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Effect of freezing and freeze-drying on the viability and storage of Lilium longiflorum L. and Zea mays L. pollen
CHYOBIOLOGY
12,
81-88 (1875)
Effect of Freezing and Freeze-Drying on the Viability and Storage of Mum iongiflorum L. and Zea mays L. Pollen x J. NATH Dir;ision
AND
J. 0. ANDERSON z
of Plant Sciences, West Virginia University, Morgantown, West Virginia 26506
Viability of pollen after freezing and storage at low temperature and low relative humidity has been reported by several investigators (4, 5, S, 12). For example, Visser reported the preservation of apple and pear poIIen after freezing at various temperatures. However, preservation by freeze-drying gave negative resuhs ( 12). Pfeiffer obtained some viable lily pollen upon freeze-drying, but viability percentages were higher for non-freeze-dried pollen stored at low reIative humidity (8). Layne and Hagedorn obtained similar viability percentages for freeze-dried and vacuum-dried pea pollen after storage at -25°C (6). Wood and Barker preserved blueberry pollen by freeze-drying at -60°C and by storing in dry air at -20°C (13); however, there was a progressive decrease in pollen viability with storage. Ching and Ching found that freeze-drying reduced the viability of Douglas fir pollen if free water was not removed before freeze-drying (1). King suggested that the residua1 moisture content of freeze-dried pollen is critica for preservation (5). He has demonstrated that the response of pollen to freeze-drying and
vacuum-drying varies with species. Davies and Dickinson attributed the decrease in germination of freeze-dried IiIy pollen to the changes in the differential permeability of plasma membrane (2). Rehydration of freeze-dried or vacuumdried pollen affects viability although little research has been done in this area, Pfeiffer observed that viability of freeze-dried lily pollen stored at 5°C and 65% reIative humidity was higher than viability of poIIen stored under a less humid atmosphere (8). King also reported high germination vaIues for freeze-dried pine pollen exposed to 60% relative humidity for 48 hr at 5°C (41. The objectives of this study were to determine optimum rates of freezing, thawing, and rehydration for maximum pollen preservation. The study also was aimed at obtaining the optimum freeze-drying temperature, freeze-drying duration, storage temperature and storage environment for pollen preservation. hlATERIALS
L&urn 1ongifEorum L. var. Ace and Zea mays L, var. NJ-8 were grown in staggered plantings in the greenhouse at West Virginia University. Lily pollen was collected by removing mature anthers with a cylcone separator developed by Tervet et al. (11). Mature corn pollen was obtained by shaking the tassel over a large sheet of white
Received January 4, 1974. 1 Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 130%. 2 Present address: U.S. Regional Pasture Research Laboratory, University Park, Pennsylvania 16802. 81 CvpJright
1975 by Academic Press, Inc.
All eight4D? reproductionin any form reserved,
AND METHODS
82
NATH AND ANDERSON
paper. Pure poIlen was then placed in dry vials and stored at 05°C. Corn and lily pollen thus collected, were stored at 05°C until used for experimentation. Fresh samples of approximately 0.1 g each of corn and lily pollen were placed in A/S Nunc Serum Test Tubes (Vangard International Inc.) for freezing and freeze-drying. Pollen viability was assayed by observing pollen germination on a medium of 15% sucrose, 10 ppm HsBO3, 300 ppm Ca (NOa)z. 4 HzO, 100 ppm KNOZ and 2% agar on petri dishes (Quebec-style counting grids ) and in double depression microscope slides (3). Al1 dishes and slides were incubated for 1 hr at 30°C in a 60% relative humidity. To obtain percentages of viable (germinable) pollen, 100 grains in five grids of the petri dish and 100 grams within both cavities on the slides were counted and averaged. In large experiments, germination counts were done by photomicrography. In all experiments, control viability counts were made on untreated pollen stored in refrigerator at 0-5°C. In experiments involving length of storage, control counts were made on day one. Freezing rates of OS-%“C/min were obtained with a Neslab Model TE-9 circulating bath with a PBC4 cooler and a Model TP-2 temperature programmer. The bath was filled with 95% ethanol and a wire cage assembly containing sample vials and thermocouple was suspended in it. Freezing rates of 7%lBO”C/min were obtained in a liquid nitrogen vapor freezer mounted over the opening of a Linde L-35 liquid nitrogen refrigerator. Freezing rates of ZOO”C/min were produced by direct immersion of samples in liquid nitrogen. Thawing rates of 2.545”C/min were obtained with the Neslab TE-9 circulating bath with a TP-2 programmer. The portable bath cooler was turned off for the faster rates of lOOthawing rates. Thawing 218”C/min were obtained by placing the samples in water baths at temperatures
to 30°C ranging from melting ice (04°C) until equilibration to room temperature. All temperature measurements were recorded on a Honeywell Electronic 15 recorded with 3 ml copper-constat.an thermocouple embedded in the pollen sample. Frozen samples were transferred to a freeze-drier designed in this laboratory. The freeze-drying chambers were precooled to the desired freeze-drying temperature and maintained there by an electronic controller-recorder which activates liquid nitrogen pumping. A final freeze-drying pressure of approximately IO+ Torr was achieved with a high speed mechanical vacuum pump. Sublimed water was collected in two liquid nitrogen traps. TO determine whether or not a specimen at a given temperature was dry, the vapor pressure of the material was used as an index of moisture content. This was accomphshed by isolating the specimen chamber from the rest of the apparatus for 4-5 hr and recording the vapor pressure increase. The specimen was considered dry if the increase was less than 5 pm. Freeze-dried pollen samples were rehydrated by opening the sealed vials under controlled humidity conditions. Rapid rehydration and slow rehydration were done at 5°C in a glove box with relative humidities of 90 and IO%,, respectively. Rehydsation, in experiments other than those invoIving rapid and slow rehydration, was done at SO-SOY, relative humidity at 2022°C. Freeze-dried samples were rehy drated for 24 hr under the above conditions, resealed and stored at 05°C.
RESULTS
Freezing and Thawing To determine the optimum freezing and thawing rates for pollen preservation, corn and lily pollen were frozen to -196°C at rates between 200”C/min (rapid) and 0.5”C/min (slow). Thawing rates of 218”C/min (rapid) and 2,5”C/min (slow)
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FIG. 1. Viability of lily pollen upon rapid thawing (218”C/min) after freezing at varied rates, Control samples unfrozen, stored at 0-5”C. X - X control; [J - - 0 ZOO”/min; A-A 18O”/min; A - - LJ 15O”/min; A -A lOO/“min; A --A 75”/min; 1 - n 25”/min; W- - I lO”/min; l - l 2..5”/ min; 0 -- l 0.5”/min.
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FIG. 3. Viability of lily pollen upon rapid freezing (BOO”C/min) and varied thawing rates. ContraI samples unfrozen, stored at 0-5”C. X - X contro1; l - - 0 218”jmin; 0 - 0 lSO”/min; l-J-- IJ 12O”/min; A-A 1100”/min. 45”j min, 2O”/min, 15”/min, lO”/min, 2,5”/min.
were employed in this study. A comparison of Figs. 1 and 2 shows that for corresponding freezing rates and days of storage, rapid thawing resulted in higher percentages of polIen viability. In general, freezing rates above 15O”C/min yielded more viabIe pollen than slow freezing rates of 0.5-lOO”C/ min. Corn pollen was more sensitive to freezing and thawing damage than lily pollen (figures omitted to avoid redundancy), A slow thawing rate (2.5”C/min) yielded very little germinable pollen for
both species even when combined with the most rapid freezing rate (ZOO’C/min). Figures 3 and 4 show data for lily and corn polIen frozen at the optimal freezing rate of 200”C/min and thawed at rates ranging from 2.5 to 218”C/min. Only rapidly (IOO-218”C/min) thawed pollen was viable. The highest recovery was for pollen frozen at 2OO”C/min and thawed at 218”C/min. Pollen germination percentages were somewhat higher for lily than for corn. Slow freezing, in general, reduced pollen viability, but the reduction was not
FIG. 2. Viability of lily pollen upon slow thawing (2.5’C/min) after freezing at varied rates. Control samples unfrozen, stored at O-5%. X - X control; 17 -- jJ 2OO”/min; A -A lSO”/min; lOO”/min, 75”/min, 25”/ A - - A 15O”/min. min, lO”/min, 2.5’/min, 0.5’/min.
FIG. 4. Viability of corn ‘pollen upon rapid freezing (2OOT/min) and varied thawing rates. Control samples unfrozen, stored at O-5% X - X l - l 150’/min. control; l - - l 218”/min; 120°/min, lOO”/min, 45”/min, 25”/mm, 15”/min, 10°/min, Z.B”/min,
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and -50°C. Samples frozen to temperatures below -50°C (-100 and -196°C) yielded viabIe pollen only when these samples were rapidly thawed.
Fro. 5. Viability of corn pollen after freezing to temperatures ranging from 0 to -196°C. For all final temperatures four combinations (ABCD) of freezing and thawing rates were employed. Control samples unfrozen, stored at 04°C. Freezing + Thawing: A 0.5 + 2.5”/min; B 200+2.5”/ min; C 0.5 + 218”/min; D 200 + 218*/min.
as pronounced as that observed for slow thawing which produced pollen inviability immediately after thawing. The final freezing temperature is important in the freeze preservation of pollen ( Fig, 5), Pollen was frozen to several temperatures between 0 and -196°C. Four different freezing and thawing combinations ( ABCD) were employed. Regardless of the freezing and thawing regime employed, a general loss of viability was observed for sampIes frozen to temperatures between 0
OAYS STOR*GE
FIG. 6. Viability of lily pollen after rapid freezing (2OO”C/min) and freeze-drying at varied temperatures. Control samples, unfrozen, nonfreeze-dried, stored at 05°C. X - X control; n - - n -70°C; n - n -60°C; A - - A -50°C; A -30°C; A -A -2O’C; l - - l -10°C; Al -
a 0°C;
-
22°C.
Pollen was freeze-dried at temperatures ranging from 22 to -70°C for 48 hr. Prior to freeze-drying, samples were frozen to the respective freeze-drying temperature by rapid (2OO”C/min) and slow (O.S’C/ min), freezing rates. Rapidly frozen pollen survived freeze-drying stress better than slowly frozen pollen (Figs. 6, 7). Freezedrying temperatures above -50°C yielded pollen with low viability percentages in rapidly frozen samples and no germinable pollen in slowly frozen pollen. Corn pollen appeared slightly more sensitive to freezedrying stress than lily poIlen (figures omitted). Freeze-drying temperatures below -50°C seemed beneficial to both rapidly and slowly frozen pollen. Freeze-drying time affects the final water content of the sample. Pollen was freezedried at -60°C for varied periods of time after rapid and slow freezing. The optimal drying time for lily pollen was approximately 70 hr. Rapid freezing again yielded more viable pollen than slow freezing
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FIG. 7. Viability of lily pollen after slow freezing (O.S”C/min) and freeze-drying at varied temperatures. Control samples unfrozen, non-freeze-dried, stored at O-5%. X - X control; l - - W -70°C; I - I -60°C; A -* A -50°C. -3O”C, --2O”C, -lO”C, OT, 22°C.
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FIG. 8. Viability of lily pollen after rapid freezing (2OOT/min) and freeze-drying for varied durations. Control samples unfrozen, non-freeze-dried, stored at 0-5°C. X - X control; 100 hr; 90 hr; m-- m 80 hr; M - - W 70 hr; a --A 40 hr; A--A 60 hr; A-A 50 hr; l --e l -e 30hr;,---20hr; +-+ IOhr.
(Figs. 8, 9), The optimal drying time for corn pollen was approximately 75 hr at -60°C (figures omitted) ~ Freeze-drying for extended periods (beyond optima1 drying time) appears detrimental to pollen viability perhaps indicating that there is a critical amount of residual water or bound water that is essential for the maintenance of bioIogica1 stability. Storage Environment
Untreated pollen was stored at several temperatures between 0 and -196°C in
FIG. 10. Viability of untreated My pollen folIowing storage at -196°C (direct immersion in liquid nitrogen) in vacuum, nitrogen, air and dry air. Control samples unfrozen, non-freeze-dried, stored at O-5%. m - n vacuum; H - - I nitrogen; l - l air; l - - # dry air.
vacuum, nitrogen, air, and dry air. Storage at -190°C was accomplished by direct immersion in liquid nitrogen. Pollen of both species stored in vacuum and nitrogen maintained highest viability over longer durations at Iower temperatures (Figs. 10, 11). Storage of pollen in air yielded good viability at 0°C for both corn and lily pollen. However, beIow 0°C air-storage became ineffective (figures omitted). Dry air gave poor resuks at all temperatures perhaps indicating desiccation of pollen during storage, The effects of storage temperature (22, 0, -20, and -196’C) and environment on 80-l
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FIG. 9. Viability of lily pollen after SIOWfreezing (OS”C/min) and freeze-drying for varied durations. Control samples unfrozen, non-freeze-dried, stored at O-5X. X - X control; - - - 100 hr; A- A 90 hr; w - u 80 hr; S - - I 70 hr. 60 hr, 50 hr, 40 hr, 30 hr, 20 hr, 10 hr.
FIG. 11. Viability of unkeated corn pollen folIowing storage at --196”C (direct immersion in Iiquid nitrogen) in vacuum, nitiogen, air, and dry air. Control samples unfrozen, non-freeze-dried, stored at O-5%. See legend for Fig. 10.
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FIG. 12. Viability of rapidly frozen (ZOO”C/min) freeze-dried corn pollen stored at 22°C in vacuum, nitrogen, air, and dry air. Control samples unfrozen, X-X connon-freeze-dried, stared at O-5% trol; m - W vacuum; n - - n nitrogen; l - l air; l - - l dry air.
FIG. 14. Viability of rapidly frozen (ZOO”C/min) corn pollen freeze-dried at -50°C and rehydrated at varied rates. Control samples unfrozen, nonfreeze dried, stored at 0-5°C. X - X control; l - l rapid; l - - l slow.
Rehydration freeze dried material were also studied. Figures 12 and 13 are for storage at 22°C and -196°C; figures for storage at O°C/ min and -20°C are omitted for redundancy. Rapidly frozen (ZOO”C/min) freezedried lily pollen stored in vacuum nitrogen and air yielded substantially more viable cells at any given storage temperature than control pollen (Figs. 12 and 13). In comparison, the amount of viable pollen resulting from any given comparable treatment was somewhat lower for corn than for lily ( figures omitted).
Rehydration of freeze-dried corn and lily palIen is critical for viability. Too rapid rehydration may cause cell rupture and too slow rehydration may induce biochemical damage due to prolonged exposure of celIs to altered solute concentrations. Both corn and lily pollen have higher tolerance for slow rehydration than for rapid rehydration at 0-5°C (Figs. 14, 15). Rapidly frozen samples ( 200°C / min ) and freeze-dried yielded more viable pollen over a longer period of time than slowly frozen (O.S’C/ min) and freeze-dried sampIes (Figs. 14, 15). Rapid rehydration of freeze-dried pol-
FIG. 13. Viability of rapidly frozen (2OO”C/min) freeze-dried corn pollen stored at -198°C in vacuum, nitrogen, air, and dry air. Control samples unfrozen, non-freeze-dried, stored at 05°C. See legend to Fig. 12.
FE. 15. Viability of slowly frozen (O.S”C/min) corn poIlen freeze-dried at -50°C and rehydrated at varied rates. Control samples unfrozen, nonfreeze-dried, stored at 04°C. See legend to Fig. 14.
DAYSSTORAGE
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len produced extensive rupturing of pollen grains, especially for corn pollen (figures omitted), DISCUSSION
Since rapid freezing is less injurious to corn and lily poIlen than slow freezing, it is inferred that smal1 ice cry&& formed at rapid freezing rates cause less structural damage to the subcellular organelles and membrane systems in the pollen grains. Slow freezing may produce larger ice crystals resulting in greater structural damage in combination with dehydration of the cytoplasm. Thawing damage can lead to further loss of pollen viability. The slower thawing rates may permit ice formed during freezing to “grow” or recrystallize during the thawing process. These results are in accord with Sakai’s work on cortical cells of mulberry ( 10). He has suggested than during rapid cooling, freezable water enucleates within the cells and grows during subsequent slow rewarming into larger ice crystals. However, Dickinson maintains that pollen has a water content of Y-10% (dry weight) and a high sucrose concentration which makes it unlikely that intracellular freezing occurs ( 2 ) . Although Dickinson makes no statements of freezing rates, immersion in dry ice and acetone bath at -6o--70°C could produce a freezing rate rapid enough to produce minute ice ,crystals. Data on the combinations of the more extreme freezing and thawing rates indi,cates that rapid thawing is essential in preserving the germinability of frozen pollen. Slow freezing produces a reduction in viable polIen, but the reduction is not as pronounced as that produced by slow thawing which renders poIIen immediately nonviable. This may indicate that thawing damage is primariIy a structura1 one involving disruption of the interna structures of the cell by ice crystallization. Freezing damage, on the other hand, may involve relativeIy l.ess structural damage but more subtle biochemical alterations.
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Differential sensitivity of corn and lily pollen to freezing and thawing may in part be due to differences in morphology, water ,content and other biochemical constituents (2,9). For example, the degree of freezing damage appears ,to be related to the amount of lipid pigment associated with pollen exine. Corn poIlen which has much less lipid pigment than lily pollen, is more susceptible to freezing damage than the Iatter species. Thus, it is surmised that polIen exine associated lipid, in some unknown manner, imparts protection against freezing damage. Final freezing end point seems critical in the freeze-preservation of pollen, Final temperatures between 0 and -196°C were investigated. A significant loss of pollen germinabiIity was observed at temperatures above -50°C. Temperatures below -50°C gave the best results perhaps indicating prevention or slowing of recrystallization. The more rapidly frozen samples exhibited higher germination values than those slowly frozen to the same temperature. Slowly thawed sampIes, however, gave much lower germination values in most cases regardless of freezing rate. This indicates that recrystallization, which occurs during slow thawing, leads to a loss of poIIen viability. Freeze-dried lily and corn pollen generally remained viable at higher storage temperatures than nonfrozen or frozen pollen. Rapidly frozen and freeze-dried samples were more viabIe than slowly frozen and freeze-dried polIen. Slow freezing prior to freeze drying may itself render the cells inviabIe, or it may damage the cells in a manner that they become much more susceptible to freeze-drying stress. Freezedrying temperatures below -50°C appeared more beneficial for both lily and corn pollen regardless of their freezing rates. Loss of viabiIity at high freeze-drying temperatures perhaps indicates recrystallization damage even under reduced pressure or vacuum. At temperatures beIow
NATH AND ANDERSON
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-50°C optimum drying times for lily and corn pollen were approximately 70 and 80 hr, respectively. Slight overdrying did not appear as detrimental as underdrying. The residual free water left by underdrying could cause viability loss during storage of freeze-dried material. The storage environment may also have some effect on bound water remaining in the freeze-dried samples. The effect may be primarily on enzyme systems as has been suggested by Litt and Boyd (7). SUMMARY
The freeze-preservation of pollen is dependent on the interaction of several factors such as freezing rate, thawing rate, freezedrying temperature and duration, storage temperature and environment and rehydration rates. Changes in any of these variables affects the others directly or indirectly. Rapid freezing of pollen at rates of approximateIy 200 oC/ min maintains the highest degree of viable pollen in combination with rapid thawing rates of 218”C/min. Rapid cooling and slow rewarming resulted in a substantial loss of pollen viability. This might indicate that intracellular ice crystals formed during rapid cooling perhaps grow into larger ice masses during slow rewarming or storage at temperatures above -50°C. The germinability of pollen freeze-dried at temperatures below -50°C was also prolonged over that of the controls. Germination values for unfrozen pollen stored for 30 days at 05°C averaged 50% for lily and 20% for corn. Freeze-dried pollen stored for 30 days at the same temperature yielded considerably higher viability percentages for both lily and corn pollen. Drying time is an important factor, perhaps indicating that residual moisture is critical. Freeze-
dried pollen can be stored at higher temperatures than frozen and control pollen. Freeze-dried material stored for five months at 05”C, upon slow rehydration yielded intact grains which has average germination percentages of 25 for Iily and 15 for corn. The same pollen upon rapid rehydration showed rupturing of 2O-4O$& of the cells and practically no germination. REFERENCES 1. Ching, T., and Ching, K. Freeze-drying pine pollen. I%nt Physiol. 39, 705-709 ( 1964 ) , 2. Davies, M. D., and Dickinson, D. B. Effects of freeze-drying on permeability and respiration of germinating lily pollen. Pht Phytiol. 24, 5-9 ( 1971). 3. Dickinson, D. B. A photomicrographic study of lily pollen germination. Hurt. Sd. 1, 20 (1966). 4. King, J. R. The freeze-drying of pine pollen. Bull. Tar. Bat. Chb 86, 383-386 ( 1959). 5. King, J. R. The freeze-drying of pollens. &on. Bat. 15, 91-98 (1961). 6. Layne, R. E. C., and Hagedorn, D. J. Effect of vacuum-drying, freeze-drying and storage environment of viability of pea pollen. Crop Zci. 3, 433-436 ( 1963). 7. Litt, M., and Boyd, W. Preservation of haemocyanin. Nature (London) 181, 1075 ( 1958). 8. Pfeiffer, N. E. Effect of lyophilization of viability of La’Eilsmpollen. Contr. Boyce Thompson rn.!it. 18, 153-158 ( 1955). 9. Rosen, W. G. Ultrastructure and physiology of pollen. Ann. Rev. Plant PhysioE. 19, 435462 ( 1968). 10. Sakai, A., and Yoshida, S. SurvivaI of pIant tissues at super Iow temperatures. VI Effects of cooling and rewarming rates, Pht Physlol. 42, 1695-1701 ( 1987). 11. Tervet, I. W., Rawson, A. J., Cherry, E., and Saxon, R. B. A method for the collection of microscopic particles. Phytopathol. 41, 282-285 ( 1951). 12. Visser, T. Germination and storage of poIlen. Meded. Laludbozr. Wagen. 55, 1-68 ( 1955). 13. Wood, G. W., and Barker, W. G. Preservation of blueberry polIen by the freeze-drying process. Canad. I. Hunt Sci. 44, 387-388 (1964).
12,
81-88 (1875)
Effect of Freezing and Freeze-Drying on the Viability and Storage of Mum iongiflorum L. and Zea mays L. Pollen x J. NATH Dir;ision
AND
J. 0. ANDERSON z
of Plant Sciences, West Virginia University, Morgantown, West Virginia 26506
Viability of pollen after freezing and storage at low temperature and low relative humidity has been reported by several investigators (4, 5, S, 12). For example, Visser reported the preservation of apple and pear poIIen after freezing at various temperatures. However, preservation by freeze-drying gave negative resuhs ( 12). Pfeiffer obtained some viable lily pollen upon freeze-drying, but viability percentages were higher for non-freeze-dried pollen stored at low reIative humidity (8). Layne and Hagedorn obtained similar viability percentages for freeze-dried and vacuum-dried pea pollen after storage at -25°C (6). Wood and Barker preserved blueberry pollen by freeze-drying at -60°C and by storing in dry air at -20°C (13); however, there was a progressive decrease in pollen viability with storage. Ching and Ching found that freeze-drying reduced the viability of Douglas fir pollen if free water was not removed before freeze-drying (1). King suggested that the residua1 moisture content of freeze-dried pollen is critica for preservation (5). He has demonstrated that the response of pollen to freeze-drying and
vacuum-drying varies with species. Davies and Dickinson attributed the decrease in germination of freeze-dried IiIy pollen to the changes in the differential permeability of plasma membrane (2). Rehydration of freeze-dried or vacuumdried pollen affects viability although little research has been done in this area, Pfeiffer observed that viability of freeze-dried lily pollen stored at 5°C and 65% reIative humidity was higher than viability of poIIen stored under a less humid atmosphere (8). King also reported high germination vaIues for freeze-dried pine pollen exposed to 60% relative humidity for 48 hr at 5°C (41. The objectives of this study were to determine optimum rates of freezing, thawing, and rehydration for maximum pollen preservation. The study also was aimed at obtaining the optimum freeze-drying temperature, freeze-drying duration, storage temperature and storage environment for pollen preservation. hlATERIALS
L&urn 1ongifEorum L. var. Ace and Zea mays L, var. NJ-8 were grown in staggered plantings in the greenhouse at West Virginia University. Lily pollen was collected by removing mature anthers with a cylcone separator developed by Tervet et al. (11). Mature corn pollen was obtained by shaking the tassel over a large sheet of white
Received January 4, 1974. 1 Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 130%. 2 Present address: U.S. Regional Pasture Research Laboratory, University Park, Pennsylvania 16802. 81 CvpJright
1975 by Academic Press, Inc.
All eight4D? reproductionin any form reserved,
AND METHODS
82
NATH AND ANDERSON
paper. Pure poIlen was then placed in dry vials and stored at 05°C. Corn and lily pollen thus collected, were stored at 05°C until used for experimentation. Fresh samples of approximately 0.1 g each of corn and lily pollen were placed in A/S Nunc Serum Test Tubes (Vangard International Inc.) for freezing and freeze-drying. Pollen viability was assayed by observing pollen germination on a medium of 15% sucrose, 10 ppm HsBO3, 300 ppm Ca (NOa)z. 4 HzO, 100 ppm KNOZ and 2% agar on petri dishes (Quebec-style counting grids ) and in double depression microscope slides (3). Al1 dishes and slides were incubated for 1 hr at 30°C in a 60% relative humidity. To obtain percentages of viable (germinable) pollen, 100 grains in five grids of the petri dish and 100 grams within both cavities on the slides were counted and averaged. In large experiments, germination counts were done by photomicrography. In all experiments, control viability counts were made on untreated pollen stored in refrigerator at 0-5°C. In experiments involving length of storage, control counts were made on day one. Freezing rates of OS-%“C/min were obtained with a Neslab Model TE-9 circulating bath with a PBC4 cooler and a Model TP-2 temperature programmer. The bath was filled with 95% ethanol and a wire cage assembly containing sample vials and thermocouple was suspended in it. Freezing rates of 7%lBO”C/min were obtained in a liquid nitrogen vapor freezer mounted over the opening of a Linde L-35 liquid nitrogen refrigerator. Freezing rates of ZOO”C/min were produced by direct immersion of samples in liquid nitrogen. Thawing rates of 2.545”C/min were obtained with the Neslab TE-9 circulating bath with a TP-2 programmer. The portable bath cooler was turned off for the faster rates of lOOthawing rates. Thawing 218”C/min were obtained by placing the samples in water baths at temperatures
to 30°C ranging from melting ice (04°C) until equilibration to room temperature. All temperature measurements were recorded on a Honeywell Electronic 15 recorded with 3 ml copper-constat.an thermocouple embedded in the pollen sample. Frozen samples were transferred to a freeze-drier designed in this laboratory. The freeze-drying chambers were precooled to the desired freeze-drying temperature and maintained there by an electronic controller-recorder which activates liquid nitrogen pumping. A final freeze-drying pressure of approximately IO+ Torr was achieved with a high speed mechanical vacuum pump. Sublimed water was collected in two liquid nitrogen traps. TO determine whether or not a specimen at a given temperature was dry, the vapor pressure of the material was used as an index of moisture content. This was accomphshed by isolating the specimen chamber from the rest of the apparatus for 4-5 hr and recording the vapor pressure increase. The specimen was considered dry if the increase was less than 5 pm. Freeze-dried pollen samples were rehydrated by opening the sealed vials under controlled humidity conditions. Rapid rehydration and slow rehydration were done at 5°C in a glove box with relative humidities of 90 and IO%,, respectively. Rehydsation, in experiments other than those invoIving rapid and slow rehydration, was done at SO-SOY, relative humidity at 2022°C. Freeze-dried samples were rehy drated for 24 hr under the above conditions, resealed and stored at 05°C.
RESULTS
Freezing and Thawing To determine the optimum freezing and thawing rates for pollen preservation, corn and lily pollen were frozen to -196°C at rates between 200”C/min (rapid) and 0.5”C/min (slow). Thawing rates of 218”C/min (rapid) and 2,5”C/min (slow)
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FIG. 1. Viability of lily pollen upon rapid thawing (218”C/min) after freezing at varied rates, Control samples unfrozen, stored at 0-5”C. X - X control; [J - - 0 ZOO”/min; A-A 18O”/min; A - - LJ 15O”/min; A -A lOO/“min; A --A 75”/min; 1 - n 25”/min; W- - I lO”/min; l - l 2..5”/ min; 0 -- l 0.5”/min.
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FIG. 3. Viability of lily pollen upon rapid freezing (BOO”C/min) and varied thawing rates. ContraI samples unfrozen, stored at 0-5”C. X - X contro1; l - - 0 218”jmin; 0 - 0 lSO”/min; l-J-- IJ 12O”/min; A-A 1100”/min. 45”j min, 2O”/min, 15”/min, lO”/min, 2,5”/min.
were employed in this study. A comparison of Figs. 1 and 2 shows that for corresponding freezing rates and days of storage, rapid thawing resulted in higher percentages of polIen viability. In general, freezing rates above 15O”C/min yielded more viabIe pollen than slow freezing rates of 0.5-lOO”C/ min. Corn pollen was more sensitive to freezing and thawing damage than lily pollen (figures omitted to avoid redundancy), A slow thawing rate (2.5”C/min) yielded very little germinable pollen for
both species even when combined with the most rapid freezing rate (ZOO’C/min). Figures 3 and 4 show data for lily and corn polIen frozen at the optimal freezing rate of 200”C/min and thawed at rates ranging from 2.5 to 218”C/min. Only rapidly (IOO-218”C/min) thawed pollen was viable. The highest recovery was for pollen frozen at 2OO”C/min and thawed at 218”C/min. Pollen germination percentages were somewhat higher for lily than for corn. Slow freezing, in general, reduced pollen viability, but the reduction was not
FIG. 2. Viability of lily pollen upon slow thawing (2.5’C/min) after freezing at varied rates. Control samples unfrozen, stored at O-5%. X - X control; 17 -- jJ 2OO”/min; A -A lSO”/min; lOO”/min, 75”/min, 25”/ A - - A 15O”/min. min, lO”/min, 2.5’/min, 0.5’/min.
FIG. 4. Viability of corn ‘pollen upon rapid freezing (2OOT/min) and varied thawing rates. Control samples unfrozen, stored at O-5% X - X l - l 150’/min. control; l - - l 218”/min; 120°/min, lOO”/min, 45”/min, 25”/mm, 15”/min, 10°/min, Z.B”/min,
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and -50°C. Samples frozen to temperatures below -50°C (-100 and -196°C) yielded viabIe pollen only when these samples were rapidly thawed.
Fro. 5. Viability of corn pollen after freezing to temperatures ranging from 0 to -196°C. For all final temperatures four combinations (ABCD) of freezing and thawing rates were employed. Control samples unfrozen, stored at 04°C. Freezing + Thawing: A 0.5 + 2.5”/min; B 200+2.5”/ min; C 0.5 + 218”/min; D 200 + 218*/min.
as pronounced as that observed for slow thawing which produced pollen inviability immediately after thawing. The final freezing temperature is important in the freeze preservation of pollen ( Fig, 5), Pollen was frozen to several temperatures between 0 and -196°C. Four different freezing and thawing combinations ( ABCD) were employed. Regardless of the freezing and thawing regime employed, a general loss of viability was observed for sampIes frozen to temperatures between 0
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FIG. 6. Viability of lily pollen after rapid freezing (2OO”C/min) and freeze-drying at varied temperatures. Control samples, unfrozen, nonfreeze-dried, stored at 05°C. X - X control; n - - n -70°C; n - n -60°C; A - - A -50°C; A -30°C; A -A -2O’C; l - - l -10°C; Al -
a 0°C;
-
22°C.
Pollen was freeze-dried at temperatures ranging from 22 to -70°C for 48 hr. Prior to freeze-drying, samples were frozen to the respective freeze-drying temperature by rapid (2OO”C/min) and slow (O.S’C/ min), freezing rates. Rapidly frozen pollen survived freeze-drying stress better than slowly frozen pollen (Figs. 6, 7). Freezedrying temperatures above -50°C yielded pollen with low viability percentages in rapidly frozen samples and no germinable pollen in slowly frozen pollen. Corn pollen appeared slightly more sensitive to freezedrying stress than lily poIlen (figures omitted). Freeze-drying temperatures below -50°C seemed beneficial to both rapidly and slowly frozen pollen. Freeze-drying time affects the final water content of the sample. Pollen was freezedried at -60°C for varied periods of time after rapid and slow freezing. The optimal drying time for lily pollen was approximately 70 hr. Rapid freezing again yielded more viable pollen than slow freezing
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FIG. 7. Viability of lily pollen after slow freezing (O.S”C/min) and freeze-drying at varied temperatures. Control samples unfrozen, non-freeze-dried, stored at O-5%. X - X control; l - - W -70°C; I - I -60°C; A -* A -50°C. -3O”C, --2O”C, -lO”C, OT, 22°C.
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FIG. 8. Viability of lily pollen after rapid freezing (2OOT/min) and freeze-drying for varied durations. Control samples unfrozen, non-freeze-dried, stored at 0-5°C. X - X control; 100 hr; 90 hr; m-- m 80 hr; M - - W 70 hr; a --A 40 hr; A--A 60 hr; A-A 50 hr; l --e l -e 30hr;,---20hr; +-+ IOhr.
(Figs. 8, 9), The optimal drying time for corn pollen was approximately 75 hr at -60°C (figures omitted) ~ Freeze-drying for extended periods (beyond optima1 drying time) appears detrimental to pollen viability perhaps indicating that there is a critical amount of residual water or bound water that is essential for the maintenance of bioIogica1 stability. Storage Environment
Untreated pollen was stored at several temperatures between 0 and -196°C in
FIG. 10. Viability of untreated My pollen folIowing storage at -196°C (direct immersion in liquid nitrogen) in vacuum, nitrogen, air and dry air. Control samples unfrozen, non-freeze-dried, stored at O-5%. m - n vacuum; H - - I nitrogen; l - l air; l - - # dry air.
vacuum, nitrogen, air, and dry air. Storage at -190°C was accomplished by direct immersion in liquid nitrogen. Pollen of both species stored in vacuum and nitrogen maintained highest viability over longer durations at Iower temperatures (Figs. 10, 11). Storage of pollen in air yielded good viability at 0°C for both corn and lily pollen. However, beIow 0°C air-storage became ineffective (figures omitted). Dry air gave poor resuks at all temperatures perhaps indicating desiccation of pollen during storage, The effects of storage temperature (22, 0, -20, and -196’C) and environment on 80-l
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FIG. 9. Viability of lily pollen after SIOWfreezing (OS”C/min) and freeze-drying for varied durations. Control samples unfrozen, non-freeze-dried, stored at O-5X. X - X control; - - - 100 hr; A- A 90 hr; w - u 80 hr; S - - I 70 hr. 60 hr, 50 hr, 40 hr, 30 hr, 20 hr, 10 hr.
FIG. 11. Viability of unkeated corn pollen folIowing storage at --196”C (direct immersion in Iiquid nitrogen) in vacuum, nitiogen, air, and dry air. Control samples unfrozen, non-freeze-dried, stored at O-5%. See legend for Fig. 10.
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FIG. 12. Viability of rapidly frozen (ZOO”C/min) freeze-dried corn pollen stored at 22°C in vacuum, nitrogen, air, and dry air. Control samples unfrozen, X-X connon-freeze-dried, stared at O-5% trol; m - W vacuum; n - - n nitrogen; l - l air; l - - l dry air.
FIG. 14. Viability of rapidly frozen (ZOO”C/min) corn pollen freeze-dried at -50°C and rehydrated at varied rates. Control samples unfrozen, nonfreeze dried, stored at 0-5°C. X - X control; l - l rapid; l - - l slow.
Rehydration freeze dried material were also studied. Figures 12 and 13 are for storage at 22°C and -196°C; figures for storage at O°C/ min and -20°C are omitted for redundancy. Rapidly frozen (ZOO”C/min) freezedried lily pollen stored in vacuum nitrogen and air yielded substantially more viable cells at any given storage temperature than control pollen (Figs. 12 and 13). In comparison, the amount of viable pollen resulting from any given comparable treatment was somewhat lower for corn than for lily ( figures omitted).
Rehydration of freeze-dried corn and lily palIen is critical for viability. Too rapid rehydration may cause cell rupture and too slow rehydration may induce biochemical damage due to prolonged exposure of celIs to altered solute concentrations. Both corn and lily pollen have higher tolerance for slow rehydration than for rapid rehydration at 0-5°C (Figs. 14, 15). Rapidly frozen samples ( 200°C / min ) and freeze-dried yielded more viable pollen over a longer period of time than slowly frozen (O.S’C/ min) and freeze-dried sampIes (Figs. 14, 15). Rapid rehydration of freeze-dried pol-
FIG. 13. Viability of rapidly frozen (2OO”C/min) freeze-dried corn pollen stored at -198°C in vacuum, nitrogen, air, and dry air. Control samples unfrozen, non-freeze-dried, stored at 05°C. See legend to Fig. 12.
FE. 15. Viability of slowly frozen (O.S”C/min) corn poIlen freeze-dried at -50°C and rehydrated at varied rates. Control samples unfrozen, nonfreeze-dried, stored at 04°C. See legend to Fig. 14.
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len produced extensive rupturing of pollen grains, especially for corn pollen (figures omitted), DISCUSSION
Since rapid freezing is less injurious to corn and lily poIlen than slow freezing, it is inferred that smal1 ice cry&& formed at rapid freezing rates cause less structural damage to the subcellular organelles and membrane systems in the pollen grains. Slow freezing may produce larger ice crystals resulting in greater structural damage in combination with dehydration of the cytoplasm. Thawing damage can lead to further loss of pollen viability. The slower thawing rates may permit ice formed during freezing to “grow” or recrystallize during the thawing process. These results are in accord with Sakai’s work on cortical cells of mulberry ( 10). He has suggested than during rapid cooling, freezable water enucleates within the cells and grows during subsequent slow rewarming into larger ice crystals. However, Dickinson maintains that pollen has a water content of Y-10% (dry weight) and a high sucrose concentration which makes it unlikely that intracellular freezing occurs ( 2 ) . Although Dickinson makes no statements of freezing rates, immersion in dry ice and acetone bath at -6o--70°C could produce a freezing rate rapid enough to produce minute ice ,crystals. Data on the combinations of the more extreme freezing and thawing rates indi,cates that rapid thawing is essential in preserving the germinability of frozen pollen. Slow freezing produces a reduction in viable polIen, but the reduction is not as pronounced as that produced by slow thawing which renders poIIen immediately nonviable. This may indicate that thawing damage is primariIy a structura1 one involving disruption of the interna structures of the cell by ice crystallization. Freezing damage, on the other hand, may involve relativeIy l.ess structural damage but more subtle biochemical alterations.
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Differential sensitivity of corn and lily pollen to freezing and thawing may in part be due to differences in morphology, water ,content and other biochemical constituents (2,9). For example, the degree of freezing damage appears ,to be related to the amount of lipid pigment associated with pollen exine. Corn poIlen which has much less lipid pigment than lily pollen, is more susceptible to freezing damage than the Iatter species. Thus, it is surmised that polIen exine associated lipid, in some unknown manner, imparts protection against freezing damage. Final freezing end point seems critical in the freeze-preservation of pollen, Final temperatures between 0 and -196°C were investigated. A significant loss of pollen germinabiIity was observed at temperatures above -50°C. Temperatures below -50°C gave the best results perhaps indicating prevention or slowing of recrystallization. The more rapidly frozen samples exhibited higher germination values than those slowly frozen to the same temperature. Slowly thawed sampIes, however, gave much lower germination values in most cases regardless of freezing rate. This indicates that recrystallization, which occurs during slow thawing, leads to a loss of poIIen viability. Freeze-dried lily and corn pollen generally remained viable at higher storage temperatures than nonfrozen or frozen pollen. Rapidly frozen and freeze-dried samples were more viabIe than slowly frozen and freeze-dried polIen. Slow freezing prior to freeze drying may itself render the cells inviabIe, or it may damage the cells in a manner that they become much more susceptible to freeze-drying stress. Freezedrying temperatures below -50°C appeared more beneficial for both lily and corn pollen regardless of their freezing rates. Loss of viabiIity at high freeze-drying temperatures perhaps indicates recrystallization damage even under reduced pressure or vacuum. At temperatures beIow
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-50°C optimum drying times for lily and corn pollen were approximately 70 and 80 hr, respectively. Slight overdrying did not appear as detrimental as underdrying. The residual free water left by underdrying could cause viability loss during storage of freeze-dried material. The storage environment may also have some effect on bound water remaining in the freeze-dried samples. The effect may be primarily on enzyme systems as has been suggested by Litt and Boyd (7). SUMMARY
The freeze-preservation of pollen is dependent on the interaction of several factors such as freezing rate, thawing rate, freezedrying temperature and duration, storage temperature and environment and rehydration rates. Changes in any of these variables affects the others directly or indirectly. Rapid freezing of pollen at rates of approximateIy 200 oC/ min maintains the highest degree of viable pollen in combination with rapid thawing rates of 218”C/min. Rapid cooling and slow rewarming resulted in a substantial loss of pollen viability. This might indicate that intracellular ice crystals formed during rapid cooling perhaps grow into larger ice masses during slow rewarming or storage at temperatures above -50°C. The germinability of pollen freeze-dried at temperatures below -50°C was also prolonged over that of the controls. Germination values for unfrozen pollen stored for 30 days at 05°C averaged 50% for lily and 20% for corn. Freeze-dried pollen stored for 30 days at the same temperature yielded considerably higher viability percentages for both lily and corn pollen. Drying time is an important factor, perhaps indicating that residual moisture is critical. Freeze-
dried pollen can be stored at higher temperatures than frozen and control pollen. Freeze-dried material stored for five months at 05”C, upon slow rehydration yielded intact grains which has average germination percentages of 25 for Iily and 15 for corn. The same pollen upon rapid rehydration showed rupturing of 2O-4O$& of the cells and practically no germination. REFERENCES 1. Ching, T., and Ching, K. Freeze-drying pine pollen. I%nt Physiol. 39, 705-709 ( 1964 ) , 2. Davies, M. D., and Dickinson, D. B. Effects of freeze-drying on permeability and respiration of germinating lily pollen. Pht Phytiol. 24, 5-9 ( 1971). 3. Dickinson, D. B. A photomicrographic study of lily pollen germination. Hurt. Sd. 1, 20 (1966). 4. King, J. R. The freeze-drying of pine pollen. Bull. Tar. Bat. Chb 86, 383-386 ( 1959). 5. King, J. R. The freeze-drying of pollens. &on. Bat. 15, 91-98 (1961). 6. Layne, R. E. C., and Hagedorn, D. J. Effect of vacuum-drying, freeze-drying and storage environment of viability of pea pollen. Crop Zci. 3, 433-436 ( 1963). 7. Litt, M., and Boyd, W. Preservation of haemocyanin. Nature (London) 181, 1075 ( 1958). 8. Pfeiffer, N. E. Effect of lyophilization of viability of La’Eilsmpollen. Contr. Boyce Thompson rn.!it. 18, 153-158 ( 1955). 9. Rosen, W. G. Ultrastructure and physiology of pollen. Ann. Rev. Plant PhysioE. 19, 435462 ( 1968). 10. Sakai, A., and Yoshida, S. SurvivaI of pIant tissues at super Iow temperatures. VI Effects of cooling and rewarming rates, Pht Physlol. 42, 1695-1701 ( 1987). 11. Tervet, I. W., Rawson, A. J., Cherry, E., and Saxon, R. B. A method for the collection of microscopic particles. Phytopathol. 41, 282-285 ( 1951). 12. Visser, T. Germination and storage of poIlen. Meded. Laludbozr. Wagen. 55, 1-68 ( 1955). 13. Wood, G. W., and Barker, W. G. Preservation of blueberry polIen by the freeze-drying process. Canad. I. Hunt Sci. 44, 387-388 (1964).