In Vitro Conservation of Plant Germplasm

Transcription

1 In Vitro Conservation of Plant Germplasm P.E. Rajasekharan and Leela Sahijram 3 Abstract Biotechnology has brought about a revolution in the way that plant genetic resources can be utilized. Clonal crops cover a wide range of species from the root and tuber crops, such as potato, cassava, yam, taro and sweet potato, to fruits, such as apple, pear, citrus, banana and the cooking banana (plantain). Other miscellaneous crops including vanilla, ginger, turmeric, hops and sugarcane are also clonally propagated. In some of these cases, seed production is impossible due to sterility. In others, it is undesirable to produce seeds for conservation as this would break up highly heterozygous clonal genotypes. The foundation technologies that make up an in vitro conservation system are collection, disease eradication and indexing, culture initiation, multiplication, storage and distribution. There are two basic options for in vitro storage, slow growth for the short to medium term and cryopreservation for the long term. (Since the definition of storage time-span and the concepts of active or base storage derive strictly from motivation rather than methodology, there is no reason why, in fact, cryopreservation should not have applications in short- to medium-term conservation.) Intensive conservation efforts are needed for clonally propagated crops, constituting about 1,000 species, and for difficult-to-store seeds, constituting about 88,250 species throughout the world. In vitro approaches, including tissue culture maintenance and cryopreservation, P.E. Rajasekharan Division of Plant Genetic Resources, Indian Institute of Horticultural Research, Hessaraghatta Lake, Bangalore, Karnataka , India L. Sahijram (*) Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake, Bangalore, Karnataka , India leelas@iihr.ernet.in B. Bahadur et al. (eds.), Plant Biology and Biotechnology: Volume II: Plant Genomics and Biotechnology, DOI / _22, Springer India

2 418 P.E. Rajasekharan and L. Sahijram are recognized as useful tools for medium- to long-term conservation of these groups of species. The in vitro techniques for conserving plant biodiversity include shoot apical or axillary-meristem-based micropropagation, somatic embryogenesis, cell culture technologies and embryo rescue techniques, and a range of in vitro cold storage will be discussed in this chapter. Keywords In vitro conservation Clonal Slow growth Tissue culture Introduction Need for In Vitro Conservation Seed storage is a preferred method of conservation, but it is not feasible for germplasm from crops that are either clonally propagated or that do not produce seeds. For some genotypes, elite genetic combinations are only preserved through clonal means as their conservation is dictated by breeding strategy; this is because heterozygosity does not permit the maintenance of desired characteristics. Clonally propagated plants thus require special conservation approaches. Options include maintenance in field gene banks and the conservation in cold stores of dormant vegetative propagules (Reed 2001); however, these methods have limitations regarding efficiency, costs, security and long-term maintenance. In vitro conservation is preferentially applied to clonal crop germplasm as it also supports safe germplasm transfers under regulated phytosanitary control (IBPGR 1988). Conservation in IVGBs combines tissue culture and cryopreservation for medium-term (MTS) and long-term (LTS) storage, respectively. For MTS, subculture intervals are extended, reducing processing costs by arresting growth using reduced temperature treatments and/or growth retardants. For LTS, germplasm (usually shoot tip meristems) from in vitro propagated plants is cryobanked for long-term storage in liquid nitrogen (LN) to a minimal temperature of 196 C in the liquid phase. There are a number of crops which are normally propagated vegetatively, such as potato, sweet potato, yams, cassava, several fruit tree species and many others. In this category, the clonal material carries variable gene combinations which have been maintained by the avoidance of sexual reproduction. When these clones are maintained in field gene banks, the traditional procedures tend to be expensive due to (1) high labour costs, (2) vulnerability to environmental hazards and (3) requirement for large amount of space. An even more serious problem is the vulnerability of such clones to pests and pathogens or natural disasters to which they are almost continuously exposed. This can lead to sudden loss of valuable germplasm or accumulation of systemic pathogens, especially viruses. In such cases, in vitro conservation is complementary to field gene banks, seed gene banks and pollen/ DNA preservation which along with in situ conservation measures provide an integrated conservation strategy. In vitro gene banks, where plant material is stored in nutrient medium under artificial conditions, are being increasingly used as alternatives to conserve vegetatively propagated species and threatened plant species (Fay 1994; Bhat et al. 1995; Sharma and Chandel 1996). Much of the world s germplasm is currently maintained as breeders collections in gene banks, plantations, orchards or even in evolution

3 22 In Vitro Conservation of Plant Germplasm gardens primarily raised from seeds, such as rubber and coconut, and vegetatively propagated plant species such as citrus, cocoa, banana and many other fruits. These also include clonal collections of important staple food crops, such as cassava, sweet potato and yams, and aroids such as Colocasia and Xanthosoma. These field gene banks do not represent the entire range of genetic variability within the respective crop gene pool, and most of them represent only a fraction of the variability which should be conserved (Withers and Williams 1985). The IBPGR programme initiated a move to include fruits, vegetables and forages. Any strategy for collection and conservation of samples of crops that are normally propagated vegetatively or that produce seeds which cannot be stored using normal procedure of storage may require alternative methods. This led to the consideration of in vitro techniques and cryopreservation of seeds for germplasm conservation (Withers and Alderson 1986). Problems of in vitro storage of such material, when solved, should also relate to cycling of the material through multiplication schemes, distribution of germplasm and also its characterization and evaluation. Hence, the development of the full potential of in vitro culture storage and associated biochemical techniques could revolutionize the handling of germplasm. A range of in vitro techniques have been developed in the last few decades. The organized culture systems have a high degree of genetic stability and are more likely to be of importance for germplasm storage, especially the shoot tips or meristem cultures. In vitro techniques are employed to eliminate diseases and pests. However, some viroids and viruses particularly are not necessarily eliminated or even detected and can readily multiply in tissue culture. These can be eliminated by meristem or shoot tip cultures possibly in combination with both heat and cold therapy. The International Potato Research Center (CIP) maintains the pathogen-tested potato germplasm in the form of in vitro plantlets or tuberlets. Potato germplasm is being preserved at CIP through in vitro techniques using meristem and shoot tip culture for international exchange of germplasm. With in vitro techniques, it is now possible to provide a germplasm storage procedure which uniquely combines the possibilities of disease elimination and rapid clonal propagation (Henshaw and Grout 1977). Further, the virustested cultures could provide ideal material for international exchange and distribution of germplasm as they will be acceptable to plant quarantine authorities (Paroda et al. 1987) and comply with international quarantine regulations. In vitro conservation strategy offers an appropriate alternative and would be discussed in detail in the present chapter. With in vitro techniques, it is now possible to provide a germplasm storage procedure which uniquely combines the possibilities of disease elimination and rapid clonal propagation (Henshaw and Grout 1977). However, field gene banks have the potential risk of germplasm being lost due to disease, stress or disaster and are labour intensive. Cryogenic preservation of seeds or vegetative material is another potential mode of ex situ conservation which is still at experimental stages. Various aspects of in vitro conservation and cryopreservation were reviewed by Normah et al. (1996), Ashmore (1997), Engelmann and Takagi (2000), Reed et al. (2004), Sarasan et al. (2006) and Krishnan et al. (2011). Research requirements identified by Reed et al. (2004) were: 1. Germplasm health: virus surveys, indexing techniques, development of effective virus testing in vitro and whether viruses can be transmitted in vitro and development of indexing techniques for latent endogenous bacteria 2. Slow growth: research into the effects of plant growth regulators and growth retardants, light and light-temperature interactions, propagule type, size, growth stage (microtubers, bulbs, rooted plantlets, unrooted shoots), statistical

4 420 P.E. Rajasekharan and L. Sahijram rigour in experimental design and minimizing the use of growth retardants 3. Cryopreservation: widening its applicability to more crops and genotypes, methods developed for several localities, and use of cryotherapy 4. Genetic stability: selection pressure of in vitro maintenance, genetic variation in field compared to in vitro, field evaluations on material with known instabilities and development of markers to monitor genetic stability 22.2 In Vitro Conservation: An Overview In vitro storage of germplasm first reported two decades ago (Henshaw 1975) offers promise for conservation of threatened species of known and/ or potential medicinal and aromatic value and for species clonally propagated. The material for such species could be available once the true potential of the species is realized. The fundamental objectives of in vitro conservation technology are the maintenance and exchange of germplasm in disease-free and genetically stable state through tissue culture. The essential prerequisites for an in vitro conservation programme are: 1. Creation of special facilities including tissue culture facility, green-/glasshouse facilities, storage facility, computer facility and facility for monitoring genetic stability 2. Presence of trained scientists and technicians 3. Linkage with farmers fields Information on the in vitro multiplication and/ or conservation of the plant species is also desirable. Many laboratories and institutes in India have been engaged mainly in developing protocol for micropropagation of various threatened endemic species. The Department of Biotechnology established India s first national facility of plant tissue culture repository in 1986 at National Bureau of Plant Genetic Resources (NBPGR), New Delhi, which has made concerted efforts towards developing in vitro technology for conservation of several vegetatively propagated agri-horticultural and several threatened/rare species, especially of medicinal and aromatic value. The number of species being worked upon has increased appreciably with the initiation of DBT funded G-15 project operative at three centres, namely, NBPGR, Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, and Tropical Botanic Garden and Research Institute (TBGRI), Thiruvananthapuram. Any in vitro conservation programme mainly comprises of two stages: 1. In vitro multiplication to build up a large number of plants 2. In vitro storage/preservation The cultures may be conserved for either short, medium or long term, depending on the requirement as well as the technique applied and infrastructure availability. For short-term maintenance of cultures, regular subculture (4 8 weeks interval) may suffice. To conserve cultures for a longer period of time, two strategies normally adopted include slow growth and cryopreservation. The use of artificial seeds in combination with the above two is a more recent approach in the conservation programmes Type of Culture Systems In vitro techniques rely on the concept of totipotency of plant cells. Cultures could be initiated from two types of explants: first, explants that retain developmental integrity such as meristem shoot tips and axillary buds and, second, explants that differentiates to a more or less organized state such as somatic embryos and adventitious buds through a disorganized callus phase. One of the important requirements of in vitro conservation is to get high-frequency regeneration of plantlets from organized explants such as meristem/shoot tips, embryos, embryonic axes and plantlets as they offer the lowest frequency of genetic variation during conservation (Karp 1989). In contrast, callus, cell suspension and protoplast culture are preferred systems only when endowed with special attributes or required for biotechnological applications

5 22 In Vitro Conservation of Plant Germplasm Germplasm of threatened plants is collected from difficult areas and may be available in the form of either seeds or cuttings or vegetative propagules such as bulb, corm or tubers in limited number. In such cases, the less preferred culture system may be the only choice for conservation In Vitro Propagation Development of efficient plant regeneration protocols for clonally propagated species and threatened plants of medicinal and aromatic value is a recent phenomenon. The methods for micropropagation include stimulation of axillary bud, proliferation from shoot tip and nodal explants, induction of somatic embryogenesis using explants from juvenile or mature plants depending on the availability of material and inducing adventitious bud directly from explants or through intervening callus. Protocol for rapid multiplication involves five stages: 1. Section and preparation of stock plants 2. Establishment of aseptic cultures 3. Multiplication of propagules 4. Preparation for re-establishment in soil 5. Transfer to greenhouse and acclimatization Since multiplication is carried out under artificial conditions on a nutrient medium, plants can be produced round the year if photoperiod and temperature are properly maintained. As plants are produced under aseptic conditions, they are free from pest and pathogen. Even virus can be eliminated by meristem culture technique, that is, regeneration of plants from 0.1 to 0.2 mm shoot meristem. Various techniques have been used for micropropagation of plants. From conservation, the most useful is the propagation from existing meristem as by this method plants with desired traits are obtained. Micropropagation protocols have been developed in an increasingly large number of species. However, certain species are recalcitrant to tissue culture (Coptis), and this is a major obstacle in using tissue culture for germplasm conservation. The rate of shoot multiplication varies from 3.5-fold per 3 weeks in Saussurea lappa (Arora and Bhojwani 1989; Bhojwani et al. 1989) to as high as 150 shoots every 4 months in Coleus (Sen and Sharma 1991). High multiplication rate has major advantages for raising plans for nurseries and commercial plantings; however, for conservation programmes, very high multiplication rate is not desirable. The mode of regeneration has been either direct or through callus, in the form of shoots or somatic embryos. It is evident that in most of the cases, the propagation is through axillary branching. It is worthwhile to mention here that tissue culture methods also provide potential means of multiplying threatened species and clonally propagated with possible reintroduction into their original habitats for throated species. However, it is too early to predict the survival and fate of reintroduced tissue culture-raised endangered plants in their natural/native habitats. Sustainable utilization of this germplasm depends on the development of appropriate in vitro conservation procedure to ensure its availability for future utilization Conservation of Tissues Six major steps defined in the conservation use cycle are collection, quarantine, propagation, characterization, evaluation, monitoring, storage and distribution. The role of in vitro conservation techniques in the overall conservation strategies should be indicative of the fact that it should complement other conservation strategies within the total programme of a given species or population. The methods chosen should be carefully considered taking into account the feasibility, practicality, economy and security. Generally, field conservation of plants requires more space and is labour intensive and expensive. They also run the risk of being damaged by natural calamities and biotic stress factors. Techniques to conserve such species in vitro have recently been developed. For some species, while in situ conservation is the only option available, tissue culture systems offer advantages, which are listed below: 1. Very high multiplication rates 2. Aseptic system

6 422 P.E. Rajasekharan and L. Sahijram Free from fungi, bacteria, viruses and insect pests Production of pathogen-free stocks 3. Reduction of space requirements 4. Genetic erosion reduced to zero under optimal storage conditions 5. Reduction of the expenses in labour costs In vitro collections of species could be maintained at the same or separate site, but should have clear linkages with field gene banks. The properties required for a successful in vitro conservation system as defined by Grout are: The ability of the biological system to: 1. Minimize growth and development in vitro 2. Maintain viability of stored material at the highest possible level along with minimum risk of genetic stability 3. Maintain full developmental and functional potential of the stored material when it is returned to physiological temperatures 4. Make significant savings in labour input, materials and commitment of specialized facilities Some of the advantages favouring this conservation strategy are: 1. Collection may occur at anytime, independent of flowering periods for each species. 2. There is potential for virus elimination from contaminated tissue through meristem culture. 3. Clonal material may be produced. 4. Rapid multiplication. 5. Germination of difficult immature seed/ embryo rescue may be facilitated for breeding. 6. Distribution across borders may be safer. Issues of concern that could limit potential application of in vitro techniques for conservation of plants include: 1. The whole programme can be initially expensive, but with low recurring cost. Technology inputs as adopted by the developed nations will have to play a major role for successful implementation of this conservation strategy. 2. In vitro storage techniques, particularly cryopreservation procedures, are not yet well optimized for routine application across a wide range of species or genotypes. Although cryopreservation techniques are currently being tested and protocols optimized for gene pool components of several plant species, the rate of success is limited to only a few, where the conditions need to be carefully monitored to ensure viability, minimize genetic damage and prevent contamination by diseases and pests. 3. Somaclonal variation can be a major limitation among tissue culture regenerated plants; some of the methodological basis for variation is explant source, age of culture, hormone used, genotype, ploidy status, etc. 4. The problems of genetic stability manifested by loss of cellular integrity among most tissue culture systems pose a major obstacle in using this technique as a conservation strategy. Variation can be observed at different levels, such as morphological, karyotypic or biochemical. The use of axillary or apical meristem for micropropagation reduces the probability of genetic variation among plant tissue culture systems. The genetic stability at all stages of an in vitro conservation programme should be monitored. No welldefined techniques are available for conservation of endangered medicinal plants. Therefore, special attention is required in this regard, since it is a question of species extinction, and it is essential to retain the quality and quantities of secondary metabolites contained in the species 5. There is a need to establish the basic tissue culture competence of the plant species in question; difficulties can be encountered during culture initiation, micropropagation, rooting and establishment of plants extra vitrum. All these stages for any given medicinal plant species must be optimized. Some species show recalcitrance in culture system for which may require special attention In Vitro Collection In vitro collection involves initial disinfestations and placement of plant explants in sterile culture medium, before transport to a tissue cul

7 22 In Vitro Conservation of Plant Germplasm ture laboratory for further in vitro procedures. In vitro collection is particularly useful for species that are vegetatively propagated and for those with recalcitrant seeds or embryos, which deteriorate rapidly. The technique has much potential to facilitate the collection of germplasm of tropical and subtropical fruit species, as has already been demonstrated with cassava and coconut. Recently, 300 Musa accessions were collected in Papua New Guinea using this technique; before being transported to a collection in Australia, an added advantage of this exercise is that it complied with quarantine regulations that are in place to stop the spread of Fusarium and other diseases In Vitro Storage As with short-term storage, there have been very few attempts to apply cryopreservation techniques to tropical and subtropical fruit species, with the exception of Musa spp. (Panis 1995) and Citrus spp. (Pérez-Molphe-Balch and Ochoa-Alejo 1997). Withers (1992), in a review article, reported successful cryopreservation of the recalcitrant tropical species, Theobroma cacao (cocoa), Artocarpus heterophyllus (jackfruit), Cocos nucifera (coconut) and Nephelium lappaceum (rambutan), but provided no details. Very low survival rates have been reported when excised embryos from seeds of jackfruit, rambutan and coconut were cryopreserved (Chin 1988). No survival was achieved when excised embryos from partially dehydrated seeds of rambutan, durian and cempedak (Artocarpus integer) were cryopreserved (Hor et al. 1990). However, before cryopreservation can be universally applied to woody perennial fruit species, there is still much research, development and field testing that need to be done. For example, the issue of genetic stability is rarely mentioned. Because growth is suspended, the potential to store material for long periods without genetic variation is assumed. However, any system based on cell suspension or callus (including embryogenesis) is prone to somaclonal variation and should be field-tested before being accepted unreservedly. Field testing of tropical and subtropical fruits should be continued through to the fruiting stage, as fruit production is the primary reason for their collection and use. Unfortunately, this requires long-term projects for many species. Nevertheless, a research effort into cryopreservation of tropical and subtropical fruit species should be encouraged because of its potential for long-term preservation of germplasm. At the current rate of development, it is reasonable to assume that routine protocols for cryopreservation and subsequent regeneration of explants will eventually become available for most plant species. However, protocols must be repeatable and result in high percentages of preserved tissue being viable after thawing, before they can be used routinely for storage of germplasm Germplasm in growing state Germplasm under suspended growth Maintenance under normal growth conditions Maintenance under growth limitation Cryopreservation Technical approaches to in vitro storage

8 424 P.E. Rajasekharan and L. Sahijram In Vitro Conservation: Strategies Once cultures have been established and multiplied in sufficient number, an effective method for conservation is required. Conservation can partly be achieved by regular subculture on fresh media. However, it may not be practical due to the danger of microbial contamination and equipment failure and may be uneconomical in terms of labour, physical resources and time requirement. Additionally a few systems may have constraints such as loss of morphogenic capacity and occurrence of somaclonal variation. The main aim of in vitro conservation programmes is to reduce frequent demand for subculture, which can be accomplished in two ways: by maintaining cultures under normal growth (SCC) or by subjecting them to growth limiting strategies (for detailed reviews see Grout 1995). The latter includes slow growth and suspended growth (cryopreservation). Normal growing cultures along with those in slow growth comprise the active collections whereas those cryopreserved constitute the in vitro base collection. The expectations are high about tissue culture methods providing sound strategy for both clonal propagation and medium-term storage. Literature survey revealed that till date there is very limited documented information on in vitro conservation Normal Growth It is possible to maintain cultures virtually indefinitely under normal growth conditions provided nutrients are supplied and accidents avoided. This method is preferred for inherently slowgrowing, stable systems and for cultures for which there is no other method though it is laborious and abounds with risks of genetic alterations with time, contamination or loss through human errors; in specific cases such as tropical germplasm, it can be useful because of the following advantages: 1. It minimizes requirement of low temperature facility (particularly for developing countries saves inputs). 2. It promises avoidance of stress-induced variability. 3. It helps in getting cultures for instant multiplication and exchange Slow Growth The aim of this method is to reduce requirement for subculture without causing any damage to the tissue. It is the most direct way of restricting growth and development of in vitro materials and is usually applied to differentiated plantlets or shoot cultures. Slow growth involves one or a combination of the following techniques: 1. Type of enclosure 2. Temperature and/or light reduction 3. Use of minimal media and osmotic 4. Use of growth retardants 5. Other approaches: (i) Reduction of oxygen pressure (ii) Mineral oil overlay (iii) Encapsulation (iv) Desiccation Type of Enclosure Type of enclosure seems to have direct influence on subculture requirement of growing cultures. One of the simplest and cost-effective approaches for slowing growth rate of cultures has been replacement of the commonly used cotton plugs with polypropylene caps as culture tube enclosures (Balachandran et al. 1990; Sharma and Chandel 1992b). The increase in storage time is attributed to the reduction in evaporation of water from the medium in culture tubes. Shoot cultures of Coleus forskohlii, Rauvolfia serpentina and Tylophora indica have been conserved for months at 25 C without requiring any intermittent subculture (Chandel and Sharma 1992; Sharma and Chandel 1992b; Sharma and Chandel 1996). In Allium tuberosum and Dioscorea too, the shelf life of shoot cultures was extended for up to 9 months at 25 C. Encouraging results have been obtained in other species also in our laboratory. This technique seems to work well with species belonging to subtropical or tropical region probably due to their inherent property of growing at higher temperature

9 22 In Vitro Conservation of Plant Germplasm In most of the species of temperate region, optimum subculture period could be extended only to 5 months. The added advantage of this approach is that mostly the cultures can be visibly assessed for viability and can readily be brought back to fresh culture medium to produce plants on demand. Excised roots to Rauvolfia serpentina could be conserved for 16 years, and these retained the regenerative capacity to produce plants (Chaturvedi et al. 1991). This approach needs to be tested for other important threatened species Reduction in Temperature and/or Light The most commonly used method for reducing growth of tissues in cultures is by decreasing the temperature in which cultures are maintained. This may also be accompanied with reduction of light intensity. The basic principle in this method is that incubation at temperature lower than that required for optimum growth would reduce or decrease the metabolic activities, thereby restricting the growth of the plants. However, care must be taken to avoid temperature below freezing or where chilling injury could occur (Lyons et al. 1979). In most cases, the storage temperature is species specific. Usually, the most suitable storage temperature for temperate species is between 1 and 10 C, whereas tropical species are stored in the range of C. The light intensity can be reduced by 60 % from standard requirement, i.e. 1,000 1 and 16th photoperiod. This method is very simple and easy to use and may be applicable to a wide range of genotypes. However, uninterrupted maintenance of reduced temperature, required for the purpose, may be difficult and uneconomical particularly for tropical and developing countries. Low temperature incubation of in vitro cultures appears highly promising. This may also be accompanied by reduction in light intensity. Cultures are kept at ambient culture conditions for 1 week and subjected to screening for detection and elimination of contamination prior to low temperature incubation. Cultures exhibit reduction in growth when transferred to low temperature. Shoot culture/nodal cultures of Rauvolfia serpentina have been successfully conserved for over 15 months at 15 C, while 10 C and 5 C were found deleterious to growth of cultures (Sharma and Chandel 1992b). These in vitro conserved cultures showed shoot multiplication comparable to the cultures maintained at 25 C. The plantlets obtained from these conserved cultures could be established in soil and no morphological differences were observed in these plants. Tylophora indica have been successfully conserved from more than 12 months tested so far at 10 C (Sharma and Chandel 1996). These in vitro conserved cultures exhibited normal growth and multiplication on rejuvenation. Reduction of light along with temperature has extended shelf life of cultures in Saussurea lappa. According to Arora and Bhojwani (1989), shoot cultures of Saussurea could be successfully stored at 4 C in the dark for 12 months, with 100 % viability. Shoot tip cultures of the same species have been conserved for more than 15 months at 4 C in the dark (Sharma et al. 1995). Further experiments have indicated that cultures can be maintained for 22 months at 10 C under reduced light. However, the result has to be repeated for confirmation. An interesting observation has been that some of the cultures that were subjected to reduced temperature/ light regime exhibited a better rate of multiplication following storage as compared to cultures maintained at normal temperatures and light. In Picrorhiza kurroa, shoot cultures have been successfully conserved for 6 months at 25 C and over 9 months at 10 C. Shoot cultures of this species are reported to have been stored for 10 months at 5 C in the dark, with 70 % survival. Cold-stored shoots, on transfer to 25 C, multiplied at rates comparable to cultures maintained under normal conditions (Upadhyay 1989). Embryogenic calli of Podophyllum hexandrum cold stored for 7 months at 5 C exhibited 95 % viability and differentiated globular embryos (Bhojwani et al. 1989). However, similar studies carried out in our laboratories indicated that cold-stored calli failed to regenerate beyond the globular stage. Gentiana kurroo, another threatened medicinally important species, has been conserved for 11 months at 4 C (Sharma et al. 1993; Sharma and Chandel 1996). In Allium tuberosum, shoot

10 426 P.E. Rajasekharan and L. Sahijram cultures stored at 10 C survived for 18 months without subculture Minimal Media/Osmotica Inclusion of osmotica with or without low temperature incubation has also proved to be an important technique to prolong subculture period. Change of carbon source may have a very remarkable effect on growth rate. Use of either increased or decreased doses of sucrose yielded interesting results in some species. Inclusion of non-metabolizable, inert sugar alcohol, particularly mannitol and sorbitol, in the range of 3 6 % (w/v) has been quite effective in restricting the growth of many plant species. The major limitation of this method is that different genotypes may react differently under these conditions. However, when used in combination with reduced temperature incubation, it becomes the most realistic and cost-effective method because the combinations of these two treatments show synergistic effects on in vitro conservation of many species. Inclusion of osmotica such as sucrose and mannitol, with or without low temperature incubation, has also proved to be an important technique to prolong subculture period. There is no documented information regarding its use in case of threatened plant species. Effect of addition of mannitol in the medium to cultures of Rauvolfia, Picrorhiza, Gentiana and Saussurea has been studied in extending shelf life. Addition of mannitol led to maintenance of these species for more than 9 12 months at 25 C. Reduction of temperature further resulted in prolongation of shelf life of the same cultures up to 16 months. Experiments are underway to study the effect of mannitol and other osmotica, in other species too. Incorporation of high concentration of sucrose has also been beneficial in extending the storage time of cultures of Allium tuberosum. In Rauvolfia serpentina, half-strength medium and full-strength medium without hormones were used to test their effect on survival of cultures at 25 C (Sharma and Chandel 1992b). None of these were effective in improving the shelf life of cultures in comparison to low temperature storage, with survival rates of only % after 6 months of storage. An important observation made in author s lab is that rooted cultures survive longer probably due to roots being capable of absorbing water and nutrients from the medium more efficiently than shoot cultures especially at later stages Growth Retardants The basic principle of using these compounds is simply to reduce the overall growth rate of the in vitro plantlets and thereby enhance the subculture interval. The choice of inhibitory growth regulator necessarily depends upon the circumstances and species. Abscisic acid, N-dimethylaminosuccinamic acid, maleic hydrazide (MH), trans-cinnamic acid (TCA), chlorocholine chloride (phosphon-d), daminozide (B 995) and Cycocel (CCC) at a concentration range 2 50 mg/l have been reported to extend subculture period for 6 12 months. This method is simple, efficient and cheaper. Using these chemicals, cultures can be stored at normal culture room, thereby eliminating the requirement of low temperature facility. However, its use may pose serious problems in the germplasm conservation: 1. The plant may become stunted and show abnormal growth, thereby posing problems in regenerating normal plants. 2. The presence of these retardants may lead to selection of lines with resistance or tolerance to growth retardants. 3. Chances of induction of genetic alterations increase because some of the growth retardants have mutagenic properties Other Approaches Growth rates of in vitro culture are influenced by composition and volume of atmosphere inside the culture vessel (Gould and Murashige 1985). Mineral oil overlay and reduction of oxygen pressure in the culture vessel may extend shelf life cultures from a few weeks to months. The main problem with the mineral oil overlay is the recovery growth of shoot tips. The limitation of modification of gaseous environment is the diffi

11 22 In Vitro Conservation of Plant Germplasm culty of maintaining gas atmosphere of individual culture vessel. However, the use of gas permeable culture vessel such as star packs (Reed and Abdelnour 1991) offers the possibility of controlling the gas atmosphere of the entire incubation chamber which may be more practical. This technique therefore needs further investigation. Storage of encapsulated buds and somatic embryos at room temperature for 8 months when water content was reduced down to 15 %. Though the result of these methods is encouraging and may be useful in specific cases, the methods need further standardization for their acceptability and wider use. Thus, these may be viewed as experimental techniques with future application. It is worthwhile to mention here that the technique of applying slow growth to reduce subculture frequency is not without drawbacks, mainly of somaclonal variation. Moreover, reduction of temperature has to be used with caution for tropical species which require higher temperatures for growth. Besides cost-effectiveness of low temperature storage, maintenance of constant low temperature for desired long periods may pose a practical constraint, especially in subtropical/tropical regions Principles of Medium-Term Storage Standard culture conditions can only be used for medium-term storage of slow-growing species. However, in most cases, environmental conditions and culture medium have to be modified to induce growth reduction. This is most frequently achieved by temperature reduction, often in association with a decrease in light intensity or even its complete suppression. Temperatures in the range of 0 5 C are employed with cold-tolerant species, but higher temperatures are required in the case of tropical species, which are generally cold sensitive. It is also possible to limit growth by modifying the culture medium, mainly by reducing the sugar and/or mineral element concentration. Finally, the type of culture vessel, its volume as well as the type of enclosure influence survival of cultures. Notably, it is important to limit the evaporation of the culture medium by using a type of enclosure, which is tight enough. Medium-term conservation techniques have been developed for a wide range of plant species, but they are used routinely for the conservation of only a limited number of species such as Musa, potato or cassava (Engelmann 1997). New medium-term conservation techniques include reduction of the oxygen level available to cultures (achieved by covering explants with a layer of liquid medium or mineral oil or by placing them in controlled atmosphere), desiccation and encapsulation of explants in alginate beads (Engelmann 1997). However, these techniques are still at the experimental stage. The objective of slow growth (or minimal growth) is to reduce subculture intervals to a critical level that does not impose a long-term deleterious effect on germplasm or put at risk the stability of regenerated/regrown plants. However, slow growth treatments incur some level of stress, and it is essential to optimize MTS with respect to the timing of subculture regimes and regeneration. When this is achieved, slow growth is a successful method of securing plant germplasm in MTS (Cha-um and Kirdmanne 2007). Minimal growth storage is useful for genotypes that cannot be cryopreserved, and it is a key part of in vitro gene banks that clonally propagate crops for distribution services. Several MTS treatments are applied, either singularly or in combination to retard growth: Physical growth limitation: Low temperature Low light/restricted photoperiod Minimal containment Minimal O 2 Osmotic (water) stress Chemical growth limitation: Growth regulator retardation Growth inhibitors Nutrient limitation: Low macronutrient levels Low micronutrients levels Ashmore (1997) listed key issues and actions for future consideration

12 428 P.E. Rajasekharan and L. Sahijram In Vitro Conservation: Practical Considerations The real utilization of any conservation programme lies in carefully selecting the species, designing work plan and sincerely executing it. The first and foremost and essential step, once the infrastructure is available, is prioritization of species. In deciding the priority, the main points to be considered include threat of genetic erosion, importance of the species and potential demand for distribution/exchange for commercial utilization, besides size of collection and cost of field maintenance. It is equally important to select a strategy after assessing its economy and safety. An ideal strategy should meet the following requirements: 1. Wide and easy applicability of protocols and techniques over a range of species 2. Economy (establishment and running cost) in operation/management 3. Ensure high viability after storage 4. Low risk of genetic alteration 5. Technically less demanding techniques The material needs to be well documented and conserved under safe condition and duplicated in at least one other location/country to minimize accidental loss and to ensure its availability. Before embarking on in vitro conservation, the potential approach should be balanced against other conservation strategies. Experience with in vitro maintenance is limited; thus, time and cost compliment other conservation strategies for the same crop species (seed, field gene banks, etc.) including in situ conservation. Issues of concern in potential application of in vitro techniques for germplasm conservation of medicinal and aromatic plants include: 1. Genetic stability 2. Basic tissue culture competence Genetic Stability Maintenance of specific gene combination (genotypes) is vital for any germplasm conservation programme. The phenomenon of somaclonal variation associated with certain culture systems is perceived as major obstacle in using plant tissue culture for germplasm conservation. The instability has been documented to occur at the morphological, karyotypic and biochemical levels. The variations primarily result from the change in ploidy level, structural changes in chromosome morphology and mitotic aberrations. It has been reported that these changes may occur during culture, though genetic variation occurs spontaneously in nature also. Long preservation in culture media composition can also give rise to variations. Somaclonal variation can be enhanced, particularly where regeneration is adventitious. It has also been observed that certain in vitro storage conditions can increase the risk of integrity because of directional change in response to selection. The use of axillary bud for multiplication reduces the probability of genetic variation. Therefore, formation of callus and adventitious shoots should be avoided (Roca et al. 1989). Monitoring of genetic stability is an important aspect of in vitro conservation. Technique of monitoring of stability will depend on the need, type and nature of plant species and its economic value. The characterization may be for morphological, cytological, histological and/or biochemical traits. Some of the techniques like field testing for morphological traits cannot be avoided. Other techniques have to be chosen judiciously depending on the crop, the economic product, the life cycle of the plant and the resources available. Use of sophisticated high- technology monitoring (molecular markers/dna polymorphism) has value when there is sufficient basis to assume instability, either intrinsic or due to the culture system and storage conditions. In the case of medicinal and aromatic plants, however, it is more relevant to monitor the potentially useful metabolites of plant species. Greater understanding of the causes and nature of somaclonal variation (SCV) is needed, particularly after prolonged storage in culture and cryopreservation and in establishing safe storage procedures, developing improved markers and methods for characterizing SCV and monitoring genetic instability. Encouragement of

13 22 In Vitro Conservation of Plant Germplasm research aimed at understanding causal factors of SCV and comparative assessments of genetic stability in germplasm conserved in the field and IVGBs is recommended Reproducible and Wider Use of Slow- Growth and Cryopreservation Methods Improved, reproducible and robust protocols required for slow growth and cryopreservation optimized across genotypes held in gene banks, greater provision for species that have received limited attention, application of in vitro methods for safe movement of germplasm and prioritizing problem species. Developing methods using simple facilities, with general application, optimized, and tested in IVGBs; more information on in vitro distribution and transportation of in vitro material Management Issues Database of in vitro collections and their activities and operations are required including routine methods and guidelines, particularly addressing what are acceptable ranges for survival and amount of material to be stored per accession for in vitro storage techniques Performance Indicators for Slow Growth Performance indicators include (1) plant health, (2) extension of subculture interval, (3) contamination frequency and (4) capacity [viability, vigour, health status] to recover from stress treatments. Reed et al. (1998b, 2003) use descriptive scales of 0 and 1 5 to rate the performance of in vitro Pyrus and Humulus cultures maintained under minimal, low temperature growth: 0 = all of the plantlet is brown and no visible indication of growth. 1 = very poor, questionable viability, brown, necrotic shoots, only extreme shoot visibly green, plantlet mostly brown. 2 = poor, much browning, most shoot tips necrotic, shoot tip green, leaves and stems mostly brown, base may be brown. 3 = fair, some browning, some shoot tips necrotic, shoot tips and upper leaves green, etiolation present, base green. 4 = good, elongated shoots, shoot tips generally healthy, green leaves, stem and limited etiolation. 5 = excellent condition, dark green leaves and shoots, no etiolation. A widely used method for conservation is seed storage. Several categories of crop present problems with regard to seed storage. At present, the most common method to conserve the genetic resources of these problem species is as whole plants in the field (field gene banks FGBs). However, several serious problems such as labour-intensive field maintenance, shrinking land resources, etc. are associated with field gene banks. The conservation of rare and endangered plant species has also become an issue of concern. During the last 20 years, in vitro culture techniques have been extensively developed and applied for more than 1,000 different species (George 1996). Tissue culture techniques are of great use for the collection, multiplication and storage of plant germplasm. Storage techniques, which allow to reduce the procedures and to preserve the genetic integrity of the plant material, are urgently needed. Different in vitro conservation methods are employed depending on the storage need (Engelmann 1991). For short- and mediumterm storage, the aim is to reduce growth and to increase the interval between subcultures. For long-term storage, cryopreservation in liquid nitrogen is the only available method. Tissue culture techniques are of great interest for collection, multiplication and storage of plant germplasm (Engelmann 1991). Tissue culture systems allow propagating plant material with high multiplication rates in an aseptic environment. Virus-free plants can be obtained