In vitro Plant Propagation: A Review

Journal of Forest Science Vol. 27, No. 2, pp. 61~72, August 2011 In vitro Plant Propagation: A Review Nitish kumar1,3*, and M. P. Reddy2,3 1 Centre for Biotechnology, School of Earth, Biological and Environmental Science, Central University of Bihar, BIT campus, Patna 800014, Bihar, India 2 Plant Stress Genomics Research Centre, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia 3 Discipline of Wasteland Research, Central Salt & Marine Chemicals Research Institute (Council of Scientific and Industrial Research), Bhavnagar, Gujarat-364002, India. ABSTRACT : Micropropagation is an alternative mean of propagation that can be employed in mass multiplication of plants in relatively shorter time. Recent modern techniques of propagation have been developed which could facilitate large scale production of true-to-type plants and for the improvement of the species using genetic engineering techniques in the next century. An overview on the in vitro propagation via meristem culture, regeneration via organogenesis and somatic embryogenesis is presented. The usefulness of the plants in commercial industry as well as propagation techniques, screening for various useful characteristics and the influence of different cultural conditions in the multiplication, rooting and acclimatization phases on the growth of tissue cultured plant discussed. Keywords : Meristem culture, Micropropagation, Regeneration, Somatic embryogenesis INTRODUCTION usses the different micropropagation techniques and various factors affecting the micropropagation of plants. Micropropagation is a plant tissue culture technique used for producing plantlets and implies the culture of MICROPROPAGATION aseptic small sections of tissues and organs in vessels with defined culture medium and under controlled enviro- Principles of tissue culture nmental conditions and has become an increasingly important tool for both science and commercial applications in Plant micropropagation is an integrated process in which recent years. It is the foundation on which all biotechno- cells, tissues or organs of selected plants are isolated, logical research rests, because almost all uses of plant surface sterilized, and incubated in a growth-promoting biotechnology ultimately require the successful culture of aseptic environment to produce many clone plantlets (Alt- plants cells, tissues or organs. This technique has many man, 2000). The technique of cloning isolated single cells advantages over conventional vegetative propagation, as in vitro demonstrated the fact that somatic cells, under e.g. the propagation of a great number of pathogen-free appropriated conditions, can differentiate to a whole plant. plants in a short time with high uniformity. The success This potential of a cell to grow and develop a multicellular of micropropagation involves several factors, as the com- organism is termed cellular totipotency. This potential of position of the culture medium, culture environment, and cells or tissues to form all cell types and regenerate a genotype. The development of procedures for rapid in plant is the basic principle of tissue culture. In vitro vitro clonal micropropagation of any plants may be a culture is one of the key tools of plant biotechnology that great commercial value to the industry. This review disc- exploits the totipotency nature of plant cells, a concept * Corresponding author: (E-mail) nitishbt1@rediffmail.com, nitish@cub.ac.in 62 ‧ Journal of Forest Science proposed by Haberlandt (1902) and unequivocally demon- Micropropagation via meristem culture or axillary bud/shoot strated, for the first time, by Steward et al. (1958). Tissue tip culture and regenerarion culture is alternatively called cell, tissue and organ culture through in vitro condition (Debergh and Read, 1991). It In vitro propagation through meristem culture is the can be employed for large-scale propagation of disease best possible means of virus elimination and produces a free clones and gene pool conservation. Now a day’s large numbers of plants in a short span of time. It is a industry has applied immensely in vitro propagation app- powerful tool for large-scale propagation of plants. The roach for large-scale plant multiplication of elite superior term ‘meristem culture’ specifically means that a meristem varieties. As a result, hundreds of plant tissue culture with no leaf primordia or at most 1-2 leaf primordial laboratories have come up worldwide, especially in the which are excised and cultured. The pathway of regenera- developing countries due to cheap labour costs. However, tion undergoes several steps. Starting with isolated explants, micropropagation technology is more costly than convent- with de-differentiation followed by re-differentiation and ional propagation methods, and unit cost per plant becomes organization into meristematic centres. Upon further indu- unaffordable compelling to adopt strategies to cut down ction the cells can form unipolar structures i.e. organoge- the production cost for lowering the cost per plant. The nesis, or bipolar structures called somatic embryogenesis. micropropagation process can be divided in five different The organization into morphogenetic patterns can take stages: place directly on the isolated explant or can be expressed Phase 0: growing mother plants under hygienic conditions. only after callus formation, which is called indirect morp- It involves the production of stock plants in greenhouse. hogenesis. When shoots are developed directly from leaf Phase I: initiation of culture. The purpose of this stage or stem explants it refers to direct morphogenesis. Micro- is to initiate axenic cultures. It involves the selection of propagation is an alternative method of vegetative propa- explants, disinfestations and the cultivation under aseptic gation, which is well suited for the multiplication of elite conditions. clones. It is accomplished by several means, i.e., multip- Phase II: rapid regeneration and multiplication of num- lication of shoots from different explants such as shoot erous propagules (multiplication phase). Masses of tissues tips or axillary buds or direct formation of adventitious are repeatedly subcultured under aseptic conditions onto shoots or somatic embryos from tissues, organs or zygotic new culturing media that encourage propagule proliferation. embryos. Many commercial plants are being propagated The culture can supply shoots for the subsequent propag- by in vitro culture on the culture medium containing ation phases as well as material that is required to maintain auxins and cytokinins (Preil, 2003; Rout and Jain, 2004). the stock. Several different explants have been used for direct shoot Phase III: elongation and root induction or development formation. Mayer (1956) succeeded first time regeneration (rooting phase). This phase is designed to induce the of Cyclamen shoots from tuber segments on MS medium establishment of fully developed plantlets. It is the last supplemented with 10.7 μM NAA. Furthermore, plants period in vitro before transferring the plantlets to ex vitro have been regenerated from leaf tissues and petiole conditions. segments of Jatropha curcas (Kumar, 2009; Kumar and Phase IV: transfer to ex vitro condition (acclimatization). Reddy, 2010; Kumar et al., 2010a; Kumar et al., 2010b; Acclimatization is defined as the climatic or environmental Kumar et al., 2010c; Kumar et al., 2011a; Kumar et al., adaptation of an organism, especially a plant that has 2011b). Preil (2003) noted that the regeneration potential been moved to a new environment (Kozai and Zobayed, of isolated cells, tissue or organs and the callus cultures 2000). is highly variable. Furthermore, petiole cross sections cultivated on auxin and cytokinin containing medium give In vitro Plant Propagation: A Review ‧ 63 rise to adventitious shoots from epidermal cells and somatic embryo production in bioreactors, encapsulation, subepidermal cortex cells, never from pith cells of the cryopreservation, genetic transformation and clonal propa- central regions of the petiole. The direct shoot bud formation gation. The major limitations are genotypic dependence of without any callus phase from appropriate explants is of somatic embryo production and poor germination rate. great success for large-scale clonal multiplication of desired plants/clone. Micropropagation via somatic embryogenesis Factors affecting in vitro propagation Media Significant effect of media has been observed on plant regeneration from different parts of plant (Sharswat and Somatic embryos, which are bipolar structures, arise Chand, 2004). Various basal media like White medium, from individual cells and have no vascular connection Nitsch and Nitsch medium, B5 medium and Gamborg with the maternal tissue of the explants (Haccius, 1978). medium for micropropagation (Khan et al., 1988; Prakash Embryos may develop directly from somatic cells (direct and Gurumurtthi, 2005; Diallo et al., 2008), have been embryogenesis) or development of recognizable embryogenic employed, but most widely used culture medium is Mura- structures is preceded by numerous, organized, non-embr- shige and Skoog (1962) (MS medium), because most of yogenic mitotic cycles (indirect embryogenesis). Somatic the plants respond favorably to MS medium, since it embryogenesis has a great potential for clonal multiplication. contains all the nutrients essential for plant growth in Under controlled environmental conditions, somatic embryos vitro. Selection, strength and combination of media are germinate readily, similar to their seedling counterpart. also one of important parameter for optimizing the regen- The commercial application of somatic embryogenesis will eration protocol (Khan et al., 1988; Zukar et al., 1997; be accomplished only when the germination rate of somatic Prakash and Gurumurtthi, 2005; Diallo et al., 2008). embryos is high up to 80-85%. Considerable success has been achieved in inducing somatic embryogenesis in Type of explant many plants like Dendrathema grandiflorum (May and Type of explant is also one of the important factors in Trigiano, 1991; Tanaka et al., 2000). Castillo and Smith optimizing the tissue culture protocol. Type of explants (1997) induced direct somatic embryogenesis from petiole like leaf, petiole, cotyledonary leaf, hypocotyle, epicotyle, and leaf blade explants of B. gracilis on MS medium embryo, internode and root explant significantly effect on supplemented with 0.5 mg/ l kinetin and 2% (v/v) coconut tissue culture process of plants (Khan et al., 1988; Sujatha water. Somatic embryos were obtained with greater freq- and Mukta, 1996; Tyagi et al., 2001; Gubis et al., 2003; uency from petiole explants than from leaf blade sections. Alagumanian et al., 2004; Ali and Mirza, 2006; Kumar et Osternack et al. (1999) succeeded in achieving somatic al., 2011b). This may be due to the different level of embryos from hypocotyl tissues of E. pulcherrima on MS endogenous plant hormones present in the plants parts. medium supplemented with 2.0 mg/l IAA. About 1400 Leaf is the most commonly used explant for regeneration embryos were developed from 320 calli derived from due to more surface area available (Sujatha and Muktha, outer regions of the hypocotyls. However, only 8% 1996; Tyagi et al., 2001). Tyagi et al. (2001) used root, developed normal plantlets. Pueschel et al. (2003) succe- shoot, and leaf explant and maximum regeneration eded in plant regeneration via somatic embryogenesis of efficiency was observed from leaf expalnts in Cajanus C. persicum and maintained the regeneration ability for cajan. Sujatha and Mukta (1996) used different explant prolonged period. There are advantages and disadvantages like leaf, petiole, hypocotyle and maximum regeneration of somatic embryogenesis in large-scale plant multipl- frequency was observed from leaf explant of Jatropha ication (Jain, 2001). The major advantages are large-scale curcas. Alagumanian et al. (2004) used leaf and stem 64 ‧ Journal of Forest Science explant and maximum regeneration efficiency observed Genotype from stem explant in Solanum trilobotam. Gubis et al. Genotype is also one of the most important factors (2003) used hypocotyle, epicotyle, cotyledons, leaf, petiole, affecting regeneration (Tyagi et al., 2001; Gubis et al., internode and maximum response were obtained from 2003; Gandonou et al., 2005; Feyissa et al., 2005; Chitra hypocotyle in Tomato. Ali and Mirza (2006) used root, and Padmaja, 2005; Landi and Mezzeti, 2006; Reddy et stem, leaf and petiole but maximum responses were al., 2008; Kumar and Reddy, 2010). Genotypic effect on observed from stem explant in Citrus jambhiri Lush. An shoot regeneration and elongation has been described in example of the effect of the type of explants on regener- many species, and could be due, in part, to differences in ation in Jatropha curcas is shown (Fig. 1). the levels of endogenous hormones, particularly cytokinins Fig. 1. Plant regeneration from leaf explants of non-toxic Jatropha curcas. Plant regeneration from (A) in vitro mature leaf explant (bar 5 mm), (B) Field-grown mature leaf explant (bar 5 mm), (C) in vitro cotyledonary leaf explant (bar 5 mm) and (D) Glasshouse-grown cotyledonary leaf explants (bar 5 mm) on MS medium with 2.27 μM thidiazuron (TDZ) after 6 weeks. (E) Shoot proliferation of regenerated shoot buds on MS medium with 10 μM kinetin (Kn) + 4.50 μM 6-benzyl aminopurine (BAP) + 5.50 μM α-naphthaleneacetic acid (NAA) after 4 weeks (bar 75 mm). (F) Elongation of proliferated shoot on MS medium with 2.25 μM BAP + 8.5 μM indole-3-acetic acid (IAA) after 6 weeks (bar 5 mm). (G) Elongation of proliferated shoot on MS medium with 2.25 μM BAP + 2.5 μM indole-3-butyric acid (IBA) after 6 weeks (bar 5 mm; arrow indicate proliferation of axillary buds). (H) Elongation of proliferated shoot on MS medium with 2.25 μM BAP + 8.25 μM NAA after 6 weeks (bar 5 mm; arrow indicate formation of callus at the base). (I) Elongated shoot cultured on half strength basal MS liquid medium supplemented with 15 μM IBA + 5.7 μM IAA + 5.5 μM NAA for root induction (bar 1 mm). (J) Development of roots at the base of auxins treated elongated shoot on half strength basal MS medium with 0.25 mg/l activated charcoal after 4 weeks (bar 1 mm). (K) Regenerated plant in polythene bags after 2 weeks (bar 100 mm) (Source; Kumar et al., 2011b) In vitro Plant Propagation: A Review ‧ 65 levels during the induction period although the precise responsive or meristematic than mature plants (Teng, mechanism remains unclear (Pellegrineschi, 1997; Schween 1999; Feyissa et al., 2005) due to different level of plant and Schwenkel, 2003). Henry et al. (1994) reported that hormones present in the plants. An example of the effect genotypic differences with respect to embryogenesis and of the source of explants on regeneration in Jatropha regeneration result from quantitative or qualitative genetic curcas is shown (Fig. 1). differences. Orientation of explant Source of explant Orientation of explant in the culture medium also Source of explant i.e. in vitro and in vivo is also affects the regeneration efficiency (Sharma and wakhlu, important for regeneration (Reddy et al., 2008; Kumar et 2001; Arockiasamy et al., 2002; Kumar and Reddy, 2010). al., 2010a). In vitro explant is considered to be the most In general regeneration efficiency is higher in horizontal suitable for organogenesis (Reddy et al., 2008). The fact position as compared to vertical condition of explant due that source of explant has different capacity of regene- to little contact of explant to medium in vertical position ration are well documented (Feyissa et al., 2005). In vitro as compared to horizontal position. The initiation site, explant in general has better potential to organogenesis as polarity, and efficiency of bud regeneration were altered compared to in vivo explant (Reddy et al., 2008). The by explant orientation is well documented in Dionaea difference may be due to the level of endogenous hormones muscipula (Teng, 1999). Cotyledons placed in abaxial (lower present in the plant explant. Seedling explant is more surface facing down) orientation consistently produced better Fig. 2. Direct induction of shoot buds from petiole explants of Jatropha curcas. Direct induction of shoot buds from (A) in vitro petiole in horizontal position (bar 5 mm), (B) in vivo petiole in horizontal position (5 mm), (C) in vitro petiole in vertical position (bar 5 mm) and (D) in vivo petiole in vertical position on MS medium with 2.27 μM TDZ after 6 weeks (bar 5 mm). (E) Shoot proliferation of induced shoot buds on MS medium with 10 μM Kinetin + 4.5 μM BAP + 5.4 μM NAA after 4 weeks (bar 100 mm). (F) Elongation of proliferated shoot on MS medium with 2.25 μM BAP and 8.5 μM IAA after 6 weeks (bar 1 mm). (G) Development of roots at the base of elongated shoot on half strength of MS medium with 2% sucrose + 15 μM IBA + 5.7 μM IAA +5.5 μM NAA + 0.25 mg/L activated charcoal after 4 weeks (bar 1 mm). (H) Regenerated plants in polythene bags after 4 weeks (bar 100 mm). (I) A six month old regenerated plant in pot under natural condition (bar 100 mm) (Source; Kumar and Reddy, 2010 ) 66 ‧ Journal of Forest Science shoot regenerative response and produced greater numbers added to the culture medium supply energy for the and taller shoots compared to those inoculated in adaxial metabolism (Caldas et al., 1998). The addition of a (upper surface facing down) orientation (Bhatia et al., carbon source in any nutrient medium is essential for in 2004, 2005). An example of the effect of the orientation vitro growth and development of many species, because of explants on regeneration in Jatropha curcas is shown photosynthesis is insufficient, due to the growth taking (Fig. 2). place in conditions unsuitable for photosynthesis or without photosynthesis (in darkness). Normally, green tissues are Mineral nutrition Minerals are important components of the culture medium. There is a large choice of combinations of macro- not sufficiently autotrophic under in vitro conditions (Pierik, 1997) and depend on the availability of carbohydrates in the growing medium. and micro-salt mixtures. The most widely used culture medium is described in Murashige and Skoog (1962) (MS Growth regulators medium), because most plants react to it favorably. It Growth regulators are organic compounds naturally contains all the elements that have been shown to be synthesized in higher plants, which influence growth and essential for plant growth in vitro. It is classified as a development. Apart from the natural compounds, synthetic high salt medium in comparison to many other formulations, chemicals with similar physiological activities have been with high levels of nitrogen, potassium and some of the developed which correspond to the natural ones (Pierik, micronutrients, particularly boron and manganese (Cohen, 1997). There are several classes of plant growth regulators, 1995). Due to the high salt content, however, this nutrient as e.g. cytokinins, auxins, gibberellins, ethylene and abscisic solution is not necessarily always optimal for growth and acid. Growth and morphogenesis in vitro are regulated by development of plants in vitro (Pierik, 1997). For that the interaction and balance between the growth regulators reason, the use of dilute media formulations has generally supplied in the medium, and the growth substances prod- promoted better formation of roots, since high concentration uced endogenously (George, 1993). A balance between of salts may inhibit root growth, even in presence of auxin and cytokinin is most often required for the formation auxins in the culture medium (Grattapaglia and Machado, of adventitious shoots and roots. In tobacco cultured in 1998). The ability of rose explants to produce shoots and vitro, it was found that the formation of roots and shoots initiate roots was studied by Kim et al. (2003). They depended on the ratio of auxin to cytokinin in the culture concluded that optimum shoot proliferation was obtained medium. High levels of auxin relative to cytokinin stimu- in full-strength MS salts, while rooting improved with 1/4 lated the formation of roots, whereas high levels of strength. Sauer et al. (1985) reported that 1/3 strength MS cytokinin relative to auxin led to the formation of shoots salts proved to be suitable or rooting of rose. For globe (Taiz and Zeiger, 1991). The balance of growth regulators artichoke, 1/2 strength MS salts have been used in the depends on the objective of the cultivation in vitro (as rooting medium (Ancora, 1986; Lauzer and Vieth, 1990). e.g. shoot, root, callus or suspension culture) and on the micropropagation phase considered (initiation, multiplication Carbon source or rooting). In the multiplication phase, the level of Sucrose is by far the most used carbon source, for citokinins should be normally higher than of auxins. In several reasons. It is cheap, readily available, relatively the rooting phase, in turn, the use of cytokinin is, in some stable to autoclaving, and readily assimilated by plants. cases, not necessary and higher levels of auxins can be Other carbohydrates can be also used, such as glucose, supplemented to the culture medium (Torres et al., 2001). maltose and galactose as well as the sugar-alcohols The cytokinins are derived from adenine (aminopurine) glycerol and sorbitol (Fowler, 2000). The carbohydrates and play an important role in the in vitro manipulation of In vitro Plant Propagation: A Review ‧ 67 -1 plant cells and tissues (Torres et al., 2001). Cytokinins IAA (2.0 mg L ) Gibberellins are a group of compounds stimulate plant cells to divide, and they were shown to that is not necessarily used in the in vitro culture of affect many other physiological and developmental process. higher plants. In some species, these growth regulators These effects include the delay of senescence in detached are required to enhance and in others to inhibit growth organs, the mobilization of nutrients, chloroplast maturation, (Razdan, 1993). Gibberellic acid (GA3) is the most common and the control of morphogenesis (Taiz and Zeiger, gibberellin used. It induces the elongation of internodes 1991). Added to the culture medium, these compounds and the growth of meristems or buds in vitro (Pierik, overcome apical dominance and release lateral buds from 1997). Furthermore, the use of gibberellins in the rooting dormancy (George, 1993). The most common cytokinins medium may reduce or prevent the formation of adventi- used are kinetin, BA and 2iP (Pierik, 1997). Also auxins tious roots and shoots, although it can stimulate root (IAA, IBA, NAA or 2,4-D) are often added to the culture formation when present in low concentrations. Morzadec medium to promote the growth of callus, cell suspensions and Hourmant (1997) showed the beneficial effect for or organs, and to regulate morphogenesis, especially in globe artichoke of using gibberellin at a concentration of combination with cytokinin (George, 1993). Auxins are 1.0 or 5.0 mg L-1 GA3 in the rooting medium, resulting involved in the regulation of several physiological processes, in a rapid root expression and in the formation of high as e.g. apical dominance and formation of lateral and quality explants. An example of the effect of the plant adventitious roots. This growth regulator generally causes growth regulators on regeneration in Jatropha curcas is cell elongation and swelling of tissues, cell division (callus shown (Fig. 1). formation) and the formation of adventitious roots as well as the inhibition of adventitious and axillary shoot formation Gelling agents (Pierik, 1997). Normally, the concentration of auxin used Culture media can be classified as liquid or solid. The -1 in the culture medium varies between 0.01 and 10 mg L liquid media have the advantage of faster (and cheaper) (Torres et. al., 2001). The IAA is a natural auxin, whereas preparation than the solid ones. Furthermore, liquid media 2,4-D and NAA are synthetically produced and have are more homogeneous, since gradients of nutrients may similar effect in comparison to natural-occurring auxins. appear during tissue growing in solid media. This pheno- According to most of the studies that have been published menon is not observed in liquid media (Caldas et al., concerning the effect of auxin type and concentration in 1998). Furthermore, it has been shown that the propagation rose, low concentrations of this growth regulator should ratio of some species is higher in liquid than in solid be used in the culture medium. The rooting of rose shoots media (Debergh et al., 1981; Pateli et al., 2003). One was improved with IAA (considered a weak auxin) serious disadvantage of using liquid media for shoot growth -1 supplementation at 1.0 mg L (Kim et al., 2003), 0.1 mg -1 and multiplication is that shoots, which are perpetually L NAA (Rahman et al., 1992; Leyhe and Horn, 1994) submerged in liquid cultures, may become hyperhydric or even in absence of auxin (Ibrahim and Debergh, 2001). and will then be useless for micropropagation (George, The combination of two types of auxin can be also used 1993; Debergh, 2000). Ebrahim and Ibrahim (2000) reported to increase root formation in rose. Kosh-Khui and Sink that the solid medium should be used to overcome the (1982) found that the best combination for the production production of vitrified shoots of Maranta leuconeura and -1 -1 of rooted plants was 0.1 mg L NAA with 0.05 mg L to insure obtaining vigorous plants with higher chlorophyll of either IAA or IBA. The combination of two auxins content. Agar has traditionally been used as the preferred was more effective for root formation than either auxin gelling agent for tissue culture, and is very widely empl- alone. In globe artichoke, the most effective auxin for oyed for the preparation of semi-solid culture media -1 rooting was NAA (0.1-2.0 mg L ) also combined with (Torres et al., 2001). It is a polysaccharide extracted from 68 ‧ Journal of Forest Science species of red algae which are collected from the sea sealing of the vessels must allow sufficient ventilation to (Torres, 1999). Concerning the optimal agar concentration prevent significant accumulation of ethylene and depletion in the culture medium, large differences between two rose of CO2 (Buddendorf-Joosten and Woltering, 1994). cultivars were observed by Acker and Scholten (1995). Carbon dioxide concentrations inside the vessels alter due The cv. ‘Motrea’ preferred higher concentrations of agar to respiration and photosynthesis of the plant. In the dark, -1 (7 g L ). At this concentration, completely developed CO2 concentrations increase due to respiration, whereas shoots were formed. The cv. ‘Sweet Promise’, in turn, during the light period the concentration decreases (Budd- showed the best results with extremely low concentrations endorf-Joosten and Woltering, 1994). The utilization of -1 (4 g L ). Paques (1991) pointed out that there is a strong tightly closed vessels that reduce the gas exchange may connection between culture medium hardiness, proliferation affect negatively the normal growth and development of ratio and hyperhydration. Normally, an increase in the plants during cultivation in vitro. Several studies have agar concentration promotes a reduction in the occurrence shown the advantages of using closures with filters or of hyperhydration symptoms in plants. However, the vented vessels, which allow gas exchange, increasing the propagation rate can be drastically reduced and, conseq- photosynthetic capacity, the multiplication rate, and the uently, the efficiency of micropropagation (Debergh, 2000). survival of plants after transfer to ex vitro conditions The concentration of agar in the medium may also affect (Chuo-Chun et al., 1998; Murphy et al., 1998; Zobayed the formation of roots. Rahman et al. (1992) reported that et al., 2000; Benzioni et al., 2003). The increased avail- rooting performance of rose decreased with increasing ability of CO2 by using vessels with filter may also -1 -1 agar concentration (from 6 to 15 g L ). At 6 g L , influence the amount of photosynthetic pigments. Nicotiana optimal rooting induction was achieved. An alternative to tabacum plants grown in vessels with closures with agar is the use of a gelling agent named gelrite. Gelrite microporous vents (better supplied with CO2) had higher is a gellan gum - a hetero-polysaccharide produced by the contents of chlorophyll a, b and ß-carotene, higher photo- bacterium Pseudomonas elodea (Kang et al., 1982). chemical activity of photosystem II and electron transport Gelrite is an attractive alternative to agar for plant tissue chain. Furthermore, plants grown under this condition had culture because its cost per liter of medium is lower, and higher net photosynthetic rate, lower transpiration rate it produces a clear gel which facilitates the proper and stomatal conductance under ex vitro conditions than observation of cultures and their possible contamination plants grown in glass vessels tightly closed (Haisel et al., (George, 1993). Williams and Taji (1987) found that several 1999). Water status of cultures is influenced by the growing Australian woody plants survived best on a medium medium used, the culture vessel itself, and the physical gelled with gelrite rather than agar. environment. The medium affects water status in various ways, including the gelling agent used (or its absence), PHYSICAL ENVIRONMENT Gas exchange and relative air humidity inside the vessel the osmotic pressure (influenced by e.g. salt concentration, The response of plant tissue culture in vitro can be other constituents), and changes in the medium with time significantly affected by the gaseous constituents in and (Zimmerman, 1995). It is generally accepted that the adjacent to the culture vessel. Carbon dioxide, oxygen relative humidity in the vessel is approximately 98-100% and ethylene are the most frequently studied constituents (Altman, 2000). The plants that develop under higher of the culture atmosphere (Read and Preece, 2003). The relative humidity in vitro have more transpiration and culture vessel is usually a closed system, but some gas more anatomical abnormalities under ex vitro conditions, exchange may occur depending upon the type of vessel, which may result in high mortality rate during acclimatiz- the closure and how tightly they are sealed together. The ation. Therefore, different methods to reduce the relative amount and type of carbohydrate and quantity and type of In vitro Plant Propagation: A Review ‧ 69 air humidity inside the vessel have been tested, including no significant changes were observed with increasing the opening of culture containers for some days before light intensity. Matysiak and Nowak (1998) investigated acclimatization (Brainerd and Fuchigami, 1981; Kirdmanee the influence of CO2 concentrations (350 and 1200 μmol et al., 1996), the use of special closures that facilitates mol ) on the growth of Ficus benjamina microcuttings at water loss (Gribaudo et al., 2003) or the cooling of two light levels (50 and 150 μmol m-2 s-1) under ex vitro container bottoms, which increases the condensation of conditions. -1 water vapour on the gel surface (Ghashghaie et al., 1992). However, methods to improve gas exchange and reduce Temperature relative humidity inside the vessel should be carefully used, in order to prevent excessive water loss during Temperature influence on various physiological processes, cultivation. A study carried out with grapevine shoots such as respiration and photosynthesis, is well known and testing different hole diameters in the vessel closure it is not surprising that it profoundly influences plant showed that shoots cultivated in vented vessels were taller tissue culture and micropropagation. The most common than shoots grown in unvented ones, and had higher culture temperature range has been between 20°C and chlorophyll content. On the other hand, the largest holes 27°C, but optimal temperatures vary widely, depending (40 mm) caused an excessive water stress. Shoots became on genotype (Altman, 2000; Read and Preece, 2003). more resistant to wilting, but their growth was seriously Horn et al. (1988) studied the effect of different tempera- retarded (Gribaudo et al., 2003). tures in the multiplication phase of 14 rose cultivars. The best overall results were obtained at 18°C, but certain Light ‘thermonegative’ cultivars gave best results at 12°C, while Light is an important environmental factor that controls other ‘thermopositive’ cultivars had their optimum at 18°C plant growth and development, since it is related to or 24°C. Alderson et al. (1988) observed that the temper- photosynthesis, phototropism and morphogenesis (Read ature during the rooting phase affected the timing of root and Preece, 2003). The three features of light, which emergency, the final rooting percentage and shoot health. influence in vitro growth, are wavelength, flux density For the cv. ‘Dicjana’, roots emerged first at 25°C and and the duration of light exposure or photoperiod (George, approximately 2 and 4 days later at 20°C and 15°C, resp- 1993). Several studies showed that light enhanced root ectively, but at 25°C the final rooting percentage was formation and shoot growth (Kumar et al., 2003), whereas lower. The shoots remained green and looked healthiest at in others darkness favored root formation (Hammerschlag, 20°C. 1982). The reduced rooting in presence of light is due to the degradation of the endogenous IAA (George, 1993). CONCLUSION Some species may react positively to an increase in the photosynthetic photon flux, especially under photoautotro- Successful in vitro propagation of plants is now being phic/mixotrophic growing conditions (low sugar levels used for commercialization. Many commercial laboratories and CO2 enrichment). Limonium grown under photoauto- and national institutes worldwide use in vitro culture trophic conditions in vitro associated with a higher light system for rapid plant multiplication, germplasm conser- -2 -1 s ) had more leaves, higher vation, elimination of pathogens, genetic manipulations, chlorophyll and sugar contents, higher net photosynthetic and for secondary metabolite production. Annually, millions rate and percent survival of plants ex vitro than plants of plants are routinely produced in vitro. The great intensity (200 μmol m -2 -1 grown at lower light intensities (50 and 100 μmol m s ) potential of micropropagation for large-scale plant multip- (Lian et al., 2002). Under heterotrophic growing conditions, lication can be tapped by cutting down the cost of prod- 70 ‧ Journal of Forest Science uction per plant by applying low-cost tissue culture, which is to adopt practices and proper use of equipment and resources to reduce the unit cost of micropropagule and plant production without compromising the quality. Somatic embryogenesis facilitates cryopreservation, synseed development, mutations, and genetic transformation. Recent progress in genetic manipulation of plant cells has opened new possibilities for improvement of plants which is totally depends on tissue culture. REFERANCES Acker, C.A.M.M., Sholten, H.J. 1995. 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Plant cell Rep. 16, 775-778. (Received April 21, 2011; Accepted May 17, 2011) View publication stats