Plant Cell Biotechnology

The techniques of plant organ, tissue and cell culture have evolved over several decades (Table 1). These techniques combined with recent advances in developmental, cellular and molecular genetics and using conventional plant breeding have turned plant biotechnology into an exciting research field with significant impact on agriculture, horticulture and forestry. There has also been a growing interest in the use of suspension and immobilised plant cell cultures and organ cultures for the production of fine chemicals and some specific biotransformation reactions.

The power, promise and potential benefits of plant biotechnology in producing better and improved plants and plant products are widely recognised and appreciated. There are, however, only a few selected instances of limited value where these benefits have actually been realised. Applications of plant biotechnology to the improvement of major crop species must await intensive further research in the basic understanding of plant growth and development, and the molecular biology of plant gene structure and function, in addition to adaptation of the appropriate technologies to crops of interest. A plea is made to tone down the rhetoric and the hype of plant biotechnology and to concentrate more on developing the basic scientific competence to achieve the stated goals.

Since Carlson et al. (1972) first reported the successful recovery of somatic hybrid plants, very many protoplast fusion experiments have been done. These have ranged from experiments aimed at answering fundamental questions on the interaction of different plant genomes following fusion to practical questions regarding the improvement of crop species by fusion with wild species possessing desirable agronomic traits. The agricultural applications of protoplast fusion are now beginning to be realised and although the recent successes in plant transformation offer many new opportunities in plant genetic engineering for crop improvement, it is clear that protoplast fusion does have an important role to play in practical plant biotechnology.In this article we discuss the methodologies needed to recover somatic hybrids, their key features and some examples of somatic hybrids which are proving useful in agriculture. It should be noted that there exists a huge literature on protoplast fusion and only a limited number of examples are quoted in each section.

This paper briefly summaries the research carried out in the author’s laboratory on the cell culture of gramineous species, and highlights the importance of the developmental and physiological state of the expiant, the role of endogenous plant growth regulators in the control of morphogenic competence of the expiants, and the early recognition and selection of competent cells in obtaining efficient regeneration of plants by somatic embryogenesis. The significance of embryogenic cell cultures in the recovery of normal plants, and in the establishment of regenerable cell suspension and protoplast systems is discussed. The successful use of gramineous protoplasts in obtaining somatic hybrids, and genetic transformation is also described.

Plant cell and tissue culture constitutes an important and integral part of modern plant biotechnology including in the areas of application such as mass propagation, mass production of chemicals, and genetic engineering which will be emphasized during the course of this meeting. Most gene transfer systems, either in practice or proposed, rely on the regeneration or recovery of whole plants from single cells. It is well known that the development of in vitro culture systems for the Poaceae (Gramineae) are much less advanced than for certain other plant families, especially, the Solonaceae. However, the regeneration of rice from protoplasts, as reported recently by groups in Japan, France, and Britain represents a long-awaited breakthrough and the conquering of a major barrier in cereal cell culture technology (Coulibaly and Demarly 1986; Fujimura et al 1986; Yamada et al 1986; Abdullah et al 1986).

From the first successful in vitro culture of maize endosperm tissue, reported in 1949 (La Rue 1949), long-term cultures of callus derived from immature endosperms have been established from sweet corn varieties (Straus and La Rue 1954; Tamaoki and Ullstrup 1958), from starchy inbreds varieties (Shannon and Batey 1973) and from several endosperm mutants: ae, su2 (Shannon and Batey 1973), wx (Reddy and Peterson 1977), o2 (Manzocchi and Racchi 1986); endosperm cultures have been demonstrated suitable for metabolic studies, and have been employed for research on cell division and chromosome constitution (Boyer and Shannon 1974), starch synthesis (Chu and Shannon 1975) and an-thocyanins biosynthesis (Straus 1959; Racchi 1985).

Somatic embryogenesis has been reported in several species of Gramineae. Different expiants have been used and immature embryos, inflorescences and leaves have been cultured successfully in some of these species. Here, we report on the obtainment of somatic embryogenesis in Hordeum vulgare and Secale vavilovii.

There is currently considerable interest in developing techniques for producing somatic embryos from conifers. Somatic embryos could be used for micropropagation of trees with desirable characteristics such as improved dimension increment, better quality wood and disease resistance. The availability of somatic embryos would also provide excellent experimental material for basic studies of conifer embryo development. Finally, embryogenic callus and suspension cultures should facilitate the development of a protoplast regeneration system to be used in future genetic manipulation studies. The use of embryogenic material has previously proved successful for developing protoplast systems with other recalcitrant species (e.g. Vasil and Vasil 1986).

Tissue culture techniques could be applied to improve the genetic qualities of seaweeds. In order to apply those techniques, efficient methods for obtaining and regenerating calli are needed. The term “callus” or “calluslike” has been applied to define very different structures in seaweeds (Dixon 1963; Fries 1980; Chen 1982; Saga et al. 1982; Tsekos 1982; Polne-Fuller et al. 1984; Yan 1984). Histological studies have been performed on “tumourlike” growths induced by bacteria in Gigartina teedii (Tsekos 1982), and to our knowledge nothing is known on the cytological events preceding differentiation from true callus. In a previous paper (Garcia-Reina et al. 1987), we reported the spontaneous formation of morphogenetic calli, the different calligenic potentials among Laurencia species and primary expiants, and the lack of necessity for axenicity. Callus growth was drastically reduced after its organogenetic trigger, and the organogenetic potential seemed to decrease with time in culture.

Plant cell cultures are now acknowledged as a rich source of genetic variation. Numerous recent reviews have summarized observations on the genetic instability of cultured plant cells and the associated variability among regenerated plants (Evans and Sharp 1985; Larkin et al. 1985; Reisch 1983). Variation associated with culture conditions has also permitted the selection of numerous types of biochemical and agronomic mutants at the cellular level (Chaleff 1983). The list of agriculturally useful in vitro selected mutant plants is growing rapidly. In light of the amount of culture induced variability, it is not surprising that the addition of mutagens has not often resulted in significant increase s in mutant recovery. Yet, not all cultures are inherently variable (Krikorian et al. 1983). Before the notion that variability is inherent in the tissue culture process can be broadly accepted, a wide number of species, genotypes and culture conditions must be surveyed for conditions associated with variability.

Higher plants possess three different genomes, located respectively in the nucleus, in the chloroplasts and in the mitochondria.

Directed genetic modification of plants requires the transmission of specific foreign DNA to the host plant, where the DNA is incorporated and stably maintained in the plant nuclear genome. Although there are several DNA transmission techniques, the transfer of DNA mediated through the natural transmission of T-DNA of Ti plasmids of Agrobacterium tumefaciens has proven relatively efficient and practical. The initial processing and transmission of the T-DNA requires genes situated on the Ti plasmid and on the bacterial chromosome. The T-DNA processing genes on the Ti plasmid are located in the virulence (Vir) region. The virD2 gene of the virD operon, which appears to be involved in the formation of T-DNA intermediates by encoding a specific endonuclease, is constitutively synthesized in a permissive mutant ros of A. tumefaciens. Gene fusions to reporter genes such as the luciferase cassette of Vibrio fischeri made it possible to measure the expression of Vir genes during the on-going interaction between A. tumefaciens and carrot disks. Using phenolic inducers such as acetosyringone, virB, virC, virD and virE were identified as inducible operons of the Vir region in the presence of virA, virG and inducer. virC and virD are also controlled by the ros gene of the chromosome as mutations in the ros gene results in the constitutive expression of these operons. This has enabled us to study the formation of double stranded T-DNA intermediates in the absence of acetosyringone or other phenolic inducers. Integration of the T-DNA was directly observed in Haplopappus gracilis chromosome A by in situ hybridization analysis.

In vitro culture studies of the effect and action mechanism of plant growth regulators are relevant in establishing, the basis of prediction and understanding the “in vitro” behavior.

The genetic basis for NaCl tolerance in the tomato has been proved (Rush and Epstein 1976, 1981). Salt tolerance mechanisms are thought to be cell-based and do not depend on whole plant organization in Lycopersicon (Tal et al. 1978, Tal and Katz 1980, Taleinsk-Gertel et al. 1983). Different mechanisms for NaCl tolerance have been described: salt exclusion (Jia-Ping et al. 1981), osmotic regulation (Orton 1980) or organic synthesis (Rush and Epstein 1976). The objective of this work is to describe the characteristics of NaCl-tolerant morphogenetic calli and isolated somaclones of three tomato land races from the Canary Islands.

In the breeding of barley for resistance against barley powdery mildew (Erysiphe graminis f.sp. hordei) the so-called specific resistance genes have been utilized for many years (Torp et al. 1977).

Protoplasts have been used extensively for basic studies of plant cells and their organelles. Research has included studies of cell fusion (Gleba and Sytnik 1984), the plasma membrane (Leonard and Rayder 1985; Fowke 1986), the cytoskeleton (Willison and Klein 1982) and cell organelle isolation and characterization (Hall 1983). Recent research has shed light on the structure and function of a little known organelle in plants, the coated vesicle. Studies with plant protoplasts have clearly indicated that coated vesicles are involved in the endocytosis of externally supplied labels which are delivered sequentially to a number of membranous organelles and ultimately to the central vacuole of plants. This chapter summarizes some of the recent developments which have clarified our understanding of the function of plant coated vesicles.

The economic importance of plants is immense with plants being used as a source of food, construction materials, fabrics and paper and fuel. In addition, plants contribute to a wide array of chemicals used in industry including pharmaceuticals, foods, flavours, dyes, pigments and agrochemicals. The potential for chemical synthesis within the plant kingdom is vast, something of the order of 2 × 104 structures are known from plants. New chemical structures reported from plants each year are currently running in excess of 1500.

During the past 25 years plant cell and tissue cultures have successfully been used for basic research and biotechnological application, including the selection of agronomically and industrially important traits. As photosynthesis is a characteristic feature of plant cells, the development of photosynthetically active chloroplasts in plant cell cultures should greatly increase their applicability for diverse studies.

Stress on whole plants is generally defined as a condition which has a negative effect on the increase of the dry matter of the plant. Here, stress is defined as an external constraint influencing the secondary metabolism of cultivated plant cells. Of particular interest is the induction of enzymes of secondary metabolism. An increased yield of a target secondary metabolite is therefore considered as a positive effect of the stress even though the growth of the cells may be more or less restricted. From a biotechnological point of view stress may be an important alternative to other methods (e.g. strain selection) to increase the productivity of plant cell cultures. Stress on cultivated plant cells may be caused by a number of different cultivation conditions or modifications of the growth medium as summarized in Table 1. In addition, immobilization of cells is often considered as a stress factor. Some of the stress-inducing agents will be discussed in the following and elucidated by a few examples. Emphasis will be placed on the use of fungal elicitors to induce or enhance the production of secondary metabolites.

In the last decade cell suspension cultures of higher plants have convincingly been established as very suitable experimental systems to study the biosynthesis, the enzymology and the metabolic regulation of the accumulation of secondary plant constituents. Numerous compounds of very different chemical structure have been isolated from such cultures and in some cases cell cultures have turned out to be high accumulating systems of secondary plant products (Barz et al 1977, Barz and Ellis 1981, Yamada and Fujita 1983, Berlin 1984, Whitaker and Hashimoto 1986).

Pterocarpan phytoalexins such as medicarpin, maackiain, phaseollin, pisatin or the various isomers of glyceollin are antimicrobial compounds synthesized by plant species of the Leguminosae in response to stress, primarily caused by fungal infection (Dixon 1986, Ebel 1986).

Nicotinic acid can be regarded as an important connecting link between primary and secondary metabolism in higher plant cells. Being a constituent of the pyridine nucleotide cycle it is a precursor and a degradation product of NAD and NADP which play a decisive role as coenzymes of oxidation-reduction reactions (Waller and Nowacki 1978, Wagner and Wagner 1985).

Unorganized suspension cultures of some higher plants are known to produce various industrially important products, i.e. nicotine from Nicotiana tobacum [Al-Abta, 1978] and purine alkaloids from Coffea arabica [Frischknecht, 1977, 1980]; however, structurally organized organs or somatic embryos are needed in some cases [Butcher, 1977; Al-Abta, 1978, 1979; Yamada, 1983] for secondary metabolite production. To achieve redifferentiation, media and culture conditions sometimes need to be varied. These changes complicate the selection of optimum production conditions by introducing more manipulative variables. A three stage scheme for production of tropane alkaloids from Atropa belladonna is proposed here.

Plant cell cultures have been proved in the past to be an efficient source for the isolation of novel enzymes. Therefore, cultivated plant cells are a valuable tool für the investigation of the biosynthesis of secondary metabolites. Three examples in the alkaloid field are discussed: the enzymatic formation of the isoquinoline alkaloid berberine in the family Berberidaceae, the biosynthesis of the indole alkaloid ajmaline in Rauwolfia serpentina Benth. and the in vitro preparation of the dimeric alkaloids voafrine A and B.

Tissue cultures of Nicotiana plumbaginifolia were used to study the effect of glucose on the activity of glutamate dehydrogenase, malate dehydrogenase, lactate dehydrogenase and alcohol dehydrogenase. GDH and MHD were repressed while LDH and ADH were not affected by 2% glucose in the culture medium. The low glucose level (0.5%) or the addition of cAMP to the culture medium containing 2% glucose restored the initial level of GDH while they were ineffective on MDH activity.

Plant cell cultures are potential sources for the production of useful secondary compounds. The selection of high-producing variant cell lines is the first step for scale-up procedures. Unfortunately the maintenance of these variants is not always easy and reliable.

The large biochemical potential of plant cells cultivated in vitro for performing specific biotransformations on particular natural or synthetic substrates resulting in more complex and, from a pharmaceutical point of view, more useful products, has been well recognized in recent years. The reactions observed up until now include reductions, oxidations, hydroxylations, epoxidations, glycosylations as well as esterifications. Various phenols, coumarins, alkaloids, terpenoids, steroids and cardenolides have been used as substrates (for reviews see Furuya 1978, Reinhard and Alfermann 1980, and Kurz and Constabel 1985, among others).

Sucrose and glucose are the most effective carbon sources in supporting high growth rates and biomass yields in plant cell cultures. Media based on waste material such as milk whey and molasses have been tested also (Fowler and Stepan-Sarkissian 1985), but no growth data in these media have been reported (Berlin and Sasse 1985; Fowler 1982).

Our aim is the improvement of cereal cell cultures with regard to plant regeneration from cultured cells and protoplasts. To achieve this objecitve we modify the composition of media, baded on investigations of uptake and release of components in cell suspension cultures. This work was started with assays of sugars (glucose, fructose and sucrose) and anions (chloride, nitrate, phosphate and sulfate) in graminaceous and tobacco cell suspension cultures.

The conditioning of media is a well-known method for growing plant cells at low density (Street 1977). Stuart and Street (1969) studied the conditioning effect in more detail. They were able to prove a conditioning effect for Acer cell cultures. The critical initial cell density of such cultures could be lowered by a factor of at least 10 by using a conditioned medium. It was also attempted to define the chemical basis of the conditioning effect (Stuart and Street 1971). From experiments measuring the cell numbers after 4 weeks of culture of different initial cell densities on various (conditioned) media, the authors concluded that the carbon course, the vitamins and other growth factors as well as the growth hormones (2,4-D and kinetin) were probably not involved in the conditioning effect. Analysis of a conditioned medium showed the presence of a series of amino acids in small amounts. Addition of these compounds to a non-conditioned medium indeed resulted in a lowering of the critical initial cell density, however, not to the same level as a conditioned medium. Furthermore, a volatile factor was proven ro be involved in the conditioning effect.

Naturally occurring sesquiterpenes with α-methylene-γ-lactone units in its structure are known to exhibit a diversity of biological activities, namely cytostatic and anti-helmintic, and to act as plant growth regulators (Gross 1975; Hoffmann and Rube 1985). The mechanism of its action consists of an irreversible inhibition of sulfhydryl enzymes by a Michael-type reaction, in which the electrophilic methylene group is added to the sulfhydryl group of the enzyme.

Higher plants produce a large variety of metabolites, some of which are potentially useful compounds of commercial interest, including pharmaceuticals (steroids, alkaloids, glycosides), flavor and fragrances, sweeteners, etc.; these compounds are mainly secondary metabolites (Berlin 1986; Nickell 1980).

The premise of this article is that the rational design of a bioreactor can be used to direct plant cell culture towards desired physiological states and toward differentiation and organization. Bioreactors can be used as tools to probe stimulus-response in plant cultures and at the commercial level as devices to increase the productivity of a culture for secondary metabolite formation or production of organized tissues. However, before these aspects of bioreactor design can be discussed the reader must understand the essential principles of bioreactor design.

The species Cynara cardunculus is traditionally used in Portugal to produce tasteful and valuable cheese.

Immobilization of biocatalysts has been defined as the operation leading to their confinement in a well-defined region of space, allowing continuous or successive reuse. It is the latter part of this definition that underlies the most commonly referred advantage of immobilization. Besides this possibility of use in continuous reactors, other advantages which may be obtained are the yielding of a purer product, as the biocatalyst is easily separated from the liquid, and better reaction control.

For many years the biotransformation of digitoxin or β-methyldigitoxin to digoxin or β-methyldigoxin (Fig. 1) by cell cultures of Digitalis lanata has been studied (Reinhard 1974). This reaction is of pharmaceutical interest, since cardiac glycosides, mainly of the C-series, are among the most frequently used drugs against congestive heart failure. The enzyme catalyzing the 12β-hydroxylation was described for the first time by Petersen and Seitz (1985). It could be shown that this enzyme is another cytochrome P-450-dependent monooxygenase from higher plants. Among the best-studied cytochrome P-450-containing enzymes from plants are the cinnamic acid 4-hydroxylase (Russell 1971; Benveniste et al. 1977, and many others), the flavonoid 3′-hydroxylase (Formann et al. 1980; Larson and Bussard 1986) or the monoterpene hydroxylase (Meehan and Coscia 1973; Mady-astha et al. 1976).

A novel bioreactor has been designed to produce secondary metabolites from immobilised plant cell cultures (Mavituna et al, 1987). We have used the production of capsaicin, the hot chilli flavour, by Capsicum frutescens as a model system. This is a particularly suitable system for an immobilised cell process since capsaicin is excreted into the liquid medium.

Plant rennets are used in Portugal for the small-scale manufacture of certain varieties of cheese, namely “Serra” and “Serpa”. In the traditional preparation of these types of cheese the clotting stage is induced by immersing the flowers of the native Compositae Cynara cardunculus or Silybum marianum in the milk vats. However, the flowers are, by themselves, a potential source of microbial contamination of the product. This problem could be overcome by plant cell culture. Previous work demonstrated that in vitro cultivated cells of both species present clotting activity (Fevereiro et al. 1986, Esquîvel et al. 1986), no clotting activity being found in the supernatant (Fonseca et al. 1987).

Some of the ideas presented at the panel discussion on engineering aspects of plant cell culture are brought together, with special emphasis on mass transfer problems and on the release of intracellular products by permeabilized cells.

The ability of plant cell cultures to produce useful compounds (pharmaceutical, food additives, cosmetics) with large-scale production has been investigated by numerous authors from scientific and economical points of view. (For reviews see: Zenk and Deus 1982, Kurz and Constabel 1983, Rosevear 1984. Yamada and Hashimoto 1984, Brodelius 1985, Dougall 1985, Sahai and Knuth 1985, Collinge 1986, Fowler 1986a, Fujita and Tabata 1986, Mac Laren 1986.)

Cell aggregates of T. patulasynthesize and secrete the thiophenes BBT, BBTOH and BBTOAc (see Fig. 1), when cultured in liquid medium. There is a positive correlation between thiophene synthesis and aggregate diameter in the 1 to 12 mm range. No thiophenes are produced by smaller or larger aggregates. Differentiated, hormone-autotrophic “root” cultures, obtained from T. patulatissues after transformation by Agrobacterium tumefaciensLBA 8370, show an extended period of stable BBT and BBTOAc production. The results support the hypothesis that morphological differentiation is required for thiophene biosynthesis. They also show the value of differentiated cell cultures of T. patulafor the production of natural biocides by plant cell biotechnology.

The study of plant secondary metabolism, with the aim of bio-technological exploitation using tissues grown in vitro, has traditionally tended to use dispersed cell cultures or callus. The cultures usually have a strong tendency to be genetically and biochemically unstable and often synthesize very low levels of secondary products. Recently we have begun to study the potential of “hairy root” cultures, resulting from the infection of dicotyledonous plants with Agrobacterium rhizogenes, for the production of secondary products in vitro. We have found that such cultures produce the secondary products synthesized in the roots of the plant species in question in qualitative proportions typical of the parent plant and in quantitative levels at least as high as those found in the plant. Such cultures grow very rapidly in vitroin simple media devoid of phytohormones. They are biochemically stable and we have evidence that their chromosome number remains stable for at least a year in culture.Such cultures are amenable to genetic mainipulation, raising the possibility of increasing the secondary product synthesizing capacity of hairy roots grown in vitro. Coupled with the inherent biochemical and genetic stability of such tissues, these methods may facilitate the biotechnological exploitation of hairy roots as a source of valuable secondary products.

Biotechnology has precipitated a vast change in the way in which plants are utilised. Plant tissue culture techniques and genetic engineering provide a new approach to plant breeding. Furthermore large scale in vitroculture is a medium through which plant metabolism can be exploited to provide products of potential benefit to industry. These developments have also affected the applications and needs of plant germplasm conservation, these can be categorised into several main areas: 1.The conservation of important genotypes (including cultures used in genetic engineering and industrial processes).2.The commercial exploitation of cultures which need conserving for patenting purposes.3.The control of time-related change (e.g. somaclonal and culture variation).4.Reduction of handling risks (contamination hazards of long-term routine culture work).5.Reduction of costs (maintenance costs of keeping plants in long-term culture at normal growth rates).

The plant tissue culture (ptc) technique has grown spectacularly since the 1930’s when it was demonstrated that aseptic plant organs and explants could be subcultured (Gautheret 1983). A dramatic example of this growth is the large number of ptc research articles published in 1985, 4,200, which is seven times greater than that published in 1965 (Bhojwani et al 1986). Another even more significant growth indicator is the attendance of more than 1,500 scientists at the 1986 International Association of Plant Tissue Culture (IAPTC). The number attending exceeded by approximately one-third that attending the 1982 IAPTC meeting. It does appear that the ptc technique has evolved from an art to that of a science with principles that assure reasonable experimental reproducibility. However, the question of ptc as an art or a science is irrelevant as it has dramatically affected the practice of the plant sciences in academia, the government, and industry.

Recently, the interest of international pharmaceutical industries has been directed more and more to high molecular compounds (peptides, proteins) with distinctive biological activities as forerunners of a new therapeutic era. On this bases some people are even dreaming of a new biosocietyaround the turn of this century (Howink 1985).

Research on the production of useful secondary metabolites by plant cell culture (pcc) has been going on for the past 40 years, during which time technology has prgressed remarkably, resulting in the large-scale production of shikonin by cell cultures of Lithospermum erythrorhizon (Fujita et al. 1981a,b, 1982a,b, 1985 a,b). But, the fact that shikonin is still the only plant metabolite that is commercially produced, in spite of energetic studies by researchers throughout the world, indicates that the technology for industrial purposes is still at an immature stage.

The following thoughts should not be regarded as the authorized opinion of an expert in plant biotechnology: the author of this exercise is neither an expert in plant biotechnology nor a person authorized to express himself on behalf of the scientific community. He can only avail himself of a recent 5-year experience in trying, with others, to promote research and development in plant biotechnology by working on a scale which could hopefully circumvent some of the most penalizing academic and geographical barriers of the community. This contribution is therefore not as focused as the others on scientific demonstrations, but it will also, at the request of the organizers, address questions of a more general nature: questions which arise at the interface between the research domain of plant biotechnology, and some major development partners in biotechnology for the economic world. This is where the community dimension enters the scene, a concept which, for the purpose of this meeting, will be understood in its broad sense of “societal dimension”; which will be illustrated, however, against the background of European experience, and in the framework of the EEC in particular.

At the beginning of the present decade, the International Board for Plant Genetic Resources (IBPGR) became aware of the considerable potential for the application of in vitro technology in plant genetic conservation. However, it was clear that this potential was under-exploited (Withers 1980; Withers and Williams 1982). There was a lack of published research on basic techniques and little liaison between tissue culturists and conservation workers. Before any future work could be directed appropriately, there was a need for a state of the art review of current research. To this end, IBPGR supported a survey by questionnaire of institutes working on tissue culture techniques with particular reference to genetic conservation. Information was sought on IBPGR priority crops such as staple roots and tubers, tropical and temperate fruits, and palms with respect to clonal propagation, research problems, characterization, storage and in vitro exchange. The survey showed that much valuable information, especially on storage techniques and biological problems encountered in research, was left unpublished. The results were published in report format (Withers 1982).