CRYOPRESERVATION OF CELL CULTURES AND EXPILANTS OF GRASSES
ANTOINE'TTE SHASHIKALA GNANAPRAGASAM
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
DEDICATED TO MY LATE BELOVED FATHER, S.P. GNANAPRAGASAM
I have great pleasure in acknowledging my sincere thanks to Dr. I.K. Vasil for his valuable guidance throughout my research and for his support and the laboratory facilities. I would also like to thank my committee members, Dr. H.C. Aldrich, Dr. D.J. Cantliffe, Dr. R.J. Ferl, and Dr. L.C. Hannah, for their suggestions in shaping up my thesis. To the lab colleagues I owe the friendly atmosphere we shared during the course of my work. In particular I would like to thank Dr. Vimla Vasil, Mr. Luis Pedrosa and Mr. Mark Taylor for their cooperation and help in my laboratory work.
To my family, who have been encouraging and supporting me throughout, I owe a deep sense of love and gratitude. Last, but not the least, my deepest gratitude and thanks go to my loving husband, Shan, for his support and encouragement in helping me complete my research at the earliest.
Finally, I recall with fond memories all the friends in Gainesville for making my stay here a pleasant experience.
TABLE OF CONTENTS
A CK N O W LE D G M E N TS.................................................................................................. iii
A BSTR A CT .......................................................................................................................... vii
1 IN TR O D U CT IO N ............................................................................................ 1
2 LITE R A T U R E R E V IE W ............................................................................... 5
Freeze Injury ................................................................................................. 6
Pregrow th ........................................................................................................ 8
Cryoprotection .............................................................................................. 9
Freezing ....................................................................................................... 12
Storage ......................................................................................................... 14
Thaw ing........................................................................................................ 15
R ecovery ...................................................................................................... 16
D eterm ination of V iability ........................................................................ 17
Cryopreservation of Cell Suspension
C ultures ................................................................................................ 19
Cryopreservation of C allus Cultures ....................................................... 21
Cryopreservation of Protoplasts ............................................................... 23
Cryopreservation of Shoot-Tips ............................................................... 24
3 ULTRASTRUCTURAL STUDIES USING A CELL SUSPENSION
OF PANICUM MAXIMUM TO DETERMINE THE CHANGES
THAT OCCUR DURING THE PROCESS OF
M aterials and M ethods .............................................................................. 29
R esults.......................................................................................................... 32
D iscussion ....................................................................................................75
4 OPTIMIZATION OF THE CRYOPRESERVATION
PROCEDURE FOR STORAGE OF CELL SUSPENSIONS
M aterials and M ethods..............................................................................81
R esults.......................................................................................................... 88
D iscussion .................................................................................................. 111
5 CRYOPRESERVATION OF AN EMBRYOGENIC CALLUS
CULTURE AND A CELL SUSPENSION CULTURE OF A
COMMERCIAL SACCHARUM HYBRID
M aterials and M ethods............................................................................119
D iscussion .................................................................................................. 142
6 PLANT REGENERATION FROM CRYOPRESERVED
IMMATURE EMBRYOS OF WHEAT (TRITICUMAESTIVUM L.) Introduction ............................................................................................... 147
M aterials and M ethods ............................................................................ 147
D iscussion ..................................................................................................163
7 SUMMARY AND CONCLUSIONS........................................................168
REFERENCES ............................................................................................. 171
BIOGRAPHICAL SKETCH ...................................................................... 188
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
CRYOPRESERVATION OF CELL CULTURES AND EXPLANTS OF GRASSES
ANTOINETTE SHASHIKALA GNANAPRAGASAM August 1991
Chairman: Indra K Vasil
Major Department: Botany
Methods were developed for the successful cryopreservation of whole immature embryos, embryogenic callus and cell suspension cultures of several gramineous species. The application of a pregrowth treatment, the use of cryoprotectants and the cooling rate of the tissue were all essential to success, but different cultures showed different requirements that had to be optimized for each.
A combination of cryoprotectants was needed for the cryopreservation of cell suspensions, but using a single cryoprotectant was best for the cryopreservation of callus and immature embryos. A pregrowth period in a liquid culture medium supplemented with an osmoticum was essential for recovery of callus cultures. Slow cooling was suitable for all types of cultures. Rapid cooling by direct immersion in liquid nitrogen was always lethal. Washing was invariably detrimental, and for callus cultures it was essential to plate the tissues with the cryoprotectants.
Plants were regenerated from a cryopreserved embryogenic cell suspension and a callus culture of a sugarcane commercial hybrid and from immature embryos of wheat. All were grown to maturity in the greenhouse and found to be morphologically similar to control plants.
A Panicum maximum cell suspension was used to study ultrastructural changes during the process of cryopreservation using light and electron microscopy.
Growing the cells in high osmoticum caused a reduction in cell size and vacuolar volume. Dilation of the ER and mitochondria was also observed at different stages of the cryopreservation procedure. Extensive damage was observed in cells that were frozen slowly without any cryoprotection or frozen by direct immersion after treatment with cryoprotectants. Osmiophilic granules were abundant in lethally damaged cells.
Cell cultures have to be routinely maintained for extended periods of time by periodic subculture, a time consuming and labor intensive process which can result in loss of the cultures due to microbial contamination, equipment failure or human error. Long term maintenance by subculture can also result in mutations including changes in the structure and number of chromosomes (Heinz and Mee 1971; Sheridan 1975; Sunderland 1977; Bayliss 1980; D'Amato 1985), and the consequent loss of morphogenetic potential (Murashige and Nakano 1967; Torrey 1967; Nag and Johri 1969; Meyer-Teuter and Reinert 1973; Smith and Street 1974; Reinert et al. 1977).
Crop improvement by conventional selection and breeding depends on reliable germplasm storage of the source material and improved genotypes (Withers 1988). For species that produce orthodox seeds, germplasm storage is achieved by gene-banks mostly composed of seed stores held at reduced temperature and humidity. Maintaining them in storage without physiological deterioration is an important but difficult task (Stanwood 1985). This method is not useful for species with recalcitrant seeds (coffee, coconut, oil palm, cocoa, rubber, mango, walnut etc.), which lack a dormancy period and are therefore incapable of surviving low temperatures and dehydration, and also for vegetatively propagated crops (Withers
1986). Such materials are normally stored in field gene-banks which are expensive to maintain and susceptible to environmental and pathogenic risks (Withers 1988).
Isolated shoot-tips (meristems) are often used for clonal propagation and production of virus free plants (Kartha and Gamborg 1975; Quak 1977; Roca et al. 1982; Kartha 1985a; Sakai 1985). Such cultures, and plants derived from them, cannot be maintained in a disease free and genetically stable condition for extended periods of time.
In vitro cultures are being increasingly used for the synthesis of secondary products which are of pharmaceutical and nutritional importance (Zenk 1978; Kurz and Constabel 1979; Staba 1980; Deus and Zenk 1982; Fowler 1986; Constabel and Vasil 1988). Therefore, periodic reselection or even the establishment of new cultures becomes necessary. The tendency of such cultures to lose the capacity to synthesize secondary products (Dhoot and Henshaw 1977) can result in the loss of valuable cell lines.
Haploid plants are important in the induction of mutations, identification of useful recessive genes, production of isogenic lines, and biochemical/genetic studies (Han 1983). They can be obtained from in vitro culture of microspores/anthers. They are often highly unstable, and rapidly become aneuploid and polyploid.
An in vitro method which could store material in a stable state for indefinite periods of time, in addition to being applicable to all types of cultures, would be ideal for overcoming most of the problems mentioned above. Two methods have been used for the storage of plant cell and tissue cultures: (1) reduction in the rate of growth (slow growth), and (2) suspension of growth.
Slow growth can be achieved by a reduction in incubation temperature or available oxygen, or by the application of osmotic inhibitors or hormonal retardants (Bridgen and Staby 1981; Withers 1985a, 1986). The most widely used method of slow growth is the reduction in culture temperature (Bannier and Steponkus 1972;
Meyer-Teuter and Reinert 1973; Hiraoka and Kodama 1982, 1984; Jacques et al. 1982). Plant cultures are normally grown at temperatures of 20 to 250C, and lowering the temperature by 10-150C results in a marked decrease in the rate of growth (Withers 1985a).
The mineral oil overlay technique, which reduces growth rates by limiting the availability of oxygen, is commonly used for storage of microbial cultures, and can be used to a limited extent for higher plant cell cultures (Caplin 1959; Augereau et al. 1986). Subculture intervals can be extended from weeks to months by a decrease in the growth rate by at least a factor of four (Dougall 1980). Osmotic (mannitol) or hormonal (ABA) inhibitors can be added to the culture medium to retard growth, as an alternative or in combination with reduced growth temperatures (Withers 1985a). Slow growth can be used for short to medium term storage, but long term storage requires periodic renewal, at least every one to two years. Therefore the problems encountered in maintaining the cell cultures by repeated subculture are not eliminated by growth limitation, but rather extended over a longer period of time (Withers 1985a). In addition, there is increased risk of selecting new variant cell lines, due to the stress imposed by the condition of culture (Withers 1984a). Slow growth has been mostly successful for storage of shoot cultures (Mullin and Schlegel 1976; Lundergan and Janick 1979; Dale 1980). It has shown only limited success with callus cultures (Bannier and Steponkus 1972, 1976; Hiraoka and Kodama 1984), and cannot be applied to cell suspension cultures (Rose and Martin 1975).
For complete avoidance of subculturing, the cultures have to be stored under conditions which involve the suspension of all metabolism (Withers 1983). This can be achieved by either freeze-drying or cryopreservation. Freeze drying, which is extensively used for conservation of microorganisms (Grout et al. 1990), is not adaptable to higher plant tissue cultures (Withers 1983). Furthermore, mutation
events associated with freeze drying have been documented (Ashwood-Smith and Grant 1976; Tanaka et al. 1979).
Cryopreservation, which eliminates all requirements for periodic transfer or viability testing by storing material at liquid nitrogen temperatures (liquid at -1960C and vapor at -150'C), can in theory store material for indefinite periods of time. Unlike slow growth, it is applicable to all types of cultures from protoplasts to plantlets, but unorganized cultures generally fare better than organized material (Withers 1987). At liquid nitrogen temperatures the kinetic energy levels are too low to allow the necessary molecular motions for the normal cellular chemical reactions to occur (Grout et al. 1990). Changes such as formation of free radicals and macromolecular damage due to ionizing radiation can still occur at liquid nitrogen temperatures (Grout et al. 1990), but the risk of occurrence of genetic instability in the cultures is negligible (Withers 1985a).
Embryogenic cell suspension cultures have proven very useful for the isolation of protoplasts and genetic transformation studies (Vasil and Vasil 1979, 1980; Vasil et al. 1988, 1990; Potrykus 1990). They are normally obtained from callus cultures initiated from leaves, immature embryos and inflorescences. These callus and suspension cultures are routinely maintained by subculturing to fresh medium every month and every week, respectively. As formerly discussed, this is a time consuming and labor intensive process and can lead to genetic variation. The explants used to initiate the callus are not available throughout the year. It would thus be very useful if explants used to initiate the callus as well as the cell lines derived from them could be stored by cryopreservation, to be used whenever the need arises, and to serve as a permanent source of unique cells.
The objectives of this project are to study the factors involved in the cryopreservation of immature embryos, callus and cell suspension cultures of graminaceous species, and to regenerate plants from the cryopreserved tissues.
Experimental work on freezing of biological material has been recorded from the mid 19th century. Significant progress, however, has been made only after the middle of this century (Withers and Street 1977a; Meryman and Williams 1985). Cryopreservation has proven to be a valuable tool for the storage of valuable genomes in microbiology, medicine and animal husbandry (Meryman 1966; Ashwood-Smith and Farrant 1980). Cryopreservation of plant germplasm is a more recent development that emerged both from studies of cold hardiness and freezing injury in plants (Levitt 1966; Li and Sakai 1978, 1982), and from the cryopreservation of animal cells and microbes.
Successful freeze preservation of plant cell cultures was first reported by Quatrano in 1968. A cell suspension of Linum usitatissimum L. (flax), stored at
-500C for up to one month using 10% (v/v) DMSO (dimethyl sulfoxide), retained a viability of only 14%, because the temperature employed was not low enough for stable long term storage.
True cryopreservation of in vitro cultures of higher plants was first reported in 1973 by Nag and Street, and Sakai and Sugawara, in two independent studies. Suspension cultures of carrot (Daucus carota) were frozen to liquid nitrogen temperature using 5% DMSO as a cryoprotectant, and survival rates of up to 68% (based on fluorescein diacetate test) were obtained. No decline in survival was observed even after 100 days of storage in liquid nitrogen, and when transferred to
an appropriate medium, healthy plants of normal morphology were recovered (Nag and Street 1973). Callus cultures of Populus euramericana cv. gelrica, cold acclimated prior to freezing, survived liquid nitrogen temperatures in the absence of cryoprotectants (Sakai and Sugawara 1973).
Different hypotheses have been put forward to help explain the mechanisms of cell injury during freezing. The two-factor hypothesis of Mazur et al. (1972) states that cell injury results from either the concentration of solution by extracellular ice, producing "solution effects" (i.e. solute concentration, changes in pH, and reduction in cell volume), or by formation of intracellular ice which causes mechanical injury to the plasmalemma and organelle membranes (Lovelock 1953; Lusena 1965; Mazur 1966; Sukumaran and Weiser 1972; Steponkus and Wiest 1978; Steponkus et al. 1982a; Hofmo and Berg 1989). It has also been suggested that alterations in membrane properties, rather than their mechanical breakdown, cause freezing injury (Heber 1967, 1968; Palta et al. 1977a,b; Palta and Li 1980). Palta et al. (1982) suggested that membrane proteins are the sites of membrane alterations while membrane lipids remain unaltered in spite of freezing injury.
According to the "minimum volume hypothesis," for every cell there is a minimal volume beyond which it cannot be reduced without injury (Meryman 1974). Any further volume decrease will cause stress in the membranes resulting in increased permeability, loss of membrane material, or irreversible mechanical disintegration (Meryman and William 1985).
Freeze damage can be prevented in tissues either by "freeze hardening," or by treatment with cryoprotective additives. The two methods have many aspects in common. Both involve a decrease in cellular water content and an increase in
specific solutes (Finkle et al. 1985b; Delvallee et al. 1989). Freeze hardened cells are no more resistant to osmotic dehydration than freeze sensitive ones, but they have evolved mechanisms to cool to lower temperatures before achieving the same degree of cell dehydration (Meryman et al. 1977; Steponkus and Wiest 1978; Meryman and William 1985). This is done in a variety of ways, including synthesis of additional intracellular solutes that increase the nonaqueous volume of the cell, "binding" of water, mechanical resistance to plasmolysis, and membrane leak (Williams and Meryman 1970; Williams and Williams 1976; Meryman et al. 1977).
Studies with cold-hardened and non-hardened rye mesophyll cells indicated that during extracellular freezing to lethal temperatures, membranes of nonhardened cells "roll up" and fuse to form multilayered vesicles, and eventually lose their phospholipid lamellar lattice altogether. No fusion, membrane roll up or loss of lamellar lattice was observed in cold-hardened cells (Singh and Miller 1985). Studies using protoplasts isolated from non acclimated and acclimated rye (Secale cereale L. cv Puma) leaves showed formation of endocytotic vesicles and exocytotic extrusions during freezing, respectively. During osmotic expansion following thawing the endocytotic vesicles remained in the cell and the protoplasts lysed before reaching their original volume (Gordon-Kamm and Steponkus 1984a), a phenomenon referred to as "expansion induced lysis" (Steponkus and Wiest 1978, 1979; Steponkus 1985a), whereas the exocytotic extrusions were drawn back into the surface of the protoplast thereby preventing lysis (Gordon-Kamm and Steponkus 1984b). In acclimated tissue freeze injury occurs at a lower temperature, predominantly by the complete loss of osmotic responsiveness following cooling (Steponkus et al. 1982a). These findings have been confirmed by direct cryomicroscopic observations (Dowgert and Steponkus 1984; Steponkus and Lynch 1989), and micro-osmotic manipulation (Dowgert et al. 1987)
According to Siminovitch and Levitt (1941), the plasma membrane of plants that survive very cold conditions is more resistant to damage by dehydration and mechanical breakdown. These differences are due to modifications in membrane composition, including substantial changes in the lipid composition of the plasma membranes during cold-hardening (Lynch and Steponkus 1987; Steponkus and Lynch 1989).
Freeze damage can also be reduced by the use of cryoprotective compounds which protect the cell from freeze injury by decreasing the freezing point of the cytoplasm, by reducing the size and growth rate of ice crystals, by protecting the cells against high intra- and extra-cellular solute levels and by stabilizing the membrane components (Withers 1980).
Cryopreservation of plant cell cultures includes a number of steps:
1. Pregrowing the cells in a medium supplemented with cryoprotectants or
osmotic compounds (when appropriate).
2. Addition of cryoprotective compounds prior to freezing.
3. Freezing to ultra-low temperatures.
4. Storage of frozen cultures at liquid nitrogen temperatures.
5. Thawing of frozen cells.
6. Determination of viability.
7. Reculture on appropriate medium.
8. Induction of growth and regeneration of plants.
Freeze tolerance can be increased prior to freezing either by cold-hardening (Sakai and Sugawara 1973; Seibert and Wetherbee 1977; Chen and Gusta 1982; Delvallee et al. 1989) or by growing the cells in a culture medium supplemented
with osmotic additives such as mannitol (Withers and Street 1977a,b; Pritchard et al. 1986), sorbitol (Chen et al. 1984a,b; Pritchard et al. 1986; Kartha et al. 1988), trehalose (Bhandal et al. 1985), various sugars (Latta 1971; Bannier and Steponkus 1972; Finkle et al. 1985b), proline (Withers and King 1979a,b) or cryoprotective compounds (Kartha et al. 1980, 1982). Osmotically active compounds probably act by inhibiting cell expansion, resulting in reduction in cell size, vacuolar volume and consequently water content (Withers and Street 1977b; Withers 1985b; Pritchard et al. 1986). Sixty percent reduction in vacuolar volume was observed when sycamore cells were grown in the presence of 6% mannitol for seven days (Pritchard et al. 1982).
Cryoprotective compounds can be of two types. Small molecular weight compounds such as DMSO, glycerol, methanol and acetamide penetrate the cells and act by colligative action causing a freezing point depression. Therefore, at a given temperature there is an increased volume of liquid available as solvent to minimize the deleterious action caused by electrolyte concentration due to dehydration during freezing (Finkle et al. 1985b).
Large molecular weight compounds such as polyvinylpyrrolidone (PVP), polyglycol, dextran, lactose and sucrose (Rowe 1966) do not penetrate the cell but act by osmotic action (Sakai 1962; Mazur 1970; Finkle et al. 1985b). Excess water is removed from the cell during the initial phases of cooling, particularly between
-10ï¿½C and -20 oC (McGann 1978). If dehydration is carried out too far, accumulation of excess amounts of damaging solutes in the cell can cause "solution effects."
Disulfide bond formation, one of the reasons for frost injury (Levitt 1962), can also be prevented by cryoprotectants which inhibit the formation of these S-S bonds (Andrews and Levitt 1967). Cryoprotectants also interact with the cell membrane, either directly or indirectly, and stabilize its water-lipid-protein complex tertiary structure (Rowe 1966).
Most commonly used cryoprotectants are DMSO and glycerol. The successful use of DMSO as a cryoprotectant for animal cells was first reported by Lovelock and Bishop in 1959, but its use in the freezing of plant tissue was not reported until 1968 (Quatrano 1968). DMSO is an efficient cryoprotectant because of its low molecular weight, easy miscibility with water, lack of toxicity at low concentrations, rapid penetration and easy removal from the cells (Bajaj and Reinert 1977). Farrant (1972) reported that DMSO prevents cell damage during freezing by postponing both the shrinkage of the cell and the onset of cation leaks to higher osmolalities.
The optimal concentration of DMSO is found to be between 5-10% (Latta 1971; Nag and Street 1973, 1975a; Dougall and Wetherell 1974; Seibert and Wetherbee 1977; Sakai and Sugawara 1978; Sala et al. 1979). At high concentrations it is known to exert toxic effects by interfering with membrane permeability, and RNA and protein synthesis (Bajaj et al. 1970; Morris 1976; Barnett 1978; Zavala and Finkle 1980, 1981; Kartha et al. 1982; Finkle et al. 1985b). Concentrations ranging from 5% to 20% were found to exert the same level of toxicity on cells of Picea glauca, but towards the end of the regrowth period the cells recovered and a growth rate of 80-90% of untreated control was attained irrespective of the concentration used (Kartha et al. 1988).
Exposing cells to DMSO for long to medium term may also be damaging to the cells (Dougall and Wetherell 1974; Nag and Street 1975a; Morris 1980; Kartha et al. 1982). There is some indication that in higher plants DMSO protects cells
against genetic damage from radiation occurring at ultra-low temperatures (Ashwood-Smith 1967; Kaul 1970; Ashwood-Smith and Friedmann 1979; Finkle et al. 1985b; Withers 1988).
First successful use of glycerol was reported by Polge et al. in 1949. It has been used extensively for the cryopreservation of microbial and animal cells. Glycerol is not very effective in preventing freeze damage when used alone. Reduced uptake at the temperature of application could even cause injury by excessive plasmolyzation (Latta 1971; Nag and Street 1975a; Towill and Mazur 1976; Withers 1985b). It acts either as a penetrating compound or a nonpenetrating compound depending on the temperature of application (James 1983). The optimal concentration of glycerol when used alone is 5-10% (Latta 1971; Nag and Street 1975a,b).
Using a mixture of cryoprotectants at low concentrations is better than using a single cryoprotectant at higher concentrations. An additive cryoprotective effect of the combined compounds is obtained while the concentration of toxic compounds is reduced (Finkle and Ulrich 1978, 1979; Hauptmann and Widholm 1982; Withers 1982, 1985b; Bajaj 1983; Chen et al. 1984a,b; Finkle et al. 1985b; Kartha et al. 1988). Combinations of cryoprotectants are used at a total concentration of 0.5-2 M (Withers 1980). Cryoprotectants are applied to cells during the period immediately preceding cooling. They are normally prepared in culture medium except in a few instances where they were dissolved in water with or without sugar (Uemura and Sakai 1980; Watanabe et al. 1983), and the pH is adjusted to the standard pH of the medium used. Because of the high concentrations of solutes, such mixtures are prone to caramelization if autoclaved, hence they should be sterilized by filtration (Withers 1986).
Cold application (0OC) of cryoprotectants is beneficial over application at room temperature. Toxic effects of the cryoprotectants are probably reduced due to
slower metabolic activities of the cells at lower temperatures (Finkle and Ulrich 1982). Sudden mixing of the cryoprotectant with the specimen may lead to damage by plasmolysis, therefore the cryoprotectants should be added to the culture gradually over a period of time (Nag and Street 1975a; Towill and Mazur 1976; Withers and Street 1977a,b; Bajaj 1979a). Cryoprotectants are added at a reduced temperature (0-4oC), since an exposure to the cryoprotectants at room temperature has adverse effects on the tissue.
Cells can be frozen by slow freezing, stepwise freezing, and rapid freezing. Freezing as previously mentioned can cause injury due to "solution effects" caused by extracellular freezing, or mechanical damage caused by intracellular freezing (Mazur et al. 1972). Intracellular freezing is lethal and to prevent this cooling should be such that all freezable water flows out of the cell before intracellular freezing ensues, and yet rapid enough to prevent solution effects (Mazur 1970; Bajaj 1979a). Slow cooling is the most widely used procedure especially for cryopreservation of cell suspensions and protoplasts (Withers and Street 1977a; Chen et al. 1984b). However, too slow a rate of cooling can be damaging due to excessive dehydration which results in solution effects (Mazur 1969, 1970; Meryman et al. 1977; Withers 1984a). Cooling rates of 0.5ï¿½-4ï¿½C/min give best results (Withers and Street 1977a), with the optimum at around 20C/min (Dougall and Wetherall 1974; Henshaw 1975; Nag and Street 1975b; Bajaj 1976a,b).
Stepwise freezing is achieved by exposing the specimens to one or more intermediate temperatures (Sugawara and Sakai 1974; Sakai and Sugawara 1978). At -300C all freezable water is removed from the cell due to extracellular freezing, and they are not injured by exposure to extremely low temperatures (Sakai 1960).
This temperature may vary with the degree of frost-hardiness (Sakai 1965). A combination of slow and stepwise freezing, in which the specimen is cooled at a slow rate to ultra-low holding temperatures, and held for up to one hour at this temperature before plunging into liquid nitrogen, has been found to be very useful (Farrant et al. 1977; Withers 1985b). Heszky et al.(1990) reported that cell survival could be considerably improved by holding suspension cultures of Puccinellia distans
(L.) Parl at sub-zero temperatures for 10-30 minutes before plunging into liquid nitrogen. Cooling to too low or too high a transfer temperature or prolonged exposure to suboptimal temperatures before transfer to liquid nitrogen, may be injurious due to excessive cellular dehydration and intracellular freezing (Withers 1985c; Kartha et al. 1988).
Rapid freezing at rates up to > 1000ï¿½C/min is achieved by plunging the specimen into liquid nitrogen directly. This is not recommended for cell suspensions (Bajaj 1976a; Kartha 1985b), but has been successful for organized structures (Seibert 1976). Even though intracellular ice crystals are formed, they do not have enough time to grow to damaging sizes because of the high rates of cooling (Bhojwani and Razdan 1983; Withers 1984a). Also, during rapid thawing they melt before attaining damaging sizes (Sakai and Sugawara 1973; Sugawara and Sakai 1974). Harmful effects of ice crystals are determined by the amount, crystal size and location of the ice (Withers 1987). Farrant et al. (1977) reported that it is the amount rather than the size of intracellular ice crystals that determines survival during thawing. Rapid freezing is the method of choice for pollen and meristems which have very low water content and are adversely affected by solution effects during slow freezing (Nath and Anderson 1975; Withers 1978a). Withers (1979) used a dry freezing method for cryopreservation of clonal plantlets of Daucus carota, where the specimens were blotted dry and enclosed in foil envelopes.
Presterilized, polypropylene screw cap ampoules of different capacity are used for freezing of the samples. Flame sealed glass ampoules are not recommended because incomplete sealing could result in liquid nitrogen penetration during storage and explosion upon thawing (Withers 1986).
The storage temperature should be low enough to stop all metabolic activity and prevent biochemical injury. Storage at higher temperatures causes structural damage to cells as a result of progressive recrystallization of ice, resulting from the migration of water in the solid state (Withers 1985b). Temperatures in the range of
-4oC to -700C are not recommended for long term storage, since deterioration of the cells is observed at such temperatures (Bajaj 1983; Withers 1984a). At -200C protein denaturation takes place because of metabolic activity, leading to death of cells. In addition, changes in concentration of solutes and subsequent variation in pH also take place at these temperatures (Bajaj 1979b).
For short to medium term storage, a temperature of -80aC may be adequate. Although intracellular ice is maintained at this temperature, structural damage and consequent progressive deterioration in viability occurs due to recrystallization of ice (Nag and Street 1973; Nei 1973; Withers 1980, 1986). Recrystallization of ice cannot be prevented when storage temperatures above -1300C are used (Henshaw 1975; Bhojwani and Razdan 1983), hence for long term storage, temperatures of liquid nitrogen are recommended. Liquid nitrogen and suitable storage containers are available commercially, and a container holding about 4000, 2 ml capacity ampoules normally consumes 20 - 25 1 of liquid nitrogen per week (Withers and Street 1977a).
Even though metabolic activity is completely stopped at liquid nitrogen temperatures, molecular changes due to ionizing effect of radiation may lead to cumulative damage (Withers 1987), but this may not pose a major problem at least for decades of storage (Whittinghamrn et al. 1977).
Thawing can be achieved either rapidly at rates of 500-7500C/min by plunging the frozen sample into water at 37-400C (Chen et al. 1984b), or slowly by exposing the vials containing the specimens to blown warm air, air at room temperature, or to liquid nitrogen vapor followed by air at room temperature (Sakai and Sugawara 1973; Dougall and Wetherell 1974; Withers 1978b, 1979, 1980, 1983). Contents of the ampoule should be thoroughly mixed to prevent local heating while thawing proceeds and the ampoule should be transferred to an ice bath as soon as the ice plug in each ampoule disappears, which could be anywhere from 60-90 seconds when thawed rapidly, and would take up to 20 minutes when thawed slowly (Withers 1980).
Recrystallization of ice crystals occurs during slow thawing, particularly at temperatures above -45ï¿½C, resulting in the formation of progressively larger ice masses which cause damage to cell membranes and cytoplasmic organelles. This zone of recrystallization is passed so rapidly during rapid thawing that the ice crystals melt before they have the opportunity to recrystallize (Sakai and Otsuka 1967; Sakai and Yoshida 1967; Bajaj and Reinert 1977).
Rapid thawing is more advantageous than slow thawing only at higher transfer temperatures. When specimens are cooled to temperatures beyond a transfer temperature of -40ï¿½C, the thawing rates do not have any effect on recovery
rates (Withers 1985b), probably because of the reduction of water content of the cell to an optimal level (Bhojwani and Razdan 1983).
Thawed cultures can be recovered by three methods:
1. Removing the cryoprotectants by washing before returning to culture.
2. Diluting the cryoprotectants out by using liquid medium.
3. Inoculating into semi-solid medium directly without washing.
Post-thaw washing was routinely used in early studies to avoid deleterious effects of cryoprotectants (Latta 1971; Nag and Street 1973; Dougall and Wetherell 1974; Withers and Street 1977b; Sala et al. 1979; Kartha et al. 1982). Stepwise dilution may be advantageous for avoidance of deplasmolysis injury (Towill and Mazur 1976; Withers and Street 1977a). The temperature of the washing solution is important in the survival of the specimen, and 0OC is generally employed (Kartha et al. 1982). Sugarcane and rice cultures showed a higher viability when washed at room temperature than at 00C, probably due to the differences in membrane fluidity at these temperatures (Finkle and Ulrich 1982). Recent studies have shown that washing may be more deleterious than prolonged exposure (Withers and King 1979b; Chen et al. 1984b). Freezing and storage at low temperatures could result in loss of water, ions, sugars and amino acids due to increased membrane permeability (Siminovitch et al. 1964; Lyons 1973; Palta et al. 1977a,c). Washing or diluting with liquid medium would result in removal of these compounds which may be required for the recovery of the thawed cells (Withers 1979; Withers and King 1979b). Washing or diluting may also result in deplasmolysis injury resulting from the loss of membrane material during freeze dehydration (Wiest and Steponkus 1978; Withers and King 1979b; Withers 1985c). Supplementing the washing medium with an
osmoticum such as mannitol or sorbitol, and removing the cryoprotectants by stepwise dilution have been employed to minimize deplasmolysis injury (Towill and Mazur 1976; Withers and Street 1977a; Maddox et al. 1983).
Placing the cells without washing on semi-solid medium similar to the composition of the liquid medium, has been found to be better than washing or diluting with the culture medium, by allowing the toxic cryoprotective compounds to gradually diffuse out (Withers and King 1980; Finkle et al. 1985b).
Frozen and thawed cells, when returned to culture, may start to grow within a day or two (Sala et al. 1979), or enter a prolonged lag phase of even up to 1-6 months (Nag and Street 1973; Bajaj 1976a). This could be due either to the revival and repair that take place in the cold shock and partially damaged cells or due to the suppression of growth by the cryoprotectants (Bajaj and Reinert 1977). Cella et al. (1982) reported that freeze-thawed cells of rice showed a number of physiological alterations, including reduction in respiration and glucose uptake, loss of intracellular ions, decrease in cellular levels of key metabolites and fragility of the protoplast, all of which were repaired during a lag period of 2-4 days. Cells may fail totally to enter growth if the viable cell density falls below the minimum inoculum level (Stuart and Street 1969, 1971; Maddox et al. 1983).
Determination of viability
Viabilities of cryopreserved and thawed cells can be determined by various means. For a reliable estimation of viability, growth parameters such as mitotic index, cell number, packed cell volume, increase in fresh and dry weights and plating efficiencies should be employed. For rapid processing, and to screen large numbers of specimens, growth parameters cannot be used. In such instances, staining
reactions are employed for the determination of viability, especially for cell suspensions.
TI'C (2,3,5-triphenyl tetrazolium chloride) test involves the reduction of tetrazolium salts by mitochondrial activity, to a water insoluble red compound, formazan. Formazan can be extracted in ethanol and examined spectrophotometrically (Steponkus and Lanphear 1967; Towill and Mazur 1975). The amount of formazan produced is proportional to the number of viable cells present, and can be used to estimate viability of cells (Towill and Mazur 1975).
Flourescein diacetate test, developed by Widholm (1972), is based on the ability of viable cells to uptake and break down flourescein diacetate by esterase activity. Only the viable cells can be visualized under UV light by their fluorescence (Withers 1984b). Cells that emit intermediate levels of florescence and cell aggregates where the central cells cannot be clearly seen are complicating factors for this method (Withers 1980).
Evans blue staining is based on the ability of the viable cells to exclude a dye (Gaff and Okong'O-Ogola 1971). Cells are treated with a dye and observed after a few minutes. The viable cells remain colorless and the dead cells show blue coloration. The same principle applies for phenosafranine staining which is also used for the estimation of viability of cell suspensions (Widholm 1972). The drawback of these methods is that if left too long in the stain all the cells become colored (Bajaj 1979a).
Staining reactions should not be used as the sole means of estimation of viability, since there may be cells present which show positive reaction immediately after thawing but later die in culture, or cells in a state of cold shock giving a negative reaction but later reviving in culture (Bajaj 1976a, 1979a).
Cryopreservation of Cell Suspension Cultures
Cell suspension cultures are widely used in physiological studies and for plant regeneration, protoplast isolation, genetic transformation, and secondary product synthesis. Cryopreservation has been used successfully for the storage of cell suspensions which cannot be achieved by slow growth (Rose and Martin 1975).
Age, nature and the physiological state of cells considerably influence their survival during cryopreservation (Bajaj 1979a, 1983). Generally, with cell suspensions, small non-vacuolated thin walled cells survive better than larger highly vacuolated thick walled cells (Nag and Street 1975a; Bajaj 1976a; Withers and Street 1977b).
Cells in the late lag or early exponential phase are more resistant to freeze damage (Sugawara and Sakai 1974; Withers and Street 1977b; Sakai and Sugawara 1978). A decrease in viability is generally observed in stationary phase cells (Sala et al. 1979), probably due to the higher vacuolation and water content associated with increased cell size and consequently a smaller surface area to volume ratio, which impede protective dehydration (Withers and Street 1977b; Withers 1978c). Lag and stationary phases can be effectively eliminated by rapid subculture, which also causes a reduction in cell size (Withers and Street 1977a; Withers 1978c). Mitotic stage also influences the survival of suspension cultures (Withers 1985b). Experiments on synchronized cultures of Acer pseudoplatanus by nitrate starvation revealed that cells in G1 or GO phase were more tolerant to freeze damage, while the highest viability loss was observed during cytokinensis or S phase (Withers 1978c). Synchronization and selection of suitable cell stage is not recommended in cryopreservation of cell suspensions, because of the relatively brief periods of freeze tolerance and the technical demands involved.
Filtered cell suspensions consisting of free cells showed poor survival compared to actively growing suspensions consisting of highly cytoplasmic cells in
small colonies (Bajaj 1976a). Survival is also reduced in large aggregates where the proportion of highly meristematic, rapidly dividing cells is reduced (Withers 1983). The location of the cells in an aggregate also determines their survival, the central cells being more susceptible due to differences in freezing rates, and the degree of protective dehydration (Withers and Street 1977a; Withers 1978b; Withers 1983).
Cell suspensions are commonly grown for a minimum of 3-4 days in a medium supplemented with osmotic additives to increase freeze tolerance prior to freezing (Withers 1983, 1984b, 1985b). Mannitol is the most widely used pregrowth additive for cryopreservation of cell suspensions, and concentrations up to 6% (v/v) have been successfully used (Withers 1985b). Mannitol did not enhance the survival of Nicotiana sylvestris cells whereas the closely related compound sorbitol increased their survival considerably (Maddox et al. 1983). Pregrowing the cells in the presence of cryoprotective additives such as DMSO is found to be of little value for cryopreservation of cell suspensions (Withers 1983).
For cryopreservation of cell suspensions, the cryoprotectants are prepared in double strength and normally added gradually to an equal volume of cell suspension at low temperatures. The cell suspension is often concentrated before cryoprotectant addition to provide sufficient cell density during recovery (Stuart and Street 1969; Sala et al. 1979). Mixtures of cryoprotectants are generally more successful than single compounds. Mixtures of 0.5 M DMSO, 0.5 M glycerol and 1 M sucrose or proline adapted by Withers and King (1980), sorbitol and DMSO by Chen et al. (1984a,b), or glycerol and DMSO (Nag and Street 1975a,b; Hauptmann and Widholm 1982; Maddox et al. 1983), are some of the combinations commonly used.
Slow freezing to sub-zero temperatures and then immersion in liquid nitrogen is the most widely used mode of freezing for cryopreservation of cell suspensions. Rapid freezing is not recommended. Rapid thawing is found to be
more beneficial than slow thawing (Sugawara and Sakai 1974; Nag and Street 1975b; Seibert and Wetherbee 1977; Withers and Street 1977a; Withers 1978b; Withers and King 1980; Kartha et al. 1982). Dougall and Wetherall (1974) reported that slow thawing could be used successfully for cell suspensions of carrot, but the viability was never more than that with rapid thawing.
Staining reactions are commonly employed for a quick estimation of viability.
-TC test is found to be reliable for cell suspensions (Sugawara and Sakai 1974; Towill and Mazur 1976; Finkle and Ulrich 1979), although there are reports in which TTC test results could not be faithfully correlated with estimates obtained from other tests (Bajaj and Reinert 1977; Withers 1980; Sala et al. 1979).
Flourescein diacetate test, another commonly used staining reaction for cryopreserved cell suspensions, has been found to be reliable in some studies (Nag and Street 1975a; Withers and Street 1977a). However, unreliability by overestimation (Pritchard et al. 1986) as well as under-estimation (Withers and Street 1977b) have been documented.
Cell are returned to semi-solid medium rather than liquid medium for rapid recovery (Withers and King 1979b). Washing is not recommended and the potentially damaging cryoprotectants can be gradually diluted out by plating the cells on a filter paper layered on a semi-solid culture medium, and transferring to fresh medium after a period (Chen et al. 1984b).
Cryopreservation of Callus Cultures
Short term storage of callus cultures by slow growth has been attempted by both reduction in temperature (Bannier and Steponkus 1972, 1976; Hiraoka and Kodama 1982, 1984), and reducing the availability of oxygen by mineral oil overlay (Caplin 1959; Bridgen and Staby 1981; Augereau et al. 1986). In some strains, the
morphogenetic capacity and secondary metabolite production were retained during storage by slow growth, whereas in others they were lost (Hiraoka and Kodama 1982, 1984).
Cryopreservation of callus cultures is not as widely explored as cryopreservation of cell suspensions, and only limited success has been reported. Callus cultures have the same requirements for cryopreservation as cell suspensions, but they are less amenable to freezing, because protective dehydration and cryoprotectant penetration are impaired by the size and heterogeneity of callus tissue (Withers 1980). Fractionating the callus into small pieces before freezing has been employed in some cases to avoid this problem (Watanabe et al. 1983).
Unlike suspension cultures, callus cultures are generally not pregrown on medium supplemented with cryoprotective additives or high osmoticum, because of their growth habit (Withers 1986). Pregrowth treatment involving cold acclimation is also not commonly employed, but has been found to be useful for cryopreservation of callus cultures of Populus euramericana cv. gerica in the absence of cryoprotectants (Sakai and Sugawara 1973).
Combinations of cryoprotectants such as 10% polyethylene glycol, 8% glucose and 10% DMSO (Ulrich et al. 1979, 1984a,b; Finkle et al. 1980, 1983, 1985a,b; Tisserat et al. 1981; Finkle and Ulrich 1982), 0.5 M sorbitol and 5% DMSO (Chen et al. 1985), 0.5 M DMSO, and 0.5 M glycerol with 1 M sucrose (Hahne and Lorz 1987) have been successfully used.
Slow freezing at rates from 0.5 - 3ï¿½C/min to sub-zero temperatures, followed by storage in liquid nitrogen, rapid thawing and reculture with or without washing is the routine method used (Ulrich et al. 1979, 1984b,c; Chen et al. 1985). Rapid cooling by direct immersion into liquid nitrogen was reported to be deleterious (Ulrich et al. 1979; Hahne and Lorz 1987). Blotting dry the specimen before freezing and omitting the washing after thawing improved the survival rates
considerably (Hahne and Lorz 1987). Viability is determined by regrowth potential. Staining reactions are generally not employed (Ulrich et al. 1979, 1984b,c; Tisserat et al. 1981; Chen et al. 1985; Hahne and Lorz 1987). A short lag period is observed in some cases (Tisserat et al. 1981; Hahne and Lorz 1987) and retention of morphogenetic and biosynthetic capacity after cryopreservation have been documented (Tisserat et al. 1981; Watanabe et al. 1983; Ulrich et al. 1984c; Chen et al. 1985; Hahne and Lorz 1987).
Cryopreservation of Protoplasts
Freezing of protoplasts is mainly used as a model system to investigate the effects of freezing and thawing on the structure and function of the plasma membrane in the absence of complicating factors from the cell wall (Singh 1977, 1979a; Wiest and Steponkus 1977a,b, 1978; Siminovitch et al. 1978; Steponkus and Wiest 1978; Levin et al. 1979; Bartolo et al. 1987). They have also been used for long-term storage of germplasm (Takeuchi et al. 1982). However, because of the difficulties encountered in regenerating plants from protoplasts of most species, the usefulness of protoplasts for germplasm conservation is negligible. Cell walls of protoplasts begin to regenerate within minutes of culture, and to prevent this, the method employed for storage should be such that all metabolic activity is at a standstill.
Isolated protoplasts are suspended in a medium supplemented with an osmotic stabilizer, and cryopreservation usually follows immediately to prevent the regeneration of cell wall (Withers 1985b), except in one study where a period of equilibration was required (Mazur and Hartmann 1979). Requirements for successful cryopreservation of protoplasts are similar to those of cell suspensions, but they cannot be exposed to any pregrowth conditions because of their delicate
and ephemeral nature (Withers 1985b, 1986). A method involving cryoprotection with a single or a mixture of cryoprotectants, followed by freezing at 1-20C/min to
-40ï¿½C, storage in liquid nitrogen and rapid thawing has been used for cryopreservation of plant protoplasts (Hauptmann and Widholm 1982; Takeuchi et al. 1982; Withers 1983). Freezing the protoplasts in heat sealed foil envelopes improved the viabilities considerably, probably due to uniform freezing rates obtained from better heat conductivity (Takeuchi et al. 1980).
No special post-thaw treatment is required (Withers 1983), and a quick estimation of viability can be obtained from staining reactions (Hauptmann and Widholm 1982). Cell wall regeneration could be observed within a few hours by using the fluorescent dye, Calcofluor White. Cryopreserved protoplasts retained their morphogenetic capacity (Takeuchi et al. 1982).
Cryopreservation of Shoot-Tips
Shoot-tip cultures, commonly used for elimination of viruses (Ingram 1973; Kartha and Gamborg 1975; Walkey 1976; Grout et al. 1978; Kartha 1985a), are ideal for the conservation of vegetatively propagated plants (Henshaw 1975; Morel 1975; Bajaj 1979a; Kartha 1985a). They are genetically more stable than either callus or suspension cultures, and thus eminently suited for germplasm storage and conservation (D'Amato 1975).
Storage of shoot-tips by slow growth has been successful for short to medium term, but long term storage requires periodic renewal (Mullin and Schlegel 1976; Lundergan and Janick 1979; Monette 1986). Cryopreservation of shoot-tips is more difficult, because of the diversity of the cells and the need to maintain their organization (Henshaw 1982).
The first report of cryopreservation of shoot-tips was by Seibert in 1976. Aseptically excised shoot-tips of Dianthus caryophyllus were frozen to -1960C using 5% DMSO as cryoprotectant, and viabilities up to 33% were obtained. Cryopreservation of shoot-tips of in vitro maintained plantlets was first reported by Kartha et al. (1980).
Prior to freezing, the shoot apical meristem region, which is amenable to freezing, should be excised from the shoots which in general cannot be frozen intact (Withers 1985c). The shoot meristem at the early stages of development is best for survival (Benson et al. 1989), and 0.5-1 mm specimens consisting of the apical dome and 2-4 intact leaf primodia are ideal for cryopreservation (Seibert and Wetherbee 1977; Grout and Henshaw 1978).
Shoot-tips are normally frozen after a pregrowth period of 1-3 days after excision (Grout and Henshaw 1978, 1980; Haskins and Kartha 1980; Kartha et al. 1980; Henshaw et al. 1985; Benson et al. 1989). The increase in viability after pregrowth can be attributed to the healing of the dissection wound and/or the achievement of an optimum unit size (Withers 1985c). Supplementing the pregrowth medium with nontoxic levels of DMSO improved the percentage of recovery (Kartha et al. 1979, 1980; Manzhulin et al. 1983; Kartha 1985a). Coldhardening is also employed to enhance survival to freezing (Seibert and Wetherbee 1977). Reed (1989) reported that by in vitro cold-hardening of plants of Vaccinium corymbosum for three or more weeks prior to excision, survival can be increased from 6% to 58%. However, Kartha et al. (1979) reported that freeze hardening at 4ï¿½C prior to freezing was not as satisfactory as pregrowing them in the presence of cryoprotectants.
DMSO at 5-15% is generally employed for cryopreservation of shoot-tips (Seibert 1976; Seibert and Wetherbee 1977; Grout and Henshaw 1978, 1980; Grout et al. 1978; Kartha et al. 1979; Haskins and Kartha 1980; Kartha 1984; Withers et al.
1988; Benson et al. 1989), whereas glycerol has been found to be inappropriate (Kartha et al. 1979; Grout and Henshaw 1980). Combinations of cryoprotectants have also been found to be useful (Bajaj 1982). Shoot-tips of apple (Malus domestica Borkh. cv. Fuji) survived liquid nitrogen temperatures following prefreezing to sub-zero temperatures in the absence of any cryoprotectant treatment (Katano et al. 1983).
Slow (Kartha et al. 1979, 1980, 1982; Towill 1981), intermediate (Seibert and Wetherbee 1977), and rapid cooling have been successfully used for the cryopreservation of shoot-tips (Grout and Henshaw 1978, 1980). Rapid cooling achieved by immersing the shoot-tips directly in liquid nitrogen with the aid of a hypodermic needle was used for the cryopreservation of shoot-tips of Solanum goniocalyx, while slow and intermediate cooling rates were ineffective (Grout and Henshaw 1978, 1980).
Rapid thawing is generally employed for cryopreserved shoot-tips (Grout et al. 1978; Grout and Henshaw 1980; Kartha et al. 1979; Haskins and Kartha 1980; Kartha 1984; Benson et al. 1989). The most important aspect after thawing seems to be the composition of the medium, and post-thaw washing seems to have no deleterious effects (Grout et al. 1978; Haskins and Kartha 1980; Withers 1985c). Post-freezing recovery was found to be significantly affected by alternating light conditions during culture (Benson et al. 1989), and the medium used in the recovery phase (Withers et al. 1988).
Microscopical studies show evidence of damage in randomly scattered sites composed of single cells or small groups of cells in the shoot-tip, but their presence did not have any detrimental effect on the developmental potential of the shoot primodium (Grout and Henshaw 1980). The survival of the dome entirely or partly after freezing is not essential for regeneration of plants from the shoot-tip, as surviving cells located laterally on the dome and on the shoot apical meristem have
the capacity to form shoot meristems (Haskins and Kartha 1980). Plantlets have been produced from cryopreserved shoot-tips of many species (Kartha et al. 1979, 1980; Grout and Henshaw 1980; Haskins and Kartha 1980).
ULTRASTRUCTURAL STUDIES USING A CELL SUSPENSION OF
PANICUM MAXIMUM TO DETERMINE THE CHANGES THAT OCCUR
DURING THE PROCESS OF CRYOPRESERVATION
Successful cryopreservation of plant tissue cultures have been reported in the past decade. However, the mechanism of freeze injury is far from understood. To optimize the technique of cryopreservation in such a way that it can be applied to any type of culture with high survival rates, it is important that the factors controlling freeze injury be well understood.
Membranes are known to be a major cause of freeze injury which can manifest in many different forms such as intracellular ice formation, loss of osmotic responsiveness during cooling, expansion-induced lysis during warming and altered osmometric behavior (Steponkus 1984, 1985a,b; Steponkus et al. 1982a,b ).
More than 90% of Panicum maximum suspension culture cells can withstand ultra-low temperatures when pregrown in MS2C medium supplemented with 6% mannitol, and cryoprotected with 0.5 M sorbitol and 5% DMSO before being subjected to sub-zero temperatures (See chapter 4). In the absence of any cryoprotective treatment, cells did not survive the freezing process. Therefore, cells from the above mentioned treatments were used to study the differences at the ultrastructural level that enable the cells to withstand such cold temperatures.
Materials and Methods
Cell suspension culture of Panicum maximum was used to determine ultrastructural changes that occur during different stages of cryopreservation. The suspension was pregrown in MS (Murashige and Skoog 1962) basal medium supplemented with 3% sucrose, 2 mg/1 2,4-D (2,4-dichlorophenoxyacetic acid), 100 mg/1 inositol and 5% coconut water (MS2C medium) at weekly intervals. The pH of the medium was adjusted to 5.8 before autoclaving. Prior to freezing the suspension was pregrown in MS2C medium supplemented with 6% (w/v) mannitol for three days, treated with an equal volume of pre-chilled, filter sterilized cryoprotectant mixture composed of 10% DMSO and 1 M sorbitol, and cooled in a Cryomed 1010 Micro Computer Programmable Freezer Unit at a rate of 0.5"C/min to -400C before transfer to liquid nitrogen. Cells not subjected to pregrowth or cryoprotectant treatment were also frozen as above, as well as by direct immersion into liquid nitrogen.
Rapid thawing was employed in all cases, where the ampoules containing the cells were removed from liquid nitrogen and dropped directly into a water bath at 40'C and swirled until the ice melted. The cells were then plated without washing on semi-solid MS2C medium for recovery.
Cells were fixed at the following stages for light and electron microscopical studies.
1. Three day old control cells without any pregrowth or cryoprotective
2. After three days in liquid MS2C medium supplemented with 6%
3. After treatment with the cryoprotective additives.
4. Immediately after thawing.
5. Two days after thawing.
6. Ten days after thawing.
7. Cells frozen rapidly without any pregrowth or cryoprotectant treatments,
thawed and fixed immediately.
The cells were fixed with 4% glutaraldehyde in 0.1 M sodium cacodylate buffer for two hours at room temperature, and then rinsed three times with 0.1 M sodium cacodylate buffer for a total of one hour. They were then treated with a secondary fixative consisting of 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for two hours at room temperature. Cells were rinsed with deionized water for a total of one hour to remove the osmium.
The cells were then dehydrated gradually by treating with 25%, 50%, 75%, 95% and 100% ethyl alcohol for twenty minutes each, and then transferred to a mixture of 1:1 absolute ethanol:absolute acetone for 30 minutes followed by another 30 minutes in 100% acetone. All dehydration steps were carried out at 0-4C.
Cells were infiltrated in Spurr's resin (Spurr 1969). They were treated overnight with 30% plastic and 70% acetone, followed by 70% plastic and 30% acetone, and finally with 100% plastic. The cells were then transferred to fresh 100% plastic, poured into plastic molds and left overnight in a 60C oven for polymerization. All infiltration steps were carried out at room temperature.
Cells with and without any cryoprotective treatments were cooled at a rate of 0.50C/min and freeze-fixed according to the method described by Mackenzie et al. (1975), and adapted by Singh (1979b). A mixture of 2% osmic acid and 18% NaCI was prepared and stored at its equilibrium freezing point of -100C. Suspension cultures of Panicum maximum with and without cryoprotectant treatments were transferred to polypropylene ampoules with their bases cut off and replaced with Parafilm. Excess water was removed and the ampoules were cooled to -10C at a rate of 0.50C/min. The cells were then transferred into the osmic acid mixture by
removing the Parafilm base, and stored at -10ï¿½C overnight. The osmic acid mixture was then removed and the cells were washed with three changes of distilled water over a one hour period. Dehydration and embedding were done next, as described previously.
For light microscopy semi-thin sections were cut on a Sorvall MT2-B ultramicrotome with a DuPont diamond knife, and the sections were picked up and placed on a drop of water on a gelatine coated slide prepared according to Hayat (1981). A drop of xylene was added to stretch the sections and the slide was kept on a hot plate at 60ï¿½C until the xylene evaporated. The water was then removed carefully by using a piece of tissue paper. A drop of toluidine blue in 0.1% NaCO3 at pH 11.1 (Trump et al. 1961) was added next and the slide was left on the hot plate until a golden ring was observed along the edge of the drop of stain. The specimen was then rinsed with water, dried and dry mounted with permount. Cells were observed under a Zeiss light microscope under normal and phase contrast lenses.
For electron microscopy thin sections were picked up on formvar coated (0.35%), 100 or 200 mesh grids, and post stained with 1% uranyl acetate (aqueous) for 15 minutes followed by lead citrate (Reynolds 1963) for 5 minutes. A Joel 100CX transmission electron microscope operating at 60 KV was used for taking photographs of the specimens.
Differences in vacuolar volume as well as differences in sizes of the organelles were determined at different stages of the cryopreservation procedure by using morphometry as described by Toth (1982). The volume of a single mitochondrion in cells subjected to different treatments was compared by using point counting and then dividing the values obtained by the total number of mitochondria found in each treatment. The volume and area of the ER were determined in relation to the volume of the cell between different treatments. The number of interceptions per unit length of test line (NL) was determined for each
cell. The surface area to volume ratio of the ER volume was obtained from the equation 4 x NL (Toth 1982).
When observed under light and electron microscopes, the control tissue contained cells with a large nucleus and a few large vacuoles. Organelles such as mitochondria, golgi, amyloplast with starch granules and ER were clearly visible. Oil globules were also present in the cytoplasm (Figs. 1-5). The plasma membrane was relatively smooth (Figs. 6, 7), or had minor undulations (Fig. 8).
Cells pregrown in mannitol were smaller in size with dense cytoplasm. The cells either lacked a vacuole or the central vacuole was replaced by numerous smaller ones (Figs. 9, 10). The plasma membranes differed markedly from those of the control cells. Invaginations were frequently found in the multivacuolated cells (Figs. 11-14). The average number of invaginations found in one section was five. In some sections up to 12 invaginations could be observed. These invaginations were completely absent in control cells as well as cells fixed at all other stages of the cryopreservation procedure. A reduction in cell size was also observed in cells pregrown in mannitol, the volume being 89% of the control cells. Vacuolar volume was also considerably reduced in these cells. The vacuole of the control cells on average occupied 58% of the cell volume, whereas in mannitol pregrown multivacuolated cells it occupied only 23% of the cell volume.
Dilation of organelles also occurred in mannitol pregrown cells. Average volume of the mitochondria increased by 8%. The ER occupied 1% of the volume of the control cell, whereas in mannitol pregrown cells the volume of the ER was 1.2% in relation to the volume of the cell. The area of the ER in control cells was
Figs. 1 and 2. Groups of cells from a three day old suspension of
Panicum maximum, observed under a light microscope.
Cells are large and highly vacuolated.
1. x 280 2. x 200
Figs. 3 and 4. Electron micrograph of a three day old suspension
culture of Panicum maximum observed under an electron microscope showing nuclei, Iarge vacuole, plastics with starch granules, mitochondria, ER and
3. x 8.5K
4. x 13K
Fig. 5. Cells from a three day old suspension showing the
presence of nucleus, large vacuole and organelles.
Fig. 6. Plasma membrane of a control cell showing the
relatively smooth appearance. Mitochondria with cristae, plastids with starch granules, ER and oil
globules are also seen. x 26K
Figs. 7. Plasma membrane of a control cell showing smooth
appearance. Organelles are also clearly seen. x 32K
Fig. 8. Plasma membrane of control cells with minor
undulations. x 13K
Fig. 9. Light micrograph of three day old cells grown in MS2C
medium supplemented with 6% mannitol. Small cells with dense cytoplasm are seen along the periphery and highly vacuolated larger cells are found at the center.
The multivacuolar nature of the cells is also apparent.
Fig. 10. Electron micrograph of a cell pregrown in the presence
of mannitol showing the presence of many small vacuoles throughout the cytoplasm. Nucleus, plastids, mitochondria, ER and golg are also seen in the
cytoplasm. x 10K
2'~' ~ ~
Figs. 11 and 12. Plasma membrane of cells grown in high osmoticum
showing the presence of invagminations.
11. x 16K 12. x 16K
~ii *.> j~ -~
~ V ~
Figs. 13 and 14. Presence of invaginations in cells grown in a medium
supplemented with mannitol.
13. x 26K 14. x 26K
found to be 13.86 Mm2/Mm3, whereas in mannitol pregrown cells it was 18 /Lm2/m3. The volume of the ER in relation to the control was 124%.
After treatment with the cryoprotectants the dilation of organelles was observed to be more pronounced. The average volume of the mitochondria was found to be 161% of those found in the control cells. ER was more prominent and could be clearly seen (Figs. 15, 16). The volume occupied by the ER in the cell was 1.7%. The area of the ER was found to be 26.6 Am2//pm3. The volume of the ER was 186% of the control tissue. Multimembranous structures were also observed in the cytoplasm (Fig. 17). The tonoplast also formed invaginations (Fig. 18), which eventually formed vesicles found inside the vacuole (Fig. 19).
Survival of cells after cryoprotectant treatment was determined by TITC (2,3,5-triphenyl tetrazolium chloride) assay as well by regrowth potential (See chapter 4), to determine whether any viability loss has occurred due to treatment with the cryoprotectants. The percent survival obtained was 98% according to TC test and 83% according to regrowth potential.
Membranous structures were found to be intact in cells subjected to pregrowth and cryoprotectant treatments and freeze-fixed at -10ï¿½C (Figs. 20, 21). Mitochondria and membrane bound nucleus could also be observed (Fig. 22). Again the mitochondria and ER were dilated, the volume of a mitochondrion being 265% of those from the control tissue. The volume of the ER was 1.3% of the total volume of the cell and 172% when compared to that of the control. The area was 21 pm2/pm3. The ER cisternae dilated and appeared vesicular and could be distinguished by the presence of ribosomes along the membrane. Damage to the plasma membrane and leakage of cell content were observed in some cells. Osmiophilic granules were observed along the plasma membrane in some cells that were lethally damaged during the freezing process (Fig. 23).
Figs. 15 and 16. Cells after treatment with the cryoprotectants.
15. x 16K 16. 16K
Fig. 17. Cell after treatment with cryoprotectants showing
formation of multi-membranous structures. x 25K
Fig. 18. An invagination found in the tonoplast (arrow) after
treatment with cryoprotectants. Nuclear envelope, ER
and mitochondria are also seen. x 32K
Fig. 19. A membrane bound vesicle is found inside the vacuole
(arrow) of a cell after treatment with cryoprotectants.
Fig. 20. Cells pregrown in a medium containing mannitol and
cryopreserved with 0.5 M sorbitol and 5% DMSO, and freeze-fixed at -10ï¿½C after cooling at a rate of 0.50C/min. Numerous mitochondria, nucleus, and ER
can be distinguished. x 13.2K
o* f -
Fig. 21. A cell freeze-fixed at -10C after treatment with the
cryoprotectants. Dilated mitochondria and ER cisternae
are seen x 16.6K
Fig. 22. Same as Fig. 21. Membrane bound nucleus,
mitochondria and ER are seen. x 16.6K
- iJ * N
Fig. 23. A cell freeze-fixed at -10C after treatment with
cryoprotectants. The cell is lethally damaged, and osnmiophilic granules are observed along the periphery
of the plasma membrane. x 13.2K
Fig. 24. A cell freeze-fixed after slow freezing without any
cryoprotection. The cell is plasmolyzed. Nucleus with a
nucleolus is present. x 10K
When cells were freeze-fixed without any treatments prior to freezing, the cells were lethally damaged. Very few, if any, intact organelles or membranous structures were observed. The cells also exhibited signs of plasmolysis (Fig. 24). Disruption of the membrane and plasmodesmatal connections could also be seen (Fig. 25).
When cells were observed immediately after thawing, organelles and membranous structures could still be seen (Figs. 27-31). Nuclear membrane was intact (Fig. 28), and mitochondria, chloroplast and ER could be clearly distinguished. The average volume of the mitochondria was 217% of those in the control tissue. ER cisternae dilated extensively and formed vesicles and occupied 3% of the total volume of the cell. The area of the ER was 18.2 gm2/ttm3. The volume of the ER was 336% of the control. Osmiophilic granules were noticed along the membrane in very few cells (Figs. 30-33). They were normally found on the outer surface of the membrane facing the cell wall (Figs. 30, 32), which could be clearly seen in cells where the plasma membrane was pulled away from the wall (Fig. 33). When the viability of cells was tested immediately after thawing, it was found to be 99% according to TIC test and 103% according to regrowth potential. The cells resumed growth after a lag period of two days, and no browning was observed (See chapter 4).
In cells fixed two days after thawing, the organelles were no longer dilated. The plasma membranes were smooth (Figs. 34, 35), and osmiophilic granules were absent. After ten days the cells looked similar to the control cells except for the absence of a central vacuole (Fig. 36).
Cells frozen rapidly by direct immersion into liquid nitrogen without any cryoprotective treatment were lethally damaged and when thawed lacked most membranous structures (Fig. 37).
Fig. 25. Cells freeze-fixed at -10ï¿½C without any cryoprotection.
Disruption to the plasma membrane and
plasmodesmatal connections can be seen. x 13.2K
Fig. 26. Cells immediately after thawing showing the presence
of intact nucleus. Mitochondria and ER cisternae are
also seen. x 10K
Figs. 27 and 28. Contents of cells immediately after thawing. The cells
were cryopreserved using the optimized procedure.
27. x 16K 28. x 16K
Fig. 29. A cell immediately after thawing. Plasma membrane
with plasmodesmatal connections is seen. Mitochondria
and dilated ER are also seen. x 26K
Fig. 30. Osmiophilic granules (arrow) are seen along the plasma
membrane in a cell immediately after thawing. Plastids
and mitochondria can also be seen. x 26K
Fig. 31. A Cell fixed immediately after thawing showing the
presence of dilated ER and mitochondria.x 16K
Fig. 32. Presence of osmiophilic granules (arrow) along the
surface of the membrane in a cell immediately after
thawing. x 26K
*44 4 ï¿½
Fig. 33. Osmiophilic granules are clearly seen on the outer
surface of the membrane. The plasma membrane is
pulled away from the cell wall. x 26K
Figs. 34. Cells fixed two days after thawing showing membrane
bound nuclei, nucleoli, plastids, ER, mitochondria, golgi
and vacuoles. x 10K
Fig. 35. Cells fixed two days after thawing showing nuclei,
nucleoli, mitochondria and ER. x 10K
Fig. 36. Cells fixed ten days after thawing showing nuclei,
nucleoli, mitochondria and ER. x 10K
4. ~ I
Fig. 37. Cells frozen rapidly without any cryoprotective
treatments. Cells is lethally damaged and tiny vesicles
are the only membranous structures present. x
Fig. 38. Same as Fig. 37. Damage to the plasma membrane
(arrow) can be seen. Some membranous structures are
seen in the cytoplasm. x 48K
Fig. 39. Presence of osmiophilic granules along the plasma
membrane in a cell frozen without any cryoprotection.
Plasma membrane was not clearly visible, and even in cells where it could be observed damage to the plasma membrane was clear (Fig. 38). Osmiophilic granules were seen along the plasma membrane in some cells (Fig. 39). When plated on culture medium these cells did not recover.
Cell injury can manifest in a number of ways during freezing of biological material, and alterations in the membrane properties play a major role in causing this injury (Steponkus 1985a,b). Panicum maximum cells, when frozen slowly or rapidly without any pregrowth or cryoprotective treatments did not survive the freezing process. When pregrown in 6% mannitol, cryoprotected with 0.5 M sorbitol and 5% DMSO and cooled slowly at a rate of 50C/min, marked improvement in survival was obtained, and more than 90% of the cells resumed growth when thawed and plated (See chapter 4).
Changes were observed in the ultrastructure of the P. maximum cells during different stages of the cryopreservation procedure. When pregrown in mannitol, a reduction of 11% was observed in the volume of the cells. The control cells were larger with a central vacuole, whereas the cells in mannitol either lacked a vacuole or were multivacuolar in nature. A considerable reduction in vacuolar volume was also observed. The vacuole occupied only 23% of the volume of the cell instead of the 58% observed in the control cells. Reduction in the vacuolar volume has also been reported in sycamore (Acer pseudoplatanus) cells grown in the presence of mannitol (Pritchard et al. 1982). A Catharanthus cell line with high freeze tolerance was also reported to be of multivacuolar type (Kartha et al. 1982).
Cell injury can be either mechanical from the formation of intracellular ice or by "solution effects" arising from the concentration of solutes in the cells due to the formation of extracellular ice (Mazur et al. 1972). Protective dehydration is essential for prevention of freeze injury, whereby the cell water content is reduced to a level where excess water is not present to cause structural damage by formation of intracellular ice crystals, but not severe enough to cause damage by "solution effects."
Mannitol acts as an osmoticum causing removal of water from the cell, hence exerting protective dehydration. By redistribution and reduction of the vacuolar volume the amount of water is reduced. Major changes were also observed in the plasma membrane of the cells grown in mannitol. Invaginations were observed from the plasma membrane into the cytoplasm. These probably form to accommodate the volume reduction caused during growth in mannitol containing medium.
Protoplasts isolated from acclimated and non-acclimated plants of rye (Secale cereale L. cv. Puma) behaved differently when observed using a cryomicroscope during cooling to sub-zero temperatures (Dowgert and Steponkus 1984). Temperature of nucleation was found to be reduced in acclimated tissue (Steponkus et al. 1982a; Dowgert and Steponkus 1983). During osmotic contraction protoplast from non acclimated plants produced endocytotic vesicles which were found in the cytoplasm, but during osmotic expansion the vesicles remained in the cytoplasm and the cell lysed before it reached its original volume, a process referred to as expansion induced lysis. Protoplasts from acclimated tissue produced exocytotic extrusions, which were drawn back into the surface of the protoplast during osmotic expansion and the cell regained its original volume (Dowgert and Steponkus 1984). Loss of osmotic responsiveness was the predominant form of injury in acclimated tissues.
Protoplasts of non-acclimated and acclimated tissue, when subjected to hypertonic solution contracted osmotically, and when transferred back to an isotonic solution they expanded, a process similar to cooling and rewarming. Therefore micro-osmotic manipulation was also used to study the mechanisms involved in cryopreservation (Wiest and Steponkus 1977a,b, 1978; Gordon-Kamm and Steponkus 1984a,b; Dowgert et al. 1987).
Using thin sections and fluorescein-Con-A labelling techniques along with micro-osmotic manipulation it was confirmed that the vesicles found in protoplasts of rye during osmotic contraction were derived from the plasma membrane (Gordon-Kamm and Steponkus 1984a), and that exocytotic extrusions were also bound by the plasma membrane with an osmiophilic interior which was composed of lipid material preferentially lost from the plasma membrane during osmotic contraction (Gordon-Kamm and Steponkus 1984b). They concluded that with nonacclimated protoplasts entire regions of the plasma membrane were pinched off during osmotic contraction and numerous vesicles were liberated into the cytoplasm, but during osmotic expansion the vesicles were not readily reincorporated into the membrane, hence causing lysis of the cell (Gordon-Kamm and Steponkus 1984a). In protoplasts of acclimated tissue the exocytotic extrusions never completely separated from the membrane and were drawn back into the plasma membrane during osmotic expansion, thereby preventing lysis of the cell (Gordon-Kamm and Steponkus 1984b).
The stresses experienced by protoplasts during osmotic contraction and expansion may be different in the presence and absence of the cell wall. After osmotic manipulation of intact cells of cold hardened and non-hardened winter rye, Singh (1979b) reported the occurrence of osmiophilic granules in cells of both hardened and non-hardened tissue, but it occurred at a higher osmotic stress in hardened cells. When these tissues were frozen to -100C, presence of osmiophilic
granules was observed in the non-hardened cells, whereas they were absent from the hardened tissue, where 100% survival was obtained after exposure to this temperature. In non-hardened cells, the cellular membrane rolled up forming multibilayered vesicles, which eventually lost their lamellar lattice forming osmiophilic bodies, whereas invaginations were observed in the plasma membrane of hardened tissue (Singh and Miller 1985). According to Singh (1979b), osmiophilic bodies were formed by the irreversible loss of membrane material, which caused the cells to lyse during reexpansion.
As the cells are plasmolyzed by protective dehydration, the area of the membrane is not conserved (Steponkus and Wiest 1978, 1979; Wiest and Steponkus 1978; Gordon-Kamm and Steponkus 1984a). Cells underwent plasmolysis when pregrown in mannitol, and the plasma membrane probably formed invaginations to accommodate the reduction in the cell volume. The evidence is not enough to determine whether they eventually form vesicles that are liberated into the cytoplasm.
The cells frozen by the optimized procedure for cryopreservation of P. maximum cell suspension (See chapter 4), retained most of the membranous structures and the organelles. Nuclear membrane, organelles and plasmodesmatal connections were seen in cells immediately after thawing. Dilation of the mitochondria and ER occurred throughout the cryopreservation procedure, the ER cisternae forming vesicles due to the dilation. Dilation of the organelles has also been reported in carrot and sycamore cells during the process of cryopreservation (Withers 1978b; Withers and Davey 1978). Some damage to the membranous structures as well as leakage of cytoplasmic contents was observed in some cells. When plated on semi-solid culture medium the cells resumed growth after a lag period of two days and grew at rates comparable to the control. Therefore, the damage incurred during the cryopreservation procedure probably gets repaired
within a very short period of time. This was confirmed by fixing cryopreserved and thawed cells after two days in culture. The cells were found to be similar to the control cells without any apparent damage to the ultrastructure.
The presence of osmiophilic granules in lethally damaged cells indicated that they could not have formed as an adaptation to prevent freeze damage. Although they are formed along the membrane, at this point their origin is not determined.
Cells freeze-fixed at -100C without any cryoprotection showed signs of plasmolysis. The plasma membrane was starting to pull away from the cell wall, and in some cells the disruption of the plasma membrane and plasmodesmatal connections could be observed (Fig. 25). When cells were frozen without any cryoprotection, protective dehydration could not have taken place. This would have resulted in the formation of intracellular ice crystals during freezing, and the consequent structural damage.
When cells were frozen rapidly by direct immersion into liquid nitrogen without any cryoprotective treatment, all the membranous structures including the plasma membrane were extensively damaged. When cells were frozen without any cryoprotective treatments protective dehydration did not take place. In addition when frozen at rapid rates, there is not enough time for the water to leave the cell, and intracellular ice crystallization takes place. These may not damage the cells in the frozen state but may injure the cells during the rewarming process (Farrant et al. 1977). Suspension cultures of Sycamore (Acer pseudoplatanus L.), when frozen rapidly or slowly without treatment with cryoprotectants suffered lethal damage by intracellular ice formation or cellular dehydration (Withers and Davey 1978). Osmiophilic granules were observed in lethally damaged cells of Panicum maximum further indicating that it is not an adaptation to prevent freeze damage in cells. Their presence was not observed in all the cells, and at this point it is not known whether they are reincorporated into the plasma membrane.
Therefore it can be inferred that protective dehydration, and cryoprotectant treatment are very important for the cells to survive sub-zero temperatures. The extend of damage to the cell varies depending on the treatment employed. When frozen by the optimized procedure, although some damage was apparent the membranous structures were still intact. The cells could repair the damages in a short period of time and resume growth. The cells frozen without any cryoprotection were extensively damaged. This damage could not be repaired and the cells did not recover when plated on semi-solid medium.
OPTIMIZATION OF THE CRYOPRESERVATION PROCEDURE FOR STORAGE OF CELL SUSPENSIONS
Cell suspension cultures are used extensively in secondary product synthesis, studies of physiology and morphology, isolation of mutant cell lines, protoplast isolation and genetic engineering (Vasil and Vasil 1980; Withers 1983; Hauptmann et al. 1987; Vasil 1987, 1988; Vasil et al. 1988, 1990). Their maintenance is a labor intensive process which could result in the loss of cell lines, in addition to loss of morphogenetic potential and accumulation of mutations.
It will be very useful, therefore, if cell suspensions can be stored at stable conditions for extended periods of time, eliminating the requirements for periodic subculture. Cryopreservation has been extremely successful for storage of a number of cell suspension cultures. It involves a number of steps, each of which is important to attain high survival rates. Panicum maximum and Pennisetum americanum cell suspensions were used in this study to optimize the cryopreservation procedure at each stage involved.
Materials and Methods
Cell suspensions of Panicum maximum (established in 1985 from leaf tissues as described in Lu and Vasil 1981a,b), and Pennisetum americanum (established in 1986 from immature embryos as described in Vasil and Vasil 1981) have been
routinely maintained by transferring 12 ml of the cell suspension into 35 ml of fresh MS2C medium in 250 ml Erlenmeyer flasks at weekly intervals. Both these cell lines were used to study and optimize each step of the cryopreservation procedure.
Effect of pregrowth treatments and cryoprotectants on survival
Cells were pregrown in MS2C alone, or supplemented with either 6% (w/v) mannitol or 6% (w/v) sorbitol prior to cryopreservation. Twelve ml of the seven day old culture was transferred to 35 ml of the pregrowth medium, and used for cryopreservation experiments after three days in culture.
Two different combinations of cryoprotectants consisting of 0.5 M DMSO + 0.5 M glycerol + 1 M sucrose or 0.5 M sorbitol + 5% DMSO were used, in combination with each of the pregrowth treatments. Cryoprotectants were prepared in double strength, filter sterilized and chilled on ice. The compounds were dissolved in water except in one experiment where they were dissolved in MS2C medium.
Within a single experiment, the same packed cell volume (PCV) of cells was used to avoid differences in response that may arise due to the differences in cell densities. Fifteen ml of the suspension was dispensed into sterile centrifuge tubes and spun at 100 x g for 3 minutes. The PCV was then adjusted to the required value. It was taken into account that the final PCV obtained was half of the initial value, due to the addition of an equal volume of cryoprotectant solution. A final PCV of 20 % was used in most experiments unless stated otherwise. The suspension was then transferred to a 125 ml Erlenmeyer flask and chilled on ice. Prechilled cryoprotectant prepared in double strength was added to the cell suspension in ten increments over a one hour period, while the suspension was still being maintained on ice on a rotary shaker. After addition the cells were left for one more hour on the shaker to facilitate the uptake of cryoprotectants.
One ml of the cell suspension was dispensed into 1.2 ml polypropylene, screw cap ampoules and cooled in a Cryomed Model 1010 Micro Computer Programmable Freezer Unit at a rate of 0.5ï¿½C/min. Temperature of the sample was monitored by inserting a thermocouple into one of the ampoules. The ampoules were cooled to a transfer temperature of -400C, and held at that temperature for 40 minutes before transfer to liquid nitrogen. The ampoules were stored for at least two weeks in liquid nitrogen, before thawing to determine viability. Thawing was carried out rapidly by plunging the ampoules into a water bath at 400C with stirring. The ampoules were removed when the ice had just disappeared, and wiped with 95% ethyl alcohol before opening.
Viabilities were determined by TTC reduction assay as well as by regrowth potential. For TTC reduction assay the contents of the ampoules were transferred to 15 ml centrifuge tubes and 3 ml of the TEC solution was added and left in total darkness for 16 hours. The tubes were then centrifuged and the supernatant was removed. The cells were rinsed with double distilled water and the supernatant was again removed after centrifugation. Seven ml of 95% ethyl alcohol was added next, and the tubes with the contents were heated for 5 minutes in a water bath at 80'C. After cooling, the volume of each tube was adjusted to 10 ml using 95% ethyl alcohol. Absorption was measured at 530 nm using a Beckman DU-40 Spectrophotometer and the viabilities were determined by comparing the values obtained from the cryopreserved cells with those of the control cells.
Viabilities were also determined by regrowth potential, which was based on the increase in the fresh weight of the cryopreserved cells during culture. Filter papers sterilized by autoclaving were placed on Petri dishes containing 25 ml of MS2C medium solidified with 0.2% gelrite. The weight of the filter papers was determined once they were saturated with water. The thawed ampoules were opened after wiping with alcohol, and contents of each ampoule were poured onto
the filter paper layered on MS2C medium. The filter paper along with the cells was weighed again after 30 minutes and every two days thereafter. Fresh weight of the cells was calculated by deducting the weight of the filter paper alone, from the weight of the filter paper with the cells growing on it. Growth curves were drawn by plotting the fresh weight of the tissue against time. The combination of pregrowth and cryoprotectants that gave the highest viability was used hereafter in all experiments to further optimize viability.
Rate of cooling
For optimization of cooling rates a final PCV of 20% was used. Cells were prepared for freezing as described before, and cooled at rates of 50C/min, l1C/min and 0.50C/min to -400C and held at that temperature for 40 minutes before plunging into liquid nitrogen. After storage they were thawed rapidly and the viabilities were determined by TTC reduction assay. The rate of cooling which resulted in the highest survival was used hereafter in all experiments.
Cells at a final PCV of 20% were cooled at a constant rate of 0.50C/min to different transfer temperatures and plunged immediately into liquid nitrogen without a holding period. The transfer temperatures employed were -200C,
-400C and -60'C. Survival was determined by TTC reduction assay once the cells were thawed rapidly after two weeks in liquid nitrogen.
Packed cell volume and holding time
Cells with final packed cell volumes of 10%, 20% and 40% were cryopreserved by the same procedure, using a cooling rate of 0.50C/min. For all three experiments the same transfer temperature of -400C was used, but the holding time at this temperature varied. Some ampoules were transferred immediately into liquid nitrogen once they reached -400C, some were held at this temperature for 40 minutes as in all previous experiments and the rest were held for a total of 80 minutes. The cells were thawed rapidly and the viabilities were determined by TTC reduction assay.
Cryoprotectants dissolved in water vs. in culture medium
Sorbitol (0.5 M) and DMSO (5%) were dissolved in water as well as in liquid MS2C medium to determine effect on survival. PCV of 20% was used and the cells were frozen at 0.50C/min to -40'C and plunged immediately into liquid nitrogen without a holding period. Viabilities were determined by TTC reduction assay after rapid thawing.
In all previous experiments, after the addition of cryoprotectants cells were left for an hour to enhance the uptake of cryoprotectants before freezing. To determine whether prolonged exposure has any deleterious effects on survival, cells were frozen immediately after the completion of addition of cryoprotectants as well as after a one hour uptake period. PCV of 20% was used, and cells were frozen at 0.50C/min to -400C and plunged immediately into liquid nitrogen. Survival was determined by TTC reduction assay after thawing.
The cryopreservation procedure was optimized from the results obtained from all the experiments described above. The cells were again cryopreserved by combining all the factors that gave the highest viability in each stage. Cells were pregrown in mannitol and the PCV was adjusted to 80% so that a final PCV of 40% could be obtained. An equal volume of double strength cryoprotectant (0.5 M sorbitol and 5% DMSO) prepared in water, was added next over a one hour period, and the suspension was transferred to ampoules without an uptake period. The cells were cooled at a rate of 0.50C/min to -400C and immediately transferred to liquid nitrogen without any holding period.
Cells were thawed rapidly by plunging the ampoules into a water bath at 400C, and slowly by allowing the ampoules to thaw in air at room temperature (about 15 min). Survival was determined by TIC test and regrowth potential at different stages of the cryopreservation procedure, such as, after treatment with cryoprotectants, after cooling to -40'C, and after rapid and slow thawing to determine whether any viability loss occurred at any of these stages. Viability was also checked at different time intervals, over a period of three years to determine whether long term storage in liquid nitrogen caused progressive deterioration in the survival.
The procedure optimized from the results obtained was used for cryopreservation of various cell lines.
Cell suspension of Panicum maximum was used for isolation of protoplasts from both control and cryopreserved cells. Cryopreserved cells were thawed and grown on semi-solid MS2C medium for a month, and then transferred into 35 ml of liquid MS2C medium in 250 ml Erlenmeyer flasks. The cryopreserved cell line was
maintained by transferring 12 ml of the cell suspension into 35 ml of fresh MS2C medium at weekly intervals.
Three day old control and cryopreserved cells were transferred into fresh culture medium and used for protoplast isolation on the second day. The PCV of both cell lines was adjusted to 40% to standardize the experiment.
Enzyme mixture was prepared by dissolving 1% Cellulase R S (Yakult Honsha), and 0.8% Pectinase (Serva) in MES buffer, and the pH was adjusted to 5.6 before filter sterilization. A small volume (1.5 ml) of each suspension was transferred to 15 ml of the enzyme mixture in Petri dishes and incubated at room temperature on a shaker for 1-2 hours, followed by overnight incubation at 120C without shaking.
The protoplast-enzyme mixture was first filtered through a Miracloth, followed by 100 and 50 pm stainless steel filters. The mixture was then transferred to 15 ml centrifuge tubes and spun at 100 x g for three minutes. The supernatant was removed and the volume was brought to 10 ml with MES buffer and mixed well. A drop from the protoplast buffer mixture was used to determine the yield by hemacytometric counting. The protoplasts were washed two more times with MES buffer. The supernatant was removed after the last wash and centrifugation, and Kao and Michayluk's modified nutrient medium (Vasil and Vasil 1980) was added to the protoplasts. The volume of the culture medium was adjusted so that a final protoplast density of 1x105/ml was achieved. The protoplasts in the culture medium were transferred to 35x10 mm Falcon Petri dishes and incubated in total darkness at 270C. The plating efficiencies of protoplasts isolated from control and cryopreserved cells were determined after 10 days in culture. Plating efficiencies were calculated from the percentage of cultured protoplasts that formed colonies.
The optimized procedure was used for the cryopreservation of cell suspensions of Pennisetum purpureum, Saccharum hybrids (SCH and SH2). Percent survival was determined from regrowth potential on semi-solid culture medium.
When cells of Panicum maximum and Pennisetum americanum were cryopreserved by using different pregrowth and cryoprotectant treatments, marked differences were observed in their viability (Tables 1, 2; Figs. 40, 41).
In Panicum maximum highest survival was obtained when the cells were pregrown in MS2C medium supplemented with either 6% mannitol or 6% sorbitol (Table 1). Rate of growth was determined for each treatment between days where the growth was found to be linear. The percent survival was calculated by comparing the rate of growth of the cryopreserved cells with that of the control. Highest survival was 71-75% based on TTC assay and 74-86% based on regrowth potential (Table 1; Fig. 40). Cells from Treatment 4 where a 28% survival was obtained based on TIC test, failed to resume growth in culture.
In Pennisetum americanum percent survival based on TTC test were considerably lower than those from regrowth potential (RP). A highest survival of 66% (TTC) and 91% (RP) was obtained when cells were pregrown in MS2C medium supplemented with 6% mannitol and cryoprotected with 0.5 M sorbitol and 5% DMSO, as with Panicum maximum cell line (Table 2; Fig. 41). When these cells were returned to culture a lag phase of about 6 days was observed, which may explain the lower results obtained from TTC test. The cells cryopreserved without any pregrowth treatments, especially those cryoprotected with 0.5 M sorbitol and 5% DMSO failed to survive when plated on semi-solid medium. The rate of growth
was determined from the difference in fresh weights between day 8 and day 18 during which time the growth was linear.
The viabilities varied considerably when cells subjected to the same pregrowth and cryoprotectant treatments were cooled at different rates to the same terminal freezing temperature before transfer to liquid nitrogen (Table 3). A cooling rate of 0.50C/min gave the highest viability with both cell lines, and the viabilities decreased with increased rates of cooling.
Terminal freezing temperature
The optimum terminal freezing temperature for both cell lines was found to be -40'C (Table 4). Survival was very low when cells were transferred to liquid nitrogen after a terminal freezing temperature of -200C. Cooling beyond -40' also decreased viability.
PCV and holding time
Improved recovery of Panicum maximum and Pennisetum americanum cells was observed with increased PCV (Tables 5, 6). Final PCV of 40% gave the highest recovery. Within each PCV, recovery varied with the holding time, when all other parameters were kept constant. Highest survival was obtained when cells were transferred to liquid nitrogen, immediately after they reached -400C, and decreased with increase in the holding time. This was found to be consistent within each PCV used.
Survival of Panicum maximum cells, based on TIC test and regrowth potential (RP) subjected to different pregrowth and cryoprotectant treatments (averages of two experiments; there were five replicates for each treatment, with 1 ml of cells).
Treat- Pregrowth Cryoprotectants % survival * ment medium TITC RP
1 MS2C 0.5 M sorbitol 31 ï¿½ 1.86 52ï¿½ 4.66 5% DMSO
2 MS2C + 0.5 M sorbitol 75ï¿½ 1.59 86ï¿½5.71
6% mannitol 5% DMSO
3 MS2C + 0.5 M sorbitol 72ï¿½ 1.47 81 5.48
6% sorbitol 5% DMSO
4 MS2C 0.5 M DMSO 28 ï¿½ 0.50 00 ï¿½ 0.00
0.5 M glycerol
1 M sucrose
5 MS2C + 0.5 M DMSO 60ï¿½2.79 74ï¿½ 7.20
6% mannitol 0.5 M glycerol
1 M sucrose
6 MS2C + 0.5 M DMSO 71ï¿½1.57 78ï¿½4.10
6% sorbitol 0.5 M glycerol
1 M sucrose
Numbers represent the values of the mean ï¿½ standard error of the mean.
Survival of Pennisetum americanum cells, based on TIC test and regrowth potential (RP) subjected to different pregrowth and cryoprotectant treatments (averages of two experiments; there were five replicates for each experiment, with 1 ml of cells).
Treat- Pregowth Cryoprotectants % survival * ment medium TI'C RP
1 MS2C 0.5 M sorbitol 19 ï¿½ 0.35 00 0.00 5% DMSO
2 MS2C + 0.5 M sorbitol 66ï¿½ 1.62 91ï¿½3.48
6% mannitol 5% DMSO
3 MS2C + 0.5 M sorbitol 56ï¿½ 1.33 78ï¿½ 1.44
6% sorbitol 5% DMSO
4 MS2C 0.5 M DMSO 24ï¿½ 0.45 04ï¿½ 1.03
0.5 M glycerol
1 M sucrose
5 MS2C + 0.5 M DMSO 42ï¿½ 0.99 69 6.20
6% mannitol 0.5 M glycerol
1 M sucrose
6 MS2C + 0.5 M DMSO 30ï¿½2.00 19ï¿½4.14
6% sorbitol 0.5 M glycerol
1 M sucrose
Numbers represent the values of the mean + SEM
Fig. 40. Regrowth curves of control and cryopreserved cells of
Panicum maximum cells plated on MS2C medium.
Cells from Treatment 4 did not resume growth.
(Averages of two experiments, five replicate plates for
each treatment, with 1 ml cells/plate).