Plant tissue culture. Biotechnology. Milestones

Summary The progress in the development of the technologies of plant tissue and cell culture over the past four decades has been remarkable. This article covers my personal reflections on the various topics and is based on my involvement in the field during that period. There are three fundamental technologies which constitute most of what is referred to as plant in vitro technologies or tissue culture. The origin and some of the key persons involved in the development of each of these procedures will be discussed. The technology that is most common is growing plant tissue on gel-solidified nutrient media. That technology is being used in the most vital procedures, namely the regeneration of plants from cultured cells. The culture of plant cells in liquid suspension was developed very shortly after that, and has become a very effective technology for plant regeneration by somatic embryogenesis. The method of meristem culture arose out of a need for developing plants that were virus-free. In many species the technique is now being used to produce virus-free crop plants. Another important technology is the culture of anthers and microspores for producing haploid and homozygous plants. Included with plant tissue culture is the development of the plant protoplast and cell fusion technologies for the production of new plant hybrids. The final aspect of the development concerns the integration of tissue culture with molecular genetics, which has developed into the rapidly expanding field of biotechnology.     

Influence of abiotic stress signals on secondary metabolites in plants

Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and industrially important biochemicals. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. Secondary metabolites play a major role in the adaptation of plants to the environment and in overcoming stress conditions. Environmental factors viz. temperature, humidity, light intensity, the supply of water, minerals, and CO₂ influence the growth of a plant and secondary metabolite production. Drought, high salinity, and freezing temperatures are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops. Plant cell culture technologies have been effective tools for both studying and producing plant secondary metabolites under in vitro conditions and for plant improvement. This brief review summarizes the influence of different abiotic factors include salt, drought, light, heavy metals, frost etc. on secondary metabolites in plants. The focus of the present review is the influence of abiotic factors on secondary metabolite production and some of important plant pharmaceuticals. Also, we describe the results of in vitro cultures and production of some important secondary metabolites obtained in our laboratory.     

Initiation, growth and cryopreservation of plant cell suspension cultures

Methods described in this paper are confined to in vitro dedifferentiated plant cell suspension cultures, which are convenient for the large-scale production of fine chemicals in bioreactors and for the study of cellular and molecular processes, as they offer the advantages of a simplified model system for the study of plants when compared with plants themselves or differentiated plant tissue cultures. The commonly used methods of initiation of a callus from a plant and subsequent steps from callus to cell suspension culture are presented in the protocol. This is followed by three different techniques for subculturing (by weighing cells, pipetting and pouring cell suspension) and four methods for growth measurement (fresh- and dry-weight cells, dissimilation curve and cell volume after sedimentation). The advantages and disadvantages of the methods are discussed. Finally, we provide a two-step (controlled rate) freezing technique also known as the slow (equilibrium) freezing method for long-term storage, which has been applied successfully to a wide range of plant cell suspension cultures.     

In vitro chili pepper biotechnology

Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.     

Constitutive caspase-like machinery executes programmed cell death in plant cells

The morphological features of programmed cell death (PCD) and the molecular machinery involved in the death program in animal cells have been intensively studied. In plants, cell death has been widely observed in predictable patterns throughout differentiation processes and in defense responses. Several lines of evidence argue that plant PCD shares some characteristic features with animal PCD. However, the molecular components of the plant PCD machinery remain obscure. We have shown that plant cells undergo PCD by constitutively expressed molecular machinery upon induction with the fungal elicitor EIX or by staurosporine in the presence of cycloheximide. The permeable peptide caspase inhibitors, zVAD-fmk and zBocD-fmk, blocked PCD induced by EIX or staurosporine. Using labeled VAD-fmk, active caspase-like proteases were detected within intact cells and in cell extracts of the PCD-induced cells. These findings suggest that caspase-like proteases are responsible for the execution of PCD in plant cells.     

In vitro selection for salt tolerance in crop plants: Theoretical and practical considerations

Summary In recent years attempts have been made to supplement traditional breeding for the production of salt-tolerant plants with variability existing in cell culture. The potential causes suggested as an explanation for the limited success of the in vitro approach include: a) lack, or loss during selection, of regeneration capability; b) the development of epigenetically adapted cells; c) lack of correlation between the mechanisms of tolerance operating in cultured cells and mechanisms that operate in cells in the intact plant; and d) multigenicity of salt tolerance. The recent successful production of healthy, fertile, and genetically stable salt-tolerant regenerants from cells obtained from highly morphogenic explants which are selected early in culture (using one-step or short-term strategies) for salt tolerance, together with the demonstration that salt-sensitive plants can become tolerant by mutations in one or few genes, suggest that some of the potential limitations can be overcome and that some of them may not exist at all.     

Nitrogen deposition and its contribution to nitrogen cycling and associated soil processes

TIP1 is a gene defined by an X-ray induced allele tip1–2 and a previously described EMS-induced allele tip1−1 . TIP1 is involved in plant cell growth. tip1–2 plants display growth defects throughout the plant and exhibit defects in both root-hair and pollen-tube growth. tip1–2 plants are partly male sterile resulting from a combination of pollen germination and pollen-tube defects; their root-hairs are short, exhibit a tendency to branch and 2–4 hairs can initiate from each hair cell. They are also slightly dwarf in stature as a result of a general decrease in cell growth indicating that TIP1 activity is required for general cell growth. We propose a role for TIP in both the initiation and maintenance of growth in tip-growing cells. In addition TIP1 activity is required for normal cell expansion (non-tip cell growth) indicating that TIP1 is not exclusively involved in tip-growth.     

The potential role of immobilization in plant cell biotechnology

It has long been hoped that the unique biosynthetic capacity of plants could be exploited in vitro using culture systems analogous to microbial fermentations. However, the characteristics of both the growth and metabolism of plant cells in vitro differ considerably from those of microbial cells and plant cell suspension culture systems have met with limited success. Immobilizing the cells creates a new set of options for the plant biotechnologist to explore. Improvements in some process criteria are apparent although evaluating the potential of immobilized plant cells for producing commercial compounds will only be possible when the biological problems have been overcome.     

Potential of plant cells in culture for cosmetic application

Plants and plant derived ingredients are common and of major importance in the fields of pharmacy, food and cosmetics. The cosmetic industry is a fast moving market. Products have short life-cycles and the industry has to come up with innovative products constantly. Most cosmetic products and their applications are defined by active ingredients. These active ingredients may derive from either synthetic sources or from plant sources. Beside this, no other origin like human or animal are accepted or allowed in cosmetics nor are genetically modified plant sources. The whole cosmetic research and development society is therefore desperately seeking for new innovative plant ingredients for cosmetic application. Unfortunately, new plant derived ingredients are limited because several plants of cosmetic interest are not to be used due to following facts: the plants contain toxic metabolites, the plants grow too slow and a seasonal harvesting is not possible, the concentration of plant constituents differ from harvest to harvest or the plant is endangered and not allowed to harvest. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plant derived actives. Due to all these findings, we decided to risk the step into plant cell culture derived cosmetic active ingredient production. This article describes the successful establishment of an apple suspension culture producing a high yield of biomass, cultured in disposable, middle-scale bioreactors. The use of a bioactive extract out of these cells for cosmetic application and the efficacy of this extract on mammalian stem cells is also outlined in this article. To obtain a suitable cosmetic product we used the high pressure homogenization technique to decompose the plant cells and release all the beneficial constituents while encapsulating these components at the same time in liquid Nanoparticles. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plants derived actives.     

Genetic changes induced in higher plant cells by a laser microbeam

Introducing foreign genes into higher plants has proved to be complicated, with the exception of transformation of protoplasts of some plants (Negrutiu et al. 1987). In particular, culture of protoplasts and regeneration to plants are difficult in many monocotyledonous crops. Therefore, it would be desirable to avoid extensive tissue culture by introducing cloned genes directly into cells. A laser microbeam can perforate plant cell walls, thus facilitating uptake of genes into cells.     

Bioprocessing of plant cell cultures for mass production of targeted compounds

More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered. This paper is dedicated to Prof. Dr. Mladenka P. Ilieva on the occasion of her 70th birthday.     

TISSUE CULTURE IN THE PRODUCTION OF NOVEL DISEASE-RESISTANT CROP PLANTS

1. Plant tissue cultures form the basis of a number of techniques which have been developed to effect genetic changes in plants. Progress is being made in the application of these techniques in breeding new, disease-resistant cultivars. 2. It is possible to induce and select for mutants among populations of cultured plant cells. Novel disease-resistant plants of a small number of species have been regenerated from cells selected in culture for their resistance to toxins produced by pathogens, both with and without prior exposure to mutagens. It is not known whether such procedures are widely applicable, and the nature of the genetic changes involved has not yet been determined. 3. The tissues of plant species which are propagated vegetatively are normally genetic mosaics with regard to many characteristics, including resistance to disease. Thus, some of the plants regenerated from cultured cells of such species are more resistant to pathogens than the parent plants. Novel plants produced in this way are already being used in some breeding programmes. 4. Many attempts have been made to modify the genomes of cultured plant cells by means of exogenous nucleic acids. The evidence for integration and replication of this genetic material is equivocal. The technique, therefore, offers no immediate prospects for the development of novel disease-resistant plants, but may be important in the long term as methods are perfected for using plasmids and other agents as carriers of useful genes. 5. Steady advances are being made in producing somatic hybrids of crop plants by fusion of isolated protoplasts. In the long term it may be possible to use protoplast fusion to transfer desirable disease-resistance traits between related species which cannot be hybridized by conventional breeding methods. 6. The culture of excised embryos may be used to grow interspecific and inter-generic hybrid plants in cases where incompatibility occurs after normal fertilization. The technique is already being used by breeders in the production of disease-resistant hybrids of crop species. 7. It is concluded that tissue culture has a limited but useful role to play in the development of novel disease-resistant crop plants.     

Crop improvement through tissue culture

Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology. Tissue culture has been exploited to create genetic variability from which crop plants can be improved, to improve the state of health of the planted material and to increase the number of desirable germplasms available to the plant breeder. Tissue-culture protocols are available for most crop species, although continued optimization is still required for many crops, especially cereals and woody plants. Tissueculture techniques, in combination with molecular techniques, have been successfully used to incorporate specific traits through gene transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic variation in the breeding lines, often via haploid production. Cell culture has also produced somaclonal and gametoclonal variants with crop-improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens from planting material and thereby dramatically improve the yield of established cultivars. Large-scale micropropagation laboratories are providing millions of plants for the commercial ornamental market and the agricultural, clonally-propagated crop market. With selected laboratory material typically taking one or two decades to reach the commercial market through plant breeding, this technology can be expected to have an ever increasing impact on crop improvement as we approach the new millenium.D.C.W. Brown is with Agriculture and Agri-Food Canada, Central Experimental Farm, Plant Research Centre, Ottawa, Ontario, K1A 0C6, Canada. T.A. Thorpe is with the Plant Physiology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada     

First encounters--deployment of defence-related natural products by plants

Plant-derived natural products have important functions in ecological interactions. In some cases these compounds are deployed to sites of pathogen challenge by vesicle-mediated trafficking. Polar vesicle trafficking of natural products, proteins and other, as yet uncharacterized, cargo is emerging as a common theme in investigations of diverse disease resistance mechanisms in plants. Root-derived natural products can have marked effects on interactions between plants and soilborne organisms, for example by serving as signals for initiation of symbioses with rhizobia and mycorrhizal fungi. They may also contribute to competitiveness of invasive plant species by inhibiting the growth of neighbouring plants (allelopathy). Very little is known about the mechanisms of release of natural products from aerial plant parts or from roots, although there are likely to be commonalities in these processes. There is increasing evidence to indicate that pathogens and symbionts can manipulate plant endomembrane systems to suppress host defence responses and facilitate accommodation within plant cells. The relationship between secretory processes and plant interactions forms the focus of this review, which brings together different aspects of the deployment of defence-related natural products by plants.     

Totipotency and embryogenesis in plant cell and tissue cultures

Summary An important development in the field of plant cell and tissue culture has been the demonstration in the past decade of the totipotency of higher plant cells. Isolated single cells were first successfully grown on a nurse tissue separated by a filter paper and gave rise to a callus tissue. Later, completely isolated single cells of tobacco were grown in microchambers to form small clumps of cells which then could be differentiated to form adult tobacco plants. Indirect evidence of the totipotency of higher plant cells has also been provided in a number of other plants. Embryo-like structures (or embryoids) or whole plants, or both, have been obtained from such highly differentiated cells as the pollen grains (gametic and haploid), photosynthetic palisade cells in leaves, epidermal cells from the hypocytyl, and the triploid endosperm cells; all of these cell types perform very highly specialized functions in the plant. Plant protoplasts (cell wall is digested with enzymes) have also been cultured to give rise to normal adult plants. In many instances embryoids have been produced in vitro from several species of flowering plants which do not show such asexual activity in nature. These embryoids are normally indistinguishable morphologically from embryos produced by gametic fusion, often follow the same pattern of cell divisions and differentiation as the developing zygote, and are economically important as they provide clonal populations. Early work in this area emphasized the necessity of dissociating tissues into single cells and providing a nutritional environment identical to that of the zygote in the embryo sac (usually by supplementing the medium with liquid endosperm from coconuts), before the cells could be released morphogenetically to express their totipotency by forming embryoids. Much of the recent work, however, has shown that perfect development of embryoids can be obtained in completely synthetic media in callus tissues as well as in suspension cultures. This paper is dedicated to the memory of the late Professor Philip R. White, a dear friend who provided much counsel and inspiration to us both. for his pioneering work, valuable contributions and untiring efforts in developing the science of plant cell, tissue, and organ culture. Florida Agricultural Experiment Stations Journal Series No. 4699. Presented in the Symposium on Functional Differentiated Systems at the 23rd Annual Meeting of the Tissue Culture Association, Los Angeles, California, June 5–8, 1972.     

Gene network analysis in plant development by genomic technologies

The analysis of the gene regulatory networks underlying development is of central importance for a better understanding of the mechanisms that control the formation of the different cell-types, tissues or organs of an organism. The recent invention of genomic technologies has opened the possibility of studying these networks at a global level. In this paper, we summarize some of the recent advances that have been made in the understanding of plant development by the application of genomic technologies. We focus on a few specific processes, namely flower and root development and the control of the cell cycle, but we also highlight landmark studies in other areas that opened new avenues of experimentation or analysis. We describe the methods and the strategies that are currently used for the analysis of plant development by genomic technologies, as well as some of the problems and limitations that hamper their application. Since many genomic technologies and concepts were first developed and tested in organisms other than plants, we make reference to work in non-plant species and compare the current state of network analysis in plants to that in other multicellular organisms.     

Non-host plants: Are they mycorrhizal networks players?

Common mycorrhizal networks (CMNs) that connect individual plants of the same or different species together play important roles in nutrient and signal transportation, and plant community organization. However, about 10% of land plants are non-mycorrhizal species with roots that do not form any well-recognized types of mycorrhizas; and each mycorrhizal fungus can only colonize a limited number of plant species, resulting in numerous non-host plants that could not establish typical mycorrhizal symbiosis with a specific mycorrhizal fungus. If and how non-mycorrhizal or non-host plants are able to involve in CMNs remains unclear. Here we summarize studies focusing on mycorrhizal-mediated host and non-host plant interaction. Evidence has showed that some host-supported both arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) hyphae can access to non-host plant roots without forming typical mycorrhizal structures, while such non-typical mycorrhizal colonization often inhibits the growth but enhances the induced system resistance of non-host plants. Meanwhile, the host growth is also differentially affected, depending on plant and fungi species. Molecular analyses suggested that the AMF colonization to non-hosts is different from pathogenic and endophytic fungi colonization, and the hyphae in non-host roots may be alive and have some unknown functions. Thus we propose that non-host plants are also important CMNs players. Using non-mycorrhizal model species Arabidopsis, tripartite culture system and new technologies such as nanoscale secondary ion mass spectrometry and multi-omics, to study nutrient and signal transportation between host and non-host plants via CMNs may provide new insights into the mechanisms underlying benefits of intercropping and agro-forestry systems, as well as plant community establishment and stability.     

Conservation In vitro of threatened plants—Progress in the past decade

Summary In vitro techniques have found increasing use in the conservation of threatened plants in recent years and this trend is likely to continue as more species face risk of extinction. The Micropropagation Unit at Royal Botanic Gardens, Kew, UK (RBG Kew) has an extensive collection of in vitro plants including many threatened species from throughout the world. The long history of the unit and the range of plants cultured have enabled considerable expertise to be amassed in identifying the problems and developing experimental strategies for propagation and conservation of threatened plants. While a large body of knowledge is available on the in vitro culture of plants, there are limited publications relating to threatened plant conservation. This review highlights the progress in in vitro culture and conservation of threatened plants in the past decade (1995–2005) and suggests future research directions. Works on non-threatened plants are also included wherever methods have applications in rare plant conservation. Recalcitrant plant materials collected from the wild or ex situ collections are difficult to grow in culture. Different methods of sterilization and other treatments to establish clean material for culture initiation are reviewed. Application of different culture methods for multiplication, and use of unconventional materials for rooting and transplantation are reviewed. As the available plant material for culture initiation is scarce and in many cases associated with inherent problems such as low viability and endogenous contamination, reliable protocols on multiplication, rooting, and storage methods are very important. In this context, photoautotrophic micropropagation has the potential for development as a routine method for the in vitro conservation of endangered plants. Long-term storage of material in culture is challenging and the potential applications of cryopreservation are significant in this area. Future conservation biotechnology research and its applications must be aimed at conserving highly threatened, mainly endemic, plants from conservation hotspots.