCUS CAROTA L.)
L.H. JONES
Unilever Research Laboratory Colworth/Welwyn, Colworth House, Sharnbrook, Beds. (Great Britain) (Received September 25th, 1973)
SUMMARY
Regeneration of normal carrot plants has been achieved from cultures kept in an inactive state for up to 2 years. Suspensions of }mbryoids plated onto an agar mineral medium lacking sucrose remained viable under growth room conditions (25 °, 16 h photoperiod) but did not grow. On supplying sucrose, spherical and heart stage embryoids germinated. In some cases torpedo stage embryoids formed fresh embryoids at the root pole, but did not themselves germinate. Callus did not survive. Over the 2-year period the parent cultures, sub-cultured at approximately 6-weekly intervals, lost the ability to regenerate plants.
INTRODUCTION
The ability to preserve useful genotypes for propagation by plant tissue culture is a vital part of any propagation programme. Continued sub-culture frequently leads to cytological abnormalities and to loss of regenerative potential in the culture. Recently, techniques for freezing plant cell cultures [1--3] have demonstrated the feasibility of this form of preservation, and the observations of Latta on freezing of carrot cultures have been confirmed in this laboratory. However, this method requires complex equipment for controlled cooling rate, and relies on continued supplies of liquid nitrogen. A cheaper and simpler method is the storage of embryoids on minimal media. This report describes the storage of carrot embryoids for u~J to two years on a sucrose free medium and their subsequent germination when sucrose was supplied.
Abbreviations: CH, casein hydrolysate; 2,4-D, 2,4-dichlorophenoxy acetic acid; MS, Murashige and Skoog; RH, relative humidity (ies). 221
MATERIALS AND METHODS
Cultures were initiated from the secondary phloem of carrot roots (Daucus carota c.v. Amsterdam Forcing). Explants were cut from 2-ram thick slices with a 2-ram canula and grown in Murashige and Skoog [4] (MS) medium, modified by the omission of potassium iodide and the inclusion of 10% coconut milk and 2,4-D (0.2 rag/l). 80 explants per flask were used in 200 ml medium in Steward's 1-1 turreted flasks [5], and rotated at 1 rev./min at 20 ° in continuous light. (Two Philips 20 W TL 55 fluorescent tubes mounted 10 cm from the wheel perimeter). After 8 weeks the highly embryogenic cell suspension was transferred to MS medium lacking 2,4-D and coconut milk but containing 200 mg/1 CH (casamino acids). Rapid embryoid development occurred in this medium, and embryoids were plated onto 75 ml of MS basal salts agar medium without sucrose in 250-ml Erlenmeyer flasks. The flasks were capped with aluminium foil and kept in a growth cabinet at 25 ° in 16 h days at approx. 15 000 lux. Later they were transferred to a lower light intensity (less than 400 lux) in a growth room, where they remained for up to two years. RESULTS The observations described were from control (zero sucrose) flasks in an experiment designed to test the stage at which carrot embryoids became autotrophic. In all cases where sucrose was present, the embryoids germinated quickly and plants were established. The zero sucrose controls failed to grow, and chlorophyll quickly bleached from greening cotyledons of torpedo stage embryoids. 1 year after plating, 4 control flasks remained containing white embryoids and small callus aggregates. At this time 5 ml 2% sucrose was injected into 2 of the flasks through a sterile filter. Within 3 weeks growth became apparent at many centres, and green cotyledons quickly developed. Growth apparently occurred from spherical and heart stage embryoids, a few of which germinated normally, but most proliferated to form fresh groups of embryoids. In general, torpedo stage embryoids did not grow. None germinated directly to form plantlets, but in some cases fresh embryogenesis occurred from the root pole. Callus did not grow. After 8 weeks, plantlets were transferred to fresh basal medium (with sucrose) and after a further 4 weeks were planted out in John Innes No.1 compost and placed in a mist propagator. The resulting plants appeared to be perfectly normal. The 2 remaining flasks were kept for a further year, and the operation was repeated with similar results. Subculture of the original parent culture into 2,4-D liquid medium was continued at approx. 6-week intervals for the first year. By this time embryogenic petential had been lost and no plantlets were obtained on plating onto basal medium with CH. 222
During seed maturation considerable desiccation takes nlace, and it was therefore decided to test whether embryoids might also be capable of surviving drying. Embryoids were allowed to equilibrate on filter papers to controlled RH. After 3 weeks they were plated onto MS/CH agar. Optimum survival was obtained after storage between 80% and 90% RH, none survived below 60%, and survival was slightly reduced at saturation. There is thus some indication of a tolerance to water loss, but not to severe desiccation. DISCUSSION
Embryoids are thought to follow the ontogeny of normal zygotic embryos. In some species storage products typical of the true seed are laid down [6]. It seems likely therefore that they should have similar physiological attributes to true seed embryos. These are characterised by the ability to survive for long periods in a metabolically inactive state. Tbe seed typically contains mainly insoluble storage products (starch, protein, oil) and the hnal stages of maturation generally involve rapid disappearance of sugars and drying out. The removal of sucrose from the medium and desiccation could be analogous to the normal seed maturation processes, and it is perhaps significant that the spherical and heart stage embryoids survived better than torpedo stage embryoids which, with green cotyledons, could be thought of as starting to germinate. It was also apparent that in freezing experiments the embryoids at these stages had a higher survival rate than either callus or older embryoids, again probably reflecting the physiological status of the seed-like embryoid well adapted for survival at a low level of metabolic activity. The observations of the effects of zero sucrose on embryoid survival were made on control flasks from an experiment designed for a different purpose. There is therefore no numerical information on survival rates, or on the effects of changing the concentration of nutrients other than sucrose. The flasks were kept in the standard growth-room environment, and no investigations have been made into optimal light or temperature regimes for long-term survival. The observation that regeneration of embryoids could be obtained after 2 years' storage in conditions probably very far from optimal, while continued subculture of the pare~t line led to complete loss of regenerative potential, could have great 1,,portance in cell culture propagation programrues and warrants wider investigation. In this context it is probabL l,~pormnt to recognise stages of embryoid development to find that most capable of survival. REFERENCES
1 2 3 4 "5
R. Latta, Can. J. Bot., 49 (1971) 1253. R.S. Quatrano, Plant Physiol., 43 (1968) 2057. L.J. Barmier and P.L. Steponkus, Hort. Sci., 7 (1972) 194. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473 F.C. Steward, Scientific American, 209 (1963) 104. 223
L.H. Jones, Plant cell culture and biochemistry; studies for improved vegetable oil production, FEBS Special Meeting, Dublin, 1973, in Symposium Volume, NorthHolland, Amsterdam, in press.
224
LONG TERM SURVIVAL OF EMBRYOIDS OF CARROT (DA UCUS CAROTA L.)
L.H. JONES
Unilever Research Laboratory Colworth/Welwyn, Colworth House, Sharnbrook, Beds. (Great Britain) (Received September 25th, 1973)
SUMMARY
Regeneration of normal carrot plants has been achieved from cultures kept in an inactive state for up to 2 years. Suspensions of }mbryoids plated onto an agar mineral medium lacking sucrose remained viable under growth room conditions (25 °, 16 h photoperiod) but did not grow. On supplying sucrose, spherical and heart stage embryoids germinated. In some cases torpedo stage embryoids formed fresh embryoids at the root pole, but did not themselves germinate. Callus did not survive. Over the 2-year period the parent cultures, sub-cultured at approximately 6-weekly intervals, lost the ability to regenerate plants.
INTRODUCTION
The ability to preserve useful genotypes for propagation by plant tissue culture is a vital part of any propagation programme. Continued sub-culture frequently leads to cytological abnormalities and to loss of regenerative potential in the culture. Recently, techniques for freezing plant cell cultures [1--3] have demonstrated the feasibility of this form of preservation, and the observations of Latta on freezing of carrot cultures have been confirmed in this laboratory. However, this method requires complex equipment for controlled cooling rate, and relies on continued supplies of liquid nitrogen. A cheaper and simpler method is the storage of embryoids on minimal media. This report describes the storage of carrot embryoids for u~J to two years on a sucrose free medium and their subsequent germination when sucrose was supplied.
Abbreviations: CH, casein hydrolysate; 2,4-D, 2,4-dichlorophenoxy acetic acid; MS, Murashige and Skoog; RH, relative humidity (ies). 221
MATERIALS AND METHODS
Cultures were initiated from the secondary phloem of carrot roots (Daucus carota c.v. Amsterdam Forcing). Explants were cut from 2-ram thick slices with a 2-ram canula and grown in Murashige and Skoog [4] (MS) medium, modified by the omission of potassium iodide and the inclusion of 10% coconut milk and 2,4-D (0.2 rag/l). 80 explants per flask were used in 200 ml medium in Steward's 1-1 turreted flasks [5], and rotated at 1 rev./min at 20 ° in continuous light. (Two Philips 20 W TL 55 fluorescent tubes mounted 10 cm from the wheel perimeter). After 8 weeks the highly embryogenic cell suspension was transferred to MS medium lacking 2,4-D and coconut milk but containing 200 mg/1 CH (casamino acids). Rapid embryoid development occurred in this medium, and embryoids were plated onto 75 ml of MS basal salts agar medium without sucrose in 250-ml Erlenmeyer flasks. The flasks were capped with aluminium foil and kept in a growth cabinet at 25 ° in 16 h days at approx. 15 000 lux. Later they were transferred to a lower light intensity (less than 400 lux) in a growth room, where they remained for up to two years. RESULTS The observations described were from control (zero sucrose) flasks in an experiment designed to test the stage at which carrot embryoids became autotrophic. In all cases where sucrose was present, the embryoids germinated quickly and plants were established. The zero sucrose controls failed to grow, and chlorophyll quickly bleached from greening cotyledons of torpedo stage embryoids. 1 year after plating, 4 control flasks remained containing white embryoids and small callus aggregates. At this time 5 ml 2% sucrose was injected into 2 of the flasks through a sterile filter. Within 3 weeks growth became apparent at many centres, and green cotyledons quickly developed. Growth apparently occurred from spherical and heart stage embryoids, a few of which germinated normally, but most proliferated to form fresh groups of embryoids. In general, torpedo stage embryoids did not grow. None germinated directly to form plantlets, but in some cases fresh embryogenesis occurred from the root pole. Callus did not grow. After 8 weeks, plantlets were transferred to fresh basal medium (with sucrose) and after a further 4 weeks were planted out in John Innes No.1 compost and placed in a mist propagator. The resulting plants appeared to be perfectly normal. The 2 remaining flasks were kept for a further year, and the operation was repeated with similar results. Subculture of the original parent culture into 2,4-D liquid medium was continued at approx. 6-week intervals for the first year. By this time embryogenic petential had been lost and no plantlets were obtained on plating onto basal medium with CH. 222
During seed maturation considerable desiccation takes nlace, and it was therefore decided to test whether embryoids might also be capable of surviving drying. Embryoids were allowed to equilibrate on filter papers to controlled RH. After 3 weeks they were plated onto MS/CH agar. Optimum survival was obtained after storage between 80% and 90% RH, none survived below 60%, and survival was slightly reduced at saturation. There is thus some indication of a tolerance to water loss, but not to severe desiccation. DISCUSSION
Embryoids are thought to follow the ontogeny of normal zygotic embryos. In some species storage products typical of the true seed are laid down [6]. It seems likely therefore that they should have similar physiological attributes to true seed embryos. These are characterised by the ability to survive for long periods in a metabolically inactive state. Tbe seed typically contains mainly insoluble storage products (starch, protein, oil) and the hnal stages of maturation generally involve rapid disappearance of sugars and drying out. The removal of sucrose from the medium and desiccation could be analogous to the normal seed maturation processes, and it is perhaps significant that the spherical and heart stage embryoids survived better than torpedo stage embryoids which, with green cotyledons, could be thought of as starting to germinate. It was also apparent that in freezing experiments the embryoids at these stages had a higher survival rate than either callus or older embryoids, again probably reflecting the physiological status of the seed-like embryoid well adapted for survival at a low level of metabolic activity. The observations of the effects of zero sucrose on embryoid survival were made on control flasks from an experiment designed for a different purpose. There is therefore no numerical information on survival rates, or on the effects of changing the concentration of nutrients other than sucrose. The flasks were kept in the standard growth-room environment, and no investigations have been made into optimal light or temperature regimes for long-term survival. The observation that regeneration of embryoids could be obtained after 2 years' storage in conditions probably very far from optimal, while continued subculture of the pare~t line led to complete loss of regenerative potential, could have great 1,,portance in cell culture propagation programrues and warrants wider investigation. In this context it is probabL l,~pormnt to recognise stages of embryoid development to find that most capable of survival. REFERENCES
1 2 3 4 "5
R. Latta, Can. J. Bot., 49 (1971) 1253. R.S. Quatrano, Plant Physiol., 43 (1968) 2057. L.J. Barmier and P.L. Steponkus, Hort. Sci., 7 (1972) 194. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473 F.C. Steward, Scientific American, 209 (1963) 104. 223
L.H. Jones, Plant cell culture and biochemistry; studies for improved vegetable oil production, FEBS Special Meeting, Dublin, 1973, in Symposium Volume, NorthHolland, Amsterdam, in press.
224