Plant Cell Tissue and Organ Culture 39: 105-108, 1994. ~) 1994KluwerAcademicPublishers. Printedin the Netherlands. Introduction

Automation of plant propagation I n d r a K. Vasil Laboratory of Plant Cell and Molecular Biology, 1143 Fifield Hall, University of Florida, Gainesville, FL 326110690, USA

There are two principal pathways of plant regeneration in vitro. The most common, popularly described as micropropagation, involves the abolition of apical dominance resulting in the derepression and multiplication of axillary buds. In the other, called somatic embryogenesis, cotyledonary embryos with a rootshoot axis are formed from somatic cells. Both result in the production of non-chimeric and true-to-type plants that comprise clonal populations. The origins of micropropagation can be traced to the early and pioneering studies of Georges Morel on the development of virus-free plants from cultured shoot meristems, and the elucidation of the role of cytokinins in shoot morphogenesis by Folke Skoog and Carlos Miller, and in the inhibition of apical dominance by Kenneth Thimann and his associates. During the past four decades, these observations have been used successfully to develop highly efficient and reliable methods for the rapid clonal propagation of a wide variety of herbaceous dicotyledonous species as well as many species of evergreen deciduous trees (Vasil 1986, 1991; Zimmerman et al. 1986; Debergh & Zimmerman 1991; Ahuja 1993; Vasil & Thorpe 1994). The formation of embryos from somatic cells was first described in carrot independently by Jacob Reinert, and Frederick Steward and his associates, in 1958. Since then somatic embryogenesis has been reported in scores of species, including the economically important cereals and grasses, woody angiosperm and gymnosperm tree species, palms, bananas, etc. Indeed, the formation of somatic embryos is the predominant mode of in vitro regeneration for a large number of woody and monocotyledonous species which were previously considered to be highly recalcitrant. This was generally achieved by placing tissue explants from young plant organs that comprised largely of meristematic and undifferentiated cells on media contain-

ing high concentrations of strong auxins (such as 2,4dichlorophenoxyacetic acid), and sometimes certain cytokinins. The primary focus of discussion in this volume is on the mass production of plants in vitro, particularly by the formation of somatic embryos. It should be recognized, however, that at the present time micropropagation is the most widely and successfully used technology for large scale propagation of plants. This raises a number of pertinent questions. For example: - What is the need for mass propagation and automation? - What are the comparative advantages/disadvantages of somatic embryogenesis and micropropagation? -Is automation of somatic embryogenesis and micropropagation technically feasible?

Need for mass propagation and automation

Tissue culture methods are used by nearly 600 companies throughout the world to produce more than 500 million units annually from almost 50,000 varieties of plants. At the present time, almost all of these units are produced by the process of micropropagation. Large scale propagation through somatic embryogenesis is either unknown or an exception. Although the propagation of ornamental plants dominates the industry, a few vegetable and fruit species, and plantation crops such as oil palm and bananas, are beginning to enter the market. In spite of the above, the industry has stagnated during the past two decades largely because of two reasons. First, the limited number of individual units that can be produced and second, the high cost of production (currently at US$ 0.10-0.15/unit). This has discouraged the adaptation of the technology for the propagation of most vegetable and forest tree species

106 where, to be successful and attractive, the cost may have to be brought down to US$ 0.01-.05/unit. Mass propagation of vegetable and tree species at such low cost will create a market for billions of units annually. This market demand can be met only by a drastic reduction in the cost of production, combined with a multi-fold increase in production capacity. The high costs of production are linked to the labor intensive nature of plant propagation in vitro, and the limited and stagnant market (Levin & Vasil 1989; Vasil 1991). Estimates of labor costs run from 50% to as high as 85% of production costs. For this reason, commercial plant propagation has rapidly moved from Western Europe and North America, where labor is expensive, to countries in Asia, Latin America and Eastern Europe, where cheap labor abounds. This trend is expected to continue with the continued use of the current technology. Automation of the propagation process, which will result in a drastic reduction of labor use, should therefore result not only in low costs, but also in producing virtually unlimited numbers of plants and opening the market to many vegetable, fruit and tree species.

Micropropagation and somatic embryogenesis At the present time there is a much better understanding of the biology of micropropagation than that of somatic embryogenesis. Furthermore, micropropagation is a well established process which is practiced widely on a commercial scale. Technically it is a rather simple procedure and does not require highly skilled labor force. The biology of somatic embryogenesis, on the other hand, is at best poorly understood, and a great deal of fundamental work must be carried out before large scale production of somatic embryos can become as routine and widely used, as micropropagation. Biologically, as well as technically, it is a very complex process. It is for these reasons that although somatic embryogenesis has been studied and described in more than 200 plant species during the past 35 years, the technical know how for the production of unlimited numbers of somatic embryos, which are uniform in size and shape and retain the capacity to give rise to normal plants, is not yet available for any species. Even in species like carrot, alfalfa and celery, which have been extensively used as model systems, the numbers and overall quality of somatic embryos formed are too small and not suitable for automation and commercial

production. Although many of the somatic embryos formed in culture give rise to normal rooted plants, there is considerable variation in their size and shape, in the number and nature of the cotyledons formed, and in the timing and efficiency of their maturity and germination. These unsolved and difficult biological problems must be resolved before somatic embyos can be grown in bioreactors and used for the production of synthetic seed. Recent advances in the experimental synchronisation of somatic embryo development, the nature and genetic control of the cell cycle, and the identification and characterization of genes and molecular controls of embryogenesis, seed maturation and germination, are all quite promising, and will be of invaluable help in understanding the biology of somatic embryogenesis. An important requirement for plants produced in vitro is that the plants must be genetically identical to the source plants. Plants regenerated via micropropagation or somatic embryogenesis are derived from characteristic organized meristems or meristematic cells. These cells are by nature genetically stable and less prone to mutational changes. Indeed, there is increasing evidence that there is a strong selection in favor of genetically normal cells during somatic embryo development. Consequently, plants derived from micropropagation, as well as somatic embryogenesis, give rise to truly clonal populations. Both systems, therefore, are well suited for the mass production of plants.

Automation of somatic embryogenesis and micropropagation Traditional methods of plant propagation in vitro include several distinct phases: preparation and placement of the primary explants in a nutrient solution, meristem or tissue proliferation, organization of somatic embryos, rooting of shoots or germination of somatic embryos, and finally the transplanting of plantlets to soil (Zimmerman et al. 1986; Debergh & Zimmerman 1991). A variety of strategies are being developed to automate either a single most labor intensive phase or the entire process. Strategies for the automation of micropropagation being developed by several groups in Japan depend on robot-assisted cutting and selection of nodal explants containing axillary buds by computerized image analysis, followed by robotic transfer to fresh agar nutrient media (Kurata & Kozai 1992). The process depends on

107 complex and rather expensive high technology,which requires a specially trained and skilled labor force. Only a few large companies may be able to afford such expensive systems, that may not necessarily reduce the costs significantly. More importantly, the process does not lend itself to growth and multiplication in bioreactors. A completely integrated, mechanical/biological and automated micropropagation process (the Vitromatic process), from growth and multiplication to planting in soil, has been developed in Israel (Levin et al. 1988; Levin & Vasil 1989). It uses bioreactors to grow large and compact masses of shoot meristems. The meristems are processed, sorted and distributed automatically, and then allowed to develop into tiny rooted plantlets. The latter are transplanted automatically into soil by a planting machine. The system neither uses robotics nor image analysis. Although the same system can be used for the production of somatic embryos, for the present time it offers a very practical method for the automation of micropropagation, with a substantial reduction in labor input and cost of production. Indeed, it should be possible to further reduce the costs by using simple and disposable low cost bioreactors, rather than the expensive commercial models that have many unnecessary features. Knowledge of the biological, physical and engineering components of bioreactor systems for the growth of plant cells has been greatly improved during the past decade. It is thus possible to grow 50-100 litre batches of plant cells in bioreactors for the production of secondary metabolites (Constabel & Vasi11987, 1988). The same technology can be readily adapted for the growth of regenerable cells and tissues in liquid media in bioreactors, as demonstrated by the successful mass propagation of shoots in a 500 litre bioreactor by Akita et al. (1994). They harvested 64.6 kg of shoots of Stevia rebaudiana from an initial inoculum of 460 gm, a 140-fold increase. Similarly, a 10 litre jar fermenter was used for the production of potato tubers, which could be used directly as seed tubers (Akita & Takayama 1994). Somatic embryos produced in culture can be germinated directly to obtain plants. However, for practical purposes it will be much more useful if they can be handled, stored and transported like seeds. For this reason, many attempts have been made during the past decade to develop efficient methods for the encapsulation of somatic embryos, or even shoot buds, for the production of 'synthetic seeds' (Redenbaugh et al. 1991). Such seed may be particularly important for the

propagation of high value specialty crops, hybrid seed, etc., although their use is not practical for major field crops, where the volume of required seeds is enormous, and the costs per seed are a fraction of in vitro produced propagules. Conversion rates of synthetic seeds to plants, which have generally been rather poor thus far, must be improved and brought to acceptable levels before they can become a commercial reality. In this regard, very encouraging results have been obtained by the controlled desiccation of the somatic embryos. The actual design and construction of the synthetic seed varies from simple encapsulation into a variety of gels to the fabrication of more complex structures. Simple encapsulation of somatic embryos has not proven to be very useful thus far. Dupuis et al. (1994) have overcome many of these problems by using a pharmaceutical type water-soluble capsule covered on its inner surface by a water-tight polymer to make synthetic seeds of carrot that have a high conversion efficiency. Finally, commercially available seed planting machines can be easily adapted for the sowing of synthetic seeds. In conclusion, it can be said that the most difficult and intractable problems in the use of bioreactors for large scale somatic embryogenesis (or for organogenesis) are in the biology of the system and not in engineering. This is the area where much of the future work should be directed and concentrated. Resolution of the biological problems discussed above may eventually make it feasible to produce large clonal populations of plants by somatic embryogenesis (or organogenesis) in bioreactors. These advances, by reducing the cost but increasing the rate of production, will inevitably result in the expansion of the market to include vegetable and tree species, which is where the future of the industry really is. The various chapters in this volume document the significant advances in the field and the state-of-the art today. They also make it clear that much further work, leading to a far better understanding of somatic embryogenesis, will have to be done before automated mass production of somatic embryos can become commercially viable. In the mean time, it may be worthwhile to further refine and test the systems which have shown much promise for micropropagation, such as those described by Levin et al. (1988) and Akita et al. (1994), in order to reduce the costs and extend micropropagation to vegetable and tree species. Automated, bioreactor-based micropropagation may be the method of choice for the mass propagation of plants until similar systems can be developed for the

108 production of somatic embryos and viable synthetic seeds.

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Dupuis J, Roffat C, De Rose R T & Molle F (1994) Pharmaceutical capsules as a coating system for artificial seeds. Bio/Technology 12: 385-389. Kurata K & Kozai T (1992) Transplant Production Systems. Kluwer Academic Publishers, Dordrecht Levin R, Gaba V, Tal B, Hirsch S, DeNola D & Vasil I K (1988) Automated plant tissue culture for mass propagation. Bio/Technology 6:1035-1040 Levin R & Vasil I K (1989) Progress in reducing the cost of micropropagation. IAPTC Newsletter 59:2-12 Redenbaugh K, Fujii JA & Slade D (1991) Synthetic seed technology. In: Vasil I K (Ed) Cell Culture and Somatic Cell Genetics of Plants, Vol 8. Scale-Up and Automation in Plant Propagation (pp 36-61). Academic Press, San Diego Vasil I K (1986) Cell Culture and Somatic Cell Genetics of Plants. Vol 3. Plant Regeneration and Genetic Variability. Academic Press, Orlando Vasil I K (1991) Cell Culture and Somatic Cell Genetics of Plants. Vol 8. Scale-Up and Automation in Plant Propagation. Academic Press, San Diego Vasil I K & Thorpe T A (I 994) Plant Cell and Tissue Culture. Kluwer Academic Publishers, Dordrecht Zimmerman R H, Griesbach R J, Hammerschlag F A & Lawson R H (1986) Tissue Culture as a Plant Production System for Horticultural Crops. Martinus Nijhoff Publishers, Dordrecht