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Strategies for 'minimal growth maintenance' of cell cultures: a perspective on management for extended duration experimentation in the microgravity environment of a space station.

II. Introduction to the Problem

Publication of a review typically reflects that a topic has matured enough to merit an evaluation of its status and has a sufficient base to allow analysis and integration of published findings. Ideally, models for future research are developed. The topic undertaken here, however, is far from mature. Moreover, it might appear at first glance so specialized as to attract the interest of perhaps only a few. After all, any kind of Space biology investigation could seem as if it represents an unusual, even esoteric, activity, far removed from mainstream research concerns. But although the number of investigations carried out on plants in the Space environment is not large, it is not negligible (Halstead & Dutcher, 1987; Halstead & Scott, 1990; Halstead et al., 1991; Merkys & Laurinavicius, 1991; Nechitailo & Mashinsky, 1993; Schaeffer et al., 1993; Dutcher et al., 1994, and references cited therein). And there is considerable optimism that this area, perhaps better viewed under the umbrella designation of "gravitational and Space biology," will in the future attract an increasing number of investigators who will take advantage of the unique environmental conditions for experimentation that Space offers.

A. SPECIAL FEATURES OF THE SPACE ENVIRONMENT

First and foremost, the Space environment [even the environment of low Earth orbit (LEO) at 120 to 450 nautical miles - 223.3 to 833.4 km - above Earth's surface] is free of gravitational influences, free of tidal forces and the cyclical events of celestial mechanics, and free of the Earth's magnetic fields. Life on Earth has evolved, of course, under the influence of each of these and perhaps other fields of force which are as yet unknown or unappreciated (see Nechitailo & Mashinsky, 1993; Markert & Krikorian, 1993, and references cited therein). While there has been some work on the effects of the absence or presence of magnetic fields and the quality and quantity of electromagnetic radiation on plants, Earth-based biological studies have generally been limited to increases in the field above Earth-normal gravity (1-G), i.e., hypergravity (Gray & Edwards, 1971; Kelly & Smith, 1974; Nakazawa, 1989; Brown, 1992, 1996).

In orbit, centrifugal force balances the gravitational force so that the mass of a satellite or Space vehicle is often thought of as existing in a state of "apparent zero gravity." The term "microgravity" ([Mu]-G) has been used to reflect more precisely the small levels of G that generally exist even in free-fall orbit. Even light exerts pressure. For example, the amount of pressure imposed by sunlight on a spacecraft is on the order of 1 x [10.sup.-9] G (e.g., see Krikorian & Levine, 1991: 494; Nechitailo & Mashinsky, 1993: 96). Simulation of the near-weightlessness of Space cannot be accurately simulated on Earth. Until recently, attempts to compensate for or to neutralize asymmetry or the G vector, by appropriate rotation of organisms on horizontal or even 3-D clinostats, so as to "null" or "time-average" the G force were seen as experimental approximations of "zero G" (Larsen, 1962; Hoshizaki, 1973; Smith, 1974, 1975; Gruener, 1985; Smith et al., 1992). But this view is increasingly being discredited, since on clinostats there may be vibrational stress and thus an increase in stirring and convection at the cellular level rather than a lowering or elimination, as would, in theory, occur in weightlessness (Silver, 1976; Albrecht-Buehler, 1991, 1992; Kessler, 1992).

Indeed, in the near-weightless environment of Space (here weightlessness is defined as [10.sup.-6] to [10.sup.-4] G), there are no convective currents due to G, there is no buoyancy, and surface tension dominates. Moreover, with respect to experiments in Space involving a fluid phase - namely, water in all biological systems - although the laws of classical physics such as momentum and mass conservation, energy conservation, and Maxwell's equations still apply, the relative importance of the G force to other forces changes (Ostrach, 1982; Netter & Weiss, 1992; Polezhaev & Ermakov, 1992). Also, the fact that Space experiments are conducted in a non-inertial frame becomes more important, and certainly the effects of variation of G become important (see Bjorkman, 1988; Krikorian & Levine, 1991; Halstead et al., 1992; Ding & Pickard, 1993; Ingber, 1993, and references therein).

NASA has made some effort at developing a system for mass culture of cells on Earth that simulates some characteristics of [Mu]-G, with the intent of being able ultimately to establish systematically the capability of the system to mimic some of the above-mentioned consequences of the Space environment. A very low shear environment culture vessel, a so-called slow turning lateral bioreactor vessel (STLV) - with a volume of about 250 ml, "zero headspace," a lateral or horizontal cylindrical configuration, an air pump for gentle continuous gas flow, and an internal silicone gas-exchange membrane - has been designed primarily to allow growth of anchorage-dependent human or other animal cells on micrometer-diameter microbeads (see Lewis et al., 1987, 1993, for photographs and schematic diagrams of the system). But the STLV system seems to have some potential for growing plant cells and even seedlings as well. Reactor vessels such as the STLV nominally provide to cells some biomechanical aspects of microgravity. These include minimal-impact cell collisons, low mechanical shear stress, enhanced culture homogeneity, and altered substrate distribution. The vessel design allows operation over a wide range of shear forces and hence allows any thresholds to be determined. Nevertheless, only after protracted use in Space and comparison of results with those obtained on Earth will it be possible to gauge the extent to which the effects of Space conditions on cells are partially or wholly simulated by such bioreactors.

B. SOME OBJECTIVES OF SPACE EXPERIMENTATION

A major goal of spaceflight experimentation with plants is to seek answers to questions such as these: How do plants detect and respond to gravity? What are the short- and long-term effects of near-weightlessness on plants? Can plants adapt successfully to long-term weightlessness? Can countermeasures such as centrifugation be utilized to eliminate any untoward effects of the Space environment? Before direct and far-reaching questions like these can be effectively addressed, a number of technologies need to be developed and perfected. One is the deceptively simple technology of growing plants reliably in Space. Procedures that are routine on Earth often need to be handled in imaginative ways and will need to be made consistent under novel and still poorly defined conditions. Some progress has been made in this area, but there is a long way to go before many outstanding questions are answered and problems are resolved. Most of the scientific community is unaware of the ongoing practical difficulties encountered in attempting to satisfactorily grow plants in Space (see Krikorian & Levine, 1991; Salisbury, 1991a, 1991b, and references there cited). Only now are some of the questions and hypotheses being sufficiently refined to be tested effectively. In addition to seeking answers to questions such as the fundamental ones just posed, there is also an increasing awareness of the need to address these issues in as much detail as possible. This includes not only describing responses at the organismal, organ, tissue, and cell level (Keefe & Krikorian, 1983; Halstead & Scott, 1984, 1990; Halstead & Dutcher, 1987; Halstead et al., 1991; Schaeffer et al., 1993; Dutcher et al., 1994; Nechitailo & Mashinsky, 1993, and references cited therein) but also understanding the biochemical basis and molecular mechanisms underlying the cellular, tissue, and organismal responses (Markert & Krikorian, 1993).

While there are a number of long-appreciated examples of gravitropism involving intact higher plants (Larsen, 1962; Firn & Myers, 1987; Salisbury, 1993) and higher fungi (Moore, 1991), and even explanted organs such as the pulvini of grasses (Kaufman et al., 1995), that may be identified as candidate test systems for studying differences after exposure to changes in gravity, there is relatively little known about cells outside the plant body (Moroz, 1984; Todd, 1989, 1991). For example, one would like to know whether [Mu]-G has an effect on developmental, physiological, and biochemical processes and whether "gravity unloading" affects cells that are specialized for G-sensing (Sievers & Volkmann, 1979; Bjorkman, 1988; Sack, 1991, 1993) in ways that are different from those that are not specialized for G-sensing (Dennison, 1961; Varju et al., 1961; Sulzman et al., 1984; De Groot et al., 1990; Kawasaki et al., 1991; Todd, 1993). It is anticipated that carefully selected cell and tissue systems growing in [Mu]-G can be used to probe some of the questions that relate to either of these cell types (Table I). Additionally, there is considerable interest in growing and utilizing cultured cells for biotechnology purposes (Cogoli & Tschopp, 1982; Keefe & Krikorian, 1983; Krikorian, 1985; Morrison, 1987; Zimmermann et al., 1988).

For example, because of gravity, on Earth the contents of bioreactors must be mixed in order to obtain a proper distribution of nutrients, oxygen, temperature, and pH environment (e.g., see Jensen, 1979: fig. 2; and the bioreactor designs in Prenosil & Pedersen, 1983; Scheld et al., 1985; Scragg et al., 1987; Kargi & Rosenberg, 1987; Moo-Young & Chisti, 1988; Scragg, 1990; Constabel & Tyler, 1994). This mixing creates a harsh hydrodynamic shear environment detrimental to sensitive cells (Prokop & Rosenberg, 1989; Papoutsakis & Michaels, 1993). If not mixed properly, cells tend to congregate and, by zone sedimentation, fall to the bottom of the culture vessel or bioreactor. Furthermore, the requirements for oxygenation frequently creates a foaming in the bioreactor which also tends to perturb and otherwise damage cells. These factors often limit the uniformity of the concentration and density of the bioreactor nutrient culture medium. On Earth, it is known that concentration and density of the solution are directly linked to the optimal performance of bioreactors: the higher the density, the more cost-effective the bioreactor "run" (Staba, 1985; Scragg et al., 1987; Fowler et al., 1990; Doran, 1993; Wilson & Hilton, 1995).

In [Mu]-G, zone sedimentation essentially disappears, which should reduce the aggregation of cultured cells. Moreover, only gentle mixing is required to distribute nutrients and oxygen. For instance, [Mu]-G is predicted to alter the mass transfer coefficients for transfer of oxygen into the medium and from the medium to a cell or enzyme. These factors should permit higher concentrations and densities to be achieved in a low-G environment. Additionally, since the cells do not need to maintain the same surface forces that they require in Earth-normal gravity, they should be able to divert more energy sources for growth (Cogoli & Gmunder, 1991; Tairbekov, 1991) and differentiation and, in theory at least, the biosynthesis of more product, or even novel products the production of which would be unpredictable. Because, again in theory, one can impose different levels of G force on these cell systems by centrifugation (e.g., see Waterman et al., 1978; Stolzenburg et al., 1984) and other means, one can test in Space the consequences of increasing or decreasing G (i.e., hyper-G to hypo-G, respectively) on metabolic pathways such as secondary product biosynthesis (see DiCosmo & Towers, 1983; Neumann et al., 1985; Robins & Rhodes, 1988; Verpoorte et al., 1991; Endres, 1994).

Some work has already been carried out on a variety of plant cells in Space that indicates that metabolism, productivity, and differentiation characteristics are altered (see Mesland, 1987; Cogoli & Gmunder, 1991; Krikorian et al., 1992; Dutcher et al., 1994). Whether this might be due to decreased cell interactions (contacts) when cells are freely suspended is not known. Clearly, the many opportunities to study these responses should eventually lead to a better understanding of the mechanisms by which plant cells control production of secondary metabolites and other cell products. With this knowledge, control of enhanced, sustained production of product by cells might be possible.

Somewhat parenthetically, earlier suggestions that unicellular algae (Krauss, 1962) or aseptically grown plant cell cultures might be amenable to use as a food source directly (Byrne & Koch, 1962; Hildebrandt et al., 1963) have long since been abandoned, although there may be some specialized situations where this might be done (see Sidorenko et al., 1983; Staba, 1980; Lembi & Waaland, 1988). A good example of the latter is the production of tomato fruits from aseptically cultured thin layers (Compton & Veilleux, 1991, 1992). Even so, it may turn out that unexpected biosynthetic potential may be expressed in the Space environment (DiCosmo & Towers, 1983; Collin, 1987; Morrison, 1987; Chasan, 1991; Petersen & Alfermann, 1993). The beauty of Space investigation is that virtually everything is unexplored and there is much to learn. Sometimes one forgets that growing plants on Earth is one of humankind's most ancient "biotechnologies"; yet plant biologists are in the unprecedented predicament of having to learn to grow plants as efficiently as possible in a novel environment thus far rudimentarily tested from the biological point of view (Halstead & Scott, 1984; Krikorian & Levine, 1991; Merkys & Laurinavicius, 1991; Salisbury 1991a, 1991b; Krikorian et al., 1992; Nechitailo & Mashinsky, 1993; Schaeffer et al., 1993, and references cited therein).

In order to evaluate any of these potentials in each of these instances, whether real or hypothesized, one obviously needs to have well-defined conditions for maintaining and growing not only intact plants in Space but also their cells, tissues, and organs in vitro in Space.

C. LOGISTICAL CONSTRAINTS

Given the special physical features of the Space environment, together with the many technical constraints imposed on researchers by the realities of carrying out experiments in a complicated and sophisticated setting, those seeking to design experiments to be carried out in the [Mu]-G environment will be confronted with a number of constraints at both the planning level and the implementation level. It may be anticipated, moreover, that these constraints will need to be taken into consideration for the foreseeable future no matter the specific growing environment for a given system or the exact nature of the Space vehicle or orbiting Space platform (Krikorian & Levine, 1991; Cogoli & Gmunder, 1991). There will be relatively limited access to power on all spacecraft since energy is not yet available in generous amounts even in relatively large facilities, such as the Space Shuttle or the Space Station, due to solar array technology and a hesitancy to rely on nuclear energy. Certainly, power will more than likely always be limiting in smaller orbiting satellites (Krikorian, 1990). Only a few astronauts or payload specialists will be available to carry out manipulations or experimental procedures on behalf of a great many investigators whose interests span a range of scientific and engineering disciplines. It is certain that they will be on strict schedules and that access to their services will be highly competitive. Equally important, for the foreseeable future there will be only a few scheduled visits a year from various space vehicles such as the Space Shuttle to the Space Station, and there will be substantial intervals between these visits. This means that materials from experiments terminated in Space - i.e., those that must be collected and returned to Earth for subsequent study, analysis, and interpretation - need to be retained on orbit after completion. There is certain to be considerable competition for storage room in Space, and one has to ensure that materials will withstand the rigors and possible hazards of storage and their ultimate return to Earth. Live samples must be kept alive, frozen samples must be kept frozen, fixed materials must be maintained in satisfactory condition. Similar problems will also have to be dealt with in the course of initiating an experiment destined for the Space environment as well. In some cases, the biological materials for an experiment will need to be established and set up on Earth, transferred to a launch vehicle, and ultimately put in place at its final destination (e.g., the Space Station). Experiments will need to be prepared on Earth and launched into Space. In the United States, experiments are generally prepared at the Kennedy Space Center, Florida, in the Life Sciences Biological Research Support Facility. Experiment preparations are initiated at a specified time prior to what is referred to as "turn-over," i.e., when the experimental material set up or installed in appropriately designed "hardware" leaves the hands of the investigator and is taken to the launch pad to be put on board the Space Shuttle. For technical reasons related to safety and engineering concerns, the very latest that this "turn-over" can occur is 16 hours before launch (so-called L minus 16). This means that cells and tissues must be maintained in an appropriate state from the time they leave the investigator to the time of actual lift-off. While there have been many lift-offs that have met scheduled timetables, there are many cases of launch delays (termed "slips") when all the "launch commit criteria" are not met. There is an official procedure that is followed in the event that slips are required for reasons of weather, engineering problems, and so forth, and it is a real possibility that an experiment may need to be prepared de novo a number of times to ensure the quality of material being launched. Obviously, the more tolerant a given system is to insults and delays, the less worrisome these "scrub turn-arounds" become.

This is all to say that the simpler and more self-contained or automated an experiment can be, the better. This applies not only to experiments using cultured cells and tissues but to all experiments. But the level of automation required for cell culture and its complexity, and hence the associated cost (especially for the kind of automation that will provide redundancy so as to permit trouble-free operation over extended periods), presently argues against full automation. Moreover, it is expected that building up to complete automation will be a gradual process, since scientists and engineers will inevitably have to learn through experimentation, both in the laboratory on Earth and under "real" conditions of spaceflight, the best way to accomplish desired objectives [ILLUSTRATION FOR FIGURE 1 OMITTED]. Justifiably, there is considerable concern about "dedicated" versus "generic" hardware, the latter being equipment that is not experiment-specific but widely applicable and hence usable by many investigators. There is also concern that there be a prudent balance between simplicity and flexibility. In any case, there is little justification in carrying out potentially costly designs and redesigns unless there is considerable evidence that what is being developed has a reasonable chance of working well for a significant number of researchers. In the very least, plant cell culture experiments (and animal cell culture ones, for that matter) should be designed so they can be activated and terminated in Space at will (by the nature of the experimental design, by automatic means, or by human intervention).

The more usual means of taking samples during the course of an experiment or terminating it will, of course, involve fixation for histological examination and such or the rapid freezing of samples for subsequent (bio)chemical analysis upon return to Earth. But there will be instances when investigators may wish to carry out an experiment, perform fixations and freezings, and retain live samples as well. But while initiating and terminating an experiment on Earth under aseptic conditions may seem easy enough, there are many situations where even achieving, much less maintaining, asepsis is not that simple, and it becomes even more difficult when one needs sterility for extended periods (Tiner, 1963; Wallhausser, 1982). Similarly, performing fixations and freezing of samples is routine enough on Earth, but accomplishing these tasks safely in [Mu]-G is no trivial feat, given the need to isolate redundantly or "triply contain" toxic materials such as glutaraldehyde from the open environment and the challenge of keeping things frozen (Schulze et al., 1992).

The task of keeping cultures healthy and vigorous is especially demanding if an experiment is to last longer than a relatively brief period - longer than, say, a matter of several days or a couple of weeks. But a major advantage afforded by an orbiting vehicle like Space Station Alpha is that biological scientists (and others) will have access to extended duration [Mu]-G environments for investigating the role of gravity in various systems. This may, for example, take the form of a given experiment being carried out to term over a long period with samplings made at appropriate intervals; it could also be an experiment of relatively short duration that is initiated, sampled periodically, terminated, and re-initiated using fresh materials that have been "withdrawn" along the line. Such "repetitive" or "end-to-end" experiments provide not only multiple samples for statistical purposes but also an opportunity to study adaptation of biological systems to [Mu]-G and other Space conditions over a long period without the logistical challenge of providing an appropriate growing environment for an extended period. Some would argue, in fact, that more reliable experimentation could be carried out if it were initiated, sampled over time, terminated, and re-initiated. Of course, the experimental objective(s) would determine the exact protocol. Seen from a perspective of the need to manage cell cultures, one would perforce be controlling cells in preflight activities, in-flight activities, and post-flight activities.

But the subject is arguably more important and much broader than any of the foregoing implies. Indeed, the area to be covered in this review should have wider appeal than only to workers interested in gravitational and Space biology. The challenge presented by dealing with plant cell cultures during extended spaceflight has considerable practical applications on Earth. For example, most plant tissue culture laboratories, both research and commercial, are faced with the chore of maintaining cumbersome inventories and are continually frustrated by the ongoing and labor-intensive activity of initiating cultures de novo on a routine basis, carrying out transfers and subcultures (see George & Sherrington, 1984; George, 1993). While most commercial micropropagation or aseptically based plant multiplication utilizes relatively large explants such as stem tips or lateral buds as the starting material for clonal expansion, there is increasing interest in evaluating the potential for utilizing embryogenic cells and bioreactors to achieve truly massive numbers (Krikorian, 1982; Ammirato & Styer, 1985; Krikorian et al., 1986; Preil et al., 1988; Gray & Purohit, 1991; Preil, 1991). Both research and commercial workers would welcome insights into devices or strategies that permit the task of conserving various cell and tissue lines for long periods but without risk of diminished potential for regrowth and regeneration of cultures or even of plants at a much later date. The problem of managing cultures for a practical end must, however, rely on a judicious combination of basic scientific principles and technology, for it ultimately comes down to the task of utilizing facts of cell and tissue physiology for the management of growth. It is for this reason that some classic references are included despite the fact they were written, in some cases, some time ago.

Against the above background and despite the fact that the topic both is quite novel and has a relatively small literature, it is appropriate now to attempt a synthesis and analysis of potentially useful strategies. It is hoped that (1) attention will be drawn to problems that still need solution and hence the analysis will serve a heuristic purpose, and (2) researchers will be made aware of some of the major scientific and logistical challenges that they will face in performing experiments with plant cells and tissues in Space.

It will not be easy but it is obviously better to approach the challenge armed, as it were, with as much information as possible.

III. Concepts and Semantics of "Minimal or Zero Growth"

Understandably, work with cell cultures of higher plants (or of lower plant forms, for that matter) is seldom framed in the context of maintaining cells with "minimal or no growth" at the beginning of, during, or at the end of an experiment. One can, of course, facetiously interpret "zero growth" as the state of being dead; indeed, Preyer, ca. 1891, referred to "zero growth" as being in an anabiotic state, i.e., lifeless and viable or lifeless and not viable, or "dead" (Leblos und lebensfahig = anabiotisch; Leblos und lebensunfahig = tot) (cited in Keilin, 1959). What is meant by "zero growth" in this review is that cells can grow but their potential to do so has been interrupted or severely curtailed. It follows that zero growth could be due to a shutdown of the system, for instance, for lack of an essential nutrient, a critical respiratory gas (e.g., oxygen or carbon dioxide), or a physiologically appropriate temperature.

The term "anabiosis," or "return to life," from the German Wiederbelegung, was applied years ago to the condition of "resuscitation, or resurrection of completely lifeless but viable organisms" (Keilin, 1959: 24). The term was extended to mean the "state of viable lifelessness." Similarly, "abiosis" was used for a while, but the term was viewed as "too close to the terms abiotic and abiogenesis which are used in a very different sense . . . dealing with the problem of the origin of life" (Keilin, 1959). In a now classic paper, Keilin (1959) summarized the historical relationships [ILLUSTRATION FOR FIGURE 2 OMITTED] of these terms and coined a new word, "cryptobiosis," meaning "latent life, for the state of an organism when it shows no visible signs of life when its metabolic activity becomes hardly measurable, or comes reversibly to a standstill."

In the context of recognizing the range of conditions that "can bring about different states of biosis, including latent life or cryptobiosis," Keilin (1959: 26) went on to list "loss of water, lowering of temperature, absence of oxygen and high salt concentration, or any combination of these factors" and summarized the relationships in the scheme in Figure 3. Halvorson (1961) early adopted Keilin's terminology but emphasized that "among various forms that exhibit reduced metabolism to a remarkable degree, there is no sharp dividing line between a state of hypometabolism and ametabolism. Halvorson (1961) emphasized that "in dealing with the state of cryptobiosis . . . one must necessarily refer to some of the borderline cases. A state of cryptobiosis is certainly not limited to any single area of biology. One can find good examples among plants and animals and micro-organisms . . . the seeds of plants; plant buds; the dehydrated forms of all kinds of micro-organisms, such as protozoa; bacteria; fungi and algae; viable forms rendered inactive by freezing; the cysts of protozoa; and the spores of bacteria." Certainly, plant cells grown in vitro qualify for inclusion in the list. Understandably, as a bacteriologist, Halvorson (1961) seems to have preferred the term "dormancy"; indeed, this term seems quite appropriate for microbial spores. But to most plant biologists, especially those dealing with the problems of dormancy, the word normally suggests problems associated with seed germination and bud break (Crocker, 1948; Vegis, 1964; Clutter, 1978).

Despite the above range of terminology, a literature search by computer using the words in Figures 2 and 3 as key words turns out to be unsuccessful in disclosing relevant literature that might shed light on the problem at hand. It may be that the various databases simply do not use these somewhat archaic terms. Similarly, there is very little, if anything, published in the Space biology literature that can directly guide a prospective investigator in experimentation with cultured cells in an environment such as a Space station. The field is simply too new and the average length of most Space biology experiments performed to date much too short to be of real value. It is clear, then, that the solution to our technical problems will have to be derived or adapted from areas peripheral to the one that might have, on the surface at least, been viewed as most closely related.

It is of no little interest that the literature of germplasm conservation, cryopreservation, secondary product biosynthesis, immobilization, and bioreactors provides the best leads. But before attention is directed to the specific strategies that might be utilized for our stated objectives, it will be worthwhile to analyze what one is perforce dealing with in terms of the biology of the cell cultures. Although this may seem obvious, certain points are critical to the emergence of any effective solutions.

IV. Plant "Cell" Cultures: Again a Case of Semantics

Aseptic culture techniques are more widely used now than ever in basic and applied plant research (see Krikorian, 1982; Mantell & Smith, 1983; George & Sherrington, 1984; Dixon, 1985; Han & Yang, 1986; Dalton & MacKenzie, 1987; Packer & Douce, 1987; Gelvin et al. 1988; Ammirato, 1989; Christen et al., 1989; Bhojwani, 1990; Carman, 1990; Chen et al., 1990; Ishimaru et al., 1990; Kysely & Jacobsen, 1990; Pollard & Walker, 1990; Rhodes et al., 1990; Zimmerman & Debergh, 1991; Negrutiu & Gharti-Chhetri, 1991; Hashimoto & Yamada, 1991; Payne et al., 1991; Compton & Veilleux, 1992; Hammerschlag & Litz, 1992; Lindsey, 1992; George, 1993; Herman, 1993; Redenbaugh, 1993; Armstrong, 1994; Dixon & Gonzales, 1994; Endres, 1994; Vasil & Thorpe, 1994; Bajaj, 1995). With its widespread reduction to a nominally routine technique and adoption as a common laboratory tool, plant tissue and cell culture per se has become somewhat less of a methodological preoccupation than it once was. But the fact remains that, despite a plethora of handbooks and laboratory guides, there is a considerable amount of what might best be termed elusive cell biology or art that is inevitably involved with the growing of plant cells in vitro. This in itself constitutes a limitation to plant cell and tissue culture being reduced to a "simple" technique, achievable with "routine" procedures. A serious consequence of the view that plant tissue culture is merely a tool is that many so-called systems are being accepted as highly reliable. Indeed, some cultures are widely and confidently accepted as "systems" (see De Vries et al., 1988; De Jong et al., 1992; Komamine et al., 1992; Kiyosue et al., 1993; Sato et al., 1995).

Inevitably, plant cell cultures vary in their physiological, growth, and developmental parameters and capacities. Indeed, cultures can and often do show tremendous heterogeneities because it is not easy to initiate and sustain cultures, and selection processes inevitably take place during cultivation and subculture (Steward et al., 1975; Yeoman, 1976; Dix, 1986, 1990; Krikorian & Smith, 1992; George, 1993). While attention is frequently drawn to the nominal advantages of uniformity, the frequently clonal nature of cell cultures, and so on, it is only much less often that the potential lack of uniformity and drawbacks of cell cultures, are mentioned, much less emphasized (Steward et al., 1958; Laetch, 1971; Torrey, 1971; Krikorian, 1982, 1988, 1989).

A. "CELL" CULTURES

When plant tissues are cultured in suitably agitated liquid media, single cells and small clusters of cells may frequently be found suspended in the liquid. This fact, established in the 1950s, enabled the question to be raised whether these suspensions, when filtered free from the large clusters of cells, could be manipulated by methods used routinely with microorganisms. Certain types of investigations previously conducted with microorganisms were viewed as being approachable with higher plant cells, and problems peculiar to higher plants were viewed as being approachable in new ways using simple microbiological techniques (Muir et al., 1954, 1958; Steward et al., 1958; Bergmann, 1959, 1960; Nickell, 1962; Ball & Joshi, 1965; Gibbs & Dougall, 1963, 1965; Blakely, 1964; Konar, 1966).

Although analogies were early drawn between plant cell suspensions and microorganisms, and these have since been repeated in the plant biological literature so often as to have become dogma to the unwary, it is well to underscore several very important differences. Many thousands of "average-size" bacteria could occupy the same volume as one average-size aseptically cultured carrot (Daucus carota) or Haplopappus gracilis cell. For example, most bacterial cells of the cocci type have a volume of about 1 [[[micro]meter].sup.3]; rods might be some three times that (e.g., see Salle, 1967: 66). Although there is tremendous variation in cell size among a population of "free" cells of plants, an average size would be, say, on the order of 100,000 [[[micro]meter].sup.3], in a spherical cell 60 [[micro]meter] in diameter or in a very elongated cell 20 [[micro]meter] in diameter and 300 [[micro]meter] long. Generation times in bacteria may be as short as 20 minutes (Salle, 1967), whereas in cultured plant cells, average generation times are more commonly measured in several hours or even days (Yeoman, 1976; Rost & Gifford, 1977; Gould, 1984; Francis, 1992, and references therein). Microorganisms are generally more uniform in size and shape than free plant cells. It may even be hard to find two cells exactly alike in a suspension of plant cells. It is often not difficult to find two cells of a suspension, in a small sample of cells, that differ in length by a factor of 10 or more (see Torrey, 1957, 1966; Steward et al., 1958; Ball & Joshi, 1965). For example, many "free" cells of certain carrot strains tend to be cigar-shaped, but oval to nearly isodiametric cells are also present. Even among elongate cells, it is a simple matter to distinguish cells from one another by one or more morphological features. It is a truism that cells in clusters tend to be smaller and more isodiametric than free single cells. Table II provides a summary of some of the more important differences between bacteria and plant cells in vitro.

A comprehensive account of the variation in the morphology of cultured cells would serve no useful purpose here. Suffice it to say that it would be ideal, for some purposes, if daughter cells separated immediately upon completion of cell division, and suspensions consisted entirely of single cells. However, as a plant cell divides, the daughter cells do not separate completely but tend normally to remain more or less firmly attached to the middle lamella. Methods are not yet available that completely circumvent this condition (Steward et al., 1958, 1975; Torrey & Reinert, 1961; Torrey et al., 1962; Street & Henshaw, 1963; Yeoman, 1986; Constabel & Shyluk, 1994). Indeed, it may be argued that it is more than likely doing violence to the inherent biology of a multicellular plant to expect that its constituent tissues and cells should be reducible in vitro so as to grow and persist in the form of single cells (see Barlow & Carr, 1984; Steeves & Sussex, 1989; Lyndon, 1990; Sachs, 1991; Fosket, 1994). But there are some plant cell cultures that are said to grow and multiply predominantly but not exclusively from single cells (see Street et al., 1971 for a description of a so-called nonadhesive "mutant line" of sycamore maple, Acer pseudoplatanus). Interestingly, addition of a polygalacturonase such as Macerozyme to a level of 0.05% w/v and a cellulase like Onozuka P1500 (0.01% w/v) did not prevent the subculturability of such cultures and allowed a "persistent high level of cell separation" (Street et al., 1971: 22). (It may be noted, however, that after some 37 years of personal experience with plant cell suspensions from many different species and visits to many tissue culture laboratories scattered throughout the world, I have never encountered one cell culture that could be sustained from absolutely single cells.)

[TABULAR DATA FOR TABLE II OMITTED]

Another inaccurate analogy that has been sometimes drawn between plant cells in suspension and bacteria and other microorganisms and unicellular algae is that cultured plant cells are genetically homogeneous (clonal) and that they generally express genetic information in ways comparable to those occurring in intact plants (see D'Amato, 1977; Krikorian et al., 1982; Snowcroft, 1985; Yamada & Mino, 1986; Karp, 1989, 1991, 1994, for genetic instability in cultured cells). In the latter supposition especially, in those instances where successes have been achieved, it was only because haploidy or a single dominant gene was involved, there was straightforward expression of the trait in question, and there was adequate plating efficiency and plantlet recovery from the "cells" (Negrutiu et al., 1984). It is true, on the other hand, that unless specific tailor-made precautions are taken to ensure that a culture is otherwise (e.g., Krikorian, 1982; Krikorian et al., 1983b; Fitter & Krikorian, 1988), higher plant cells have a tendency to be genetically diverse (see Larkin & Snowcroft, 1981; Krikorian et al., 1982; Snowcroft, 1985; Yamada & Mino, 1986; Larkin, 1987; Larkin et al., 1989; Peschke & Phillips, 1992; Kaeppler & Phillips, 1993; Karp, 1989, 1991, 1994, and references cited therein).

B. "UNITS"

There are strong indications that the adhesions between daughter cells weaken upon cell enlargement (Torrey et al., 1962; Street & Henshaw, 1963; Wallner & Nevins, 1974; Hayashi & Yoshida, 1988; Knox, 1992; Liners et al., 1994). Thus, it is to be expected that some cell lines are more friable and can release smaller units than others. In the late 1950s and early 1960s, when pioneer "cell suspension" and nominally "free cell" culture work was being carried out at Cornell University in the laboratory of F. C. Steward, terminology was adopted to handle the reality that "cells" of multicellular plants did not grow in suspension in ways that precluded the presence of cell clusters or aggregates (Blakely, 1964). More precisely, cultures very rarely, if ever, grow as single cells. "Unit," an umbrella term designating that each discrete member of a suspension, be it a single cell or a small group of cells, seemed to fit the need (Blakely & Steward, 1964). Adrian Srb, a Cornell geneticist and close friend of Steward, had used the term "unit" for discrete members of haploid yeast suspensions; it emphasized that microorganisms in suspension often occurred in small groups as well as single cells (Srb, 1956). For example, in haploid yeast (Saccharomyces cerevisiae), only some 31% comprised single cells, 45% were clusters of 2-8 cells, while some 24% of the "units" consisted of 9 or more cells.

Whereas it has long been recognized that it is virtually impossible to obtain cultures of higher plant cells that consist exclusively of, and are propagated or grown as, single cells, investigators appear to have been more reluctant to admit that this presents a hindrance to certain kinds of experimentation. At the very least, if suspensions were to consist only of single cells, certain experimental results could be interpreted in more precise terms. For instance, even counting cells in a given volume of suspension becomes more than a casual activity (Brown & Rickless, 1949; Brown & Broadbent, 1950; Letham, 1962).

Single graviresponsive cells would, of course, offer a unique and valuable system with which to study the cellular mechanisms involved, for instance, in gravitropism. In the roots of higher plants, many cell types participate in gravitropism, and the sites of graviperception and gravicurvature are generally substantially separated (e.g., in the rootcap and in the stelar zone of elongation, respectively). Both the size and the complexity of such systems make it difficult to analyze precisely the temporal, spatial, and biochemical factors that may or may not affect gravitropism. However, in single-cell systems, the gravitropic response is contained entirely within one isolated cell, and thus the range of possible influencing factors is substantially limited. Graviresponsive single-cell systems also possess a number of other characteristics that make them preferred subjects for the analysis of gravitropism. Single cells are excellent subjects for both light and electron microscopy. For example, it is much easier to obtain an undisturbed and sharper view of the protoplast of single cells using Nomarski optics than with multicellular units or tissues, because single cells are thinner and because of the proximity of their cytoplasm to the cell surface. For the same reasons, the native organization of a cell should be preserved by freezing techniques, such as freeze substitution, better than it is in multicellular units, tissues, or organs (Lancelle et al., 1986). Similarly, graviresponsive single cells are more amenable to experimental treatment such as microinjection and bathing with (bio)chemicals. Finally, because graviperception and curvature occur in close proximity in single-cell systems, it is possible to integrate the analysis of those processes. As already mentioned, however, there are a number of reasons other than the study of gravitropism at the cell level to warrant the study of higher plant cells in the space environment. Even here, however, the smaller the unit size being exposed to a discrete environment, the better. From the perspective of experimentation in [micro]-G, it is readily arguable that a single cell floating in a nutritive environment is perhaps best viewed as receiving external stimuli equally from all directions. In a unit consisting of two or more cells, a cell receives two different types of stimuli: those from the external medium and those from the adjacent or neighboring cell(s) [ILLUSTRATION FOR FIGURE 4 OMITTED]. All of this means that experiments must be carefully framed and interpretation of results rigorously limited to the facts of the status of cells being tested.

Similarly, although genetic heterogeneity may be expected to exist in any long-established cell suspension culture (D'Amato, 1977; Krikorian et al., 1982; Karp, 1991, 1994), it is a fact that the cells in any single, free group or unit could have derived from a single cell in the recent history of the unit. Small groups of cells in a suspension may arise as a result of division in free single cells, but this is recognized as a very rare occurrence, and it occurs only under special culture conditions (e.g., see Komamine et al., 1992). Much more commonly, small groups of cells fragment off as units from larger ones, sometimes through a process resembling abscission. In larger groups, any genetic heterogeneity would tend to be distributed among sectors, as a consequence of the manner in which plant cells divide. The cells resulting from a series of related divisions tend to remain attached and localized. When small groups split off from a larger, genetically sectored group, they would more frequently derive from within sectors than they would from the more restricted areas overlapping two genetically different sectors, purely on the basis of chance. This would be analogous to the behavior in intact plants where, as Neilson-Jones (1937: 546) has stated, the sectorial chimera "may be comparatively stable, so far as the branch is concerned, but is not so in respect to lateral branches, the character of any branch depending on the position in which the bud producing it arose on the main stem. Only those buds arising at the junction between the components will have a chimaeral structure; the rest, forming the majority, will be composed entirely of one or the other component" (see also D'Amato, 1977: 82 et seq.; Poethig, 1989).

C. PROTOPLASTS

Enzymatic isolations, en masse, of viable wall-less cells (protoplasts) from cells, tissues, and organs have been familiar since 1960 (see Krikorian, 1982; Fitter & Krikorian, 1983; Fowke & Constabel, 1985; Whitney & Beuschel, 1988; Power et al., 1989; Bajaj, 1994, and references cited therein). Cellulases and pectinases, generally derived from certain wood-degrading fungi, capable of dissolving the intercellular components and cell wall, are routinely used to release protoplasts from the tissues and organs of different plants and especially their aseptically derived and cultured tissues and cells (Fitter & Krikorian, 1983; Evans & Bravo, 1983). Having reconstituted walls around them, they are able to divide, proliferate, and, in some instances, eventually yield plants (Packer & Douce, 1987; Puite, 1988; Roest & Gilissen, 1989, and references cited therein). In still other cases, having obtained naked protoplasts, and having overcome the barrier to cell fusion inherent in the cell wall, fusion of protoplasts may lead to non-heterokaryotic nuclei and production of reconstituted cells from which new plants can grow. If the protoplasts were from haploid cells (as from anther or ovule culture), new diploid cells could be produced in a sort of artificial syngamy (parasexual or somatic hybridization). In select cases one envisions the exploitation of the methods in the production of novel plants, even between evolutionarily disparate organisms, by fusion breeding techniques (see Hammerschlag & Litz, 1992). Successful production and growth of protoplasts into plantlets is frequently the key feature in the implementation of genetic transformation/engineering studies (Gelvin et al., 1988; Filatti, 1990; Grierson, 1991; Negrutiu & Gharti-Chhetri, 1991; Kung & Wu, 1992). The environmental requirements for regeneration, chemical composition, and biochemical activity of walls are not well understood (Showalter, 1993), and regeneration media are often complex and must have a high osmoticum base in the form of additives like mannitol, sorbitol, inositol, sucrose, and such to prevent bursting of the wall-less cells (Fitter & Krikorian, 1983; Puite, 1988). Once walls are formed, the resultant cells are grown in the way specific for "cells" of the plant species in question. Bornman and Zachrisson (1982) were able to show that anchoring to microcarriers, such as those used with animal cells, works reasonably well with plants.

Again, it is possible to envision investigators wishing to maintain and grow cell cultures and attempt to regenerate and grow plant protoplasts or to study their products such as extracellular matrix. Iversen and co-workers (Iversen, 1985; Rasmussen, 1987; Iversen et al., 1990) and Zimmermann et al. (1988) have given considerable attention to protoplasts in the context of their vision of plant cell biology in Space, termed by them "Space biotechnology" but just as readily termed "plant cell biology" (see Mesland, 1987, 1992; Cogoli et al., 1989). Zimmermann thus far has been concerned primarily with electrofusion of higher-plant protoplasts (Vorhoek-Kohler et al., 1983). A major perceived advantage of carrying out fusions in [micro]-G includes the idea that it should be possible to use lower field strengths of the alternating electric field during the alignment or pre-fusion phase because thermal convection and mixing problems encountered at 1-G are minimized. Also, [micro]-G should allow improved performance of the electrofusion process once the breakdown pulse is applied. All this means that [micro]-G can theoretically be exploited to position and fuse plant protoplasts under minimal stress conditions.

Attempts have been made to grow protoplasts in Space to gain "basic knowledge of mechanisms of growth and development under microgravity" (Rasmussen et al., 1989). Results of an experiment flown in September 1989 on Kosmos 2044 (Biokosmos 9), by a collaborative group of Scandinavian and Soviet workers using fresh protoplasts prepared from suspensions of embryogenic carrot cells and from hypocotyls of rape (Brassica napus), are of particular interest to us in the context of this review, since they used the technique of protoplast immobilization in 1% agarose (alginate solution) along with a low temperature of 4 [degrees] C to slow down wall regeneration. Specifically, at 4 [degrees] C only 6% of the cells had regenerated walls, as opposed to 60%, at 25 [degrees] C (Rasmussen et al., 1990). In that experiment, a significant number of "flight" protoplasts did not regenerate walls (in carrot 56% and in rape 82%, compared to ground controls, regenerated walls and grew). Moreover, cellulose analyses showed a decreased amount. Similarly, peroxidase and total protein levels were much reduced as well in the flight samples. A significant decrease in plantlet regeneration was observed (Rasmussen et al., 1989, 1990). Unfortunately, 1-G centrifuge controls (however imperfect they might be) were not performed, and hence it is not possible to unequivocally attribute the results to [micro]-G. It could well be that indirect factors or spaceflight conditions not readily simulated on Earth (Mesland, 1992) were responsible (Table III).

Special but relatively unsophisticated apparatus was designed by the Scandinavian group to accommodate the protoplasts (for photographic and diagrammatic details of this apparatus see Rasmussen, 1987: 109, [ILLUSTRATION FOR FIGURE 3 OMITTED]; Rasmussen et al., 1989: [ILLUSTRATION FOR FIGURE 6 OMITTED]; Iversen et al., 1990: [ILLUSTRATION FOR FIGURE 1 OMITTED]; Rasmussen et al., 1990: [ILLUSTRATION FOR FIGURE 1 OMITTED]). The "Zimmermann apparatus" is more specialized (Zimmermann, 1982) and was at one time commercially available.

V. Low Temperature Strategies

A. CRYOPRESERVATION

It has been known for many years that plant cells can tolerate chilling and even freezing (see Molisch, 1897 [in translation, 1982]; Levitt, 1972, 1980; Withers & Street, 1977; Withers, 1980; Kartha, 1982, 1985; James, 1983; Li, 1984; Grout & Morris, 1987). The enormous potential of applying zero-growth or minimal-growth strategies (routinely used for gene banks; see Ellis et al., 1985) for maintaining nonseed germplasm has been recognized for many years (Withers, 1980, 1983, 1985, 1986; Kartha, 1982, 1985; Seitz et al., 1985; Adams & Adams, 1991; Kartha & Engelmann, 1994).

At the outset of the research work on cryopreservation, efforts in this area were focused equally on callus, suspension cells, somatic embryos, and shoot tips or apical meristems. Attention was paid to solving such problems as (1) which cryoprotectants to use, e.g., dimethylsulfoxide (DMSO) or glycerol or polyethylene glycol (PEG) or sugars such as glucose or sucrose and various combinations thereof, sometimes including proline etc.; (2) cooling rates, ranging from very slow at, say, 1 [degrees] or 2 [degrees] C per rain for cell suspensions, callus, and organized small propagules, to 50 [degrees] to [greater than]1000 [degrees] C per min for certain stem tips; (3) details of the final quenching of initially slowly frozen samples; (4) maintaining very low temperature in liquid nitrogen (order of -150 [degrees] or -196 [degrees] C in the liquid or vapor phases, respectively); and (5) rapid thawing (usually with a warm water bath at 35-40 [degrees] C). Efforts to cryopreserve in vitro cultured and multiplied [TABULAR DATA FOR TABLE III OMITTED] shoot tips were typically constrained by low levels of success; certain species, especially those of tropical and subtropical genera, were frequently designated as more or less completely recalcitrant (see Engelmann, 1991). Also in that period, the number of recoverable apices after thawing even in those cases where freezing was nominally successful was very unpredictable (Sakai, 1984).

Now there is a vast literature that deals with cryopreservation of apical meristems of higher plants, and there has been considerable attention given to a fairly broad range of cell types, including various stage somatic embryos and even protoplasts (Kartha, 1985; Kartha & Engelmann, 1994; Benson, 1995; Day & McLellan, 1995 and references cited therein). While progress at the applied level has been relatively slow until recently, investigator persistence shows signs of paying off. For example, a conventional freezing protocol is described by Gnanapragasam and Vasil (1990) for embryogenic suspension cultures of sugarcane, a tropical species very sensitive to low temperatures (Moore, 1987). Centrifuged suspensions were chilled on ice, and pre-chilled cryoprotectants were added to the cells over a period of one hour with agitation. Aliquots of this suspension were dispensed in small cryogenic ampules and frozen in a commercially available apparatus at a rate of 0.5 [degrees] C per min to -40 [degrees] C. After 45 min at -40 [degrees] C they were stored in liquid nitrogen. Cells were reestablished and somatic embryos regenerated. The success with sugarcane emphasizes that nominally recalcitrant tropical species can yield even to "traditional" cryogenic methods.

It would appear reasonable that cryopreservation could provide a major solution to some of the problems outlined here in section II. For the time being, however, conventional cryopreservation - i.e., preservation in the frozen state at very low temperatures, namely, above solid carbon dioxide or dry ice (-79 [degrees] C), in low-temperature deep freezers (80 [degrees] C or below), in vapor phase (ca. -140 [degrees] C), or in liquid nitrogen (-196 [degrees] C) - has been all but excluded for the time being from consideration in the context of managing plant cell and tissue cultures for experimentation in Space. Reasons for this are that power requirements are presently prohibitive and the logistics of carrying out necessary procedures impractical (Reinhoud et al., 1995). Moreover, while the number of species amenable to cryopreservation is increasing, it is still relatively small (see Kartha & Engelmann, 1994: tab. 1). Perhaps of greatest importance is that there is the very real biological problem of what reestablishment of cultures from cryopreserved cells means in the context of what is currently technically feasible. For our purposes, cryopreserved cells should ideally, upon regrowth in aseptic culture, show minimal change physiologically and biochemically from the cells as they were initially "stopped." This is no modest objective, and to expect it to be achievable more or less readily might be doing basic violence to the known facts of plant cell growth in vitro. In virtually all cases of regeneration from cryopreserved cultures, little if any attention has been paid to how much new growth needs to occur in order for regeneration to be achieved. For example, cryopreserved stem tips can sometimes regenerate new plants, but apical growth is curtailed in many instances and is replaced by callus proliferation, which, in turn, can sometimes be manipulated to regenerate plants. Thus, materials so generated are not directly descended from the apical material initially frozen. In the context of achieving zero-growth maintenance for Space plant cell biology experimentation using cultured cells, it is obviously undesirable to regenerate plants indirectly from cells and tissues that may only poorly reflect what they had experienced in Space. The more direct the regeneration the better.

B. "VITRIFICATION" AND "HYPERHYDRICITY" IN THE TISSUE CULTURE LITERATURE

More recent successes in cryopreservation derive from an approach termed "vitrification." Before "true" vitrification is discussed, however, it should be pointed out that the term has been used extensively in the plant tissue culture literature to refer to a condition or physiological state of cells typified by a "glassy" look. Tissues or organs such as leaves, especially in micropropagated plants, become translucent or almost transparent and in many cases abnormal organ histology is observable: decrease or lack of palisade layer (especially in young leaves), presence of a poorly formed or even totally absent cuticle, and poor vasculature development (Debergh et al., 1981; Kevers et al., 1984; Paques & Boxus, 1987; Ziv, 1991). Necrosis is not an uncommon consequence of this condition; consequently, there is considerable concern in the commercial micropropagation sector, for it is virtually impossible to establish vitreous plants ex vitro.

The causes of vitrification at the cellular level are poorly understood, but there is considerable evidence that laboratory manipulations and technique are involved in fostering its manifestation (Debergh et al., 1981; Kevers et al., 1984; Phan & Hegedus, 1986; Leonhardt & Kandeler, 1987; Leshem et al., 1988). Some laboratories encounter the problem more than others (Ziv, 1991, and references cited therein). Whatever the cause(s), there are some empirical measures that can be put in place to avoid the condition (Paques & Boxus, 1987). All of the measures are more or less based on efforts to bring about an increase of the water potential of the medium in which materials are grown. These include, for example, improved aeration (Jackson et al., 1991) and reduction of water absorption by increasing the concentration of agar in a medium (Bornman & Vogelmann, 1984; Feito et al., 1994). Sigma Chemical Co. (St. Louis, Missouri) even offers a so-called Antivitrifying Agent (catalog EM2, A-0807). Little information, other than a rough elemental analysis, is provided about this proprietary preparation used to protect against the vitreous condition. Presumably its activity is due to its ability to adjust the osmotic environment.

More recently, there has been an attempt to replace the noun "vitrification" with "hyperhydricity" (Debergh et al., 1992). The latter term, along with its adjectival form "hyperhydric," was used many years ago in connection with plant tissue culture (e.g., Paupardin & Gautheret, 1954). Resurrection and widespread adoption of the term should allow vitrification to be restricted to its proper place in the context of cryobiology.

C. "VITRIFICATION" IN A CRYOBIOLOGICAL CONTEXT

Fahy et al. (1984) define vitrification as "the solidification of a liquid brought about not by crystallization but by an extreme elevation in viscosity during cooling. During vitrification the solution is said to become a glass; translational molecular motions are significantly arrested, making the effective end of biological time but without any of the changes brought about by freezing. An organ capable of being vitrified need no longer satisfy classical constraints of optimal cooling and warming rates, but instead can neatly escape both 'solution effects' injury and the dangers of intercellular cooling. Vitrification of relevant aqueous solutions using cooling rates that are realistic for whole organs requires the presence of high concentrations of a crycprotective agent. The primary challenge that must be met in order to successfully vitrify organs, therefore, is to make the required concentrations of cryoprotectant non-toxic to the organism" (for an in-depth analysis of the principles and methodology of vitrification, see also Hirsch, 1987; Steponkus et al., 1992; Kartha & Engelmann, 1994).

Vitrification procedures thus eliminate the need for slow freezing and enhance the possibility of materials being cryopreserved by direct transfer to liquid nitrogen. This in turn obviates the need for controlled freezing apparatus and expensive equipment. Nishizawa et al. (1993) have shown that cryopreservation of embryogenic cells of asparagus and subsequent plant regeneration are achievable using vitrification procedures. These workers used a mixture of glycerol and sucrose in water as a cryoprotectant; cells were dehydrated, frozen in liquid nitrogen, thawed at 40 [degrees] C, and recovered at a level higher than 80%. Although the regenerated plants "locked" similar to control plants, additional studies are acknowledged as being needed to confirm this. Matsumoto et al. (1994) have reported high regeneration of Japanese horseradish (Wasabia japonica) apical meristems cryopreserved by vitrification. Apices of many potato varieties can now also be cryopreserved and new growth re-initiated from the same frozen apex without intervening callus. A cryopreservation project with the objective of placing the entire germplasm collection of the International Potato Center (CIP) in Lima, Peru, has been undertaken (P. Steponkus, pers. comm.).

Carefully selected, even novel, cryoprotectants and strategies for preconditioning of materials will play increasingly significant roles in the achieving of success in the foreseeable future (Benson, 1995). Clearly, the treatment(s) preceding freezing are very important, and such techniques as culturing under "hardening" conditions or prefreezing to an intermediate temperature prior to liquid nitrogen often are essential. The cell type and time of sampling is also very important. For instance, there is a higher survival of small, densely cytoplasmic cells in contrast to those that are highly vacuolated. In terms of their stage in the cell cycle, cells in late lag and early exponential phase seem to survive better. Cells in G1 reportedly have a better survival potential (Francis, 1992). Cell density, freezing rate, thawing rate, and the nature and concentration of the cryoprotectants used are also major considerations (Chmiel et al., 1988; Grout, 1995). For purposes of experimentation in Space, it would be preferable (as has already been emphasized) to have as direct a regeneration as possible.

The logical conclusion from all the above is that, despite promising developments, the requirements for cryopreservation via vitrification are still demanding and it is not apparent whether procedures can be readily automated or whether there is a potential to simplify the process for use during or at the end of a Space experiment. Cryopreservation by vitrification needs to be carefully followed so that significant new developments are quickly recognized and appreciated for what they are worth vis-a-vis their potential for incorporation into management schemes for Space biology cell culture experiments. It may well turn out that an adaptation of the process or "intermediate" procedure in which the principle of vitrification is capitalized upon but liquid nitrogen is avoided may provide a valuable avenue for investigation.

D. CHILLING OR LOW TEMPERATURE FOR MINIMAL- OR ZERO-GROWTH MAINTENANCE

Attention will now be focused on chilling vs. freezing (see Graham & Patterson, 1982; Grout & Morris, 1987). Brauner and Hager (1958) showed that the gravitropic stimulus to roots could be "stored" in 3-4-day-old sunflower seedlings for some 12 hours at 4 [degrees] C and that the stimulus is expressed as soon as the material is exposed to room temperature (ca. 20 [degrees] C). Respiratory intensity is diminished considerably by lowering of temperature. A rule of thumb seems to be lowering of temperature by 10 [degrees] C lowers respiratory intensity by about 2.5 times (see, e.g., Burzo, 1980). In 1974, Krikorian initiated studies aimed at managing embryogenic cells of wild carrot, or Queen Anne's lace (Daucus carota subsp. carota), for an experiment destined to be carried out in November 1975 on the Soviet biosatellite Cosmos 782 (see Krikorian & Steward, 1978, 1979; Krikorian, 1991). Embryogenic cells, when distributed in a nutrient medium made semi-solid with 1% agar and distributed in small (50 mm diam.) plastic petri dishes, were able to withstand 4 [degrees] C [+ or -] 2 [degrees] C for well over a month with virtually no progression in development. Representative plated carrot cells that were pretreated at 4 [degrees] C for 0, 7, 14, 21, and 28 days in darkness in a refrigerated incubator and subsequently removed to 22 [degrees] C for evaluation of subsequent growth and development for the same duration (i.e., 7, 14, 21, and 28 days) are shown in Figure 5. The low temperature did not interfere with the ability of the cells to continue development when they were transferred to the permissive temperature (Steward & Krikorian, 1978). Carrot is, of course, a biennial species and its cells, even in the in vitro state, might well be expected to tolerate cold temperatures better than cells of some other plants (see Dix & Street, 1976; Dix, 1977, 1979). Also, resumption of development from embryogenic initial cells curtailed in their progression of growth was at the beginning of the experiment cycle. There is less certainty whether somatic embryos maintained in the cold during the experiment or at the end would have necessarily behaved in a similar fashion. Genetic propensity to adapt to cold (Graham & Patterson, 1982; Thomashow et al., 1990; Thomashow, 1994) and the level of prior development will influence the ability to tolerate exposure to cold, and would be expected to be related to the dimensions of the units or structures in question. Younger, less developed, and hence more vulnerable materials are the most sensitive; those more advanced would be more tolerant (Krikorian & Weidenfeld, unpubl.).

The idea of using low temperature for minimal-growth storage of organs has been exploited with various but generally morphologically fully developed in vitro systems such as stem tips for some time (Westcott, 1981; Withers, 1983; Englemann, 1991; Kartha & Englemann, 1994, and references cited therein). Even here, in each of the instances studied, the level of efficiency has been highly variable and survival has in some cases been very poor. Even in good cases, recovery has never been anywhere near complete, and in many cases the growth in vitro has been only retarded. However, in those cases where recovery has been followed and evaluation made; after growth in the field, the indication is that this kind of conservation effort has promise despite its lack of universal applicability.

In the case of minimal-growth storage with undifferentiated callus tissues, Hiraoka and Kodama (1982) showed that the ability to produce secondary metabolites was not altered in most of their tissue culture strains tested for growth and biosynthetic potential after storage for up to 6 months in an ordinary refrigerator at 4 [degrees] C [+ or -] 3 [degrees] C. While recovery and biochemical fidelity was often excellent in many cases, Hiraoka and Kodama (1982) concluded that "the ability of callus to tolerate low temperature depends highly on a callus strain." Also of interest is that these workers were able to achieve rooting in a culture of Bupleurum, an umbellifer that ordinarily would have lost capacity to regenerate roots after a few passages.

For the German Spacelab mission (so-called D-1 mission on Space Transport System-61A, October 1985), Theimer et al. (1986) were able, like Krikorian and Steward (1978), to utilize low temperature to maintain their embryogenic cell cultures of anise (Pimpinella anisum), a biennial member of the Umbelliferae, in a quiescent state. Cells from sieved and washed suspensions were distributed onto sterile filter paper soaked with nutrient medium, and the whole was placed on a medium made semi-solid with 0.7% w/v agar. Cultures were chilled to 6 [degrees] C in a "cooling box" and stored in a mid-deck locker in the Space Shuttle, and removed for exposure to [micro]-G at 26 [degrees] C to activate the experiment.

Chilling was also used in the D-1 mission to retard the growth of the protozoan Paramecium tetraaurelia (Richoilley et al., 1986, 1988). Cultures were kept at 9 [degrees] C and then exposed to 22 [degrees] C [+ or -] 0.5 [degrees] C in the European Space Agency (ESA) "Biorack incubator." Their experimental protocol involved using a plastic-bag culture apparatus to grow paramecia in 0.65 ml liquid medium samples. Although their experimental procedures called for fixation of the material, no specific mention was made of the ability to recover live material from the flight. Even so, it may be understood that the low temperature had no adverse effects on the paramecium cultures, compared to growth data for ground controls (Richoilley et al., 1986: [ILLUSTRATION FOR FIGURE 2 OMITTED]. For their so-called Cytos 1 experiment which was set up in Moscow, chilled and flown to the Baikonour launch site, launched and transferred to Soyuz 27, and maintained until Soyuz-Salyut 6 docking in Space, these same workers (Planel et al., 1990) used a low temperature of 8 [degrees] C to halt growth prior to exposure to a permissive growth environment in Space of 25 [degrees] C [+ or -] 0.1 [degrees] C. While the precise duration of chilling was not reported, I estimate that it was on the order of about a week.

Chilling the slime mold Physarum polycephalum was also studied from the perspective of experiment management for the D-1 mission. Development of plasmodia was stopped by slow cooling to 6 [degrees] C; 14 hours before flight, the cultures were stored in a so-called passive thermal control unit (PTCU). The experiment was activated in flight on the fourth day by placing cultures in the Biorack incubator at 22 [degrees] C. Earlier investigations on Earth had shown that the effects of rapidly changing temperature (14 [degrees] to 30 [degrees] C at intervals of 2 [degrees] per minute or so) on plasmodial contraction were completely reversible when contraction frequency was at least between 10 [degrees] and 28 [degrees] C (Wohlfarth-Bottermann, 1977). A nomogram provided by Block et al. (1988: fig. 10) emphasizes how important it is in the case of Physarum to have precise control over the temperature if one is to achieve minimal adverse effect on level of survival. Also, Block et al. (1988: 65) point out that "the samples had been stored in the Biorack cooler, some parts of which also had temperatures slightly below 4 [degrees] C. . . . This was close to the time-temperature limit for survival for Physarum, [and] only a few plasmodia survived this treatment. . . . For the planned . . . Biorack II, a real 5 [degrees] C PTCU will be provided to overcome the difficulties with the storage temperature." One can anticipate that cultured higher plant cells will show the same kinds of sensitivity to temperature excursions. The more closely controlled the temperature, the better.

Finally, it may be recalled that protoplasts were chilled as part of a management strategy for Space experimentation by Iversen and co-workers (see Rasmussen et al., 1990). Clearly, chilling offers considerable opportunity to manage plant cell and tissue cultures and other organisms. In some instances it is simple and straightforward and can be performed at a single reduced temperature level; in other cases a fluctuating regime of various low temperatures seems to be useful or even required. In still others, lowered temperature is more of a problem. This emphasizes that while chilling affords considerable opportunity to stop or dramatically slow down growth, this must be closely tied to the biology of the system. It follows that any engineering of hardware associated with chilling must be precise enough to maintain temperature tolerances within specified limits.

VI. Sub-Optimal Oxygen Strategies

A. REDUCED OXYGEN

Unlike the extensive literature that applies to microorganisms (Onken & Liefke, 1989; Levett, 1991), there is much less detailed documentation that aeration rates affect growth of plant cells in suspension (e.g., see Steward & Bidwell, 1958; Ammirato, 1983; De-Eknamkul & Ellis, 1984; Hegarty et al., 1986; Chen et al., 1987; Shape et al., 1989; Tate & Payne, 1991; Gao & Lee, 1992; Jay et al., 1992; Schlatmann et al., 1994; Schripsema & Verpoorte, 1995). Cultured cells generally seem to profit by elevated oxygen tensions and hence respond adversely to oxygen limitation by not growing; this tendency could perhaps be developed in special cases as a means of conserving cells (Mitz, 1979; Bridgen & Staby, 1981; Furuya et al., 1984; Forster & Estabrook, 1993). Altered aeration has considerable impact on secondary metabolism (DiCosmo & Towers, 1983). But as has been emphasized, the single most important objective of conserving plant cells in culture in Space and their subsequent return to Earth is to be able to examine them in as unperturbed a metabolic and morphological state as possible. Significant metabolic changes are sure to occur in cells when the gaseous environment is changed (see Bullough, 1952; Stiles, 1960; Hook & Crawford, 1978; Snape et al,, 1989; Jackson et al., 1991; Pell & Steffen, 1991; Gao & Lee, 1992; Crawford, 1992; Lainbers & van der Plas, 1992), although in some cases, such as breaking bud dormancy and germination of certain species, there are beneficial effects of anoxia or severe hypoxia, i.e., less than 1-2% oxygen (see Come et al., 1991). Thus, any strategy that uses gaseous oxygen deprivation alone as a way to manage cells for zero- or minimal-growth maintenance, especially at the end of an experiment, needs to be carefully considered for its ability to effect unwanted change (van't Hof, 1970; Prince, 1989).

Even modification of the ambient gaseous environment with regard to low temperature storage of fruits such as apples is linked with delaying senescence and the onset of the climacteric and hence does not seem relevant here. This is especially so since even in the case of intact organs and plant parts with well-formed epidermis and cuticle there are many potentially undesirable effects and physiological "disorders" and consequences due to modification but incomplete suppression of the respiratory rate via change in the ambient gaseous environment. (For a historical treatment of the use of gases in the storage of fruit, especially apples, see Smock, 1970; also Kader, for a physiological treatment of postharvest biology, see Kader, 1986, 1992; for a discussion of modified atmosphere packaging of horticultural commodities, see Prince, 1989.) In the case of aseptically cultured cells and tissues with minimally developed or totally absent epidermis, the gaseous relationships become even more difficult to control and assess.

It will be seen below ("Mineral Oil Preservation or Inactivation of Cultured Plant Cells"), however, that the principle of reduced oxygen plays a role but is generally most effective when coupled with decreased temperature. Also, there is some indication that low oxygen partial pressures have potential value for stem tip conservation (Bridgen & Staby, 1981), but very little is known about the mechanism of action or its effects on metabolism of cells (Jamieson, 1980a, 1980b; Andre & Richard, 1986; Calderon & Barkai-Golan, 1990).

For our purposes, then, it may be argued that no special efforts should be made to investigate the direct use of reduced gaseous oxygen as a means to conserve plant cell cultures. Attention will, of necessity, be paid to gaseous environment when specifications for oxygenation or aeration are stipulated (Hulst et al., 1985; Prince, 1989; Prince et al., 1991).

B. MINERAL OIL PRESERVATION OR INACTIVATION OF CULTURED PLANT CELLS

The use of mineral oil (paraffin oil) as an overlay on agar slants of fungi as a means of slowing down growth and preventing desiccation has, over the years, been reported to be successful in a number of species. With the availability of lyophilization and cryopreservation, mineral oil overlay has faded out of the picture fully in terms of microorganisms. The technique of mineral oil overlay is said to have originated in 1918 with Ungermann, who initiated conservation of pathogenic bacterial cultures in dilute sera by covering them with a layer of mineral oil. The method was extended to semi-solid media by Michael in 1921 (see Morton & Pulaski, 1938, for references and a list of 45 diverse bacterial cultures so conserved for long periods). Sherf (1943) used the technique on filamentous fungi and was able to keep Fusarium and Alternaria without losing their pathogenicity for 6 months and even longer. Buell & Weston (1947) provided a comprehensive summary of mineral oil conservation work carried out on an extensive and diverse (some 1800 species) "Tropical Fungus Culture Collection" at the Harvard Biological Laboratories, isolated in connection with the study of "tropical deterioration" of Quartermaster Corps materiel during World War II.

The mineral oil technique offers a significant advantage in that it is simple and "low-tech," and hence should be relatively straightforward to implement. Fungal material to be conserved, especially non-sporulating mycelia, is first grown on an appropriate nutrient medium agar slant, and then heavy mineral oil is applied. Buell and Weston (1947) state "Parke-Davis' heavy mineral oil of exceptional purity, high Saybolt viscosity of not less than 330 at 100 [degrees] F [38 [degrees] C] and specific gravity around 0.8-0.9, autoclaved for 45 min in half-filled 250 ml cotton plugged Erlenrneyer flasks is applied." (They also point out that hot air is acceptable for sterilization: 150 [degrees] C for 1.5 hours.) These authors note that the oil was "often cloudy as a result of moisture vapor accumulated during autoclaving but was easily cleared either by letting the flasks stand for 1-2 days, or by heating them for a few minutes over an electric stove." The work of Buell and Weston (1947) emphasized that "if the organism is submerged under too deep a layer of oil, it may be smothered. Therefore, it is most important that the oil be poured in to a depth of about 1 cm above the tip of the slant." Successful storage for a couple of years was not uncommon under these conditions. The mineral oil method was also tested at Harvard by J. T. Bonner on the slime mold Dictyostelium discoideum, an organism said to profit by frequent transfers; cultures were able to last some 12 months and yielded vigorous cultures. Similarly, Blakeslea trispora, another Phycomycete and reputedly a very fastidious organism, remained viable and could be conserved under oil for 10 months; material that had been kept 12-16 months did not survive, however (Buell & Weston, 1947).

It is significant that each of these workers emphasized that no undesirable modifications were encountered on subculture and that all cultures gave typical, vigorous development and sporulation. It is also significant that they kept the oil-overlaid culture "upright at room temperature or preferably at about 10 [degrees] C." Unfortunately, no data were provided as to the advantage of using cold in combination with the mineral oil overlay, but one can see that this could offer an effective strategy for dealing with our problem of conserving cell and tissue cultures in Space.

Recovery of the culture to a subculturable mode is merely stated as having been achieved by pouring off the oil. Sherf (1943) made a special point of mentioning that "before streaking, the loop was held against the inside wall of the test tube above the agar to remove much of the oil adhering to the cells."

Edwards et al. (1947) showed that the oxygen consumption of a fungus culture submerged under 1 cm oil mineral oil "was about 10% of normal within a few hours, and thereafter declines very gradually over a period of many months." These authors emphasized that the mineral oil not only prevented the evaporation of water from the agar medium but also served to reduce the supply of available oxygen to the culture, thus reducing the respiratory rate and bringing about a retardation of growth. Also, staling products, "ordinarily increasingly harmful as a culture ages, will accumulate less rapidly and it seems probable that when formed, they will not oxidize so quickly or to the degree that they would in the usual type of culture."

Table IV provides solubility data for carbon dioxide, oxygen, and nitrogen in extra-heavy (specific gravity 0.890-0.895 at 15 [degrees] C), white medicinal mineral oil (United States Pharmacopoeia, U.S.P. grade) (Kubie, 1927). Even though the values are low, they are considerably higher than might have been guessed. Kubie (1927) pointed out that the slow rate of diffusion of carbon dioxide through mineral oil when it is saturated with 1 atmosphere of carbon dioxide, and presumably the rate of diffusion of the other gases, is the main cause of the "protective virtue" of the oil when it is used to separate a physiological fluid from air.

Rodnight (1954) gives the solubility of oxygen at S.T.P. in heavy mineral oil (sp. gr. 0.835) at 38 [degrees] C as 0.098 [+ or -] 0.0017. Since higher plant cells are normally grown at lower temperatures (say 20-28 [degrees] C), the solubilities that concern us are closer to the values given by Kubie (1927).

Caplin (1959) reported being able to conserve cultures derived from secondary phloem carrot root grown on semi-solid White's nutrient medium at 26 [degrees] C [+ or -] 1 [degrees] C using heavy U.S.P. grade mineral oil in amounts ranging from about 4 mm depth to 45 mm depth. Subsequent ability to grow after prolonged submersion under oil of some 3-5 months was not adversely affected. In fact, growth under mineral oil was better than growth under ordinary liquid nutrient medium superimposed on callus tissues grown on an agar medium. Caplin (1959) was also able to show that growth of tissue under mineral oil could reach levels equivalent to controls in air, provided the oxygen supply over the mineral oil was enhanced by flushing with 100% oxygen. Because the volume of dry oxygen that can dissolve in water under standard pressure and a temperature of 26 [degrees] C is 0.028 (about 4 times the solubility of oxygen in water), Caplin (1959) concluded that the reason for better growth of cultures under mineral oil than under stationary liquid nutrient medium was the oxygenation. Caplin (1959) states that some 30 "strains" of plant tissue culture had been "maintained in the laboratory under mineral oil, subcultured at intervals [TABULAR DATA FOR TABLE IV OMITTED] of 3-5 months, for longer than 3 years without apparent change in growth characteristics after transfer to culture medium in air."

Caplin (1959) also reported that light mineral oil was as good as heavy mineral oil. Liquid petrolatum, known as heavy liquid petrolatum, liquid paraffin, white mineral oil (U.S.P. grade) has a specific gravity of not less than 0.860 and not more than 0.905. Its kinematic viscosity is not less than 38.1 centistokes at 37.8 [degrees] C. Light mineral oil or light liquid paraffin, on the other hand, is supposed to have a kinematic viscosity of not more than 37 centistokes at 37.8 [degrees] C and a specific gravity between 0.828 and 0.880. Otherwise, both materials are nominally identical and either may contain 10 mg/l (10 ppm) of [Alpha]-tocopherol as a stabilizing agent. Both are listed in the Sigma catalog: Heavy white oil, Cat. Number 400-5, is listed as having a viscosity of 340-360 Saybolt Universal seconds and density of 0.88 gm/ml; the light white oil, "suitable for nujol mulls for infrared spectroscopy," Cat. Number M 3516, has a density of 0.84 gm/ml. These values are emphasized since if in a given situation it turns out that the light mineral oil is equivalent to the heavy in effectiveness for plant cells and tissues, then its more fluid qualities could be advantageous, e.g., for addition or removal of oil or for pumping oil through narrow-diameter tubing, etc.

It is significant that more recently, some investigators interested in in vitro conservation have noted the great potential of mineral oil overlay in low-tech situations (Englemann, 1991; Constabel & Shyluk, 1994). Augereau et al. (1986) have also carried out more recent studies with mineral oil overlay using a range of germplasm. While they are strong advocates of the mineral oil method, they noted that tissues under oil could show higher chlorophyll content than controls showed. They offer the hypothesis that "the carbon dioxide released during fermentation accumulates on the callus surface transfer of C[O.sub.2] through oil is very slow. Some small bubbles can be observed on the callus surface, it is probably C[O.sub.2] accumulation and it could induce and/or increase photosynthetic activity. The absence of [O.sub.2] forbids direct use of the sucrose present in the media. Therefore, photosynthetic activity induced by C[O.sub.2] accumulation produced by a sugar fermentation allows the growth."

VII. Constraining Gels

A. IMMOBILIZATION OR ENTRAPMENT

Interest in producing commercially valuable secondary plant products or metabolites in bioreactors has fostered research on entrapment or immobilization of cultured cells in constraining gels of various sorts. The fact is that the whole field of evoking tissue or organ-specific biosyntheses in vitro is fraught with many difficulties (cf., e.g., Weber et al., 1992; Schripsema & Verpoorte, 1995) and few systems have proven themselves economically viable. [It should be pointed out at the outset that few details are available on the effects of immobilization on plant cell cycle kinetics and metabolism (see Cabral et al., 1983; Brodelius, 1983a, 1983b, 1985, 1988; Hulst et al., 1985; Rhodes, 1985; Webb et al., 1986; Rosevear & Lambe, 1985, 1986; Constabel & Vasil, 1987; Mavituna et al., 1987; Robins & Rhodes, 1988; Chmiel et al., 1988; Charlwood & Rhodes, 1990; Karel et al., 1990; Tabata, 1991; Christen & Gibson, 1987; Furusaki & Seki, 1992; Kutney, 1993; Constabel & Tyler, 1994).]

It has been suggested that entrapment in and of itself imposes physical stress on plant cells and this in turn fosters secondary product synthesis and excretion (Fukui et al., 1983; Rhodes, 1985; Ketel et al., 1987; Bringi & Schuler, 1990; Hahn-Hagerdahl, 1990; Hamer, 1990). It is in this context of stimulating production that would otherwise not occur or would occur to a lesser extent than desired that investigators have sought to use immobilized cells (Fowler, 1987; Doran, 1993)! Various cell immobilization technologies hold some promise for our objectives (Brodelius, 1983a, 1983b, 1985, 1988; Schuler et al., 1983; Fink et al., 1983; Mattiasson, 1983; Prenosil & Pedersen, 1983; Rosevear & Lambe, 1985, 1986; Lindsey & Yeoman, 1985, 1986; Yeoman, 1987; Christen & Gibson, 1987; Bramble et al., 1990; Lambie, 1990; Scragg, 1990; Yeoman et al., 1990; Furusaki & Seki, 1992).

Lambie (1990: 270-271) provides a succinct summary of this area of research and draws attention to the advantages and disadvantages of immobilization or entrapment, especially from the perspective of handling cells that produce secondary products in bioreactors: "Three methods of entrapment have been described: (1) the cells may be entrapped on films or webs of material; (2) the cells may be entrapped in gels."

I will interrupt here with a quote from Brodelius (1985: 32) on the three basic categories of gel-forming entrapment procedures: "Gel formation by ionic cross-linking of a charged polymer. Gel formation by cooling of a heated polymer. Gel formation by chemical reactions. Alginate is an example of the first, agarose and agar of the second, and gelatin (cross linked with glutaraldehyde) and polyacrylamide of the third group. Carrageenan is a combination of the first and second groups. Up to at least 50% wet weight of plant cells can be readily immobilized in these various gels."

Continuing from Lambie (1990):

(3) the cells may be entrapped in porous or three-dimensional reticulates, e.g. like the material used for pan scrubbers. The entrapment of cells on webs or films results in low cell density in the reactor. Moreover, the cells may be easily detached. The second method has been used most widely, namely entrapping the plant cells in gel beads. The gels used include agar, agarose, gelatin, carrageenan, alginate, and polyacrylamide: alginate is the most widely used. However, these beads lack physical strength, they may be weakened by substances in the nutrient medium, e.g. phosphate will cause calcium alginate gels to break down. Methods of achieving the desirable strength with synthetic polymers usually adversely affect the viability of the plant cells therein. Moreover, owing to the encapsulation of the cells by the gel, transport problems frequently arise. Finally continued growth results in cells growing out and becoming detached whereupon they may lodge in other parts of the system, grow and cause blockages. The most effective method is the entrapment of the cells in a three-dimensional reticulate e.g. plastic foam, pan scrubbers, and steel knit mesh are examples of such reticulates. The application of this technique is reviewed by Yeoman (1987). Once cells are lodged in the voids of the reticulates they may multiply until the aggregate is entrapped.

A key point for us is that, while there are the above-mentioned and unmentioned problems, the "problems" of those seeking to use immobilized cells as systems to promote biosynthesis might be the means for us to solve our objectives. For instance, cell growth is generally diminished after entrapment or immobilization. It has been stated that "when immobilized, the cells can only exhibit properties which are inherent within them; immobilization merely provides a convenient method of exploiting these [biosynthetic] properties. Furthermore, although the immobilization process may seriously stress the cell, subsequent productivity and longevity are more likely to be features of the cell itself rather than the immobilization method used. The latter simply affects the number of viable cells and the ease with which they can be manipulated and is of little consequence if the chosen cell line selected is unstable or genetically unsuitable for high productivity" (Roseyear & Lambe, 1985: 44).

This suggests that immobilization per se has high potential to slow down plant cell growth and that there is minimal perturbation on its biochemical potential. Coupled with reduced temperature, this could offer substantial opportunities for long-term storage. Indeed, there is much literature that indicates that relatively extended storage of even very delicate cells such as protoplasts can be achieved without adverse effects on morphology or physiology. For example, using stomatal guard cell protoplasts of fava bean (Vicia faba), Schnabl et al. (1980) were able to show that calcium alginate-entrapped "cells" retained their spherical shape over a period of 14 days and kept their ability to show appropriate osmotic properties typical of guard cells. These authors further point out that "immobilized guard cell protoplasts can be sent over long distances by train without any apparent changes in morphology, membrane integrity, or cellular functions.... Thus, it is obvious that stomata protoplasts which are otherwise very fragile and cannot be transported in suspension can be subjected to transportation using this technique" (Schnabl et al., 1980: 282; see also Schnabl et al., 1983).

It was mentioned early in the context of immobilization that one can utilize a number of kinds of entrapment gels (for a discussion of various structural and mechanical properties of various polymer gels, see Clark &: Ross-Murphy, 1987; Skjak-Braek & Martinsen, 1991); each of these has advantages and disadvantages (Cabral et al., 1983; Rhodes, 1985). Even protein gels such as gelatin may have a place (Ziegler & Foegeding, 1990). Much work suggests that calcium alginate offers a number of advantages (Draget et al., 1988). The alginate can be dissolved in calcium chelating agents such as ethylenediaminetetraacetic acid (EDTA), polyphosphate, or citrate. It is a relatively mild process. Also, agarase(s) can be used to collapse gels noninvasively (Yaphe, 1957; Craigie, 1991); the same is true of alginase (Williams & Eagon, 1962). Sodium alginate can be sterilized by autoclaving, and, depending on the grade and composition of alginate used and how concentrated it is (say 4% w/v), it is quite viscous but still should be pourable and hence can be "dispensed" into a sterile solution of, say, ca. 0.2 M calcium chloride. The advantage here would be that the material intended as the immobilizing gel could be presterilized by autoclaving or preferably by filter sterilization and "stored" ready for use once cells were placed in it.

One potentially significant physiological/cell biological/biochemical problem is that the procedure of entrapment or immobilization in alginate gel involves the use of calcium chloride. Rosevear and Lambe (1985: 45) write: "Cells are suspended in a solution of sodium alginate which is then extruded through a defined orifice using either a pump or dropping pipette. The resulting droplets are gelled on contact with a solution of calcium chloride (50-100 mM) which can be made up in a nutrient medium to minimize shock to the cells. The viscosity of the alginate limits the practical range of polymer to concentrations to 2 to 3%, although it should be noted that autoclave sterilization permanently reduces the viscosity of alginate solutions. The alginate gels can be formed with many di-valent and polyvalent cations and a number of microbial cells have been immobilized in aluminum, and barium alginate. The toxicity of some metal ions causes some problems with plant cells. Since calcium is a constituent of nutrient media it will inevitably replace any other ion in time and so is routinely chosen for most purposes." (Ammonium alginate is not readily available through chemical supply houses, but it has its usefulness as well.)

Rosevear & Lambe (1985) point out that "an outer skin of gel forms around the droplet immediately upon entering the salt solution giving a clean, well defined bead. However, complete gelation takes several minutes, particularly with the dilute solutions being used to minimize osmotic shock. The cells are evenly distributed in the gel."

In connection with discussing problems with the alginate gel methods, Rosevear and Lambe (1985:45 et seq.) go on to state that a "major problem with this technique is associated with the metal ion used for cross-linking. Calcium ions can be important triggers of many biological mechanisms, and are known to affect cell/cell adhesion in plant systems" (see, e.g., Jansen et al., 1990; Marchant et al., 1993). They conclude, however, that "despite these limitations, the calcium alginate gel remains the simplest and most popular method of immobilization, particularly for short term studies."

Tamponnet (1986) pays considerable attention to the use of entrapment gels for Euglena gracilis. Entrapment in alginate coupled with refrigerator cold temperatures is now routinely used in many laboratories for storing Euglena lines (Leland Edmunds, pers. comm.). One consideration for us in the context of growing plant cells in a cell culture unit of one sort or another (and that may not be very serious) is that sterilization is needed for all these carrier gels. Provision will need to be made to keep materials sterile and in solution until needed, i.e., during or at the end of the experiment.

Table V lists advantages and disadvantages of various immobilization matrices for plant cells in the context of a "cell culture apparatus" or whatever one chooses to call it, of hypothetical design.

The emphasis is on agar/agarose, but even ammonium alginate (which is likely to be less toxic) is mentioned, as is gelatin.

Of special significance in the context of Table V is that "no agent added," i.e., liquid culture, puts investigators at a tremendous disadvantage, especially over the longer term, since cells in liquid are likely to be relatively unprotected and vulnerable to significant damage. It is, of course, for this reason that appropriate strategies such as immobilization need to be identified and developed for use. While the use of "constraining gels" or "matrices" is emphasized here, other strategies such as use of liquid "anti-freezes," even though it is probably premature to mention them here, should not be overlooked.

In its brevity, Table V seeks to cut through the details of procedures used and methods adopted in various laboratory studies by the author and in the literature.

Two points must be made. First of all, no single matrix will accommodate all, or even a wide range of, plant cells. Each individual system that an investigator seeks to use will probably have to be worked out in detail. Nevertheless, it is hoped that future work and reduction of findings will allow common ground for a wider range of cells and species than currently seems possible.

[TABULAR DATA FOR TABLE V OMITTED]

Second, one will need to have a relatively wide and precisely maintained temperature range from at least as low as 4 [degrees] C and perhaps a bit lower. A higher temperature limit for "cooling" might end up being 15-17 [degrees], even up to 20 [degrees] C. Upper limits of temperature for growing will probably be represented by 28 [degrees] C, but 22 [degrees] C will probably be the preferred growing temperature for many cell types.

Clearly there is high potential for the use of immobilization using entrapment gels. Before one gets too optimistic about the use of this method for unorganized cell cultures, however, it will be important to perform studies to determine the ease with which the procedures are amenable to use in the context of a cell culture unit destined for use in Space. There seems to be more optimism for use of the system with embryogenic cell cultures but even here similar problems obtain. In any case, entrapment per se does not stop growth but may retard it significantly. Also, depending on a number of factors such as the temperature at which the entrapment gels are used, further curtailment of growth is possible. Combined use of this entrapment technology in combination with low temperature may well afford opportunities for cell culture management that either one by itself cannot achieve fully.

B. SYNTHETIC "SEEDS"

The concept of synthetic "seeds," comprising encapsulated somatic embryos or minute shoot buds, has attracted considerable interest in the plant biotechnology industry (Gray & Purohit, 1991; Redenbaugh, 1993). Desiccated coated and uncoated somatic embryos, or somatic embryos in fluid gels, are the basis of this developing technology (see Redenbaugh, 1993, and references cited therein). While the emphasis has been on encapsulation of mature somatic embryos, clearly the principles have significance for our objectives using cells. Indeed, if the objective of a given plant cell culture experiment is the study of [Mu]-G or other space-flight factors on the development of somatic embryos, this technology could have direct application. Nevertheless, as in the case of entrapment or immobilization, this specialized approach will require development.

VIII. Critical Osmotic Adjustment

A. USE OF "NON-METABOLIZED" OSMOTICA

There is a large literature on the use of osmotic agents in the preparation and recovery of protoplasts (see Fitter & Krikorian, 1983; Dalton & MacKenzie, 1987; Nishimura et al., 1987; Roest & Gilissen, 1989; Power et al., 1989; Bajaj, 1994, and references cited therein), and, there has been substantial experience in osmotic adjustment in connection with vitrification procedures. Osmotic agents like sucrose (Torrey & Galun, 1970; Michel, 1972), mannitol (Trip et al., 1964; Jackson, 1965; Brown et al., 1979; Litz, 1986; Roberts-Oehlschlager & Dunwell, 1990), sorbitol (Chong & Taper, 1972, 1974; Ahmad et al., 1979; Pua & Chong, 1985; Kavikishor & Reddy, 1986; Reuveni et al., 1991; Leva & Muleo, 1993), melibiose (Dracup et al., 1986), inositol (Lin & Staba, 1961; Loewus, 1990; Durzan, 1987), polyethylene glycol (Jackson, 1962; Michel, 1970, 1972; Michel & Kaufmann, 1973; Basra et al., 1988), and glycine-betaine (Wyn Jones, 1984) have been used as osmotica and to retard plant cell growth (e.g., see Michel, 1970, 1972; Brown et al., 1979) or to inhibit metabolism (Jones, 1969; Cress, 1982). Osmotic increase to retard growth is only one aspect of the strategy that leads to preservation (Stiles, 1960; Crafts, 1968; Crowe & Clegg, 1973).

The other aspect is that osmotic level may be maintained by adding nominally inert osmotica (for example, partially or fully replacing sucrose with sorbitol or melibiose) to lower availability of carbon substrate for metabolism and growth. Partial or full replacement of sucrose with sorbitol has been used with a number of cell cultures and been part of management schemes for embryogenic carrot cells in this laboratory for some time (Ammirato & Steward, 1971; Steward et al., 1975). It is significant, however, that these nominally poorly metabolized sugar alcohols may be less inert than supposed (e.g., see Dracup et al., 1986; Thompson & Douglas, 1986; Einspahr & Thompson, 1990; Loewus, 1990; Stoop et al., 1995). In any case, manipulating osmotic relations will inevitably play a role in cell culture technology either alone or in connection with vitrification procedures.

B. NUTRIENT STARVATION

This category could well have been set up as a distinct method or even included in section X ("Growth-Retarding Compounds"), but since it is not being advocated as a strategy of choice, it is being subsumed under the general discussion of nutrient withdrawal or the strategy of replacing important nutrients such as sugars with largely metabolically inert sugar alcohols, etc. Withdrawal of sucrose from a growing system can reduce cell proliferation dramatically (van't Hof, 1966). The entire field of plant nutrition has been geared to the effects of deficiencies on plant growth, development, and metabolism (Amino et al., 1983; Thomas & Griefson, 1987; Sauerbeck & Helal, 1990; Dunlop & Curtis, 1991; Kabata-Pendias & Pendias, 1992; Marchner, 1994). Use of such things as nitrogen and phosphate limitation has been studied from the perspective of effects on secondary metabolism (Gershenzon, 1983; Mizrahi, 1988; Dunlop & Curtis, 1991; Schlatmann et al., 1994, and references cited therein). There may be special circumstances in which critical lowering or depletion of a particular nutrient might be useful for cell culture management. There is a considerable variety of organic acids that interfere with growth of cultures (Hildebrandt & Riker, 1949). Several amino acids that inhibit growth are known as well (Riker & Gutsche, 1948; Steward et al., 1958).

The best-studied of the amino acid type-inhibiting agents is perhaps hydroxyproline (Steward et al., 1958; Varner, 1987; Showalter & Rumeau, 1990; Robinson et al., 1990; Basile, 1990; Basile & Basile, 1990). An example of its dramatic ability to retard growth may be drawn from its action on excised maize embryos. Barrales (1959) showed that hydroxyproline at 200 mg/l was sufficient to cause severe curtailment of growth but 100 mg/l of proline could reverse the inhibition. Any slight growth that occurred in the presence of hydroxyproline was attributed to endogenous levels of proline; when the pools were used up, the inhibition by hydroxyproline was complete. The inhibition of growth by hydroxyproline and its reversal by proline are common features that apply to carrot explants (Steward & Pollard, 1958; Steward et al., 1958) and Zea embryos (Barrales, 1959). The fungus Trichophyton also shows this inhibition (Robbins & McVeigh, 1946). Ethionine and mercaptoethanol interfere with growth as well (Longevialle, 1970); methionine is a good antagonist to ethionine.

For the same objections raised above in the discussion of oxygen starvation, it seems that this is not the preferred option for storage of cells at the end of an experiment. It might, however, have value prior to the initiation of an experiment where growth has been kept to a minimum and "activation" occurs when replacement of limiting or inhibiting nutrients is carried out (e.g., see Kanabus et al., 1985; Amino & Tazawa, 1988; Hagen et al., 1991; Schnapp et al., 1991a, 1991b; Zhang & MacKown, 1992).

IX. Low pH

A. LOW pH AND MAINTENANCE OF EMBRYOGENIC CELLS

When embryogenic "cell" cultures are to be utilized, low pH offers significant potential for management in a very specific yet significant context. Work done in this laboratory using cultivated carrot has shown that embryogenic cells can be kept in a state of limited embryogenic development. The significance of this is that investigators might wish to activate an experiment by elevating the pH to a level at which somatic embryo-genesis can proceed. One could easily envision a scenario, for example, where embryogenic initials were kept in the nonadvancing mode, growing minimally at low pH to the extent that more nonadvanced preglobular stage somatic embryos are being produced. The medium would then be changed by elevating the pH a few units, which would lead to a "permissive" mode. Since the developmentally nonadvanced somatic embryos (those prevented from progressing in their development by the low pH) are clonal, one can be assured the material represents a reasonable sample.

In this context, one could readily conceive of an experiment wherein the pH is kept low from the outset of the setup; and, either alone or coupled with chilling, the experiment could be carried out by elevating the temperature and changing the pH. Some facts will be presented that should help a prospective investigator better to understand the low pH system and to ascertain whether the strategy might serve that investigator's needs [ILLUSTRATION FOR FIGURE 6 OMITTED].

Some facts of importance to us here:

(1) Somatic embryo development in carrot is prevented at an external medium pH of 4, is severely reduced at pH 4.5, and occurs at a high level at pH 5.7.

(2) The first-formed somatic embryos always develop at least to the globular stage even if the external medium pH is falling due to the presence of N[[H.sub.4].sup.+] as the sole nitrogen source.

(3) The first-formed somatic embryos will continue development into later embryo stages, without continued secondary embryo formation, if the medium pH is maintained above 4.5.

(4) The establishment of cultures consisting entirely of preglobular stage proembryos (PGSPs) is a slow process. The first-formed somatic embryos multiply in the beginning, as globular stage embryos, only when the pH of the medium is allowed to fall during the culture period. During each successive culture period (each 2-3-week period), the volume per tissue mass made up of globular stage embryos decreases while the volume of PGSPs increases. A total culture period of 2-3 months - i.e., 4-6 transfers of the entire tissue mass after the initiation of somatic embryos - is required to establish a culture consisting entirely of PGSPs.

(5) Establishment of PGSPs can be hastened by repeated mashing or wounding of the first-formed globular stage embryos at the time transfers are made.

(6) The growth rate of PGSPs maintained at pH 4 can be nearly doubled by the addition of a synthetic auxin (i.e., 4.5 [[micro]molar] 2, 4-D).

(7) Transfer of cultures consisting of "undifferentiated" callus and PGSPs - initiated and maintained in medium at pH 5.7 with the synthetic auxin 2,4-D - to hormone-free medium at pH 4 results in continued multiplication of PGSPs but not "undifferentiated" callus.

(8) PGSPs cannot be maintained as such if the medium pH is maintained at or above 4.5.

(9) Once PGSPs have been allowed to develop to the globular stage by culture on medium buffered above pH 4.5, 2-3 months' time is again required to reestablish PGSP cultures transferring the globular stage embryos back to a medium of lower pH.

(10) Continued development of PGSPs requires a source of reduced nitrogen in the medium. Ammonium at 1-5 mM is an excellent nitrogen source, whereas nitrate as the sole nitrogen source will not support continued embryo development (Smith & Krikorian, 1990a, 1990b, 1990c, 1991).

It remains to be tested whether lowering pH at the end of an experiment is sufficient to maintain it at zero growth with minimal change in qualities and characteristics.

It is not understood why low external pH allows embryogenic carrot cells (PGSPs) to multiply without development into later embryo stages on hormone-free medium. There is no evidence from other plant systems to draw on that could directly suggest a mechanism behind this low-pH phenomenon. There is, however, much information that can be drawn on for indirect comparison. There is a growing number of reports that show that challenge with low external pH, especially below 4.5, can lower the intracellular pH of cultured plant cells. Also, several nonplant systems were observed to be influenced in their morphology by changes in external pH; external pH changes probably exert control indirectly via changes in internal pH (Minocha, 1987). Changes in intracellular pH occur and are thought to influence the cell cycle, division, and growth (Busa & Nuccitelli, 1984). Particular attention has been placed on the pH changes occurring in the egg after fertilization in many systems such as sea urchin (Lytechinus pictus) and frog (Xenopus laevis) (Felle, 1989, and references cited therein).

Amoebae of Dictyostelium discoideum in the presence of cyclic AMP (cAMP) can be directed to stalk formation in a medium at low pH, whereas spore formation is favored on medium of higher pH (Town et al., 1987). Lowering of external medium pH was shown to lower the intracellular pH, and the drop in intracellular pH was first thought to direct the morphological changes (Van Lookeren Campagne et al., 1989). However, it was found that an increased intracellular pH in the absence of cAMP did not induce prespore gene expression. Furthermore, the presence of cAMP and blockage of rise in intracellular pH does not inhibit expression of prespore genes. Van Lookeren Campagne et al. (1989) therefore postulated that the main function of cytoplasmic pH change could be to provide or reduce substrate for H+-requiring enzymes. This suggestion is supported by the fact that the major response to cytoplasmic acidification is the activation of H+ ATPase (Felle, 1989). Therefore, acidification could provide the substrate (H+) or biochemical conditions needed for conformational changes necessary to activate/deactivate as-yet-undiscovered mechanisms. In fact, it has been shown that in oat roots there is a plasma membrane-associated ATPase that is activated by phosphorylation via a calcium-stimulated pH-sensitive protein kinase (Schaller & Sussman, 1988).

Other systems in higher plants reflect morphological changes due to external pH changes. An optimum pH of 5.4 was shown for somatic embryogenesis in carrot (Wetherell & Dougall, 1976). That study, however, focused on the numbers of embryos formed. At pH values below 4.5, PGSPs did not organize into later-stage embryos, and this was interpreted as a poor response. Last, in cultures of the moss Bartramidula bartramiodes it was shown that formation of gametangia, or organs in which the sex cells are produced, did not occur in medium below pH 5 but did occur with increasing frequency at pH levels up to 7 (Rahbar & Chopra, 1982).

Exogenously added auxins have been shown to affect intracellular pH most often by lowering it (Felle, 1989), but in some cases the addition of auxins has resulted in no detectable drop in intracellular pH (Kutschera & Schopfer, 1985). In these cases it has been argued that localized pH changes may occur at the inner surface of the plasmalemma especially if the result of added auxin is directed cell elongation (Kurkjian & Guern, 1989). That reported differences are probably due to the developmental and physiological states of the cells being examined has been stressed by Kurkjian & Guern (1989). This idea is reinforced by observations that an external pH of 4 blocks the initial formation of somatic embryos from wounded zygotic embryos, whereas a pH of 4 then becomes critical for maintaining the PGSPs that were derived from the first-formed somatic embryos.

That external pH below 4.5 lowers the internal pH of PGSPs to a critical level, thus yielding the response of continued multiplication without further development, seems plausible. Therefore, several points on what a lowered internal pH does may be instructive. An external pH change of 1 pH unit has generally been shown to drop the internal pH 0.1 unit (Kurkdjian & Guern, 1989).

The conclusion is that lowered pH may offer an avenue for cell culture management in a context broader than has been hitherto anticipated. It seems reasonable to believe that, provided the biology of the system is amenable to it, low pH can provide a tool for cell culture management at the outset of an experiment in a cell culture unit. It is not as likely that it will have major value by itself in the course of, or at the end of, an experiment.

There seems to be a fair amount of evidence that major metabolic changes are effected when pH is changed. This probably means little at the outset of an experiment. Nevertheless, one should test whether lowering pH at the end of an embryogenesis experiment is able to maintain cells at zero growth with minimal change in qualities and characteristics, i.e., with no significant change insofar as capacity for later development into plantlets is concerned. In short, it may be a valuable technique for morphological experiments but have less value for certain kinds of metabolic or biochemical investigations.

X. Growth-Retarding Compounds

A. GENERAL GROWTH RETARDANTS

The growth of cultured plant cells and tissues in vitro is often dependent upon exogenously added growth substances (see Steward & Krikorian, 1971; Fry & Street, 1980; Gray & Conger, 1985; Yopp et al., 1986; Fellman & Read, 1987; Jackson et al., 1987; Krikorian et al., 1987; Takahashi et al., 1990; Tiburcio et al., 1990; Horgan & Scott, 1991; Kaminek et al., 1992; Mok & Mok, 1994; Mok, 1994; Krikorian, 1995, and references cited therein). Indeed, Kisouye et al. (1990, 1993) have drawn attention to the fact that normally toxic heavy metal ions and hypochlorite, when used in "low" doses can initiate or foster somatic embryogenesis and thus, in this context, can be regarded as "growth substances." There is, moreover, a massive, more conventional literature on growth regulators used in practical settings to retard growth (Frenkel & Haard, 1973; Nickell, 1982; Scheel & Casida, 1985; Flores et al., 1990; Halmann, 1990; Davis & Curry, 1991; Rademacher, 1991; Abeles et al., 1992; Davies, 1995), but use of these would more than likely have to be experiment-specific (Grossmann & Jung, 1984). Arguably, the plant growth regulator ethylene is much too general a growth retardant (Meijer, 1989; Auboiron et al., 1990; Rademacher, 1991; Abeles et al., 1992) and the same may be said of abscisic acid (see Ammirato, 1983; Davies & Jones, 1991). Even though both are naturally occurring hormones, they normally have many adverse side effects (Smith et al., 1989), so it seems that their use should be avoided. They (especially abscisic acid) could conceivably be used in managing cultured cells at the outset of an experiment. That is, presence of abscisic acid in the nutrient medium at the beginning, followed by flushing it out to activate cell culture growth, might be a workable strategy, but it would again be so experiment-specific as to not merit our concern. It should be pointed out, however, that abscisic acid has been used in combination with osmotic agents such as mannitol or sorbitol for the tissue culture storage of potato germplasm grown as shoot tips (Westcott, 1981). In vitro shoots and plantlets become very stunted, but normal growth ensues upon return to retardant-free medium and withdrawal of osmotic agents.

Plant biology investigators will be aware of the potential for use of abscisic acid as a retardant in a given system. If it is appropriate and meets their needs, they will be able to draw upon it.

B. CELL CYCLE INHIBITORS

The cyclical events of cell multiplication involve both morphological events and biochemical syntheses - the replication of visible structures and organelles and of macromolecules (Ito & Komamine, 1993; Hemerly et al., 1993). Classical cytology of the nucleus and the wealth of modern knowledge that deals with cells in division and in interphase comprises a literature too vast to be more than superficially referred to here (Sharma & Sharma, 1980; Phillips, 1981; Francis, 1992; Murray, 1992; Ormrod, 1993; Singh, 1993). Obviously, such considerations are fundamental to any understanding of the different ways by which chemical substances may intervene to affect cell growth and cell division and to know how and when they act in relation to the cell cycle (see Wilson, 1965; Rost & Gifford, 1977; Gould, 1984; Davidson, 1991; Francis, 1992).

Figure 7 provides a simple scheme of areas points at which there might be potential for having an impact at specific control points for cell multiplication. Mitoclastic agents, or antimitotics (i.e., chemicals that interrupt the normal course of mitosis), have been known for a very long time (Wilson, 1965). The first of these studied was chloral hydrate, [Cl.sub.3]CHOH [(OH).sub.2], used in aqueous solution at about 0.75%. An excellent summary of this early literature may be found in Mangenot (1941). Various plant alkaloids such as cinchonamine, quinine, strychnine, brucine, papaverine, narcotine, nicotine, and purines such as caffeine and theophylline were early shown to have varying degrees of mitoclastic effect. The best known of these is colchicine, which over the years has been the agent of choice for use in plant cytology. (Methyl colchicine, colcemid, which differs from colchicine by having an N-methyl group instead of a N-acetyl group, is widely used by animal cell workers but less so by plant cytologists, and the same is true of the alkaloid vinblastine.) Extremely low concentrations of all arsenicals (e.g., dimethyl sodium arsenate, sodium cacodylate) can act like colchicine. Mercuric ethyl phosphate is another mitotic inhibitor. While all such compounds stop mitosis, their effects are toxic enough to render them inappropriate for our needs.

There is an extensive literature that deals with substances like colchicine, steganacin, maytansine, podophyllotoxin, vinblastine, taxol, etc. (see Vaughn & Vaughan, 1988; Devine et al., 1993). The main emphasis on these substances has been their use in treating human cancer cells, but in plants their interest stems primarily from use as cytostatics in chromosome procedures such as metaphase analysis (see Sharma & Sharma, 1980; Phillips, 1981; Krikorian et al., 1982; Singh, 1993). These alkaloids are problematical for the stated objectives since they disorganize the spindle components and poison the microtubules and bring about metaphase arrest, from which recovery may be incomplete (Vaughn & Vaughn, 1988; Devine et al., 1993). Short-term exposure can cause a number of metabolic responses (e.g., Hart & Sabnis, 1976; Sloan & Camper, 1981, and references cited therein). Protracted exposure may result in multiplication of the number of chromosomes and for that reason they are used to induce polyploidy in plants. It is interesting to note that animal cells are much more sensitive to colchicine than are plant cells, and the concentrations needed to disrupt microtubules in plants is typically 100 to 1000 times that needed in animal cells (usually ca. 1.25 x [10.sup.-5] to 7.2 x [10.sup.-3] M). The molecular mechanism for this resistance is not clear, but plant microtubules appear to be more cross-bridged than those of animal cells (Devine et al., 1993, and references cited therein).

There is an extensive literature on the use of various synthetic chemicals as herbicides (Fedtke, 1982). Some of these are acetohydroxyacid synthase inhibitors and hence affect amino acid synthesis (Scheel & Casida, 1985); others act as microtubule disruptors (Devine et al., 1993). It seems unlikely that use of such compounds even at low levels would serve our purposes.

Various antibiotics such as the fungistatic griseofulvin can also have cytostatic effects on plants, but these, too, are coupled with varying levels of toxicity (Schaffner, 1979; Berdy, 1980; Lo Schiavo et al., 1980; Pollock et al., 1983; Kavi Kishor & Mehta, 1988; Vyas, 1988; Robert et al., 1989; Tsang et al., 1989). (Other antibiotics may even stimulate plant growth.) Use of the antibiotic aphidicolin (from Nigrospora sphaerica) an [Alpha]-like DNA polymerase inhibitor at ca. 5-30 [[micro]gram]/ml of solution, acting at the G1/S border, would seem to have some potential (Joyce & Steitz, 1994), but further consideration indicates that it, too, has its limitations, since its effects seem to be relatively short-term and are best used in concert with colchicine to get cells synchronized (Guri et al., 1984). The same may be said for hydroxyurea, also said to act primarily but not exclusively as an inhibitor of DNA polymerase (Davidson, 1991, and references cited therein).

Aphidicolin has been only superficially studied in plant cells, and it would be worth an effort to see whether plant cells treated with it could be kept quiescent for prolonged periods without adverse consequences (Guri et al., 1984). Nevertheless, it seems that the general conclusion is justified that there seem to be few chemical compounds available that are selective enough to fulfill our requirement of being able to stop reversibly a critical stage in the plant cell cycle for a protracted period of time without the usual concomitant adverse effects. The use of mitoclastic agents, even DNA polymerase inhibitors, should not be pursued to any great extent but one should be alert to learn of new developments.

XI. Conclusions

The work discussed in this review demonstrates that if an appropriate technique is used, the potential for limiting metabolic activity and developmental progression is substantial, and that there is no a priori reason to lose culture characteristics. The major limitation is that the time elapsed between cessation and reestablishment of cell culture growth may entail a number of cell divisions and, hence, questions relating to how the "memory" of a plant cell that has been exposed to hypo-G works and how a plant cell transduces a G-stimulus into useful biochemical information may be lost or seriously obfuscated (Brown, 1992, 1996). There appears to be no single strategy that will be applicable to all systems; accordingly, any proposed strategy will have to be carefully matched with a given system and the desired objectives of an experiment. A variety of research opportunities exist to resolve issues of minimal- or zero-growth maintenance for cultured cells and tissues destined to be used as test systems in the [Mu]-G environment of Space. Keeping cells inactive or quiescent at the beginning of an experiment seems to be an easier objective to realize than keeping cultures quiescent after periodic sampling and at the end of the experiment prior to return to Earth and the execution of postrecovery analysis. Clearly, many investigators will wish to terminate a Space experiment by chemical fixation and rapid freezing. But there will be a desire or need on the part of some to get back material "alive." The following comments indicate that a combination of options might end up providing the most realistic solution(s) to the problem at hand. Research should go far toward resolving the requirements.

No special attempt should be made to investigate the direct use of reduced gaseous oxygen as a means to conserve cell cultures. Attention will, of necessity, be paid to gaseous environment when specifications for oxygenation or aeration are stipulated for a given test system.

There may be special circumstances in which critical lowering or depletion of a particular nutrient might be useful for cell culture management. This strategy is not the preferred option for storage of cells at the end of an experiment; it may, however, have value at the initiation.

Aphidicolin has been inadequately studied in plant cells, though it would be worth the effort to establish whether plant cells treated with it could be kept quiescent for prolonged periods without adverse consequences. Nevertheless, the general conclusion is that there are few substances available that are selective enough to fulfill our requirement of reversibly stopping a critical stage in the plant cell cycle for a protracted period of time without the usual concomitant adverse effects. Use of mitoclastic agents, even DNA polymerase inhibitors, should probably not be pursued to any great extent, but one should keep abreast of new developments.

Plant biologists will be aware of the potential for use of abscisic acid as a retardant in a given system. If its value is demonstrated, they will be able to draw upon it. However, use of retardants like abscisic acid would seem to be experiment-specific.

The recommendation for the moment is that the process of cryopreservation by vitrification be placed on a "technology watch" so that significant new developments may be quickly recognized and appreciated for what they are worth vis-a-vis their potential for incorporation into Space plant cell and tissue culture experiment management and implementation schemes. It is important to note that cryopreservation of cells via vitrification could have substantial significance for the initiation of an experiment in Space. Substantial problems would be encountered at the end of an experiment or "in between" samplings only because the procedures are not trivial. It may well turn out that an adaptation of the process or "intermediate" procedure, in which the principle of vitrification is capitalized upon but liquid is avoided, may provide a valuable avenue for investigation at the end of an experiment.

Osmotic adjustment can play a valuable role in plant cell culture management and will inevitably play a role in handling cells since osmotic relations are an integral part of the ability of plant cells to grow (see Stiles, 1960; Calcott, 1981, and references cited therein). Investigations should be carried out to develop and define this technology. Additionally, the method should be viewed as being a very important component of vitrification for cryopreservation (see below).

Chilling offers considerable opportunity to manage cell and tissue cultures of higher plants and other organisms. In some instances chilling is simple and can be performed at a single reduced temperature level; in other cases a fluctuating regime of various low temperatures seems to be useful or is supposedly required. In still others, lowered temperature is more of a problem. This emphasizes that (1) while chilling provides considerable opportunity to slow down growth dramatically, this must be closely tied to the biology of the system and (2) any engineering associated with chilling must be precise enough to maintain temperature tolerances within close or specified limits.

Mineral oil overlay offers considerable potential for investigating the preservation of plant cells, especially during the period when samplings are being carried out and especially at the end of an experiment. Use of it should be pursued both in isolation and in combination with chilling and low-temperature storage procedures.

There is high potential for the use of immobilization using entrapment gels. However, before one gets too optimistic about the use of this method, even for unorganized cell cultures, the ease with which the procedures can be used in a cell culture-unit context needs to be determined. There seems to be more optimism for use of the system with somatic embryo-producing systems, but even here similar problems obtain. In any case, entrapment in and of itself does not stop growth but may retard it. Combined use of this technology with low temperature should be pursued.

Low pH can provide a tool for cell culture management in specific cases at the outset of an experiment in a cell culture unit provided the biology of the system is amenable to it. It seems less likely that it will have major value by itself in the course of, or at the end of, an experiment in Space. There seems to be a fair amount of evidence that major metabolic changes are effected when pH is changed. This probably means little at the outset of an experiment before it is activated. Nevertheless, we should test whether lowering pH at the end of an embryogenesis experiment is able to maintain cells at zero growth with minimal change in qualities and characteristics at least insofar as capacity for later development into plantlets is concerned. In short, it may be a valuable technique for morphological experiments but may have less value for certain kinds of biochemical investigations.

XII. Acknowledgments

Financial support for this review was made available from Martin Marietta Services Inc., Cherry Hill, NJ, through a contract from NASA (NAS2-13227) administered through Ames Research Center, Moffett Field, CA. The interest, encouragement, and support of Dr. Gabrielle Meeker, Dr. Charles Wade and Ms. Catherine C. Johnson at ARC are appreciated. Grant support from NASA over the years has given me the opportunity to work on problems of plant growth and development using aseptically cultured plant cells and tissues and has sensitized me to the needs of the Life and Biomedical Sciences and Applications Division. This help is gratefully acknowledged.

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RELATED ARTICLE: Table I

Some expected changes in plant response as a result of exposure to [Mu]-G in the Space environment.

A. Situations in which orientation with respect to 1-G is established as altering the response

gravitropism

organs, especially roots and shoots; specialized single cells, e.g. statocytes

gravitaxis

gravimorphogenesis

formation of reaction wood; break/apical dominance; determination of position of organ emergence; determination of the type of organ or cell formed

epinasty

B. Unidentified situations where "Earth normal" (1-G) is required - that is, [Mu]-G might be expected to eliminate a response, i.e. have a qualitative effect

unknown at present, "everything" is a potential candidate

C. Situations where [Mu]-G would be expected to alter a response, i.e. have a quantitative effect

lignification? unknown at present, e.g. what effect does [Mu]-G have on cells that are not specialized for G-sensing? What effect does [Mu]-G have on developmental, physiological and reproductive processes? etc.
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