- Email: [email protected]
[8] Tissue culture: plant
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found that short photoperiods of bright light yielded the highest degree of synchrony. The synchrony developed for Skeletonema costatum, a familiar experimental species, was only slightly better than exponential growth.
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Tissue
Culture:
Plant
B y W. M. LAETSCH
The first true tissue cultures were apparently green, 1 but the use of such plant material for investigating photosynthesis and/or chloroplast development was essentially ignored until recent years. 2,a This is unfortunate, because tissue cultures offer the prospect of providing the desirable experimental features of algal cultures while possessing the unique developmental patterns of higher plants. The delineation of some of the advantages of this system will perhaps indicate some of the ways in which the following methods can be used. Techniques for handling cultured tissues are very similar to those for microorganisms, and the physical environment can be controlled in a common manner. The long-term control of temperature and illumination is, therefore, much more precise than is presently possible for either seedlings or mature plants. It is notoriously difficult, for example, to expose intact plants or detached organs to very high light intensities fo ~ an extended time period. This problem is minimized with cultured cells or tissues. The same is true for quantitative work involving light quality. Since most work on the development of higher plant chloroplasts centers on the light-induced etioplast to chloroplast transformation, the value of a well-defined illumination system cannot be underestimated. The control of the chemical environment is also susceptible to far greater precision than is possible with either whole plants or plant parts. Sterile conditions open the way to a variety of experiments, and the absence of a cuticle in cultured tissues lessens the problems of absorption of exogenous chemicals which so often plague those working with higher plant tissue. A defined substrate for cultured tissues offers opportunities which too often have been ignored. This permits the isolation of events in chloroplast development from general cell responses such as replication and growth. " is next to impossible to regulate the chemical imputs of R. J. Gautheret, C. R. Acad. Sci. 198, 2195 (1934). W. M. Laetsch and D. A. Stetler, Amer. J. Bot. 52, 798 (1965). 3L. Bergma~ and C. Berger, Planta 69, 58 (1966).
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normal leaf tissue, and a result is that "triggers" for chloroplast development usually "trigger" other processes as well. The difficulty in determining reciprocal influences usually results in most of them being ignored. In addition to a greater amount of physiological homogeneity, tissue cultures can possess greater anatomical homogeneity than do organs such as leaves and cotyledons. As a result, a host of variables encountered in "normal" tissues is minimized. A defined substrate also permits the regulation of tissue and organ differentiation. The ability to induce bud formation, for example, allows the examination of chloroplast development in "normal" tissue while still maintaining the advantages of sterility and precise control over the physical environment. Angiosperms, unlike most of the algae, do not synthesize chlorophyll in the dark, but this phenomenon has not been fully exploited experimentally because of the inability to maintain plants or organs in the dark for extended periods. As a result, experiments have been confined to seedlings with large seeds. Tissue cultures can be maintained indefinitely in the dark, and they are the only system for effectively investigating chloroplast continuity in higher plants. The electron microscope is a basic tool for studying chloroplast development, and cultured tissues have certain advantages for electron microscopy. The tissue generally responds well to chemical fixatives and is easy to section. The tissues of many leaves respond in just the opposite fashion. It has frequently been stated that many problems in the development of higher plant chloroplasts will not be solved until chloroplasts can be cultured independently of the cell. While this breakthrough is awaited, cultured tissues offer the possibility of working with simpler, better defined systems than is available with attached or detached plant organs. Summary of Investigations on Chloroplast Development in Cultured Tissues This brief review will provide some examples of how tissue cultures have been used in studying plastid development, but it does not pretend to cover the literature. A number of papers have described aspects of fine structure of developing plastids, 2-~ while others have been more concerned with the effect of growth regulators, nutrients, and light quality on synthesis and/or chloroplast development2 -17 Still others have investigated enzyme patterns during chloroplast development, ls,19 4 H. W. Israel and F. C. Steward, Ann. Bot. 31, 1 (1967). S. J. Blackwell, W. M. Laetsch, and B. B. Hyde, Amer..l. Bot. 56, 457 (1969). D. A. Stetler and W. M. Laetsch, Science 149, 1387 (1965). 7E. M. J. Jaspars, Physiol. Plant. 18, 933 (1965).
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Comparisons of photosynthetic structure and function of developing chloroplasts in cultured versus intact tissues have been made to my knowledge only in tobacco. 2,2° There are differences in the two types of tissue, but these are mostly quantitative in nature. These various studies have established some of the basic parameters influencing chloroplast development in cultured tissue and have demonstrated that the system is amenable to experimental manipulation. Most of these authors have employed callus tissue grown on solid media, but Bergman has utilized liquid suspension cultures in his attempts to induce autotrophic growth of tobacco cells. 21 Most of the previous studies have been on problems similar to those which have been or could be conducted on leaf tissue, but some recent work has been concerned with problems that can be investigated only by means of tissue culture techniques. One such problem concerns the ability of mature chloroplasts in mature leaf tissue to dedifferentiate, divide, and develop again into mature chloroplasts. 22 The leaf is a determinate organ and studies on chloroplast development usually end with the death of the leaf, but the chloroplast life cycle can be continuously generated in cultured tissue. Another recent project concerns the relationship of tissue growth to organelle differentiation in dark-grown cultures. The etioplast characteristic of dark-grown leaves of seedlings has not been found in dark-grown callus. I t has been determined in tobacco that the appearance of mature etioplasts can be controlled by regulating the growth rate of the callus. 23 The prolamellar body, for example, is induced by inhibiting tissue growth and is eliminated by stimulating tissue growth. It has previously been thought that the prolamellar body was intimately involved with the normal etioplast to choroplast transformation, but evidence from tissue cultures suggest 8S. Venketeswaran, Physiol. Plant 18, 776 (1965). I. K. Vasil and A. C. Hildebrandt, Planta 68, 69 (1966). lo G. Beauchesne and M. C. Poulain, Photochem. Photobiol. 5, 157 (1966). la L. Bergman and A. B441z,Planta 70, 285 (1966). R. Schantz, H. Duranton, and M. Peyri~re, C. R. Acad. Sci. Set. D 265, 205 (1967). 13N. Sunderland, Ann. Bot. 30, 253 (1966). it N. Sunderland, Ann. Bot. 31, 573 (1967). 1~R. Boasson and W. M. Laetsch, Experientia 23, 967 (1967). A. K. Stobart, I. McLaren, and D. R. Thomas, Phytochemistry 6, 1467 (1967). 1, p. K. Chen and S. Venketeswaran, Physiol. Plant. 21, 262 (1968). '* A. K. Stobart and D. R. Thomas, Phytochemistry 7, 1313 (1968). 19A. K. Stob~rt and D. R. Thomas, Phytochemistry 7, 1963 (1968). 20D. A. Stetler, Ph.D. Thesis Univ. of California, Berkeley, 1967. ~ L. Bergman, Planta 74, 243 (1967). W. M. Laetsch, Amer. J. Bot. 54, 639 (1967). D. A. Stetler and W. M. Laetsch, Amer. J. Bot. 55, 709 (1968).
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that its appearance is a function of the rate of cell division and that it does not play a causal role in chloroplast development. Problems Encountered with Cultured Tissue It is legitimate to ask why cultured tissues have not been more widely used for studies in chloroplast development and function. Aside from the fact that most people think it is easier to grow bean seedlings than to master tissue culture techniques, there are features of tissue cultures that make them unsuitable for certain studies. A greater appreciation for the potential of cultured tissues in studies on chloroplast development will be' obtained if the difficulties are discussed in some detail. Anatomical heterogeneity is generally less of a problem than in leaves, but genetical heterogeneity is a greater problem. Not only are the cells in a particular callus likely to have a variety of genotypes, but the longer the tissue is kept in culture the greater is the chance for increases in ploidy levels and chromosomal aberrations. 24,2~ This presents problems in duplicating experiments, because the tissue used one month is often genetically very different tissue when used several months later. This genetic variability is at least one cause of the considerable variability in physiological response which many workers have found. In spite of what has been said above about the anatomy of callus, it is a mistake to think that all tissues are completely homogeneous in terms of cell type and cell size. Meristematic rates within callus vary and there are often considerable differences in cell size. Differentiation in the form of xylemlike and phloemlike elements is frequent, and these elements are often localized in complex nodules. An important result of these various types of heterogeneity is the difference in developmental rates within the total plastid population. The chloroplasts in the cells near the surface of the callus often differentiate faster than those in the cells of the interior. The islands of meristematic cells commonly found in callus mean that division of proplastids is taking place in some cells while well developed chloroplasts are present in others. In other words, synchronous development of the chloroplast population is very rare in cultured tissues36 It might be thought that liquid suspension cultures would be the answer to some of these problems, but such cultures are generally very heterogeneous themselves since they consist of single cells and cell clumps of various sizes. Many of the cell clumps are actually smaller than many of the single cells. There are some ~4j. G. Torrey, Physiol. Plant. 20, 265 (1967). D. J. Heinz, G. W. P. Mee, and L. G. Nickell, Amer. J. Bot. 5{},450 (1969). M. M. Yeoman and P. K. Evans, Ann. Bot. 31, 323 (1967).
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cultures of tobacco which have a high proportion of free cells, but the cells will not synthesize chlorophyll. Dark-grown bean or barley leaves turn green within a matter of hours upon exposure to light. Dark-grown cultured tissue turns green very slowly and may take as long as a week for the development of mature chloroplasts. While the concentration of chlorophyll per dry weight of green tissue varies between cultures, it is usually much less than that of the leaf tissue of the corresponding tissue. In tobacco, for example, the mature leaf can have twenty times more chlorophyll on a dry weight basis than callus. ~ This is mainly a result of fewer chloroplasts per cell. The low final concentration of chlorophyll means that small differences during chloroplast development are difficult to quantify. While the leisurely synthesis of chlorophyll in cultured tissue is a handicap for those in a hurry, it can be used to advantage to study the intermediate steps in development which sometimes happen so rapidly in leaf tissue they are difficult to detect. The number of chloroplasts per unit volume of tissue is rather small, and rich yields of isolated chloroplasts are difficult to obtain. The chloroplasts are also full of starch, and, since the medium has sugar, the starch cannot be removed by placing the tissue in the dark. A solution to this problem might be effected by a continuation of the preliminary work on growing tissues on sugar-free medium. 21,27 Initiation of Cultures
General methods for establishing and maintaining tissue cultures have been provided in detail by several authors. ~s-a° A common impression is that tissue culture is a complex and difficult technique, but it is actually very simple. The basic rules of sterile technique must be followed, but other than that there is not a single "best way" to grow cultured tissue. The main problem is to place uncontaminated but live tissue on a medium that will induce cell division. Sterile tissue is obtained either by stripping away external tissue from an organ in order to excise internal uncontaminated tissue, or the external surface is sterilized and the whole organ is cut into explants. The latter method is used in the leaf sterilization method described below. Sterile transfer rooms and expensive inoculation hoods are nice to have, but are quite unnecessary for such work. The main problem in these operations is to eliminate air ~'T. Fukami and A. C. Hildebrandt, Bot. Mag. Tokyo 80, 199 (1967). R. J. Gautheret, "La Cillture des Tissues V4g6taux." Masson, Paris, 1959. P. R. White, "The Cultivation of Animal and Plant Cells." Ronald Press, New York, 1963. 'oH. E. Street, in "Techniques for the Study of Development" (F. H. Wilt and N. K. Wessells, eds.), p. 425. Crowell, New York, 1967.
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movements, and a few well arranged piecos of cardboard on a laboratory bench are as satisfactory as the glossiest transfer rooms. Stem explants containing cambial tissue are used most commonly as starting material for cultures. Leaf tissue is often more difficult to culture, although in some species such as tobacco, callus is easy to obtain from leaves. Callus is readily obtained from the roots of many species, but such cultures are probably less likely to be green than are those from stems and leaves. Herbaceous dicotyledons have been used most extensively for tissue cultures, because they have proved relatively easy to grow. Monocotyledonous tissues, and especially grasses, have traditionally been difficult to grow in culture, but recent work suggests that many of these difficulties show signs of being solved. 31 It remains to be seen whether chlorophyll synthesis in these monocot tissues will be sufficient to use them for studies in chloroplast development. Certain gymnosperms have been cultured with considerable success, but pteridophytes have not been promising material. 28 With the exception of tissue cultures of Ginko, there has only been casual mention of the ability of cultured tissues of gymnosperms to form chlorophyll?2 Many types of culture vessels are available, and the most suitable type is more a function of the arrangement of the available light system than any other factor. Culture tubes and flasks are more suitable for vertical lamps, and petri dishes are best for overhead lamps. Their optical properties make plastic petri dishes preferable to glass. A fair amount of nonsense has been written about the necessity for special types of specialized (and expensive) culture vessels, but there is little evidence that such exotic ware produces any better results than those obtained with standard flasks and culture tubes. Cultured tissues are generally defined as clumps of randomly proliferating cells, but there is no reason the term cannot apply to tissues of organs which are cultured in their "normal" state of differentiation under sterile conditions. This type of cultured tissue eliminates many of the disadvantages associated with callus tissue, and permits the exploitation of "normal" tissue in a sterile environment with a defined source of nutrition. 33 Small pieces of leaf tissue are examples of such cultures. The following method has been primarily used with tobacco tissue, but the same procedures work with bean leaves, and other species would undoubtedly provide suitable material. The same basic method can be used to surface sterilize stems, seed, cotyledons, or flowers, and the tissue can ~1y . Yamada, K. Tanaka, and E. Takahashi, Proc. Jap. Acad. 43, 156 (1967). 32W. Tulecke, Amer. J. Bot. 54, 797 (1967). 3~R. Boasson and W. M. Laetsch, Science 166, 749 (1969).
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then be maintained on a minimal medium or callus can be induced with a complete medium. Cut strips approximately 1 inch wide from the center portion of the leaf avoiding the main vein, the tip, and the base of the leaf. Etiolated leaves are treated intact. Soak the strips for 2-4 minutes in a beaker of water with a small amount of laboratory detergent. The subsequent steps should be conducted under a hood. Transfer the strips to a 4.0% hypochlorite solution (dilute commercial bleach 1:5 with water) with a small amount of laboratory detergent as wetting agent. The detergent is more effective than Tween. Soak the leaf strips for 5 minutes; stirring occasionally to remove air bubbles adhering to the leaf surface. Use a sterile forceps to transfer the strips to a beaker or flask containing sterile water and rinse by swirling the beaker. Transfer the leaf strips to petri dishes containing two pieces of sterile filter paper. Two strips are a convenienb number for each dish. Use a sterile cork borer (preferably stainless steel) to cut disks from the strips. Large veins should be avoided. The disks will collect inside the cork borer and should be pushed out with a sterile glass rod. Never sterilize the disks after they have been cut from the strips, because the large cut surface will permit serious damage by the hypochlorite. When the disks are placed on medium in petri dishes, they should be randomized; that is, the disks from one leaf should not all be placed in the same culture dish. Disks 2 mm in diameter are a convenient size, and 10 disks will fit in a 5-cm petri dish. Small disks are recommended because the cells rapidly lose stored nutrients and become dependent upon those in the medium. Large disks invariably mean the inclusion of major veins, and this means greater anatomical and physiological heterogeneity in the tissue. A minimal medium composed only of the inorganic constituents of the medium described below plus sucrose will maintain leaf tissue in the dark for as long as 3 months. It is also sufficient for normal rates of chlorophyll synthesis in disks of etiolated leaves. Cell proliferation resulting in callus formation can be induced in either etiolated or mature leaf tissue cultured on the minimal medium by transferring the tissue to a complete medium composed of the inorganic and organic components plus the growth regulators described below. Cell division will occur throughout the leaf disk, but it will be most active at the periphery. Enough callus will be formed in the latter region within 1-2 weeks to permit subculturing. There are many types of culture media, and the tissues of each species and perhaps each organ will have their special requirements. A reasonable approach is to start with a medium on which a large number of different tissues will grow reasonably well and then gradually define the requirements for specific tissues. The following medium is basically that
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of Murashige and Skoog,34 and it has proved satisfactory for a wide variety of dicotyledonous tissues. The use of undefined or complex ingredients such as coconut water, casein hydrolyzate, or yeast extract is to be avoided if at all possible. If used for the reasons described below, they should be added to the other inorganic and organic constituents before addition of the agar. Coconut water is generally used at a concentration of 10-20% (v/v). If 100 ml of coconut water is used, then the amount of water added to the organic and inorganic constituents is correspondingly reduced. The fresh coconut water should be autoclaved for a few minutes, and the precipitated proteins filtered before use in the culture medium. It is convenient to freeze unused coconut water in 50- or 100-ml portions for future use. The medium is dispensed into tubes or flasks and autoclaved at 15 lb for 15 minutes. Certain vitamins, antibiotics, etc., are filter-sterilized (the sterile plastic units with a membrane filter are most convenient) and added to the sterile medium. Experiments with tissue cultures are usually measured in weeks rather than days and desiccation of the medium is frequently a problem. This is aggravated in the light because of the greenhouse effect. Petri dishes sealed around the bottom with masking tape will not dry out for many weeks and gas exchange does not seem to be seriously affected. Flasks and tubes with metal or plastic closures can be treated in a similar fashion. Cotton plugs are the least satisfactory type of closure. Sterile sheets of polyethylene are frequently used to cover culture vessels, but their effectiveness in inhibiting desiccation and permitting the transmittance of light is counterbalanced by their difficult manipulation during tissue transfer. Control of Chloroplast Development Chloroplast development is strongly influenced by the growth and physiological state of the host cells, so the ability to control the development of cells assumes great importance. Nutrients and growth regulators are very effective control agents. Chlorophyll synthesis in light-grown tissue cultures can be markedly influenced by sugar concentrationJ ,12 High concentrations inhibit synthesis and stimulate tissue growth. Sucrose also inhibits chlorophyll synthesis in cultured tobacco leaf tissue when it is present at concentrations of 2.0% and higher2 ~ The standard sucrose concentration for tissue cultures ~ T. Murashige and F. Skoog, Physiol. Plant. 15, 473 (1962). W. M. Laetsch, Amer. J. Bot. 53, 613 (1966).
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ISOLATION AND CULTURE TECHNIQUES Stock Solution I Dissolve the following one at a time in 5 liters of glassdistilled water: NH~N03 16.5 g KNOB 19.0 g CaCI~ 2 H20 2.2 g MgSO4" 7 H20 1.85 g KH2PO4 1.7 g H,BOs 0. 062 g MnSO4" H~O 0.169 g ZnSO4.7 H~O 0.106 g KI 0. O083 g Stock Solution 2 Dissolve in lo0 ml of glass-distilled water: FeSO~.7 H20 5.57 g Na~EDTA 7.45 g Stock Solution 3 Dissolve in 100 ml of glass-distilled water: CuSO4- 5 H~O 0.025 g CoC12.6 H~O 0.025 g Stock Solution Dissolve in 100 ml of glass-distilled water: Na~MoO4 0. 025 g Stock Solution 5 Dissolve in 100 ml of glass-distilled water: Glycine 0.2 g Nicotinic acid 0.05 g Pyridoxine. HC1 0.05 g Thiamine. HC1 0.01 g Medium Combine the following to make 1 liter of medium: Stock Sol. 1 500 ml Stock Sol. 2 0.5 ml Stock Sol. 3 0.1 ml Stock Sol. 4 1.0 ml Stock Sol. 5 1.0 ml Add the following to the above: Myoinositol 0.1 g Sucrose 20 g Growth regulators are added to the above (see discussion below). Adjust the pH of the solution to 5.7-6.0 with 0.1 N NaOH. A liter of solid medium is made by melting 8 g of agar in 497 ml of glass-distilled water and adding to the above solution. A liter of liquid medium is made by adding 497 ml of glass-distilled water to the above solution.
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is 2.0-3.0%, so it is not surprising that chlorophyll synthesis in many cultures has not been very noticeable. It was early noted that chloroplast development is inversely correlated with tissue growth rate, ~ and other studies have substantiated this observation. From the inception of tissue culture studies, major emphasis has been on optimum conditions for growth, and this emphasis has led to an unconscious selection against conditions promoting chlorophyll synthesis. The use of sugar concentrations optimum for growth is a case in point. The auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), is an extremely effective promoter of cell division in cultured tissues and cultures grown on medium containing this auxin usually have little, if any, chlorophyll. A specific affect of this compound on chlorophyll synthesis has been suggested, 21 but it is more likely that this is the result of a primary effect on cell proliferation. Gibberellic acid also promotes growth in many cultured tissues, and chlorophyll synthesis in such cultures is usually inhibited. It is best, therefore, to use a medium that will not promote growth at the expense of chlorophyll synthesis. We have found a-naphthyleneacetic acid (NAA) to be the most effective auxin for such a medium, and a concentration of 0.5 mg of NAA/liter provides satisfactory results. Cytokinins are important ingredients of culture media, since they are generally required for cell division and are often regulators of organ induction. ~6 The most generally used cytokinins are kinetin and benezyladenine. The addition of either of these compounds to the above medium at 0.5 mg/liter completes the basic medium. Exogenously applied cytokinin has been found necessary for chlorophyll synthesis in light-grown tissue of at least one strain of tobacco callus2 This same strain will grow slowly without exogenously applied cytokinin, thus implying endogenous synthesis. If this strain is placed in the light on a medium without auxin or cytokinin it will not grow, but it will form chlorophyll3 5 It is felt that if growth is not limited by other factors, the endogenous cytokinin will be limiting for chloroplast development, but if growth is limited by other factors, then the available cytokinin is available to support chloroplast development. Again, the relationship between chloroplast development and cell replication and growth is extremely close. These events can be influenced by traces of cytokinin remaining after the tissue is transferred to cytokinin-free medium, so particular attention must be paid to ensuring the absence of residual growth regulator in such experiments. Kinetin can induce chloroplast replication without inducing cell replicaF. Skoog and C. O. Miller, Syrup. Soc. Exp. Biol. 11, 118 (1957).
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tion, ~3 so the involvement of cytokinins in chloroplast development in cultured tissue has a promising future. Cytokinin and auxin interactions are also important because of their effect on bud and root induction in certain tissue cultures, and the ability to regulate tissue differentiation has obvious implications for investigations of chloroplast development. Buds can be induced in tobacco tissue by increasing the cytokinin concentration and lowering the auxin concentration. The inverse relationship results in roots induction. It is best to use indoleacetic acid (IAA) for such experiments. An IAA concentration of 0.1 mg/liter and a kinetin concentration of 2.0 m ~ l i t e r will promote extensive bud induction both in our callus cultures and in leaf disk cultures. This bud induction will proceed in either light or dark. Cultures isolated from different species or varieties will have their own requirements, and it is not suggested that the stated auxin-cytokinin ratio will provide positive results in all cases. Embryoids which develop into normal plants can be induced from undifferentiated carrot tissue cultures by eliminating the 2,4-D from the medium27 The effect of 2,4-D on bud induction and chloroplast development is seen in sugar cane cultures2 s The undifferentiated tissues grown on high concentrations of 2,4-D do not form chlorophyll, but when the 2,4-D concentration is lowered, organized leaf primorida appear and chloroplast development commences. This relationship between tissue differentiation and the ability of proplastids to develop into chloroplasts is especially intriguing because of the opportunities it offers to control the development of dimorphic chloroplasts in sugar cane. 39 The kinds and ratios of growth regulators will have to be adapted to the respective requirements of tissues when they are grown on either solid or liquid media. The aim of cell liquid suspension cultures is to have cells which do not stick together, but conditions promoting friability may not promote chloroplast development. In many cultures, for example, low cytokinin concentrations, and the use of 2,4-D and gibberellic acid promote tissue friability but inhibit chloroplast development. High cytokinin concentrations promote chloroplast development and tissue compactness. The use of undefined media components, such as coconut water, 4° was described earlier, because a major purpose of tissue culture techniques is to minimize variables. On the other hand, coconut water and casein 3~W. Halperin and D. F. We~herell,Science 147, 756 (1965). ,sj. p. Nitseh. W. M. Laetsch and I. Price, Amer. J. Bot. 56, 77 (1969). W. Tuleeke, L. H. Weinstein, A. Rutner, and H. S. Laurencot, Contrib. Boyce Thompson Inst. 21, 115 (1961).
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hydrolyzate frequently stimulate chloroplast development, so their use is sometimes justified if chlorophyll synthesis cannot be induced by other means. These additives will often make it possible to culture tissues which only grow slowly, if at all, on defined media, but a tissue which requires such additives for either growth or chlorophyll synthesis must possess some unusual features of chloroplast development to offset the disadvantages of using a chemically undefined medium. Fluorescent lamps are satisfactory light sources for inducing chloroplast development in cultured tissues. The advantage of incandescent lamps in combination with fluorescent lamps has not been established. Many cultured tissues prosper in continuous light, but the optimum photoperiod should be determined for specific tissues. A light intensity of 500-1000 fc is generally the optimum. Cultures tend to become bleached at higher intensities, but this might be as much the result of high temperature within the culture tubes induced by the high light intensity as of any direct effect of the illumination. Cultures are frequently maintained at 100-200 fc, and this intensity is too low for optimum chloroplast development in many tissues. It is often difficult to arrange suitable facilities for maintaining large numbers of cultures under banks of high intensity fluorescent lamps, because the compressor capacity of most temperature-controlled rooms will not handle the high heat output of the lamp ballasts. Mounting the ballasts outside the rooms will usually solve the problem. Most temperature-controlled rooms are fairly narrow, so vertical lamps behind shelves represent the most economical use of space for exposing cultures. The use of commercial growth chambers for growing tissue cultures in the light is very uneconomical in terms of both space and money. Light intensity is controlled either by wrapping the culture vessels in cheesecloth or paper tissues, or by inserting metal screens of various mesh size in front of the lamps. Cultures are commonly grown at 25°-27 °, but some tissues might have unusual temperature requirements. Chloroplast development in cultured tissues can be controlled by influencing the growth rate of the tissue, by manipulating various components of the medium, and by regulating light and temperature. Certain tissues will still not synthesize chlorophyll in spite of such maneuvers. One approach to overcoming this problem is to supply intermediates in prophyrin biosynthesis22 Cultured tissues are frequently impoverished, with respect to their biosynthetic potential, in comparison with differentiated tissues, and it is probably infrequent that chlorophyll synthesis is blocked at only one point. Even a refractory tissue, such as sugar cane callus, will "turn on" its chloroplast development machinery as soon as an organized meristem appears. It is not necessary, therefore, to discard
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a tissue which will not produce chloroplasts, because it might well yield interesting problems because of that fact.
Cytological Techniques A number of standard techniques will be presented, because they are particularly useful for chloroplast studies with cultured tissue. The general anatomical features of cultured tissues are sometimes ignored, and this is unfortunate because an understanding of chloroplast development can be obtained only if there is adequate knowledge of cells and tissues. Histological preparations for light microscopy employ standard techniques. 41 The same can be said for electron microscopy, but since it is necessary to use the electron microscope to really see chloroplasts, the following schedule is given because it has yielded excellent preparations for a variety of cultured tissues. As with all such schedules, it is possible to vary many of the steps without courting disaster. Small pieces of tissue (1 mm or less across the longest axis) are fixed in 2.0% glutaraldehyde and 0.05 M cacodylate buffer pH 6.8-7.0 for 3-10 hours. (Prepare 4.0% glutaraldehyde and add 1:1 to 0.1 M cacodylate buffer. Prepare the buffer with 21.4 g/liter of sodium cacodylate. Adjust the pH with HC1.) The fixation should be done at 4 °. Wash the tissue in cold buffer three times with a 20-minute interval between each wash. Fix the tissue overnight at 4 ° in 2.0% Os04 in water. Dehydrate in the following acetone series: 30%, 20 minutes, 4°; 50%, 20 minutes, 4°; 70% (1.0% UrN0~) overnight at 4°; 90%, 1 minute, 4°; 95%, 15 minutes, 4°; 100%, 20 minutes, room temperature, repeat 3 times. Embed tissue in Epon 42 in the following manner: 50% Epon: 50% acetone overnight (lid on vials); replace 50% Epon in vials with 100% Epon and let stand for 4-6 hours. Fill embedding capsules with Epon and transfer tissue. Keep at room temperature for 24 hours, and at 45 ° and 60 ° each for 24 hours, respectively. The relationship between chloroplast development and the replication and growth of cells has been emphasized, and important aspects of this relationship can be obtained only by counting and measuring both chloroplasts and cells. The best method of counting chloroplasts is by making thick sections (about 40~) of paraffin-embedded material, so that at least one cell layer is included in each section. Chloroplasts can be counted by focusing through cells. Some cells in cultured tissues are very large and the cells must be separated to count the plastids. This can be done with pectinase. 43 The incubation period should be extended to ,1W. A. Jensen, "Botanical Histochemistry." W. H. Freeman, San Francisco, 1962. ~J. H. Luft, J. Biophys. Biochem. Cytol. 9, 409 (1961). 4aE. C. Humphries and P. W. Wheeler, J. Exp. Bot. I1, 81 (1960).
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3 days, and the suspension should be forced through a fine pipette several times at the end of this period. Better cell separation is obtained by the chromic acid method44 which is recommended for making cell counts. The chloroplasts cannot be counted in cells separated with chromic acid, because they are destroyed. Chlorophyll Determinations The method of Arnon 4~ or of Winterman and DeMots 46 employing acetone and ethanol, respectively, as solvents are generally used. Callus tissues usually contain more water than leaf tissues , and this introduces the possibility of error when comparing chlorophyll in the two tissues. Some callus has so much water that the use of 80% acetone will result in too dilute a solution. Another problem is that callus tissue often contains so much starch that much longer centrifugation periods are required to obtain a clear supernatant solution than is customary with leaf tissue. Photosynthetic Measurements Standard methods are easily adapted to tissue cultures. Studies on the incorporation of 14C02 in liquid cultures can be conducted in the same manner as for unicellular algae. The same methods used for leaf tissue can be applied to callus. Very thin slices or small pieces of callus should be used since the very compact tissue is resistant to C02 diffusion relative to leaf tissue. This is one factor that probably limits completely autotrophic growth of tissue cultures. Actually there has been little quantitative work on photosynthesis in cultured tissue, and the instrumentation currently available could be used to good effect. Conclusions
Many of the advantages and difficulties in using cultured tissue for investigating chloroplast development have been considered. The critical point is one that is not always realized by present or potential investigators in this field. It is that tissue cultures should be used only when they can provide a unique tool for investigating a basic problem in plastid development. They should not be used when "normal" tissues will do just as well. If cultured tissues are regarded as a potentially powerful technique for studying certain problems associated with the photosynthetic apparatus, rather than as a way of life, there is reasonable chance of the development of higher plant chloroplasts being explained in more satisfactory terms than at present. R. Brown and P. Rickless, Proc. Roy. Soc. Ser. B. 136, 110 (1949). 4~D. I. Arnon, Plant Physiol. 24, 1 (1949). ~J. F. G. M. Wintermans and A. DeMots, Biochim. Biophys. Acta 109, 448 (1965).
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found that short photoperiods of bright light yielded the highest degree of synchrony. The synchrony developed for Skeletonema costatum, a familiar experimental species, was only slightly better than exponential growth.
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Tissue
Culture:
Plant
B y W. M. LAETSCH
The first true tissue cultures were apparently green, 1 but the use of such plant material for investigating photosynthesis and/or chloroplast development was essentially ignored until recent years. 2,a This is unfortunate, because tissue cultures offer the prospect of providing the desirable experimental features of algal cultures while possessing the unique developmental patterns of higher plants. The delineation of some of the advantages of this system will perhaps indicate some of the ways in which the following methods can be used. Techniques for handling cultured tissues are very similar to those for microorganisms, and the physical environment can be controlled in a common manner. The long-term control of temperature and illumination is, therefore, much more precise than is presently possible for either seedlings or mature plants. It is notoriously difficult, for example, to expose intact plants or detached organs to very high light intensities fo ~ an extended time period. This problem is minimized with cultured cells or tissues. The same is true for quantitative work involving light quality. Since most work on the development of higher plant chloroplasts centers on the light-induced etioplast to chloroplast transformation, the value of a well-defined illumination system cannot be underestimated. The control of the chemical environment is also susceptible to far greater precision than is possible with either whole plants or plant parts. Sterile conditions open the way to a variety of experiments, and the absence of a cuticle in cultured tissues lessens the problems of absorption of exogenous chemicals which so often plague those working with higher plant tissue. A defined substrate for cultured tissues offers opportunities which too often have been ignored. This permits the isolation of events in chloroplast development from general cell responses such as replication and growth. " is next to impossible to regulate the chemical imputs of R. J. Gautheret, C. R. Acad. Sci. 198, 2195 (1934). W. M. Laetsch and D. A. Stetler, Amer. J. Bot. 52, 798 (1965). 3L. Bergma~ and C. Berger, Planta 69, 58 (1966).
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normal leaf tissue, and a result is that "triggers" for chloroplast development usually "trigger" other processes as well. The difficulty in determining reciprocal influences usually results in most of them being ignored. In addition to a greater amount of physiological homogeneity, tissue cultures can possess greater anatomical homogeneity than do organs such as leaves and cotyledons. As a result, a host of variables encountered in "normal" tissues is minimized. A defined substrate also permits the regulation of tissue and organ differentiation. The ability to induce bud formation, for example, allows the examination of chloroplast development in "normal" tissue while still maintaining the advantages of sterility and precise control over the physical environment. Angiosperms, unlike most of the algae, do not synthesize chlorophyll in the dark, but this phenomenon has not been fully exploited experimentally because of the inability to maintain plants or organs in the dark for extended periods. As a result, experiments have been confined to seedlings with large seeds. Tissue cultures can be maintained indefinitely in the dark, and they are the only system for effectively investigating chloroplast continuity in higher plants. The electron microscope is a basic tool for studying chloroplast development, and cultured tissues have certain advantages for electron microscopy. The tissue generally responds well to chemical fixatives and is easy to section. The tissues of many leaves respond in just the opposite fashion. It has frequently been stated that many problems in the development of higher plant chloroplasts will not be solved until chloroplasts can be cultured independently of the cell. While this breakthrough is awaited, cultured tissues offer the possibility of working with simpler, better defined systems than is available with attached or detached plant organs. Summary of Investigations on Chloroplast Development in Cultured Tissues This brief review will provide some examples of how tissue cultures have been used in studying plastid development, but it does not pretend to cover the literature. A number of papers have described aspects of fine structure of developing plastids, 2-~ while others have been more concerned with the effect of growth regulators, nutrients, and light quality on synthesis and/or chloroplast development2 -17 Still others have investigated enzyme patterns during chloroplast development, ls,19 4 H. W. Israel and F. C. Steward, Ann. Bot. 31, 1 (1967). S. J. Blackwell, W. M. Laetsch, and B. B. Hyde, Amer..l. Bot. 56, 457 (1969). D. A. Stetler and W. M. Laetsch, Science 149, 1387 (1965). 7E. M. J. Jaspars, Physiol. Plant. 18, 933 (1965).
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Comparisons of photosynthetic structure and function of developing chloroplasts in cultured versus intact tissues have been made to my knowledge only in tobacco. 2,2° There are differences in the two types of tissue, but these are mostly quantitative in nature. These various studies have established some of the basic parameters influencing chloroplast development in cultured tissue and have demonstrated that the system is amenable to experimental manipulation. Most of these authors have employed callus tissue grown on solid media, but Bergman has utilized liquid suspension cultures in his attempts to induce autotrophic growth of tobacco cells. 21 Most of the previous studies have been on problems similar to those which have been or could be conducted on leaf tissue, but some recent work has been concerned with problems that can be investigated only by means of tissue culture techniques. One such problem concerns the ability of mature chloroplasts in mature leaf tissue to dedifferentiate, divide, and develop again into mature chloroplasts. 22 The leaf is a determinate organ and studies on chloroplast development usually end with the death of the leaf, but the chloroplast life cycle can be continuously generated in cultured tissue. Another recent project concerns the relationship of tissue growth to organelle differentiation in dark-grown cultures. The etioplast characteristic of dark-grown leaves of seedlings has not been found in dark-grown callus. I t has been determined in tobacco that the appearance of mature etioplasts can be controlled by regulating the growth rate of the callus. 23 The prolamellar body, for example, is induced by inhibiting tissue growth and is eliminated by stimulating tissue growth. It has previously been thought that the prolamellar body was intimately involved with the normal etioplast to choroplast transformation, but evidence from tissue cultures suggest 8S. Venketeswaran, Physiol. Plant 18, 776 (1965). I. K. Vasil and A. C. Hildebrandt, Planta 68, 69 (1966). lo G. Beauchesne and M. C. Poulain, Photochem. Photobiol. 5, 157 (1966). la L. Bergman and A. B441z,Planta 70, 285 (1966). R. Schantz, H. Duranton, and M. Peyri~re, C. R. Acad. Sci. Set. D 265, 205 (1967). 13N. Sunderland, Ann. Bot. 30, 253 (1966). it N. Sunderland, Ann. Bot. 31, 573 (1967). 1~R. Boasson and W. M. Laetsch, Experientia 23, 967 (1967). A. K. Stobart, I. McLaren, and D. R. Thomas, Phytochemistry 6, 1467 (1967). 1, p. K. Chen and S. Venketeswaran, Physiol. Plant. 21, 262 (1968). '* A. K. Stobart and D. R. Thomas, Phytochemistry 7, 1313 (1968). 19A. K. Stob~rt and D. R. Thomas, Phytochemistry 7, 1963 (1968). 20D. A. Stetler, Ph.D. Thesis Univ. of California, Berkeley, 1967. ~ L. Bergman, Planta 74, 243 (1967). W. M. Laetsch, Amer. J. Bot. 54, 639 (1967). D. A. Stetler and W. M. Laetsch, Amer. J. Bot. 55, 709 (1968).
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that its appearance is a function of the rate of cell division and that it does not play a causal role in chloroplast development. Problems Encountered with Cultured Tissue It is legitimate to ask why cultured tissues have not been more widely used for studies in chloroplast development and function. Aside from the fact that most people think it is easier to grow bean seedlings than to master tissue culture techniques, there are features of tissue cultures that make them unsuitable for certain studies. A greater appreciation for the potential of cultured tissues in studies on chloroplast development will be' obtained if the difficulties are discussed in some detail. Anatomical heterogeneity is generally less of a problem than in leaves, but genetical heterogeneity is a greater problem. Not only are the cells in a particular callus likely to have a variety of genotypes, but the longer the tissue is kept in culture the greater is the chance for increases in ploidy levels and chromosomal aberrations. 24,2~ This presents problems in duplicating experiments, because the tissue used one month is often genetically very different tissue when used several months later. This genetic variability is at least one cause of the considerable variability in physiological response which many workers have found. In spite of what has been said above about the anatomy of callus, it is a mistake to think that all tissues are completely homogeneous in terms of cell type and cell size. Meristematic rates within callus vary and there are often considerable differences in cell size. Differentiation in the form of xylemlike and phloemlike elements is frequent, and these elements are often localized in complex nodules. An important result of these various types of heterogeneity is the difference in developmental rates within the total plastid population. The chloroplasts in the cells near the surface of the callus often differentiate faster than those in the cells of the interior. The islands of meristematic cells commonly found in callus mean that division of proplastids is taking place in some cells while well developed chloroplasts are present in others. In other words, synchronous development of the chloroplast population is very rare in cultured tissues36 It might be thought that liquid suspension cultures would be the answer to some of these problems, but such cultures are generally very heterogeneous themselves since they consist of single cells and cell clumps of various sizes. Many of the cell clumps are actually smaller than many of the single cells. There are some ~4j. G. Torrey, Physiol. Plant. 20, 265 (1967). D. J. Heinz, G. W. P. Mee, and L. G. Nickell, Amer. J. Bot. 5{},450 (1969). M. M. Yeoman and P. K. Evans, Ann. Bot. 31, 323 (1967).
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cultures of tobacco which have a high proportion of free cells, but the cells will not synthesize chlorophyll. Dark-grown bean or barley leaves turn green within a matter of hours upon exposure to light. Dark-grown cultured tissue turns green very slowly and may take as long as a week for the development of mature chloroplasts. While the concentration of chlorophyll per dry weight of green tissue varies between cultures, it is usually much less than that of the leaf tissue of the corresponding tissue. In tobacco, for example, the mature leaf can have twenty times more chlorophyll on a dry weight basis than callus. ~ This is mainly a result of fewer chloroplasts per cell. The low final concentration of chlorophyll means that small differences during chloroplast development are difficult to quantify. While the leisurely synthesis of chlorophyll in cultured tissue is a handicap for those in a hurry, it can be used to advantage to study the intermediate steps in development which sometimes happen so rapidly in leaf tissue they are difficult to detect. The number of chloroplasts per unit volume of tissue is rather small, and rich yields of isolated chloroplasts are difficult to obtain. The chloroplasts are also full of starch, and, since the medium has sugar, the starch cannot be removed by placing the tissue in the dark. A solution to this problem might be effected by a continuation of the preliminary work on growing tissues on sugar-free medium. 21,27 Initiation of Cultures
General methods for establishing and maintaining tissue cultures have been provided in detail by several authors. ~s-a° A common impression is that tissue culture is a complex and difficult technique, but it is actually very simple. The basic rules of sterile technique must be followed, but other than that there is not a single "best way" to grow cultured tissue. The main problem is to place uncontaminated but live tissue on a medium that will induce cell division. Sterile tissue is obtained either by stripping away external tissue from an organ in order to excise internal uncontaminated tissue, or the external surface is sterilized and the whole organ is cut into explants. The latter method is used in the leaf sterilization method described below. Sterile transfer rooms and expensive inoculation hoods are nice to have, but are quite unnecessary for such work. The main problem in these operations is to eliminate air ~'T. Fukami and A. C. Hildebrandt, Bot. Mag. Tokyo 80, 199 (1967). R. J. Gautheret, "La Cillture des Tissues V4g6taux." Masson, Paris, 1959. P. R. White, "The Cultivation of Animal and Plant Cells." Ronald Press, New York, 1963. 'oH. E. Street, in "Techniques for the Study of Development" (F. H. Wilt and N. K. Wessells, eds.), p. 425. Crowell, New York, 1967.
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movements, and a few well arranged piecos of cardboard on a laboratory bench are as satisfactory as the glossiest transfer rooms. Stem explants containing cambial tissue are used most commonly as starting material for cultures. Leaf tissue is often more difficult to culture, although in some species such as tobacco, callus is easy to obtain from leaves. Callus is readily obtained from the roots of many species, but such cultures are probably less likely to be green than are those from stems and leaves. Herbaceous dicotyledons have been used most extensively for tissue cultures, because they have proved relatively easy to grow. Monocotyledonous tissues, and especially grasses, have traditionally been difficult to grow in culture, but recent work suggests that many of these difficulties show signs of being solved. 31 It remains to be seen whether chlorophyll synthesis in these monocot tissues will be sufficient to use them for studies in chloroplast development. Certain gymnosperms have been cultured with considerable success, but pteridophytes have not been promising material. 28 With the exception of tissue cultures of Ginko, there has only been casual mention of the ability of cultured tissues of gymnosperms to form chlorophyll?2 Many types of culture vessels are available, and the most suitable type is more a function of the arrangement of the available light system than any other factor. Culture tubes and flasks are more suitable for vertical lamps, and petri dishes are best for overhead lamps. Their optical properties make plastic petri dishes preferable to glass. A fair amount of nonsense has been written about the necessity for special types of specialized (and expensive) culture vessels, but there is little evidence that such exotic ware produces any better results than those obtained with standard flasks and culture tubes. Cultured tissues are generally defined as clumps of randomly proliferating cells, but there is no reason the term cannot apply to tissues of organs which are cultured in their "normal" state of differentiation under sterile conditions. This type of cultured tissue eliminates many of the disadvantages associated with callus tissue, and permits the exploitation of "normal" tissue in a sterile environment with a defined source of nutrition. 33 Small pieces of leaf tissue are examples of such cultures. The following method has been primarily used with tobacco tissue, but the same procedures work with bean leaves, and other species would undoubtedly provide suitable material. The same basic method can be used to surface sterilize stems, seed, cotyledons, or flowers, and the tissue can ~1y . Yamada, K. Tanaka, and E. Takahashi, Proc. Jap. Acad. 43, 156 (1967). 32W. Tulecke, Amer. J. Bot. 54, 797 (1967). 3~R. Boasson and W. M. Laetsch, Science 166, 749 (1969).
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then be maintained on a minimal medium or callus can be induced with a complete medium. Cut strips approximately 1 inch wide from the center portion of the leaf avoiding the main vein, the tip, and the base of the leaf. Etiolated leaves are treated intact. Soak the strips for 2-4 minutes in a beaker of water with a small amount of laboratory detergent. The subsequent steps should be conducted under a hood. Transfer the strips to a 4.0% hypochlorite solution (dilute commercial bleach 1:5 with water) with a small amount of laboratory detergent as wetting agent. The detergent is more effective than Tween. Soak the leaf strips for 5 minutes; stirring occasionally to remove air bubbles adhering to the leaf surface. Use a sterile forceps to transfer the strips to a beaker or flask containing sterile water and rinse by swirling the beaker. Transfer the leaf strips to petri dishes containing two pieces of sterile filter paper. Two strips are a convenienb number for each dish. Use a sterile cork borer (preferably stainless steel) to cut disks from the strips. Large veins should be avoided. The disks will collect inside the cork borer and should be pushed out with a sterile glass rod. Never sterilize the disks after they have been cut from the strips, because the large cut surface will permit serious damage by the hypochlorite. When the disks are placed on medium in petri dishes, they should be randomized; that is, the disks from one leaf should not all be placed in the same culture dish. Disks 2 mm in diameter are a convenient size, and 10 disks will fit in a 5-cm petri dish. Small disks are recommended because the cells rapidly lose stored nutrients and become dependent upon those in the medium. Large disks invariably mean the inclusion of major veins, and this means greater anatomical and physiological heterogeneity in the tissue. A minimal medium composed only of the inorganic constituents of the medium described below plus sucrose will maintain leaf tissue in the dark for as long as 3 months. It is also sufficient for normal rates of chlorophyll synthesis in disks of etiolated leaves. Cell proliferation resulting in callus formation can be induced in either etiolated or mature leaf tissue cultured on the minimal medium by transferring the tissue to a complete medium composed of the inorganic and organic components plus the growth regulators described below. Cell division will occur throughout the leaf disk, but it will be most active at the periphery. Enough callus will be formed in the latter region within 1-2 weeks to permit subculturing. There are many types of culture media, and the tissues of each species and perhaps each organ will have their special requirements. A reasonable approach is to start with a medium on which a large number of different tissues will grow reasonably well and then gradually define the requirements for specific tissues. The following medium is basically that
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of Murashige and Skoog,34 and it has proved satisfactory for a wide variety of dicotyledonous tissues. The use of undefined or complex ingredients such as coconut water, casein hydrolyzate, or yeast extract is to be avoided if at all possible. If used for the reasons described below, they should be added to the other inorganic and organic constituents before addition of the agar. Coconut water is generally used at a concentration of 10-20% (v/v). If 100 ml of coconut water is used, then the amount of water added to the organic and inorganic constituents is correspondingly reduced. The fresh coconut water should be autoclaved for a few minutes, and the precipitated proteins filtered before use in the culture medium. It is convenient to freeze unused coconut water in 50- or 100-ml portions for future use. The medium is dispensed into tubes or flasks and autoclaved at 15 lb for 15 minutes. Certain vitamins, antibiotics, etc., are filter-sterilized (the sterile plastic units with a membrane filter are most convenient) and added to the sterile medium. Experiments with tissue cultures are usually measured in weeks rather than days and desiccation of the medium is frequently a problem. This is aggravated in the light because of the greenhouse effect. Petri dishes sealed around the bottom with masking tape will not dry out for many weeks and gas exchange does not seem to be seriously affected. Flasks and tubes with metal or plastic closures can be treated in a similar fashion. Cotton plugs are the least satisfactory type of closure. Sterile sheets of polyethylene are frequently used to cover culture vessels, but their effectiveness in inhibiting desiccation and permitting the transmittance of light is counterbalanced by their difficult manipulation during tissue transfer. Control of Chloroplast Development Chloroplast development is strongly influenced by the growth and physiological state of the host cells, so the ability to control the development of cells assumes great importance. Nutrients and growth regulators are very effective control agents. Chlorophyll synthesis in light-grown tissue cultures can be markedly influenced by sugar concentrationJ ,12 High concentrations inhibit synthesis and stimulate tissue growth. Sucrose also inhibits chlorophyll synthesis in cultured tobacco leaf tissue when it is present at concentrations of 2.0% and higher2 ~ The standard sucrose concentration for tissue cultures ~ T. Murashige and F. Skoog, Physiol. Plant. 15, 473 (1962). W. M. Laetsch, Amer. J. Bot. 53, 613 (1966).
104
ISOLATION AND CULTURE TECHNIQUES Stock Solution I Dissolve the following one at a time in 5 liters of glassdistilled water: NH~N03 16.5 g KNOB 19.0 g CaCI~ 2 H20 2.2 g MgSO4" 7 H20 1.85 g KH2PO4 1.7 g H,BOs 0. 062 g MnSO4" H~O 0.169 g ZnSO4.7 H~O 0.106 g KI 0. O083 g Stock Solution 2 Dissolve in lo0 ml of glass-distilled water: FeSO~.7 H20 5.57 g Na~EDTA 7.45 g Stock Solution 3 Dissolve in 100 ml of glass-distilled water: CuSO4- 5 H~O 0.025 g CoC12.6 H~O 0.025 g Stock Solution Dissolve in 100 ml of glass-distilled water: Na~MoO4 0. 025 g Stock Solution 5 Dissolve in 100 ml of glass-distilled water: Glycine 0.2 g Nicotinic acid 0.05 g Pyridoxine. HC1 0.05 g Thiamine. HC1 0.01 g Medium Combine the following to make 1 liter of medium: Stock Sol. 1 500 ml Stock Sol. 2 0.5 ml Stock Sol. 3 0.1 ml Stock Sol. 4 1.0 ml Stock Sol. 5 1.0 ml Add the following to the above: Myoinositol 0.1 g Sucrose 20 g Growth regulators are added to the above (see discussion below). Adjust the pH of the solution to 5.7-6.0 with 0.1 N NaOH. A liter of solid medium is made by melting 8 g of agar in 497 ml of glass-distilled water and adding to the above solution. A liter of liquid medium is made by adding 497 ml of glass-distilled water to the above solution.
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is 2.0-3.0%, so it is not surprising that chlorophyll synthesis in many cultures has not been very noticeable. It was early noted that chloroplast development is inversely correlated with tissue growth rate, ~ and other studies have substantiated this observation. From the inception of tissue culture studies, major emphasis has been on optimum conditions for growth, and this emphasis has led to an unconscious selection against conditions promoting chlorophyll synthesis. The use of sugar concentrations optimum for growth is a case in point. The auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), is an extremely effective promoter of cell division in cultured tissues and cultures grown on medium containing this auxin usually have little, if any, chlorophyll. A specific affect of this compound on chlorophyll synthesis has been suggested, 21 but it is more likely that this is the result of a primary effect on cell proliferation. Gibberellic acid also promotes growth in many cultured tissues, and chlorophyll synthesis in such cultures is usually inhibited. It is best, therefore, to use a medium that will not promote growth at the expense of chlorophyll synthesis. We have found a-naphthyleneacetic acid (NAA) to be the most effective auxin for such a medium, and a concentration of 0.5 mg of NAA/liter provides satisfactory results. Cytokinins are important ingredients of culture media, since they are generally required for cell division and are often regulators of organ induction. ~6 The most generally used cytokinins are kinetin and benezyladenine. The addition of either of these compounds to the above medium at 0.5 mg/liter completes the basic medium. Exogenously applied cytokinin has been found necessary for chlorophyll synthesis in light-grown tissue of at least one strain of tobacco callus2 This same strain will grow slowly without exogenously applied cytokinin, thus implying endogenous synthesis. If this strain is placed in the light on a medium without auxin or cytokinin it will not grow, but it will form chlorophyll3 5 It is felt that if growth is not limited by other factors, the endogenous cytokinin will be limiting for chloroplast development, but if growth is limited by other factors, then the available cytokinin is available to support chloroplast development. Again, the relationship between chloroplast development and cell replication and growth is extremely close. These events can be influenced by traces of cytokinin remaining after the tissue is transferred to cytokinin-free medium, so particular attention must be paid to ensuring the absence of residual growth regulator in such experiments. Kinetin can induce chloroplast replication without inducing cell replicaF. Skoog and C. O. Miller, Syrup. Soc. Exp. Biol. 11, 118 (1957).
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tion, ~3 so the involvement of cytokinins in chloroplast development in cultured tissue has a promising future. Cytokinin and auxin interactions are also important because of their effect on bud and root induction in certain tissue cultures, and the ability to regulate tissue differentiation has obvious implications for investigations of chloroplast development. Buds can be induced in tobacco tissue by increasing the cytokinin concentration and lowering the auxin concentration. The inverse relationship results in roots induction. It is best to use indoleacetic acid (IAA) for such experiments. An IAA concentration of 0.1 mg/liter and a kinetin concentration of 2.0 m ~ l i t e r will promote extensive bud induction both in our callus cultures and in leaf disk cultures. This bud induction will proceed in either light or dark. Cultures isolated from different species or varieties will have their own requirements, and it is not suggested that the stated auxin-cytokinin ratio will provide positive results in all cases. Embryoids which develop into normal plants can be induced from undifferentiated carrot tissue cultures by eliminating the 2,4-D from the medium27 The effect of 2,4-D on bud induction and chloroplast development is seen in sugar cane cultures2 s The undifferentiated tissues grown on high concentrations of 2,4-D do not form chlorophyll, but when the 2,4-D concentration is lowered, organized leaf primorida appear and chloroplast development commences. This relationship between tissue differentiation and the ability of proplastids to develop into chloroplasts is especially intriguing because of the opportunities it offers to control the development of dimorphic chloroplasts in sugar cane. 39 The kinds and ratios of growth regulators will have to be adapted to the respective requirements of tissues when they are grown on either solid or liquid media. The aim of cell liquid suspension cultures is to have cells which do not stick together, but conditions promoting friability may not promote chloroplast development. In many cultures, for example, low cytokinin concentrations, and the use of 2,4-D and gibberellic acid promote tissue friability but inhibit chloroplast development. High cytokinin concentrations promote chloroplast development and tissue compactness. The use of undefined media components, such as coconut water, 4° was described earlier, because a major purpose of tissue culture techniques is to minimize variables. On the other hand, coconut water and casein 3~W. Halperin and D. F. We~herell,Science 147, 756 (1965). ,sj. p. Nitseh. W. M. Laetsch and I. Price, Amer. J. Bot. 56, 77 (1969). W. Tuleeke, L. H. Weinstein, A. Rutner, and H. S. Laurencot, Contrib. Boyce Thompson Inst. 21, 115 (1961).
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hydrolyzate frequently stimulate chloroplast development, so their use is sometimes justified if chlorophyll synthesis cannot be induced by other means. These additives will often make it possible to culture tissues which only grow slowly, if at all, on defined media, but a tissue which requires such additives for either growth or chlorophyll synthesis must possess some unusual features of chloroplast development to offset the disadvantages of using a chemically undefined medium. Fluorescent lamps are satisfactory light sources for inducing chloroplast development in cultured tissues. The advantage of incandescent lamps in combination with fluorescent lamps has not been established. Many cultured tissues prosper in continuous light, but the optimum photoperiod should be determined for specific tissues. A light intensity of 500-1000 fc is generally the optimum. Cultures tend to become bleached at higher intensities, but this might be as much the result of high temperature within the culture tubes induced by the high light intensity as of any direct effect of the illumination. Cultures are frequently maintained at 100-200 fc, and this intensity is too low for optimum chloroplast development in many tissues. It is often difficult to arrange suitable facilities for maintaining large numbers of cultures under banks of high intensity fluorescent lamps, because the compressor capacity of most temperature-controlled rooms will not handle the high heat output of the lamp ballasts. Mounting the ballasts outside the rooms will usually solve the problem. Most temperature-controlled rooms are fairly narrow, so vertical lamps behind shelves represent the most economical use of space for exposing cultures. The use of commercial growth chambers for growing tissue cultures in the light is very uneconomical in terms of both space and money. Light intensity is controlled either by wrapping the culture vessels in cheesecloth or paper tissues, or by inserting metal screens of various mesh size in front of the lamps. Cultures are commonly grown at 25°-27 °, but some tissues might have unusual temperature requirements. Chloroplast development in cultured tissues can be controlled by influencing the growth rate of the tissue, by manipulating various components of the medium, and by regulating light and temperature. Certain tissues will still not synthesize chlorophyll in spite of such maneuvers. One approach to overcoming this problem is to supply intermediates in prophyrin biosynthesis22 Cultured tissues are frequently impoverished, with respect to their biosynthetic potential, in comparison with differentiated tissues, and it is probably infrequent that chlorophyll synthesis is blocked at only one point. Even a refractory tissue, such as sugar cane callus, will "turn on" its chloroplast development machinery as soon as an organized meristem appears. It is not necessary, therefore, to discard
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a tissue which will not produce chloroplasts, because it might well yield interesting problems because of that fact.
Cytological Techniques A number of standard techniques will be presented, because they are particularly useful for chloroplast studies with cultured tissue. The general anatomical features of cultured tissues are sometimes ignored, and this is unfortunate because an understanding of chloroplast development can be obtained only if there is adequate knowledge of cells and tissues. Histological preparations for light microscopy employ standard techniques. 41 The same can be said for electron microscopy, but since it is necessary to use the electron microscope to really see chloroplasts, the following schedule is given because it has yielded excellent preparations for a variety of cultured tissues. As with all such schedules, it is possible to vary many of the steps without courting disaster. Small pieces of tissue (1 mm or less across the longest axis) are fixed in 2.0% glutaraldehyde and 0.05 M cacodylate buffer pH 6.8-7.0 for 3-10 hours. (Prepare 4.0% glutaraldehyde and add 1:1 to 0.1 M cacodylate buffer. Prepare the buffer with 21.4 g/liter of sodium cacodylate. Adjust the pH with HC1.) The fixation should be done at 4 °. Wash the tissue in cold buffer three times with a 20-minute interval between each wash. Fix the tissue overnight at 4 ° in 2.0% Os04 in water. Dehydrate in the following acetone series: 30%, 20 minutes, 4°; 50%, 20 minutes, 4°; 70% (1.0% UrN0~) overnight at 4°; 90%, 1 minute, 4°; 95%, 15 minutes, 4°; 100%, 20 minutes, room temperature, repeat 3 times. Embed tissue in Epon 42 in the following manner: 50% Epon: 50% acetone overnight (lid on vials); replace 50% Epon in vials with 100% Epon and let stand for 4-6 hours. Fill embedding capsules with Epon and transfer tissue. Keep at room temperature for 24 hours, and at 45 ° and 60 ° each for 24 hours, respectively. The relationship between chloroplast development and the replication and growth of cells has been emphasized, and important aspects of this relationship can be obtained only by counting and measuring both chloroplasts and cells. The best method of counting chloroplasts is by making thick sections (about 40~) of paraffin-embedded material, so that at least one cell layer is included in each section. Chloroplasts can be counted by focusing through cells. Some cells in cultured tissues are very large and the cells must be separated to count the plastids. This can be done with pectinase. 43 The incubation period should be extended to ,1W. A. Jensen, "Botanical Histochemistry." W. H. Freeman, San Francisco, 1962. ~J. H. Luft, J. Biophys. Biochem. Cytol. 9, 409 (1961). 4aE. C. Humphries and P. W. Wheeler, J. Exp. Bot. I1, 81 (1960).
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TISSUE CULTURE:
PLANT
109
3 days, and the suspension should be forced through a fine pipette several times at the end of this period. Better cell separation is obtained by the chromic acid method44 which is recommended for making cell counts. The chloroplasts cannot be counted in cells separated with chromic acid, because they are destroyed. Chlorophyll Determinations The method of Arnon 4~ or of Winterman and DeMots 46 employing acetone and ethanol, respectively, as solvents are generally used. Callus tissues usually contain more water than leaf tissues , and this introduces the possibility of error when comparing chlorophyll in the two tissues. Some callus has so much water that the use of 80% acetone will result in too dilute a solution. Another problem is that callus tissue often contains so much starch that much longer centrifugation periods are required to obtain a clear supernatant solution than is customary with leaf tissue. Photosynthetic Measurements Standard methods are easily adapted to tissue cultures. Studies on the incorporation of 14C02 in liquid cultures can be conducted in the same manner as for unicellular algae. The same methods used for leaf tissue can be applied to callus. Very thin slices or small pieces of callus should be used since the very compact tissue is resistant to C02 diffusion relative to leaf tissue. This is one factor that probably limits completely autotrophic growth of tissue cultures. Actually there has been little quantitative work on photosynthesis in cultured tissue, and the instrumentation currently available could be used to good effect. Conclusions
Many of the advantages and difficulties in using cultured tissue for investigating chloroplast development have been considered. The critical point is one that is not always realized by present or potential investigators in this field. It is that tissue cultures should be used only when they can provide a unique tool for investigating a basic problem in plastid development. They should not be used when "normal" tissues will do just as well. If cultured tissues are regarded as a potentially powerful technique for studying certain problems associated with the photosynthetic apparatus, rather than as a way of life, there is reasonable chance of the development of higher plant chloroplasts being explained in more satisfactory terms than at present. R. Brown and P. Rickless, Proc. Roy. Soc. Ser. B. 136, 110 (1949). 4~D. I. Arnon, Plant Physiol. 24, 1 (1949). ~J. F. G. M. Wintermans and A. DeMots, Biochim. Biophys. Acta 109, 448 (1965).