Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the beginning of the 20th century.

Perhaps the earliest step towards plant tissue culture was made by Henri-Lous Duhumel du Monceau in 1756, who during studies on wound healing in plants, observed callus formation. Extensive microscopic studies led to the independent and almost simultaneous development of the cell theory by Schleiden (1838) and Schwann (1839). Scheilden and Schwann put forward the co-called totipotency theory, which states that cells are autonomic, and in principle, are capable of regenerating to give a complete plant. Their theory was in fact the foundation of plant cell and tissue culture.

Gottlieb Haberlandt in his address to the German Academy of Science in 1902 on his experiments on the culture of single cells. He opined that, to my knowledge, no systematically organized attempts to culture isolated vegetative cells from higher plants have been made. Yet the results of such culture experiments should give some interesting insight to the properties and potentialities that the cell, as an elementary organism, possesses. Moreover, it would provide information about the interrelationships and complementary influences to which cells within a multicellular whole organism are exposed. He experimented with isolated photosynthetic leaf cells and other functionally differented cells and was unsuccessful, but nevertheless he predicted that one could successfully cultivate artificial embryos from vegetative cells. He, thus, clearly established the concept of totipotency i.e the ability of a plant cell to perform all the functions of development, which are characteristic of zygote, i.e., ability to develop into a complete plant. And further indicated that the technique of cultivating isolated plant cells in nutrient solution permits the investigation of important problems from a new experimental approach. On the basis of that 1902 address and his pioneering experimentation before and later, Haberlandt is justifiably recognized as the father of plant tissue culture.

In 1902, Haberlandt reported culture of isolated single palisade cells from leaves in Knop’s salt solution enriched with sucrose. The cells remained alive for up to 1 month, increased in size, accumulated starch but failed to divide. But the effort failed because the cells do not have cleavage, it was allegedfailures because they do not use growth regulator substances needed for cell division, proliferation and induction of the embryo. In the year 1904, Hannig planting embryos isolated from several plant crucifers. Efforts to demonstrate totipotency led to the development of techniques for cultivation of plant cells under defined conditions. This was made possible by the brilliant contributions from Gautheret in France and P.R. White in U.S.A. during the third and the fourth decades of 20th century. In the early 1920s workers again attempted to grow plant tissues and organ in vitro. Molliard in 1921 demonstrated limited success with the cultivation of plant embryos and subsequently in 1922, Knudson and separately each Robbin conduct investment business and culture of orchid seedlings root tip. After the 1920s, the discovery and development of tissue culture techniques continues. Following the unsuccessful in vitro cultivation of root tips by Kotte, a student of Haberlandt in Germany and Robins in 1912, White (1934) developed the first permanent root and meristem cultures of Lycopersicon esculentum. White generated continuously growing culture of meristematic cells of tomato on medium containing inorganic salts, yeast extract and sucrose and 3 vitamins B (pyridoxine, thiamine, nicotinic acid) – established the importance of additives.

The first true plant tissue cultures were obtained by Gautheret from cambial tissue from Salix capraea, Robinia pseudoacacia, Populus nigra and other trees using agar-solidified medium of Knop’s solution, glucose and cysteine hydrochloride under aseptic condition. These studies continued, and in 1939, Gautheret , Nobecourt and White, published independently studies on the successful cultivation for prolonged periods of cambial tissues of carrot root (Gautheret, 1939) tobacco (White, 1939) and carrot (Nobecourt, 1939).

The Development and Improvement of Techniques The 1940s, 1950s, and 1960s proved an exciting time for the development of new techniques and the improvement of those already available. Overbeek, Conklin and Blakeslee(1941) used application of coconut water (often incorrectly referred to as coconut milk) allowed for the culture of young embryos and other recalcitrant tissues, including monocots.

During early 1950s, a number of lines of enquiry were initiated. Studies by Skoog and his collogue in 1955 showed that the addition of adenine and high levels of phosphate allowed nonmeristematic pith tissues to be cultured and to produce shoots and roots, but only in the presence of vascular tissue.

Further studies using nucleic acids led to the discovery of the first cytokinin (kinetin), as the breakdown product of herring sperm DNA (Miller et.al., 1955). The availability of kinetin further increased the number of species that could be cultured indefinitely, but perhaps most importantly, led to the recognition that the exogenous balance of auxin and kinetin in the medium influenced the morphogenic fate of tobacco callus (Skoog and Miller, 1957). A relative high level of auxin to kinetin-favored rooting, the reverse led to shoot formation and intermediate levels to the proliferation of callus or wound parenchyma tissue. This morphogenic model has been shown to operate in numerous species (Evans, Sharp and Flick 1981). Letham in 1974 discovered cytokinins in several tissues, including coconut water. The formation of bipolar somatic embryos (carrot) was first reported independently by Reinert and Steward (1958) in addition to the formation of unipolar shoot buds and roots.

Muir (1953) reported culture of single cells (and small cell clumps) was achieved by shaking callus cultures of Tagetes erecta and tobacco, and subsequently placing them on filter paper resting on well-established callus, giving rise to the so-called nurse culture. Later, single cells could be grown in medium in which tissues had already been grown (i.e., conditioned medium) (Jones et.al. 1960). As well, single cells incorporated in a 1-mm layer of solidified medium formed some cell colonies. This impotant technique was developed by Bergmann (1959).

Finally, Kohlenbach in 1959, success was achieved in the culture of mechanically isolated mature differentiated mesophyll cells of Macleaya cordata, and later in the induction of somatic embryos from the callus (1966). The first large-scale culture of plant cells was obtained by Tulecke and Nickell (1959) from cell suspensions of Ginkgo, holly, Lolium and rose in simple sparged 20-L carboys. The utilization of coconut water as an additive to fresh medium, instead of using conditioned medium, finally led to realization of Haberlandt’s dream of producing a whole plant (tobacco) from a single cell by Vasil and Hildebrandt (1965), thus demonstrating the totipotency of plant cells.

The differentiation of whole plants in tissue cultures may occur via shoot and root differentiation, or alternatively the cell may undergo embryogenesic development to give rise to somatic embryos. Differentiation of plants from callus cultures has been often been suggested as a potential method for rapid propagation. Plantlets were successfully produced by culturing shoot tips with a couple of primordia of Lupinus and Tropaeolum (Ball, 1946), but the importance of this finding was not recognized until later when this approach to obtain virus-free orchids, its potential for clonal propagation was realized (Morel 1960). The potential was rapidly exploited, particularly with ornamentals (Murashige, 1974). Early studies had shown that cultured root tips were free of viruses (White, 1934). It was later observed that the virus titer in the shoot meristem was very low (Limasset and Cornuet, 1949). This was confirmed by Morel and Martin, (1952) when virus-free Dahlia plants were obtained from infected plants by culturing their shoot tips. Virus elimination was possible because vascular tissues, within which the viruses move, do not extend into the root or shoot apex. The method was further refined by Quack (1961), and now routinely used. In the year 1962 Skoog and his student Murashige developed a high salt medium. The concentration of some salts was 25 times that of Knop’s solution. MS formulation allowed for a further increase in the number of plant species that could be cultured, many of them using only a defined medium consisting of macro- and micro-nutrients, a carbon source, reduced N, B vitamins, and growth regulators. The MS salt formulation is now the most widely used nutrient medium in plant tissue culture.

Important discoveries in the history of plant tissue culture.

1838 Totipotency theory (Schwann and Scheilden) – cells are autonomic, and in principle, are capable of regenerating to give a complete plant

1892 Plants synthesize organ forming substances which are polarly distributed (Sachs)

1902 First attempt at plant tissue culture (Haberlandt)

1904 First attempt at embryo culture of selected crucifers (Hannig)

1909 Fusion of plant protoplasts, although the products failed to survive (Kuster)

1922 Asymbiotic germination of orchid seeds in vitro (Knudson) In vitro culture of root tips (Robbins)

1925 Embryo culture applied in interspecific crosses of Linum (Laibach)

1929 Embryo culture of Linum to avoid cross incompatibility (Laibach)

1934 In vitro culture of the cambium of a few trees and shrubs failed to be sustained since auxin had not yet been discovered (Gautheret) Successful culture of tomato roots (White)

1936 Embryo culture of various gymnosperms (LaRue)

1939 Successful continuously growing callus culture (Gautheret, Nobecourt and White)

1940 In vitro culture of cambial tissues of Ulmus to study adventitious shoot formation (Gautheret)

1941 Coconut milk (containing a cell division factor) was the first time used for the culture of Datura embryos (van Overbeek). In vitro culture of crown-gall tissues (Braun)

1944 First in vitro cultures of tobacco used to study adventitious shoot formation (Skoog)

1945 Cultivation of excised stem tips of Asparagus in vitro (Loo)

1946 First whole Lupinus and Tropaeolum plants from shoot tips (Ball)

1948 Formation of adventitious shoots and roots of tobacco determined by the ratio of auxin/adenin (Skoog and Tsui)

1950 Organs regenerated from callus tissue of Sequoia sempervirens (Ball)

1952 Virus-free dahlias obtained by meristem culture (Morel and Martin). First application of micro-grafting (Morel and Martin)

1953 Haploid callus of Gingko biloba produced from pollen (Tulecke)

1954 Monitoring of changes in karyology and in chromosome behavior of endosperm cultures of maize (Strauss)

1955 Discovery of kinetin, a cell division hormone (Miller et al.)

1956 Realization of growth cultures in multi-litre suspension systems to produce secondary products by Tulecke and Nickell (Staba, 1985)

1957 Discovery of the regulation of organ formation (roots and shoots) by changing the ratio of cytokinin/auxin (Skoog and Miller)

1958 Regeneration of somatic embryos in vitro from the nucellus of Citrus ovules (Maheshwari and Rangaswamy). Regeneration of pro-embryos from callus clumps and cell suspensions of Daucus carota (Reinert, Steward)

1959 Publication of the first extensive handbook on plant tissue culture (Gautheret)

1960 First successful test tube fertilization in Papavar rhoeas (Kanta) Enzymatic degradation of the cell walls to obtain large numbers of protoplasts (Cocking). Vegetative propagation of orchids by meristem culture (Morel) Filtration of cell suspensions and isolation of single cells by plating (Bergmann).

1962 The development of the famous Murashige and Skoog medium (Murashige and Skoog).

1964 First haploid Datura plants produced from pollen grains (Guha and Maheshwari). Regeneration of roots and shoots on callus tissue of Populus tremuloides (Mathes)

1965 Induction of flowering in tobacco tissue in vitro (Aghion-Prat). Differentiation of tobacco plants from single isolated cells in micro-culture (Vasil and Hilderbrandt)

1967 Flower induction in Lunaria annua by vernalisation in vitro (Pierik).Haploid plants obtained from pollen grains of tobacco (Bourgin and Nitsch)

1969 Karyological analysis of plants regenerated from callus cultures of tobacco (acristan and Melchers). First successful isolation of protoplasts from a suspension culture of Hapopappus gracilis (Eriksson and Jonassen)

1970 Selection of biological mutants in vitro (Carlson). Embryo culture utilized in the production of monoploids in barley (Kasha and Kao). First achievement of protoplast fusion (Power et al.)

1971 First plants regenerated from protoplasts (Takebe et al.)

1972 Interspecific hybridization through protoplast fusion in two Nicotina species (Carlson et.al.)

1973 Cytokinin found capable of breaking dormancy in excised capitulum explants of Gerbera (Murashige et al.)

1974 Induction of axillary branching by cytokinin in excised Gerbera shoot tips (Murashige et al.). Regeneration of haploid Petunia hybrida plants from protoplasts (Binding). Fusion of haploid protoplasts found possible which gave rise to hybrids (Melchers and Labib). Biotransformation in plant tissue cultures (Reinhard). Discovery that the Ti-plasmid was the tumour inducing principle of Agrobacterium (Zaenen et al.; Larebeke et al.)

1975 Positive selection of maize callus cultures resistant to Helminthosporium maydis (Gengenbach en Green)

1976 Shoot initiation from cryo-preserved shoot apices of carnation (Seibert). Interspecific plant hybridization by protoplast fusion of Petunia hybrida and Petunia parodii (Power et al.). Octopine and nopaline synthesis and breakdown found to be genetically controlled by the Ti-plasmid of Agrobacterium tumefaciens (Bomhoff et al.). Successful integration of the Ti-plasmid DNA from Agrobacterium tumefaciens in plants (Chilton et al.)

1978 Somatic hybridization of tomato and potato (Melchers et al.)

1979 Co-cultivation procedure developed fro transformation of plant protoplasts with Agrobacterium (Marton et al.)

1980 Use of immobilized whole cells for biotransformation of digitoxin into digoxin (Alfermann et al.)

1981 Introduction of the term somaclonal variation (Larkin and Scowcroft). Isolation of auxotrophs by large scale screening of cell colonies derived from haploid protoplasts of Nicotiana plumbaginifolia treated with mutagens (Siderov et al.)

1982 Protoplasts are able to incorporate naked DNA; transformation with isolated DNA is consequently possible (Krens et al.). Fusion of protoplasts by electrical stimulus (Zimmermann)

1983 Intergeneric cytoplasmic hybridization in radish and rape (Pelletier et al.)

1984 Transformation of plant cells with plasmid DNA (Paszkowski et al.)

1984 Development of the genetic fingerprinting technique for identifying individuals by analyzing Polymorphism at DNA sequence level (Alec Jeffreys)

1985 Infection and transformation of leaf discs with Agrobacterium tumefaciens and the regeneration of transformed plants (Horsch et al.)

1986 TMV virus-resistant tobacco and tomato transgenic plants developed using cDNA of coat protein gene of TMV (Powell-Abel et al.)

1987 Development of biolistic gene transfer method for plant transformation(Sanford et al.; Klein et al.)

1987 Isolation of Bt gene for bacterium (Bacillus thuringiensis) (Barton et al.)