- Email: [email protected]
Effect of freeze-drying on viability and storage of pollen
314
ANNUAL MEETING ABSTRACTS
of Hedera helix at various temperatures it has been possible to isolate callus masses with varying degrees of cold tolerance. Subsequent subculturing of the selected cells followed by freezing at the same temperature resulted in tissue clones with a higher percentage of cells able to survive the given temperature. By coupling the selection process to studies determining its stability through tissue differentiation it may be possible to develop whole plants possessing increased cold hardiness. (Supported in part by NSF Grant GB-23,354). 29. Enzymatic Preservation
Changes Associated with PieezePollen. J. 0. ANDERSON. AND
of
J.
NATH (Division of Plant Sciences, West Virginia University. Morgantown, WV 26506).
Isozyme patterns of nonspecific esterases, leucine aminopeptidase, and acid phosphatase were studied to determine the nature of freeze-thawing and freeze-drying stresses on pollen. Fresh samples of Lilium longiflorum and Zea mays pollen were frozen and thawed at rates ranging between 2OO”C/ min to 0.5C/min. Freeze-drying temperatures ranged between 22 to -75°C. Freeze-dried samples were stored at 0-5°C for 5 months followed by rehydration in a controlled humidity chamber. Soluble enzymes were extracted from 0.1 g pollen in a pH 7.4 buffer at 0-4°C after the method of McCowen et al. [Plant Phys. 43, 578-582, (1968)J. Total protein was dctcrmined by t,he method of Lowry et al. [J. Biol. Chem. 193, 265275, (1951)]. The pollen extracts were analyzed by disc electrophoresis on 7.5% polyacrylamide gels and strained for cnzymcs using substrate strains. The stained gels were scored for isozyme pattern, R 1values and relative activity as expressed by densitometry. The data suggest that freezing and freeze-drying treatments are associated with isozymic damage to nonspecific esterases. A diffused staining pattern was noted for acid phosphatase which may be a reflection of conformational changes. The isozymes of leucine aminopeptidase remained stable in most treatments; however, some treatments resulted in minor damage to this isozymic system. The isozymic alterations enumerated appear to occur primarily in the free cytoplasmic enzymes. The enzymes associated with the coat seem to remain stable. 30. Xylem Sap Pressure Relationship to Seedling Survival after Freezing. G. N. BROWN, T. M. HINCKLEY,*
AND J.
estry, University 65201). Two-month
black
A. BIXBY* (School of Forof Missouri, Columbia, MO locust
seedlings
(Robinia
pseudoacacia L.) were cold hardened and seedlings at various stages of hardening were frozen to -20°C at a rate of lO”C/hr. Seedlings were used immediately after freezing, after 24 hr at 5”C, after 96 hr at 5°C and after 24 hr at 5°C plus 72 hr at 27°C. Nonfrozen control seedlings were treated under comparable conditions. Control and treated seedlings were placed in a Scholander Pressure Bomb to determine xylem sap pressures. Xylem water is under varying tensions in plant stems and as xylem water content decreases, xylem sap pressure becomes more negative. When cells are frozen intracellularly. membrane fractionation likely occurs and a rapid loss of cellular water should occur. Therefore, an increase in xylem sap pressure (less negative) should relate to cell rupture and death of t,issue. Seedling survival immediately after freezing and survival after the various recovery treatments at each stage of hardening are related t,o xylem sap pressure. A correlation was found between xylem sap pressure and later survival of seedlings after treatment. Seedlings at various stages of hardening also were frozen at 3”C-increments and tested in the Pressure Bomb for comparison with later survival. Again, a correlation was found. The Pressure Bomb map prove to bc a rapid and useful tool in determining seedling survival to freezing stress and degree of hardiness. (Contribution from the Missouri Agricultural Experiment Station). Viability and J. 0. ANDERSON (Division of Plant Sciences, West Virginia University Morgantown, WV 26506).
31. Effect of Freeze-Drying Storage of Pollen. J. NATH.
on
.~ND
Lilium longiflorum and Zea mays pollen was frozen and freeze-dried at several temperatures during storage. Vials containing 0.1 g of pollen were cooled at rates ranging between 2OO”C/min to 0.5”C/min. Freeze-drying was carried out for in an attempt to maintain a high level of viability 72 hr at several temperatures between 22 and -75°C. Freeze-dried samples were stored in vacuum. dry air, and dry nitrogen at 22, 0, -20 and -196°C for up to 2 years. Freeze-dried pollen was rehydrated by exposure to atmospheres of 90 and 10% relative humidity. Pollen viability was based on germination percentages. The germinability of pollen freeze-dried at temperatures below -50°C was prolonged over that of the controls. Germination values for unfrozen pollen stored for 30 days at 0-5°C averaged 50% for lily and 20% for corn. Freeze-dried pollen stored for 30 days at 0-5°C yielded 70% viable pollen for lily and 8% for corn. The viability of freeze-dried lily pollen is 70% at day 1 which drops to 30% by about 150 days and to approximately
ANNUAL MEETING 1% in 1 year at 0-5°C. The germination of control lily pollen drops to less than 1% in approximately 60 days at 0-5°C. The drying time is an important factor, perhaps indicating that residual moisture is critical. The data indicate that freeze-dried pollen can be stored at higher temperatures compared to controls. For example. the viability of control lily pollen stored at -196°C dropped to 20% in less than 20 days. On t,he other hand, in the freezedried samples there are at least 10% viable pollen even iu material stored at 0°C for 200 days. Freeze-dried pollen stored for 5 months at O-5°C upon slow rehydration at 5°C yielded intact grains which had an average germination rate of 25% for lily and 15% for corn. The same pollen when rehydrated rapidly at 5°C showed rupturing of 20-40% pollen and practically no germination. 32. Osmotic
Factors
of Hardiness
in Flowering
J. WILLIAMS,* AND R. J. WILLIAMS (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Rctheeda. MD 20014).
Dogwood.
Osmotic data from cortical tissue of the flowering dogwood tree (Cornzhs florida L.) were quant.itatively related to the degree of hardiness of the tissue. Cortex tissue was hardy to -25°C -t 0.5”C from March 22 to $pril 15, 1971, at which time the killing point rose to -15°C -C 0.5”C and remained constant to the end of June. Osmotic values for cell sap concentrations, “bound” water and cell solid content, and plasmolytic volume limit were categorized either hardy or nonhardy using the week of April 15 as the dividing period. The two groups of dat,a were statistically compared
315
ABSTRACTS
by the t test and were found to be significantly different. An estimate of the relative contribution of each osmotic parameter to the total hardiness of t,he tissue was made. We also observed an unreported phenomenon. Cells exposed to increasing concentration of CaCt2 reached a plateau beyond which furt,her volume reduction is not observed, though they are not injured until much higher concentrations are reached. We propose on this evidence that dogwood crlts possess some factor which provides a “mechanical” resistance to exosmosis as a mean of avoiding dehydration injury. 33.
Effects
of Glycerol
on the Survival
of Apple
D. 0. KETCHIE, AND C. MURREN (Tree Fruit Research Center, 1100 N. Western Avenue, Wenatchce, WA 98801). 7’j.ee.s.
Bark plugs 1 cm in diameter were removed from apple trees and dipped in different concentrations of glycerol. The plugs were then frozen, decreasing the temperature l”C/hr. The Tla’s (temperature 10% tissue killed) for 0, 25, 50, 100% glycrrol were -11. -11, -15, -lS”C, respectively. The Tw’s (temperature 90% tissue killed) for 0, 25, 50. 100% glycerol were -14. -14, -16, -24°C respectively. Apple trees grown in the greenhouse were sprayed with different concentrations of glycerol. Shoots from the trees were removed and frozen at the rate of l”C/hr. The Tlo’s for 0, 10, 15, 25. 50% were -5. -5, -5, -10, -lO”C, respectively. The Tso’s for 0. 10. 15, 25. 50% were -9, -9. -9, -11, -12”C, respectively. Occasionally 0.5% DMSO enhanced the action of glycerol in both the bark plugs and trees. (Supported by Washington Tree Fruit Research Commission Grant 1503).
SESSION D : BLOOD PRESERVATION 34. Glycerol
Permeation
Kinetics
in Red
Blood
A. W. ROWE, AND L. LENNY* (Laboratory of Cryobiology, The New York Blood Center, 310 East 67th Street. New York, NY 10021). Cells.
Although glycerol is effective as a cryoprotective additive for human red blood cells, its capacity for permeating cell membranes is not completely understood. Glycerol permeation in human red cells appears to follow first-order reaction kinetics. with the specific reaction rate constant (velocity constant) being a function of temperature. The osmotic stress which occurs in red cells during deglycerolization presumably results because pcrmeation of glycerol is both rate and temperature dependent. Glycerol permeation rates were determined directly by suspending intact red cells in a phosphate-buffered 0.3 M glycerol-water solution
and observed hemolysis at 675 nm as a function of time and temperature. The rate of glycerol permeation was considerably faster at 22°C than at 4°C. The age of the blood also influenced the rate of glycerol permeation, which stored (4°C) blood having a slower rate than that of fresh blood. The kinetics of glycerol addition was similar to glycerol removal based on a comparison of calculated specific reaction rate constants. Equilibrium dialysis was used to minimize the sudden osmolic shock which occurs by direct addition of diluent washes to glpcerolized cells. During removal of 14C glycerol by dialysis of red cells, a similar time and temperature relationship was noted, i.e., a warm (22°C) diluent resulted in faster deglycerolization and less cell trauma than occurred with use of a cold (4°C) diluent. Osmotic shock to red cells during rapid glycerol removal
ANNUAL MEETING ABSTRACTS
of Hedera helix at various temperatures it has been possible to isolate callus masses with varying degrees of cold tolerance. Subsequent subculturing of the selected cells followed by freezing at the same temperature resulted in tissue clones with a higher percentage of cells able to survive the given temperature. By coupling the selection process to studies determining its stability through tissue differentiation it may be possible to develop whole plants possessing increased cold hardiness. (Supported in part by NSF Grant GB-23,354). 29. Enzymatic Preservation
Changes Associated with PieezePollen. J. 0. ANDERSON. AND
of
J.
NATH (Division of Plant Sciences, West Virginia University. Morgantown, WV 26506).
Isozyme patterns of nonspecific esterases, leucine aminopeptidase, and acid phosphatase were studied to determine the nature of freeze-thawing and freeze-drying stresses on pollen. Fresh samples of Lilium longiflorum and Zea mays pollen were frozen and thawed at rates ranging between 2OO”C/ min to 0.5C/min. Freeze-drying temperatures ranged between 22 to -75°C. Freeze-dried samples were stored at 0-5°C for 5 months followed by rehydration in a controlled humidity chamber. Soluble enzymes were extracted from 0.1 g pollen in a pH 7.4 buffer at 0-4°C after the method of McCowen et al. [Plant Phys. 43, 578-582, (1968)J. Total protein was dctcrmined by t,he method of Lowry et al. [J. Biol. Chem. 193, 265275, (1951)]. The pollen extracts were analyzed by disc electrophoresis on 7.5% polyacrylamide gels and strained for cnzymcs using substrate strains. The stained gels were scored for isozyme pattern, R 1values and relative activity as expressed by densitometry. The data suggest that freezing and freeze-drying treatments are associated with isozymic damage to nonspecific esterases. A diffused staining pattern was noted for acid phosphatase which may be a reflection of conformational changes. The isozymes of leucine aminopeptidase remained stable in most treatments; however, some treatments resulted in minor damage to this isozymic system. The isozymic alterations enumerated appear to occur primarily in the free cytoplasmic enzymes. The enzymes associated with the coat seem to remain stable. 30. Xylem Sap Pressure Relationship to Seedling Survival after Freezing. G. N. BROWN, T. M. HINCKLEY,*
AND J.
estry, University 65201). Two-month
black
A. BIXBY* (School of Forof Missouri, Columbia, MO locust
seedlings
(Robinia
pseudoacacia L.) were cold hardened and seedlings at various stages of hardening were frozen to -20°C at a rate of lO”C/hr. Seedlings were used immediately after freezing, after 24 hr at 5”C, after 96 hr at 5°C and after 24 hr at 5°C plus 72 hr at 27°C. Nonfrozen control seedlings were treated under comparable conditions. Control and treated seedlings were placed in a Scholander Pressure Bomb to determine xylem sap pressures. Xylem water is under varying tensions in plant stems and as xylem water content decreases, xylem sap pressure becomes more negative. When cells are frozen intracellularly. membrane fractionation likely occurs and a rapid loss of cellular water should occur. Therefore, an increase in xylem sap pressure (less negative) should relate to cell rupture and death of t,issue. Seedling survival immediately after freezing and survival after the various recovery treatments at each stage of hardening are related t,o xylem sap pressure. A correlation was found between xylem sap pressure and later survival of seedlings after treatment. Seedlings at various stages of hardening also were frozen at 3”C-increments and tested in the Pressure Bomb for comparison with later survival. Again, a correlation was found. The Pressure Bomb map prove to bc a rapid and useful tool in determining seedling survival to freezing stress and degree of hardiness. (Contribution from the Missouri Agricultural Experiment Station). Viability and J. 0. ANDERSON (Division of Plant Sciences, West Virginia University Morgantown, WV 26506).
31. Effect of Freeze-Drying Storage of Pollen. J. NATH.
on
.~ND
Lilium longiflorum and Zea mays pollen was frozen and freeze-dried at several temperatures during storage. Vials containing 0.1 g of pollen were cooled at rates ranging between 2OO”C/min to 0.5”C/min. Freeze-drying was carried out for in an attempt to maintain a high level of viability 72 hr at several temperatures between 22 and -75°C. Freeze-dried samples were stored in vacuum. dry air, and dry nitrogen at 22, 0, -20 and -196°C for up to 2 years. Freeze-dried pollen was rehydrated by exposure to atmospheres of 90 and 10% relative humidity. Pollen viability was based on germination percentages. The germinability of pollen freeze-dried at temperatures below -50°C was prolonged over that of the controls. Germination values for unfrozen pollen stored for 30 days at 0-5°C averaged 50% for lily and 20% for corn. Freeze-dried pollen stored for 30 days at 0-5°C yielded 70% viable pollen for lily and 8% for corn. The viability of freeze-dried lily pollen is 70% at day 1 which drops to 30% by about 150 days and to approximately
ANNUAL MEETING 1% in 1 year at 0-5°C. The germination of control lily pollen drops to less than 1% in approximately 60 days at 0-5°C. The drying time is an important factor, perhaps indicating that residual moisture is critical. The data indicate that freeze-dried pollen can be stored at higher temperatures compared to controls. For example. the viability of control lily pollen stored at -196°C dropped to 20% in less than 20 days. On t,he other hand, in the freezedried samples there are at least 10% viable pollen even iu material stored at 0°C for 200 days. Freeze-dried pollen stored for 5 months at O-5°C upon slow rehydration at 5°C yielded intact grains which had an average germination rate of 25% for lily and 15% for corn. The same pollen when rehydrated rapidly at 5°C showed rupturing of 20-40% pollen and practically no germination. 32. Osmotic
Factors
of Hardiness
in Flowering
J. WILLIAMS,* AND R. J. WILLIAMS (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Rctheeda. MD 20014).
Dogwood.
Osmotic data from cortical tissue of the flowering dogwood tree (Cornzhs florida L.) were quant.itatively related to the degree of hardiness of the tissue. Cortex tissue was hardy to -25°C -t 0.5”C from March 22 to $pril 15, 1971, at which time the killing point rose to -15°C -C 0.5”C and remained constant to the end of June. Osmotic values for cell sap concentrations, “bound” water and cell solid content, and plasmolytic volume limit were categorized either hardy or nonhardy using the week of April 15 as the dividing period. The two groups of dat,a were statistically compared
315
ABSTRACTS
by the t test and were found to be significantly different. An estimate of the relative contribution of each osmotic parameter to the total hardiness of t,he tissue was made. We also observed an unreported phenomenon. Cells exposed to increasing concentration of CaCt2 reached a plateau beyond which furt,her volume reduction is not observed, though they are not injured until much higher concentrations are reached. We propose on this evidence that dogwood crlts possess some factor which provides a “mechanical” resistance to exosmosis as a mean of avoiding dehydration injury. 33.
Effects
of Glycerol
on the Survival
of Apple
D. 0. KETCHIE, AND C. MURREN (Tree Fruit Research Center, 1100 N. Western Avenue, Wenatchce, WA 98801). 7’j.ee.s.
Bark plugs 1 cm in diameter were removed from apple trees and dipped in different concentrations of glycerol. The plugs were then frozen, decreasing the temperature l”C/hr. The Tla’s (temperature 10% tissue killed) for 0, 25, 50, 100% glycrrol were -11. -11, -15, -lS”C, respectively. The Tw’s (temperature 90% tissue killed) for 0, 25, 50. 100% glycerol were -14. -14, -16, -24°C respectively. Apple trees grown in the greenhouse were sprayed with different concentrations of glycerol. Shoots from the trees were removed and frozen at the rate of l”C/hr. The Tlo’s for 0, 10, 15, 25. 50% were -5. -5, -5, -10, -lO”C, respectively. The Tso’s for 0. 10. 15, 25. 50% were -9, -9. -9, -11, -12”C, respectively. Occasionally 0.5% DMSO enhanced the action of glycerol in both the bark plugs and trees. (Supported by Washington Tree Fruit Research Commission Grant 1503).
SESSION D : BLOOD PRESERVATION 34. Glycerol
Permeation
Kinetics
in Red
Blood
A. W. ROWE, AND L. LENNY* (Laboratory of Cryobiology, The New York Blood Center, 310 East 67th Street. New York, NY 10021). Cells.
Although glycerol is effective as a cryoprotective additive for human red blood cells, its capacity for permeating cell membranes is not completely understood. Glycerol permeation in human red cells appears to follow first-order reaction kinetics. with the specific reaction rate constant (velocity constant) being a function of temperature. The osmotic stress which occurs in red cells during deglycerolization presumably results because pcrmeation of glycerol is both rate and temperature dependent. Glycerol permeation rates were determined directly by suspending intact red cells in a phosphate-buffered 0.3 M glycerol-water solution
and observed hemolysis at 675 nm as a function of time and temperature. The rate of glycerol permeation was considerably faster at 22°C than at 4°C. The age of the blood also influenced the rate of glycerol permeation, which stored (4°C) blood having a slower rate than that of fresh blood. The kinetics of glycerol addition was similar to glycerol removal based on a comparison of calculated specific reaction rate constants. Equilibrium dialysis was used to minimize the sudden osmolic shock which occurs by direct addition of diluent washes to glpcerolized cells. During removal of 14C glycerol by dialysis of red cells, a similar time and temperature relationship was noted, i.e., a warm (22°C) diluent resulted in faster deglycerolization and less cell trauma than occurred with use of a cold (4°C) diluent. Osmotic shock to red cells during rapid glycerol removal