Historical Background: In 1838, Schwann and Scheilden 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. The first attempts, by Haberlandt in 1902, at plant tissue culture failed. However, between 1907 and 1909, Harrison, Burrows and Carrel succeeded in culturing animal and human tissue in vitro. The history of in vivo culture is illustrated in the figure below.

Figure 1: Schematic representation of the history of plant cell and tissue culture. More than

100 years passed before the totipotency theory of Schwann and Schleiden was sustained.

It is now a century since Haberlandt first attempted to grow single cells isolated from a variety of plant tissues in simple nutrient solutions, which at that time were unknown composition. Despite the fact that Haberlandt attempts dated as far back as the beginning of last century, it has only been during the last few decades that rapid developments in plant cell, tissue and organ culture have occurred. Following the unsuccessful in vitro cultivation of root tips by Kotte and Robins in 1912, White (1934) developed the first permanent root and meristem cultures of Lycopersicon esculentum. In the same year, Gautheret and Nobecourt (1934) initiated the first permanent callus cultures of Daucus carota. Rapid developments in the 1950’s and 1960’s converted tissue and organ culture from an area primarily of academic interest into a major tool for the horticultural industry through micropropagation of desired plants. The horizon rapidly widened from horticulture to agriculture to cover important food and cash crops such as citrus fruits and jojoba. Following these developments, there has been a growing interest in other applications like the use of cell cultures for the production of natural production. In fact, in 1942, Gautheret observed the production of secondary metabolites in callus cultures of Daucus carota. Since

then, studies on the production of plant metabolites by callus and cell suspension cultures have been carried out on an increasing scale. With all the advances in plant cell tissue culture, eventually in 1983, Mitsui Petrochemicals concluded that shikonin from Lithospermum erythrorhizon can be produces commercially. Nowadays, with all the technological facilities and means of optimising secondary metabolite production, many other metabolites like berberine, nicotine, sanguinarine and rosmarininc acid are being commercialised because of their therapeutic values. Table 1: Important facts and dates in Plant Tissue Culture Research (1883 – 1985) 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)

Asymbiotic germination of orchid seeds in vitro (Knudson) 1922 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)

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

1934

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) Coconut milk (containing a cell division factor) was the first time used for the culture of Datura embryos (van Overbeek)

1941

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)

Virus-free dahlias obtained by meristem culture (Morel and Martin) 1952 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)

Regeneration of somatic embryos in vitro from the nucellus of Citrus ovules (Maheshwari and Rangaswamy)

1958

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) 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)

1960

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) First haploid Datura plants produced from pollen grains (Guha and Maheshwari) 1964 Regeneration of roots and shoots on callus tissue of Populus tremuloides (Mathes) Induction of flowering in tobacco tissue in vitro (Aghion-Prat) 1965 Differentiation of tobacco plants from single isolated cells in micro-culture (Vasil and Hilderbrandt) Flower induction in Lunaria annua by vernalisation in vitro (Pierik) 1967 Haploid plants obtained from pollen grains of tobacco (Bourgin and Nitsch) Karyological analysis of plants regenerated from callus cultures of tobacco (acristan and Melchers)

1969

First successful isolation of protoplasts from a suspension culture of Hapopappus gracilis (Eriksson and Jonassen) Selection of biological mutants in vitro (Carlson) Embryo culture utilized in the production of monoploids in barley (Kasha and Kao)

1970

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.) 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)

1974

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) 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.)

1976

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.) Introduction of the term somaclonal variation (Larkin and Scowcroft) 1981 Isolation of auxotrophs by large scale screening of cell colonies derived from haploid protoplasts of Nicotiana plumbaginifolia treated with mutagens (Siderov et al.) Protoplasts are able to incorporate naked DNA; transformation with isolated DNA is consequently possible (Krens et al.)

1982

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.) 1985 Infection and transformation of leaf discs with Agrobacterium tumefaciens and the

regeneration of transformed plants (Horsch et al.) Overview of secondary metabolite production in tissue culture: Plants produce a large spectrum of natural products, the secondary metabolites. Nowadays, it is accepted that although these compounds are not important for the primary metabolism of the plant, they are in many cases of great importance for the plant to survive in its natural environment. Man also uses these natural products as dyestuffs, food additives, cosmetics or medicines. But obtaining sufficient supplies of appropriate plant materials for the isolation of secondary metabolites has become more difficult and expensive in recent years. This is because many of these species are endangered due to severe overcollection. This explains why there is a great urge in establishing alternative supplies of such secondary metabolites; plant cell and organ cultures thus provide such alternatives. Over the past years, there has been an increasing interest in the use of plant tissue cultures as industrial biosynthetic sources of useful secondary products. Studies on the production of plant secondary metabolites by plant cell cultures have been comprehensively described. Several plant cell cultures have been shown to produce a large amount of secondary metabolites, but although the number of high-yielding cell cultures increases yearly, there exists recalcitrant plant cell cultures. For instance, cell cultures of Digitalis or Papaver accumulate the interesting cardenolides and alkaloids characteristics of the parent plants in only tiny amounts, or not at all. On the other hand, recent progress in the production of Paclitaxel (taxol) from Taxus species has demonstrated, very impressively, that plant cell cultures can serve as an alternative system for the production of a desired plant product, if there is an urgent demand.

Table 2: Some examples of high producing cell cultures Metabolite Species Yield

g/L Yield % DW

Reference

Berberine Coptis japonica 7.0 12 Fujita and Tabata, 1998 Jatrorrhizine Berberis wilsoniae 3.0 12 Breuling et al., 1985 Rosmarinic acid Coleus blumei 5.6 20 Ulbrich et al., 1985

Salvia officianlis 6.4 Hippolyte et al., 1992 Anthocyanins Perilla frutescens 5.8 Zhong and Yoshida, 1995

Shikonin Lithospermum erythrorhizon

3.5 12 Fujita et al., 1988

Anthraquinones Morinda citrifolia 2.5 18 Zenk et al., 1975 Raucaffricine Rauwolfia serpentina 1.6 3 Schubel et al., 1985 Cinnamoyl- Nicotiana tabaccum 1.5 13 Schiel et al., 1985 Putrescine Datura inoxia 7.1 43 - 50 Suzuki et al., 1985 Paclitaxel Taxus species 0.1 – 0.3 Yukimune et al., 1996 Concomitantly, since the detection of the paniculides in callus cultures of Andrographis paniculata by Overton and co-workers, it has been generally accepted that plant cell cultures produce secondary metabolites which do not seem to occur in the respective parent plants. Several such novel products have been reported from cell cultures. Table 3: Plant cell cultures that produce secondary metabolites hitherto not known to occur in intact plant

Species Compound Reference Andrographis paniculata Paniculide Allison et al., 1968 Picralina nitida Pericine Arens et al., 1982 Podophyllum versipelle Podoverine Arens et al., 1985 Crataegus monogyna Anthocyanins Bahorun et al., 1994 Thuja occidentalis Hinokiol Berlin, 1983 Morinda tricifolia 5,6-dihydrolucidin Inoue et al., 1981 Gardenia jasminoides Tarennosid Inoue et al., 1981 Ochrosia elliptica Epchrosine Inoue et al., 1981 Ruta graveolens Rutacultin Arens et al., 1971

Methodological approaches for the enhancement of secondary metabolites:

Plant cell culture is viewed as a potential means of producing plant products, which can be useful therapeutic agents, food additives, perfumes, etc. The dramatic decrease in plant resources, ecological problems and high labour costs for cultivation of plants have contributed to the increasing use of plant cell cultures for the production of these high value added substances. But a major problem is that cultured plant cells often produce reduced quantities of secondary metabolites when compared to an intact plant. In spite of this limitation, commercialisation of many products derived from cell cultures such as shikonin from Lithospermum erythrorhizon cell cultures and rosmarinic acid from Coleus blumei suspension cultures has becomes a reality. Nevertheless, if totipotency is assumed, secondary

metabolism can be switched on by several factors. Different strategies have been employed to increase secondary metabolite production but only the ones that have yielded the best results are outlined in the following subsections.

Nutrient formulation: Culture medium: Media components can play a vital role in stimulating the secondary metabolite production. Zenk et al in 1977 tested various well-known basal media for the production of serpentine, an indole alkaloid. Among them, Murashige-Skoog’s (MS) formulation was recognised to be the most suitable for the production of this particular alkaloid by Catharanthus roseus suspension. Moreover, cells of Daucus carota when grown in limited amount of nitrogen or phosphate gave enhanced yields of anthocyanins. 10-fold increase in anthocyanin production has also been obtained using a medium with low nitrate and high sucrose concentration in a Vitis hybrid cell culture. Sucrose is generally the main carbon source of most media. Increasing the sucrose concentration from 2 to 11% in a Catharanthus roseus cell suspension caused an increase in anthocyanin concentration of more than 3-fold in an experiment conducted by Knobloch et al, whereas a starvation of phosphate and nitrogen in MS medium of Silybum marianum (L) call cultures decreased the accumulation of flavonolignans. Hormonal formulation: Another important component of the culture medium is the growth regulator. Growth regulators are an obvious variable. In general, a low auxin concentration favors secondary metabolism. Thus plant tissue culture of Catharanthus roseus exhibited levels of ajmalicine and serpentine, which increased 103 fold when the cell culture was deprived of 2,4-D. The latter usually controls the activity of the Phenylalanine Ammonia Lyase (PAL) in Daucus carota cell cultures by depression of mRNA levels. However, production of ubiquinone – 10 by Nicotiana tabacum and diosgenin by Discorea deltoidea were stimulated by high levels of 2,4-D. Physical condition: In vitro production of secondary metabolites by plant cell cultures is largely dependent on environmental factors: light, pH, temperature, oxygen, etc. For instance, experiments on Catharanthus roseus cell cultures have shown that low-density cultures accumulated six times more ajmalicine than high density ones. Anthocyanin content was found to increase with osmotic stress in cells of Vitis vinifera. The intensity of light is variable for different cell cultures. Instead, shikonin biosynthesis in Lithospermum erythrorhizon and that of some anthraquinones in Morinda lucida are inhibited by light. Elicitation: Microbial infection of plant cell cultures as well as intact plants often elicits the synthesis of specific secondary metabolites. Fungal pathogens are potential inducers of the secondary metabolism. Although the mechanism, by which elicitors increase the productivity of secondary plant metabolites, has not been elucidated, their stimulating activity is quite significant if an appropriate elicitor is chosen to stimulate synthesis of a particular product. Furthermore, use of elicitors has much promise in industrial scale plant cell cultures because of their low cost.

Elicitation is not only used for callus cultures, but also for immobilized cell cultures. For instance, elicitation of Gossypium arboreum cells with Verticillium dahliae spores increased the gossypol production by 8 fold. Types of plant tissue culture:

Depending on the type of plant tissue used as the explant and the composition of the growth medium, a variety of different types of plant tissue cultures can be generated. These different types of plant tissue cultures have applications in basic plant research and commercial plant production.

• Callus culture • Cell suspension culture • Protoplast isolation and culture • Anther/pollen culture • Plant organ culture

Callus Culture: Callus culture refers to the growth of unorganized masses of plant cells in culture. To

generate a callus culture, an explant, usually containing a region of meristematic cells, is incubated on a growth medium containing certain plant growth regulators such as auxin and cytokinin.

The cells grow from the explant and divide to form an undifferentiated mass of cells

called a callus. This unorganized mass of cells is analogous to a plant tumor. Cells can proliferate indefinitely if they are periodically transferred to fresh growth media. However, if the callus cells are transferred to a growth medium containing a different combination of plant growth regulators, the cells can be directed to differentiate into roots and/or shoots. This process of converting unorganized growth into the production of roots and shoots is called organogenesis and it represents one means by which a whole plant cell can be regenerated from tissue culture cells. When a plantlet produced by organogenesis, it can be transferred to a large container with nutrients or soil and grown to maturity.

Callus culture. (a) An explant is incubated on growth media. (b) The cells grow and divide to form a callus. (c) The callus cells, grown on media containing new plant growth regulators, differentiate into plant parts. (d) After the plantlet is large enough, it is grown to maturity in soil.

Cell Suspension Culture: Plant cell suspension culture involves the growth of single or small groups of plant

cells in a liquid growth medium. Cell suspension cultures are usually initiated by the transfer of plant callus cells into a liquid medium containing a combination of plant growth regulators and chemicals that promote the disaggregation of the cells into single cells or small clumps of cells.

Continued cell growth requires that the liquid cultures be shaken at low speed to

promote aeration and chemical exchange with the medium. Suspension cell cultures are often used in research applications where access to single cells is important. The suspension bath can provide an efficient means for selecting out cells with desirable traits such as herbicide tolerance or salt tolerance because the bath is in uniform contact with all the cells at once. This differs from callus culture, where only those cells in contact with the solid medium can be selected by chemical additions to the medium.

Suspension cultures can also provide a convenient means for producing and collecting the plant chemicals cells secrete. These can include important plant metabolites, such as food products, oils, and medicinal chemicals. In addition, plant suspension cell cultures can often be used to produce whole plants via a process known as somatic cell embryogenesis.

Cell Suspension Culture. Plant cells can be grown as individual cells or small groups of cells in a liquid culture medium. Liquid suspension culture of plant cells ensures that most cells are in contact with the growth medium.

For some plants, this provides a more convenient means of regenerating a whole

plant after genetic engineering takes place at the single-cell level. In somatic cell embryogenesis, plant suspension culture cells are transferred to a medium containing a combination of growth regulators that drive differentiation and organization of the cells to form individual embryos. Under a dissection microscope, these embryos can be isolated and transferred to a new growth medium, where they grow into individual plants.

Protoplast Isolation and Culture: Protoplasts are plant cells that have their thick cell walls removed by an enzymatic

process, leaving behind a plant cell enclosed only by a plasma membrane. Plant protoplasts have been extremely useful in research on the plant plasma membrane, a structure normally inaccessible due to its close association with the cell wall. Within hours of their isolation, plant protoplasts usually begin to resynthesize cell walls, so this process has also been useful in studies on cell wall production in plants.

Plant protoplasts are also more easily transformed with foreign DNA using approaches such as electroporation. In addition, protoplasts isolated from different plants can be forced to fuse together to form a hybrid. If they are regenerated into whole plants, these hybrids formed from protoplast fusion can represent genetic combinations that would never occur in nature. Hence, protoplast fusion can provide an additional means of genetic engineering, allowing beneficial traits from one plant to be incorporated into another plant despite broad differences between the species.

When either single or fused protoplasts are transferred to a culture growth medium, cell wall regeneration takes place, followed by cell division to form a callus. Once a callus is formed, whole plants can be produced either by organogenesis or by somatic cell embryogenesis in culture.

P r

otoplast regeneration. Different stages in the recovery of intact plants from single plant protoplasts of evening primrose (a) Individual plant protoplasts (b) Regeneration of the cell wall and the beginning of cell division (c, d) Aggregates of plant cells resulting from cell division which can form a callus (e) Production of somatic cell embryos from the callus (f) Recovery of a plantlet from the somatic cell embryo through the process described in the figure above.

Anther / Pollen Culture: In flowers, the anthers are the anatomical structures that contain the pollen. In

normal flower development, the anthers mature and open to allow pollen dispersal. In anther culture, anthers are excised from the flowers of a plant and then transferred to an appropriate growth medium. After a short period of time, pollen cells can be manipulated to form individual plantlets, which can be grown in culture and used to produce mature plants. The development of these plantlets usually proceeds through the formation of embryos.

Plants produced by anther/pollen culture can be haploid because they were originally

derived from pollen cells that have undergone meiosis. However, these plants may be sterile and thus not useful for breeding or genetic manipulation. On the other hand, plants derived from anther/pollen culture can be treated at an early stage with chemical agents such as colchicine, which allows chromosome duplication. Chromosome duplication results in the conversion of sterile haploid plants into fertile diploid organisms. Under these conditions, plants can be produced that are homozygous for every single trait, even those that tend to be recessive traits.

On a cautionary note, not every cell exposed to colchicine becomes diploid. Some have unusual ploidy levels, and they can be screened for chromosome number. The homozygous plants are useful tools, allowing breeders to introduce a normally recessive trait.

Plant Organ Culture: Plant organs can also be grown under culture conditions and this has provided a

useful tool in the study of plant organ development. For example, pollinated flowers of a plant such as a tomato can be excised and transferred to a culture flask containing the appropriate medium. Over time, the ovular portion of the plant will develop into a tomato fruit that will eventually turn red and ripen. Sections of the plant roots can also be excised and transformed to a liquid medium. In this medium, the roots can proliferate extensively, forming both primary and secondary root branches.

Plant Organ Culture. Plant roots growing in a liquid culture medium. From small excised sections of plant roots, the roots will grow and proliferate with lateral root formation (branching).

Applications of Plant Tissue Culture: In addition to the applications already described, plant tissue cultures have a variety

of uses both in agriculture and in industry. Suspension Cultures as Biological Factories: An important industrial application of plant tissue culture involves the use of plant cells as biological factories. Large-scale suspension cultures can be grown to produce antimicrobial compounds, antitumour alkaloids, vitamins, insecticides and food flavors. Plant roots can also be grown in liquid culture, creating a mesh of roots that can produce a number of useful plant compounds. Horticultural uses: Plants with valuable traits can be mass propagated through tissue culture cloning. In this application of plant tissue culture, hundreds or even thousands of genetically identical plants can be produced by vegetative asexual propagation from one plant source. This has been extensively used in the flower industry where genetically identical plants can be produced from a superior parent plant. Propagation of plant tissue in the sterile environment of the growth medium can also help in the production disease-free plants, such as those cultured from the meristematic (apical dome) tissue untouched by viruses or other diseases because it is new growth. This approach has been particularly useful in the culture of disease free orchids and raspberries. Somaclonal Variation: Plant tissue culture has also a problematic side effect that can be used as an asset under certain conditions. During periods of extended growth of plant cells in callus or suspension cell culture, various parts of the plant genome may become more or less “active” due to the release of control over gene expression. Transposable elements may also become more active and chromosomal rearrangements may occur. Sometimes you end up with unusual numbers of chromosomes. This altered control provides a new source of genetic diversity that can result in novel traits, which were not even present in the original plant material used as the explant to start the cultures.

This increased genetic diversity following extended time in tissue culture is called

somaclonal variation. It can be problematic if the desired goal is the propagation or production of identical plant clones. However, somaclonal variation, induced by intentionally growing plant cells in tissue over a longer time period, can be very useful to generate novel plants with traits not currently present in a given gene pool. These traits can be identified either at the tissue culture stage (for example, disease resistance or heat tolerance) or following the regeneration of whole plants by either organogenesis or embryogenesis (plant size, photosynthetic rates, and so forth).

Somaclonal variation. Regeneration of plants from tissue culture can produce plants that are not similar to their parents due to chromosomal aberrations. This variability can be used to select plants with altered traits. These maize plants show evidence of somaclonal variation. (a) Yellow leaf stripe (b) Dwarf maize (c) Yellow leaf tip