What Is Plant In Vitro Propagation

Published Date: 02 Nov 2017

Introduction

India has a rich biological diversity due to its varied climatic, altitudinal variations and ecological habitats. There have been increasing rates of threats of depletion to these biological resources due to immense biotic and abiotic stresses. Indiscriminate collection of plants for their medicinal, ornamental, perfumery uses, etc. and habitat loss and degradation are potential causes of threats. Conventionally, there are some methods of conservation which includes in situ and ex situ conservation both are complementary to each other. In situ methods allow conservation to occur with ongoing natural evolutionary processes, ex situ conservation via in vitro propagation also acts as a viable alternative for increase and conservation of populations of existing bioresources in the wild and to meet the commercial requirements. A review highlighting various in vitro protocols developed for selected rare and threatened plant species of India has been done to highlight the significance of ex situ conservation in cases where regeneration through conventional methods is difficult to undertake and species are left with low population in the wild.

Technology became an essential element to evaluate available resources with respect to their utilitarian value, make them available on a sustainable basis for future generations, and convert them into products of economic value. Plant tissue culture is a sun-rise technology that can have a great impact on both agriculture and industry, through providing plants needed to meet the ever increasing world demand. Since the in vitro produced plants are usually identical with each other (i.e. clones), tissue culture has been effectively used by private and government-owned enterprises for production of plants on commercial scale. According to Bhojwani and Razdan (1996), an estimated one billion plants per year are produced by Micro propagation, in about 50 biotech companies. This number probably doubled during the past 10 years, since more companies were established during this period all the world around (Gupta & Yasuomi, 2006 and Manickam, 2007).

Recent modern techniques of propagation have been developed which could help growers to meet the demand of the horticultural industry in the next century. In vitro propagation has played a very important role in rapid multiplication of cultivars with desirable traits and production of healthy and disease-free plants. During the last several years, different approaches have been made for in vitro propagation. New challenges for refinements of protocols for high rate of shoot multiplication and development of cost effective methods has gained importance in the recent past. Importance of liquid static culture for shoot proliferation and root induction. The development of protocol for in vitro plant regeneration which is considered as most important step for successful implementation of various techniques used for plant improvement.

Well developed techniques are currently available to help growers meet the demand of the pharmaceutical industry in the next century. These protocols are designed to provide optimal levels of carbohydrates, organic compounds (vitamins), mineral nutrients, environmental factors (e.g. light, gaseous environment, temperature, and humidity) and growth regulators required to obtain high regeneration rates of many plant species in vitro and thereby facilitate commercially viable micro propagation. Well-defined cell culture methods have also been developed for the production of several important secondary products.

Experimental approaches used for propagation of plants through tissue culture can be divided into three broad categories. The most common approach is to isolate organized meristems like shoot tips or axillary buds and induce them to grow into complete plants.

This system of propagation is commonly referred to as micro propagation. In the second approach, adventitious shoots are initiated on leaf, root and stem segments or on callus derived from those organs. The third system of propagation involves induction of somatic embryogenesis in cell and callus cultures. This system is theoretically most efficient as large numbers of somatic embryos can be obtained once the whole process is standardized.

The development of reliable in vitro protocols are of great importance for conservation of rare and threatened plant species by virtue of producing uniform planting material for offsetting the pressure on the natural populations especially for medicinal and ornamental plants. Concerted international and national efforts have been initiated to conserve and to sustainably use the biodiversity. The micro propagation unit at Royal Botanic Garden, Kew is involved in propagation and maintenance of more than 3000 plant taxa, from all over the world, for over 30 years (Sarasan et al., 2006). The use of various approaches of biotechnology in conservation of biodiversity and plant genetic resources has been described by various authors (Fay, 1992; Rao, 2004; Bapat et al., 2008).

Applications:

In recent years, tissue culture has emerged as a promising technique for culturing and studying the physiological behavior of isolated plant organs, tissues, cells, protoplasts and even cell organelles under precisely controlled physical and chemical conditions. Tissue culture can be divided into three broad categories. The most common approach is to isolate organised meristems like shoot tips or axillary buds and induce them to grow into complete plants. This system of propagation is commonly referred to as micro propagation. In the second approach, adventitious shoots are initiated on leaf, root and stem segments or on callus derived from those organs. The third system of propagation involves induction of somatic embryogenesis in cell and callus cultures. The commercial technology is primarily based on micropropagation, in which rapid proliferation is achieved from tiny stem cutting, axillary buds and to a limited extent from somatic embryos, cell clumps in suspension cultures and bioreactors. This technique is being used for large scale propagation of a number of plant species viz. Rauvolfia tetraphylla (Faisal et al., 2005), Tylophora indica (Faisal and Anis, 2003), Vitex negundo (Ahmad and Anis, 2007), Pterocarpus marsupium (Husain et al., 2007), Mucuna pruriens (Faisal et al., 2006), Balanites aegyptiaca (Siddique and Anis, 2009). The present review highlighted in vitro regeneration of plants.

What is Plant in vitro propagation?

Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.[1] Micropropagation is used to multiply novel plants, such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative reproduction. Micropropagation begins with the selection of plant material to be propagated. Clean stock materials that are free of viruses and fungi are important in the production of the healthiest plants. Once the plant material is chosen for culture, the collection of explant(s) begins and is dependent on the type of tissue to be used; including stem tips, anthers, petals, pollen and others plant tissues. The explant material is then surface sterilized, usually in multiple courses of bleach and alcohol washes, and finally rinsed in sterilized water. This small portion of plant tissue, sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators (plant hormones). Usually the medium is thickened with agar to create a gel which supports the explant during growth. Some plants are easily grown on simple media, but others require more complicated media for successful growth; the plant tissue grows and differentiates into new tissues depending on the medium. For example, media containing cytokinins are used to create branched shoots from plant buds.

Multiplication

Multiplication is the taking of tissue samples produced during the first stage and increasing their number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds or thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media. If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. If the tissue is grown as small plants called plantlets, hormones are often added that cause the plantlets to produce many small offshoots that can be removed and recultured.

Methods Available for Plant Propagation

Three methods are currently available that can be utilized for mass propagation of plants, each have its advantages and limitations, including the following: 1. Somatic embryogenesis: This depends on stimulation of asexual embryos either directly from cultured organ or indirectly from callus culture derived from cultured organs (Takayama, 2002). A huge amount of embryos can be regenerated from various plant species. Such embryos can be further encapsulated with sodium alginate and treated like synthetic seeds (Re- denbaugh et al., 1991). However, wide expansion in propagation by this methods is usually hampered with the possible genetic modifications that might appear, especially if intermediate callus phase is involved. 2. Adventitious bud formation: This method is based on the stimulation of organs (stem, leaf, and root) on callus cultures through manipulation of growth regulators in the medium (Akita and Takayama, 1994). High cytokinin/ auxin ratio in the medium favors shoot initiation, while roots can be induced in the presence of high auxin/cytokinin ratio. However, a balanced cytokinin/ auxin ratio leads to the regeneration of complete plant. This method is characterized with relatively high number of regenerated plants. Nevertheless, the chances of somaclonal or other variations might be encountered in the developed plants. 3. Enhanced axillary branching in cultured shoot tips and lateral buds: This is the most widely used method in the in vitro propagation programs (Hohnle and Weber, 2007). The technique is based on the inhibition of the apical dominance by a cytokinin, followed by stimulation of growth of bud primordia in the axils of leaves within the cultured shoot tip or lateral bud. Shoots developed in this process are severed and serially propagated, then individually rooted. This method is characterized with moderate number of plant production with high genetic stability.

Stages of Mass Propagation of Plants through Tissue Culture

In 1974, Murashige outlined four stages that can be followed in tissue culture, each with specific nutritional and incubation conditions requirements (Murashige, 1974). Such stages are as follows: 1. Stage I: Initiation stage: In this stage an explants (i.e. shoot tip, lateral bud, leaf segment, etc.) is surface sterilized and cultured on nutrient medium. The objective of this stage is to obtain a clean or contamination- free cultures that can be used in the following stage, regardless of the amount of growth attained. The nutritional requirements are usually very simple and the cultures are incubated either under light or dark conditions according to the method of propagation. 2. Stage II: Multiplication stage: This is the most important stage in any propagation program since it determines the number of produced plants. In this stage, the number of propagules is multiplied by repeated sub- and reculture until the desired (or planned) number of plants is attained. The chemical formulation and physical status of nutrient medium, as well as, the incubation conditions are of prime significance. 3. Stage III: Rooting stage: Shoots produced from the previous stage are separated and individually rooted in a relatively high auxin -containing media. In this stage, a good root system is initiated and complete plants are achieved.

4. Stage IV: Acclimization Stage:

This stage may be included with the pervious stage. Plants developed through tissue culture are heterotrophic, lack cuticle on their epidermis (Adelberg et al., 2000), as well as having non-functional stomates (Czynczyk and Takubowski, 2007). Such plants cannot survive the outside unfavorable conditions. Thus, they need hardening and acclimization, where they receive a special treatment before they can be transferred to the soil in order to stimulate photosynthesis, cuticle development and their stomates starts functioning. Probably this is the most important stage in the whole process. Thus, it should be conducted under proper conditions regarding soil, light, temperature and irrigation. It is worth mentioning that each specific plant species has its own requirements in all previous stages. The following parameters needs evaluation for each plant considered for propagation through tissue culture: 1. Source of explant. 2. Nutrient media composition and physical conditions at each stage. 3. Incubation conditions regarding temperature, light intensity, duration of light and dark, and humidity. 4. Hardening conditions and transfer to soil.

In vitro propagation

In vitro culture is one of the key tools of plant biotechnology that exploits the totipotency nature of plant cells, a concept proposed by Haberlandt (1902) and unequivocally demonstrated, for the first time, by Steward et al. (1958). Tissue culture is alternatively called cell, tissue and organ culture through in vitro condition (Debergh and Read, 1991). It can be employed for large-scale propagation of disease free clones and gene pool conservation. Ornamental industry has applied immensely in vitro propagation approach for large-scale plant multiplication of elite superior varieties. As a result, hundreds of plant tissue culture laboratories have come up worldwide, especially in the developing countries due to cheap labour costs. However, micropropagation technology is more costly than conventional propagation methods, and unit cost per plant becomes unaffordable compelling to adopt strategies to cut down the production cost for lowering the cost per plant (IAEA-TECDOC- 1384, 2004).

Micropropagation via meristem culture or axillary bud/shoot tip culture:

In vitro propagation through meristem culture is the best possible means of virus elimination and produces a large numbers of plants in a short span of time. It is a powerful tool for large-scale propagation of horticultural crops including pot plants. The term ‘meristem culture’ specifically means that a meristem with no leaf primordia or at most 1–2 leaf primordial which are excised and cultured. The pathway of regeneration undergoes several steps. Starting with an isolated explant, with de-differentiation followed by re-differentiation and organization into meristematic centres. Upon further induction the cells can form unipolar structures i.e. organogenesis, or bipolar structures called somatic embryogenesis. The organization into morphogenetic patterns can take place directly on the isolated explant or can be expressed only after callus formation, which is called indirect morphogenesis. When shoots are developed directly from leaf or stem explants it refers to direct morphogenesis. Micropropagation is an alternative method of vegetative propagation, which is well suited for the multiplication of elite clones. It is accomplished by several means, i.e., multiplication of shoots from different explants such as shoot tips or axillary buds or direct formation of adventitious shoots or somatic embryos from tissues, organs or zygotic embryos. The first significant use of plant tissue culture in ornamental was made during 1920s when orchid seeds were germinated under laboratory conditions (Knudson, 1922). Micropropagation generally involves four distinct stages: initiation of cultures, shoot multiplication, rooting of in vitro grown shoots, and acclimatization. The first stage: culture initiation depends on explants type or the physiological stage of the donor plant at the time of excision. Explants from actively growing shoots are generally used for mass scale multiplication. The second stage: shoot multiplication is crucial and achieved by using Plant Growth Regulators i.e. auxin and cytokinin. The third stage: the elongated shoots, derived from the multiplication stage, are subsequently rooted either ex vitro or in vitro. In some cases, the highest root induction occurs from excised shoots in the liquid medium when compared with semi-solid medium. The fourth stage: acclimatization of in vitro grown plants is an important step in micro propagation. In vitro plants are exposed to invariably controlled growth conditions such as high amount of organic and inorganic nutrients, Plant Growth Regulators, carbon source, high humidity, low light and poor gaseous exchange. Although they may support rapid growth and multiplication, the controlled conditions induce structural and physiological changes in plants rendering them unfit to survive when transferred directly to the field. Thus, a gradual acclimatization from laboratory to field condition is necessary. The plants are gradually shifted from high humidity/low irradiance conditions to low humidity/high irradiance conditions, enabling them to survive under ‘adverse’ climatic conditions. Carbon dioxide enrichment in the greenhouse for the cultivation of ornamental plants has a positive impact on production. Increased CO2 concentration also lessens water stress of microcuttings by closing the stomata as reported by Matysiak and Nowak (1995). In CO2 enriched atmosphere (1200 μl/l) of the greenhouse and the highest level of electrical conductivity (EC=2.8 mS cm) of the medium produced the best growth of gerbera microcuttings taken from in vitro plants (Matysiak and Nowak, 2001). Photoautotropic micropropagation of ornamental plants have been reviewed (Kozai et al., 1988; Kozai, 1990a,b), and is suggested to use for reducing production costs, and automation to use robots for micropropagation process (Kozai et al., 1988; Kozai, 1991a,b). Many commercial ornamental plants are being propagated by in vitro culture on the culture medium containing auxins and cytokinins (Preil, 2003; Rout and Jain, 2004). Several different explants have been used for direct shoot formation. Mayer (1956) succeeded first time regeneration of Cyclamen shoots from tuber segments on MS medium supplemented with 10.7 μM NAA. Furthermore, plants have been regenerated from leaf tissues and petiole segments of Cyclamen (Geier, 1977; Geier et al., 1983; Schwenkel, 1991; Dillen et al., 1996), Heuchera sanguinea (Hosoki and Kajino, 2003), and Begonia (Takayama, 1983). In vitro clonal propagation of Dracaena deremensis has been reported by several groups (Debergh, 1975, 1976; Miller and Murashige, 1976; Chua et al., 1981). The first report on shoot multiplication and rooting of rose (Rosa multiflora) was made by Elliott (1970) by using shoot tip explants and later on followed by others (Hasegawa, 1979; Skirvin and Chu, 1979; Rout et al., 1989). AboEl-Nil (1983) reviewed on the large-scale production of Pelargonium by using different explants. Atta-Alla et al. (1998) reported the shoot bud regeneration from leaf and petiole explants of Anthurium parvispathum and subsequently establishment in soil. Martin et al. (2003) succeeded in direct shoot bud regeneration fromlamina explants of Anthuriumandraeanum on MS medium fortified with 1.11 μMBA, 1.14 μM IAA and 0.46 μM Kn. Furthermore, the regenerated shoots were rooted on half-strength MS medium supplemented with 0.54 μM NAA and 0.93 μM Kn. Nearly 300 plantlets of each cultivar were transferred to soil with 95% survival rate (Joseph et al., 2003). Thao et al. (2003) achieved shoots regenerated from petiolederived callus of Alocasia micholitziana "Green velvel" on MS medium fortified with 0.5 μMKn and 0.5 μM2,4- D. The regenerated shoots were rooted on hormone free MS medium and subsequently established in the field. Skirvin et al. (1990) reported rapid method of shoot multiplication and rooting of Rosa hybrida cultivars. Now shoot tip explant is being routinely used for the micropropagation of ornamental plants including Rhododendron (Ettinger and Preece, 1985;McCown and Lloyd, 1983; Brand and Kiyamoto, 1994a,b), Zantedeschia albomaculata (Chang et al., 2003) and Ebenus cretica (Hatzilazarou et al., 2001). Later on Brand and Kiyomoto (1997) accomplished shoot multiplication of Rhododendron "Montego" on woody plant medium (Lloyd and McCown, 1980) supplemented with 10–50 μM 2ip. The number of shoots increased with subsequent subcultures on the fresh culture medium. Micro-shoots were rooted in moist sphagnum moss and vermiculite (3 :1 ratio), and 90% microshoots survived and grown in the greenhouse. Several researchers have reported on clonal propagation of Spathiphyllum (Fonnesbech and Fonnesbech, 1979;

Orlikowska et al., 1995; Wated et al., 1997). Cytokinin alone in the culture medium induces shoot formation in many plants. MS medium supplemented with 3.0 mg/l BA was suitable for micropropagation of Ficus benjamina vars. Natasja and Starlight (Rzepka- Plevnes and Kurek, 2001). Jain (1997) micropropagated Saintpaulia ionantha by culturing leaf disks on MS medium containing 0.22–0.50 μM BA. Addition of auxins with cytokinins becomes essential for shoot induction and multiplication depending on the plant type. In Petunia hybrida, mass shoot multiplication was achieved on MS medium amended with 2.2 μM BA and 5.7 μM IAA within 4 weeks of culture (Sharma and Mitra, 1976). High concentration of cytokinins is unsuitable for shoot formation from leaf or petiole explants in some ornamental pot plants. Takayama and Misawa (1981, 1982) used 1.3 μM BA or 4.6 μMKn in combination with 5.4 μM NAA for shoot bud regeneration from leaf, petiole or inflorescence segments of Begonia species. Further, low concentration of cytokinins also influences high rate of shoot bud regeneration (Reuter and Bhandari, 1981; Bigot Jain (1997), 1981a,b; Roest et al., 1981; Mikkelson and Sink, 1978a,b; Welander, 1977, 1979, 1981; Simmonds, 1984; Appelgren, 1976, 1985). The addition of 1–2 g/ l activated charcoal in the culture medium increased the rooting efficiency from excised shoots of Begonia× hiemalis (Bigot, 1981a,b). Activated charcoal seems to adsorb Plant Growth Regulators, prompting to better response for rooting and even for shoot formation. I seems Begonia has high endogenous cytokinin and auxins, and by adding activated charcoal in the medium certainly promotes organogenesis. Used two cytokinins (Kn and zeatin) for regeneration of plantlets of Begonia×elatior. He suggested that two cytokinins did not affect the basic plant characteristics including flower colour. Similarly, many reports indicated mass multiplication of Ficus species by adding cytokinins in the culture medium (Gabryszewska and Rudnicki, 1997; Debergh and DeWael, 1997; Nobre and Romano, 1998). Demiralay et al. (1998) achieved shoot multiplication of Ficus carica var. Bursa Siyaki on MS medium containing 1.0 mg/l BA and 89 mg/l phloroglucinol; shoot multiplication rate was 4.43 shoots/explant; and 68.33% rooting rate of the micropropagated shoots on rooting medium containing 1 mg/l IBA. The rooting efficiency enhanced by addition of 0.05% PVP in the culture medium containing 2.5 μM IBA (Nobre and Romano, 1998). The addition of PVP helps in oxidizing polyphenols leached in the medium, and promotes high rate of organogenesis. The quality of light also influences shoot induction.

Gabryszewska and Rudnicki (1997) developed a micropropagation protocol for F. benjamina by using shoot meristems; shoot numbers increased on MS medium supplemented with 15 mg/l 2ip by red light treatment; and root initiation occurred in all light treatments (white, blue, green and red). However, the rooting and number of roots/shoot were highest in red light on the medium having 0.5 mg/l IAA. Liquid medium seems to be more effective for shoot regeneration and root induction, which is due to better aeration. Simmonds and Werry (1987) used liquid medium for enhancing the micropropagation profile of Begonia×hiemalis. Wated et al. (1997) compared performance of agar-solidified medium and interfacial membrane rafts floating on liquid medium for shoot multiplication and root induction. The results showed shoot multiplication was highest on membrane rafts floating on the liquid medium, and also plants rooted much better. Similarly, Osternack et al. (1999) succeeded in inducing somatic embryogenesis and adventitious shoots and roots from hypocotyl tissues of Euphorbia pulcherrima on cytokinin containing medium. Subsequently, Preil (2003) noted that the regeneration potential of isolated cells, tissue or organs and the callus cultures is highly variable. Furthermore, petiole cross sections cultivated on auxin and cytokinin containing medium give rise to adventitious shoots from epidermal cells and subepidermal cortex cells, never from pith cells of the central regions of the petiole. The direct shoot bud formation without any callus phase from appropriate explants is of great success for large-scale clonal multiplication of desired clone all round the year to boost the commercial floriculture.

Micropropagation via somatic embryogenesis

Somatic embryos, which are bipolar structures, arise from individual cells and have no vascular connection with the maternal tissue of the explant (Haccius,1978). Embryos may develop directly from somatic cells (direct embryogenesis) or development of recognizable embryogenic structures is preceded by numerous, organized, non-embryogenic mitotic cycles (indirect embryogenesis). Somatic embryogenesis has a great potential for clonal multiplication. Under controlled environmental conditions, somatic embryos germinate readily, similar to their seedling counterpart. The commercial application of somatic embryogenesis will be accomplished only when the germination rate of somatic embryos is high up to 80–85%. Considerable success has been achieved in inducing somatic embryogenesis in ornamental pot plants like chrysanthemum (Dendrathema grandiflorum) (May and Trigiano, 1991; Tanaka et al., 2000), Cyclamen persicum (Wicart et al., 1984; Pueschel et al., 2003), rose (R. hybrida) (Rout et al., 1991, Kim et al., 2003a), Begonia gracilis (Castillo and Smith, 1997), S. ionantha cv. Benjamin (Murch et al., 2003), and E. pulcherrima (Osternack et al., 1999). In chrysanthemum, somatic embryos were produced from leaf mid-rib explants on modified MS medium supplemented with 1.0 mg/l 2,4- D and 0.2 mg/l BA (May and Trigiano, 1991). Highest somatic embryos were produced on the medium containing 6–8% sucrose and kept in the darkness for first 28 days, followed by 10 days in the light. Twelve cultivars produced somatic embryos, but complete plantlets were recovered from only five cultivars. Castillo and Smith (1997) induced direct somatic embryogenesis from petiole and leaf blade explants of B. gracilis on MS medium supplemented with 0.5 mg/ l kinetin and 2% (v/v) coconut water. Somatic embryos were obtained with greater frequency from petiole explants than from leaf blade sections. Osternack et al. (1999) succeeded in achieving somatic embryos from hypocotyl tissues of E. pulcherrima on MS mediumsupplemented with 2.0 mg/l IAA (Fig. 1). About1400 embryos were developed from 320 calli derived from outer regions of the hypocotyls. However, only 8%developed normal plantlets. In most cases, shoots were rooted in hormone free medium. Both orientation of the petiole explants and auxin transport system are crucial factors for the induction of somatic embryogenesis of Saintpaulia (Murch et al., 2003), and TDZ helped in the development of somatic embryos. Winkelmann et al. (1998) used cell suspension culture of Cyclamen for rapid development of somatic embryos, and later on followed by Hohe et al. (2001), who developed a largescale propagation system of Cyclamen from embryogenic cell suspension cultures. Bouman et al. (2001) reported that the efficiency of embryogenic callus of Cyclamen seems to be stable for more than 5 years; however, suspension cultures can lose embryogenic potential after a number of subcultures. Therefore, it is necessary to determine the number of subcultures before embryogenic cell suspensions lose their potential of embryogenic nature. Pueschel et al. (2003) succeeded in plant regeneration via somatic embryogenesis of C. persicum and maintained the regeneration ability for prolonged period. There are advantages and disadvantages of somatic embryogenesis in large-scale plant multiplication (Jain, 2002). The major advantages are large-scale somatic embryo production in bioreactors, encapsulation, cryopreservation, genetic transformation and clonal propagation. The major limitations are genotypic dependence of somatic embryo production and poor germination rate.

Micropropagation via thin cell layer :

Thin cell layer (TCL) is a simple but effective system that relies on a small size explant derived from a limited cell number of homogenous tissue. They are excised longitudinally or transversely from different organs ranging from floral parts to root/rhizome of plants. Longitudinal TCL (lTCL) (0.5–1 mm wide and 5– 10 mm long) is used when a definite cell type (epidermal, sub-epidermal, cortical, cambial or medullar cell) is to be analysed. TCLs can be excised fromstem, leaf, vein, floral stalk, petiole, pedicel, bulb-scale, etc.As for the transverse TCL (tTCL) (0.1–5 mm), other organs (leaf blade, root/ rhizome, floral organs, meristems, stem node, etc.) can be used. The reduced cell number in TCL is important for the developmental process or the morphogenetic programme, which can be altered by making changes in organ/tissue and size to be uniformly exposed to the medium (Tran Thanh Van, 1980). Thin cell layer is the model systems and find applications in higher plant tissue and organ culture and genetic transformation (Teixeira da Silva, 2003a, 2005). Moreover, thin cell layer technology is a solution to many of the issues currently hindering the efficient progress of ornamental and floricultural crop improvement, since it solves the initial step i.e. plant regeneration problem. This technology has also been effectively used in the micropropagation of various crops including floricultural crops (Tran Thanh Van and Bui, 2000; Fiore et al., 2002; Nhut et al., 2003a,b; Teixeira de Silva and Nhut, 2003a). Recently, Teixeira da Silva (2003a) published a detailed review on the use of thin cell layer technology in ornamental plant micropropagation and biotechnology, which highlights organogenesis and somatic embryogenesis for plant regeneration and genetic improvement via transformation. Mulin and Tran Thanh Van (1989) indicated that in vitro shoots and flowers were formed from thin epidermal cells excised from the first five internodes of basal flowering branches in P. hybrida. Explants (1×10mm2) consisting of 3–6 layers of subepidermal and epidermal cells produced vegetative buds within 2 weeks of culture. Ohki (1994) reported that 100–200 shoots per tTCL (transverse thin cell layer) explants were obtained from 0.3 to 0.5mmpetiole or 3×3 mm2 lamina sections, respectively of S. ionantha within 4 weeks of culture. Over 70,000 plants were produced from a single leaf within 3–4 months. Gill et al. (1992) used tTCL hypocotyl explants (10 mm) of 1-weekold geranium (Pelargonium×hortorum) hybrid seedlings for induction of somatic embryogenesis. They observed that the development of somatic embryos was rapid and the number of embryos was about 8-fold higher than the culture of whole hypocotyl explants. Hsia and Korban (1996) achieved organogenic and embryogenic callus and subsequent regeneration from lTCL (longitudinally thincell layer) explants derived from dormant bud floral stalks of R. hybrida cv. Baccara. Thin cell layer systems could be used as a tool for in vitro regeneration and micropropagation. The efficiency is very high compared to the conventional technique of tissue culture. The TCL method is also very useful in virus elimination in combination with antiviral compounds. Recent progress in thin cell layer technology has opened new possibilities for improvement of ornamental and floricultural crops.

Mechanization of in vitro plant propagation:

The exploitation of in vitro methods for profitable plant micropropagation requires automation and scalingup, which depend on the use of liquid cultures (Takayama and Misawa, 1981). The use of bioreactors is a step forward for commercial propagation of ornamental plants. Bioreactors with computer control systems offer various advantages over conventionally produced culture due to possibilities of automation, saving labour and production cost (Aitkens-Christie, 1991; Preil, 1991; Ziv, 1991, 1995; Paek et al., 2001; Eide et al., 2003). Since microbial fermentation techniques were first used in studies on growth kinetics of higher plant cell suspensions (Tulecke and Nickell, 1959), major progress has occurred in the area of largescale liquid culture and in the development of bioreactor process control system. Since then bioreactor system was applied for meristem, embryogenic and organogeniccultures of several plant species (Levin et al., 1988; Preil et al., 1988; Takayama and Akita, 1994, 1998; Takayama, 2002; Eide et al., 2003). The various propagation aspects of several plant species in bioreactors, applications, and some of the problems associated with the operation of bioreactors have recently been reviewed (Takayama and Akita, 1998; Ziv, 2000; Paek et al., 2001). Liquid media have been used for plant cells, somatic embryos and cell suspension cells in either agitated flasks or various types of bioreactors (Smart and Fowler, 1984; Tautorus and Dunstan, 1995; Takayama, 2000; Ziv, 2000; Paek et al., 2001; Eide et al., 2003). Considerable attention has been given to automation of the repeated cutting, separation, subculture, and transfer of buds, shoots, or plantlets during the multiplication and transplanting phases (Levin et al., 1988; Aitkens- Christie, 1991; Vasil, 1994; Aitkens-Christie et al., 1995). Automation of tissue culture will depend on the use of liquid cultures in bioreactors, allow fast proliferation, mechanized cutting, separation, and automated dispensing (Sakamoto et al., 1995). These techniques were used in some plants, which involve minimal hand manipulation and thus reduce in vitro plant production costs (Levin et al., 1988; Ziv, 1991, 1992, 1995; Vasil, 1994; Aitkens-Christie et al., 1995; Curtis, 2002). Eide et al. (2003) reported two liquid culture systems for plant propagation i.e. temporary immersion systems and permanent submersion of the plant cells/tissue that requires oxygen supply through rotary shakers or bioreactors. Temporary immersion system, e.g. RITA bioreactor, seems to be better than the permanent submersion system for shoot proliferation. However, Takayama et al. (1986) demonstrated vigorous growth of organogenic cultures of Begonia in a bioreactor. The oxygen partial pressure in bioreactors helps cell proliferation and subsequent differentiation of somatic embryos from suspension cultures of C. persicum (Hvoslof-Eide and Munster, 1998, 2001). A significant high number of germinating embryos were obtained from the cultures grown at 40% pO2 than from those grown in flasks or in bioreactors at 5%, 10% and 20% pO2 (Hohe et al., 1999). Kim et al. (2003 established a large-scale propagation of chrysanthemum through bioreactor system, and obtained 5000 plantlets after 12 weeks of culture in 10 l column type bioreactor. They also found that the bioreactors maintained at 25 °C, 100 μmol/m2/s PPF and 0.1 vvm air volumes as optimal conditions for this propagation. Weber et al. (1994) reported the propagation efficiency of Clematis tangutica in a bioreactor. Preil (2003) established successfully eleven hybrid cultivars and a wild type of C. tangutica in a bioreactor (Fig. 2). This method resulted rapidly increased pro-embryogenic clusters up to 4500/ml. Later, some 200 globular embryos, 300 heart and torpedo-shaped embryos per ml were determined after 4 weeks of culture in auxin-free medium. About 500,000 cotyledonary embryos were obtained from 1 l cell suspension culture. Further, the clusters of embryos developed into plantlets differing in length. The plantlets were transferred to the greenhouse. Somatic embryos and shoot cultures could be grown in both liquid systems, embryogenesis possibly being the most suited for full automation through a synthetic seed scheme. Adapting bioreactors with liquid media formicropropagation is highly suitable due to the ease of scaling-up (Preil, 1991; Preil and Beck, 1991) and the ability to prevent the physiological disorders of shoot and leaf hyperhydricity (Ziv, 1999) and, thereby, lowering production costs. The major risk in using bioreactors for large-scale plant production is contamination.

Future presprocetives: