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Possibly the contribution of Haberlandt to the Sitzungsberichte der Wissenschaftlichen Akademie zu Wien more than a century ago (Haberlandt 1902) can be regarded as the first publication of experiments to culture isolated tissue from a plant (Tradescantia). To secure nurture requirements, Haberlandt used leaf explants capable of active photosynthesis. Nowadays, we know leaf tissue is rather difficult to culture. With these experiments (and others), Haberlandt wanted to promote a “physiological anatomy” of plants. In his book on the topic, with its 600 odd pages, he only once cited his “tissue culture paper” (page 13), although he was not very modest in doing so. Haberlandt wrote:

Gewöhnlich ist die Zelle als Elementarorgan zugleich ein Elementarorganismus; mit anderen Worten: sie steht nicht bloß im Dienste der höchsten individuellen Lebenseinheit, der ganzen Pflanze, sondern gibt sich selbst als Lebenseinheit niedrigen Grades zu erkennen. So ist z.B. jede von den chlorophyllführenden Palisadenzellen des Phanerogamenlaubblattes ein elementares Assimilationsorgan, zugleich aber auch ein lebender Organismus: man kann die Zelle mit gehöriger Vorsicht von dem gemeinschaftlichen Zellverbande loslösen, ohne daß sie deshalb sofort aufhören würde zu leben. Es ist mir sogar gelungen, derartige Zellen in geeigneten Nährlösungen mehrere Wochen lang am Leben zu erhalten; sie setzten ihre Assimilationstätigkeit fort und fingen sogar in sehr erheblichem Maße wieder zu wachsen an.

In English, this reads:

Usually, a cell is an elementary organ as well as an elementary organism—it is not only part of an individual living unit, i.e., of the intact plant, but also is itself a living unit at a lower organizational level. As an example, each palisade cell of the phanerogamic leaf blade containing chlorophyll is an elementary unit of assimilation, and concurrently a living organism—careful isolation from the tissue keeps these cells alive. I have even been able to maintain such cells living in a suitable nutrient medium for several weeks; assimilation continued, and considerable growth was possible.

With this, the theoretical basis of plant and tissue culture systems as practiced nowadays was defined. Apparently, this work was of minor importance to Haberlandt, who viewed it only as evidence of a certain independence of cells from the whole organism. Nevertheless, it has to be kept in mind that at the time Schleiden and Schwann’s theory of significance of cells was only about 60 years old (cf. Schwann 1839). Later, Haberlandt abandoned this area of research, and turned to studying wound healing in plants. A critical review is given by Krikorian and Berquam (1986).

It was not before the late 1920s–early 1930s that in vitro studies using plant cell cultures were resumed, in particular due to the successful cultivation of animal tissue, mainly by Carrell. In a paper published in 1927, Rehwald reported the formation of callus tissue on cultured explants of carrot and some other species, without the influence of pathogens. Subsequently, Gautheret (1934) described growth by cell division in vitro of cultured explants from the cambium of Acer pseudoplatanus. Growth of these cultures came to a halt, however, after about 18 months. Meanwhile, the significance of indole acetic acid (IAA) became known, as a hormone influencing cell division and cellular growth. Rehwald did not continue his studies, but based on these, Nobecourt (1937) investigated the significance of this auxin for growth of carrot explants. Successful long-term growth of cambium explants was reported at about the same time by Gautheret (1939) and White (1939).

For Gautheret and Nobecourt, continued growth could be maintained only in the presence of IAA. White, however, was able to achieve this without IAA, by using tissue of a hybrid of Nicotiana glauca and Nicotiana langsdorffii. Intact plants of this hybrid line are also able to produce cancer-like outgrowth of callus without auxin. Many years later, a comparable observation was made on hybrids of two Daucus subspecies produced by protoplast fusion, yielding somatic embryos for intact plants (Sect. 7.3) in an inorganic nutrient medium. Daucus and Nicotiana have remained model systems for cell culture studies until now, but have recently been rivaled by Arabidopsis thaliana.

In the investigations discussed so far, the main aim was to unravel the physiological functions of various plant tissues, and their contributions to the life of the intact plant. In the original White’s basal medium often used, not much fresh weight is produced, and this mainly by cellular growth. Only a low rate of cell division has been observed.

A new turn of studies was induced in the late 1950s and early 1960s by the work of the research group of F.C. Steward at Cornell University in Ithaca, NY, and of F. Skoog’s group in Wisconsin. Steward was interested mainly in relations between nutrient uptake and tissue growth intensity. To this end, he attempted to use fast and slow growing tissue cultures of identical origin in the intact plant as model systems. He was aware of the work of van Overbeck et al. (1942), who used coconut milk, i.e., the liquid endosperm of Cocos nucifera, to grow immature embryos derived from hybrids of crossings between different Datura species. Usually, the development of embryos of such hybrids is very poor, and they eventually die. Following the application of coconut milk, however, their development was accomplished. A supplement of coconut milk to the original medium of P. White induced vigorous growth in quiescent carrot root explants (secondary phloem), compared to that in the original nutrient medium. For Steward, this meant he now had an experimental system in which, by addition or omission of coconut milk, it was possible to evaluate the role played by variations in growth intensity of tissue of identical origin in the plant (Caplin and Steward 1949). The supplement of coconut milk induced growth mainly by cell division that resulted in dedifferentiation of the cultured root explants, and the histological characteristics of the secondary phloem tissue was soon lost. This probably provoked P. White, at a conference in 1961, to ask “What do you need coconut milk for?”

The observation of the induction of somatic embryogenesis in cell suspensions was an unexpected by-product of such experiments (Steward et al. 1958; see Sect. 7.3), a process described at about the same time also by Reinert (1959). Contrary to Steward, who observed somatic embryogenesis in cell suspensions derived from callus cultures, Reinert described this process in callus cultures.

At the beginning of the 1950s, the Steward group initiated investigations to isolate and characterize the chemical components of coconut milk responsible for the vigorous growth of carrot explants, after its supplementation to the nutrient medium. Similar influences on growth became known for liquid endosperms of other plant species, like Zea or Aesculus, and these were consequently included into the investigations. Some years ago, when already retired, Steward (1985) published a very good summary of these investigations, and therefore no detailed discussion of this work will be attempted here, but some highlights will be recalled.

In summary, using ion exchange columns, three fractions with growth-promoting properties have been isolated from coconut milk. These are an amino acid fraction that, to promote growth, can be replaced by casein hydrolysate, or other mixtures of amino acids. Then came the identification of some active components of a neutral fraction. This fraction contains mainly carbohydrates, and other chemically neutral compounds. Particularly active in the carrot assay were three hexitols, i.e., myo- and scyllo-inositol, and sorbitol. Of these, the strongest growth promotion was obtained with m-inositol: 50 mg/l of this as supplement induced the same amount of growth as did the whole neutral fraction of coconut milk. Actually, earlier also White (1954) recommended an m-inositol supplement to the media as a promoter of growth. Finally, there remains the so-called active fraction of coconut milk to be characterized, the analysis of which is yet not really completed. Still, the occurrence of 2-isopentenyladenine, and of zeatin and some derivatives of these have been detected, and it seems justifiable to label it as the cytokinin fraction of coconut milk. The occurrence of these cytokinins would be responsible for the strong promotion of cell division activity by coconut milk, as will be described later.

In terms of when they were discovered, cytokinins are a rather “young” group of phytohormones, the detection of which is tightly coupled with cell and tissue culture. The first characterized member of this group was accidentally detected in autoclaved DNA. Its supplementation to cultured tobacco pith explants induced strong growth by cell division, and consequently it was named kinetin (Miller et al. 1955). Chemically, kinetin is a 6-substituted adenine. In plants, this compound has not been detected yet; it should be the product of chemical reactions associated with the process of autoclaving, and deviating from enzymatic in situ reactions.

Using tobacco pith explants, Skoog and Miller (1957) carried out by now classic experiments demonstrating the influences of changes in the auxin/cytokinin 
concentration ratio on organogenesis in cultures. If auxin dominates, then the ­formation of adventitious roots is promoted; if cytokinins dominate, then the differentiation of shoot parts is observed. At a certain balance between the two hormone groups in the medium, undifferentiated callus growth results (Skoog and Miller 1957). These results are not as distinct in other experimental systems, but the principle derived from these experiments seems to be valid, and to some extent it can be applied also to intact plants.

As mentioned above, the liquid endosperm of Zea exerts a similar influence on growth as does coconut milk. Based on the work of the Steward group, Letham (1966) isolated the first native cytokinin, and fittingly it was named zeatin. Shortly after, a second native cytokinin, 2-isopentenyladenine, was identified, which is a precursor of zeatin. Since then, several derivatives have been described, and today more than 20 naturally occurring cytokinins are known, a number that will certainly grow.

In the early 1960s, the way was paved to formulate the composition of synthetic nutrient media able to produce the same results as those obtained with complex, naturally occurring ingredients such as coconut milk or yeast extracts (of unknown composition). Nowadays, mostly the Murashige–Skoog medium (Murashige and Skoog 1962) is used, with a number of adaptations for specific purposes (cf. MS medium; see tables and further information in Chap. 3). In such synthetic media, somatic embryogenesis in carrot cultures was soon also induced (Halperin and Wetherell 1965; Linser and Neumann 1968).

Another line of research was initiated by the National Aeronautics and Space Administration (NASA), which started to support research on plant cell cultures for regenerative life support systems (Krikorian and Levine 1991; Krikorian 2001, 2003). Since the early 1960s, experiments with plants and plant tissue cultures have been performed under various conditions of microgravity in space (cf. one-way spaceships, biosatellites, space shuttles and parabolic flights, and the orbital stations Salyut and Mir), accompanied by ground studies using rotating clinostat vessels (http://www.estec.esa.nl./spaceflights).

Neumann’s (1966) formulation of the NL medium (see tables and further information in Chap. 3) was based on a mineral analysis of coconut milk (NL, Neumann Lösung, or medium). The concentrations of mineral nutrients in this liquid endosperm were applied, in addition to those already used for White’s basal medium; moreover, 200 mg casein hydrolysate/l was supplemented, and kinetin, IAA, and m-inositol were applied at the concentrations given in the tables.

Using such synthetic nutrient media, it was possible to investigate the significance of each individual ingredient for the growth and differentiation of cultured cells, or for the biochemistry of the cells, including the production of components of secondary metabolism. This will be dealt with in later chapters of the book.

In the early 1960s appeared the first reports on androgenesis (Guha and Maheshwari 1964), and on the production and culture of protoplasts (Cocking 1960). Concurrently, systematic studies on components of secondary metabolism, mainly of medical interest, were initiated. At that time, cell and tissue cultures were at an initial peak of enthusiasm and popularity, which stretched from the end of the 1960s to the second half of the 1970s. The state of knowledge was such as to stimulate expectations of an imminent practical application of these techniques in many domains, e.g., plant breeding, the production of enzymes, and that of drugs for medical purposes. To this end, considerable financial resources were made ­available from governments, as well as from private companies. Potential applications seemed limitless, and included rather exotic ones such as the production of food for silkworms. These high investments were accompanied by first applications for patents (some examples from that time are given in Table 2.1). In the late 1970s, however, reality caught up—promises made by scientists (or at least by some) to sponsors, and expectations raised for an early application of these techniques on a commercial basis were not fulfilled—a “hangover” was the result.

Table 2.1 Some examples of patent applications in Japan in the 1970s
Full size table

All projects envisaged in that period had aspects related with cellular differentiation and its control. It was realized that without a clear understanding of these fundamental biological processes, enabling scientists to interfere accordingly to reach a given commercial goal, only an empirical trial and error approach was possible. In that pioneer phase in the commercialization of cell and tissue culture, a parallel was often drawn with the early days in the commercial use of microbes, i.e., the production of antibiotics with its originally low yield. It seemed to be necessary only to select high-yielding strains. Compared to microbes, however, the biochemical status of cultured plant cells is less stable, and many initially promising approaches were eventually found to lead to a technological blind alley. Furthermore, it has to be kept in mind that at the advent of antibiotics, no competitor was on the market. By contrast, for substances produced by plant cell cultures, well-established industrial methods and production lines exist. Also, the commercial production of enzymes and other proteins found solely in cells of higher plants would be based on microbes transformed by inserting genes of higher plants. Evidently, of more importance is certainly somatic embryogenesis to raise genetically transformed cell culture strains, and to produce intact plants for breeding—on condition that the transformation be carried out on protoplasts, or isolated single cells.

A first system of this kind was reported by Potrykus in 1984 at the Botanical congress in Vienna (see Sect. 13.2). Kanamycin resistance was incorporated into tobacco protoplasts, from which kanamycin-resistant tobacco plants were obtained. Here, cell culture techniques were an indispensable, integral part of the experiments. Later, these basic principles were applied in many other systems and today, after hundreds of genetic transformations, 100,000s hectares are planted with genetically transformed cultivated plants (see Sect. 13.2). An initial attempt to introduce commercially useful traits into plants was to prolong the viable storage period of tomatoes (Klee et al. 1991); these tomatoes became known as “Flavr-Savr”. In spite of being patented (Patent EP240208), commercial success was rather limited, and they were never permitted on the European market. In Chapter 13, more details will be given on gene technology.

It was known for a long time that green cultured cells are able to perform photosynthesis (Neumann 1962, 1969; Bergmann 1967; Neumann and Raafat 1973; Kumar 1974a, b; Kumar et al. 1977, 1989, 1990; Neumann et al. 1977; Roy and Kumar 1986, 1990; Kumar and Neumann 1999; see review by Widholm 1992). In the 1980s were published the first papers reporting the prolonged cultivation of green cultures of various species growing at normal atmosphere in an inorganic nutrient medium (Bender et al. 1981; Neumann et al. 1982; Kumar et al. 1983a, b, 1984, 1987, 1989, 1999; Bender et al. 1985). Subsequently, the ability of such cultures to produce somatic embryos was demonstrated (see Chaps. 7, 9). More recently, methods have been published to raise immature somatic embryos of the cotyledonary stage under autotrophic conditions, yielding intact plants (Chap. 7). It remains to be seen to which extent such material will be useful to obtain plants with special genetic transformations involving photosynthesis. Later, more details on this will be given (see Sect. 13.2).

Based on much earlier work in Knudson’s laboratory at Cornell University in 1922 (cf. Griesebach 2002), in the early 1960s Morel (1963) reported a method to propagate Cymbidium by culturing shoot tips on seed germination medium supplemented with phytohormones in vitro. At Cornell, probably the first experiments with orchid tissue culture were performed, and inflorescence nodes of Phalaenopsis could be induced to produce plantlets in vitro cultured aseptically on seed germination media. Indeed, the Knudson C medium (with some variations) is still in use for orchid cultivation in vitro. During the last 40 years, techniques have been found to propagate many plant species, mainly ornamentals, generally employing isolated meristems for in vitro culture (see Chap. 7). These methods were developed empirically by trial and error, and the propagation in vitro of many plant species is used commercially. Up to the 1960s, orchids belonged to the most expensive flowers—the low price nowadays is due to propagation by tissue culture techniques (even students can afford an orchid for their sweetheart at their first date!).

In the following, the various branches of cell and tissue cultures will be described, including methods for practical applications.