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10. Plant Genetic Resources Conservation: Recent Approaches - K.P.S. Chandel and Ruchira Pandey

Recent conservation approaches - The need
In-vitro conservation
Cryopreservation of in-vitro cultures
Cryopreservation of Seeds
New conservation strategies - Indian scenario
Future perspectives


Plant genetic resources in agri-horticultural crops and their wild relatives are of immense value to mankind as they provide food, fodder, fuel, shelter and industrial products. The plant breeders require reservoir of genetic variation (genepools) for crop improvement. The larger the reservoir of variation, the better are the chances of finding particular characters, such as resistance genes for diseases, pests and nematodes or for adaptation to wider ecological amplitudes and stress conditions. However, in the wake of spread of high yielding varieties, this genetic variability comprising landraces is gradually getting eroded resulting in the large scale depletion of variability. This situation thus demands priority action to conserve such germplasm (Frankel, 1975).

Further, the process of genetic erosion necessitates measures that germplasm must be conserved in such a manner that there are minimal losses or changes in genetic variability of the population. We need to decide on the appropriate storage methods, whether seeds, pollen, roots, tubers, bulbs, other vegetative material or cell, meristem and other tissue culture systems. Genebanks (seed banks) are generally advocated, since the storage technology is relatively simple and well known at least for most annual, orthodox seed species which are desiccation tolerant. This has already been discussed in Chapter 9 of this book. Here, emphasis will be laid on new approaches including the role of biotechnology in the conservation of plant genetic resources. Emphasis will be on the non-conventional and problematic materials, i.e., vegetatively propagated material and recalcitrant types.

Recent conservation approaches - The need

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 genebanks, the traditional procedures tend to be expensive due to: (i) high labour costs, (ii) vulnerability to environmental hazards, and (iii) 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.

Much of the world's germplasm is currently maintained as breeders collections in genebanks, plantations, orchards or even in evolution gardens primarily raised from seeds, such as rubber and coconut; vegetatively propagated plant species, such as Citrus, cacao, banana and many other fruits. They 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 genebanks do not represent the entire range of genetic variability within the respective crop genepool and most of them represent only a fraction of the variability which should be conserved (Withers and Williams, 1985). The IBPGR programme recently 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 organised 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 (Upadhya, 1988). These can be eliminated by meristem or shoot tip cultures possibly in combination with both heat and cold therapy. 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 virus-tested 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

Slow growth

Two basic approaches are followed to maintain germplasm collections in-vitro: (i) minimal growth, and (ii) cryopreservation (Scowcroft, 1984). Minimal growth conditions for short to medium term storage can be followed in several ways - reduced temperature and/or light; incorporation of sub-lethal levels of growth retardants, induction of osmotic stress with sucrose or mannitol, and maintenance of cultures at a reduced nutritional status particularly reduced carbon, reduction of gas pressure over the cultures, desiccation and mineral oil overlay. The advantage of this approach is that cultures can be readily brought back to normal culture conditions to produce plants on demand. However, the need for frequent subculturing may pose a great disadvantage including contamination of cultures as well as imposition of selection pressure with subsequent change in genetic make-up due to somaclonal variation.

Cryopreservation at the temperature of liquid nitrogen (LN) (- 196° C) offers the possibility for long-term storage with maximal phenotypic and genotypic stability (Steponkus, 1985). This method being relatively convenient and economical, large number of genotypes and variants could be conserved and thus maximize the potential for storage of genetically desirable material.

Slow growth

The first report of successful in-vitro storage (Galzy, 1969) was of shoot tips of Vitis rupestris wherein at 9° C, cultures could be stored upto 290 days. Barlass and Skene (1983) demonstrated in several Vitis species and cultivars that cultures could be maintained either as proliferating shoots or as single rooted shoots at 9.5° C for 6 to 12 months. Variation in recovery percentage was noted with different genotypes. Single shoots responded better than the proliferating shoot cultures, towards plantlet production. In recent years, 'T-bud culture' technique has been employed in Vitis spp. for storage purposes.

In Solanum species, particularly in potato, Henshaw et al. (1980) reported that there was 14 percent survival of meristems, stored for one year at 22° C, whereas it was 61 percent at 6° C for the same length of storage period. Survival rate could be increased to 83 percent by alternating day and night temperatures (12° C and 6°C, respectively). At temperatures below 6° C, no success could be achieved. Modification of culture medium has also been used (Henshaw et al., 1979, 1980) to reduce the subculture period for meristem cultures. Survival of cultures maintained at -10° C for one year could be increased from 39 to 56 percent following elevation of sucrose from 3 to 8 percent and it could be further increased to 30 percent by increasing culture volume from 3.5 to 6.0 ml. Incorporation of mannitol at 6 percent or abscissic acid (5 mg l-1) in the medium for cultures maintained at 22° C resulted in 63 and 43 percent survival respectively, compared with 14 percent without supplements, after one year's storage. In taros (Colocasia esculenta L.), cultures could be successfully stored at 9° C (Staritsky et al., 1986) in dark for three years. Recent reports indicate that taro cultures could be successfully stored by reducing growth temperature to 3-4° C. Genotypic differences to storage conditions have been encountered but slow growth storage appears generally feasible. In Xanthosoma brasiliense, reduction in temperature to 6° C in dark resulted in storage of cultures upto two months. In Beta vulgaris, lowering of temperature from 20 to 12° C could prolong culture period to 18 months. Another approach in this system has been to use flower buds to initiate adventitious shoots which could be stored in low light intensity for a year at 5° C (Hussey and Hepher, 1978). In garlic (Allium sativum), shoot tips could be stored for a period of 16 months following reduction of temperature from 25 to 4° C and an increase in sucrose concentration to 10 percent (El-Gizawy and Ford-Lloyd, 1985).

In contrast to above, in Ipomoea batatas, reduction in temperature below 14° C has not yielded any success (Alan, 1979). In cassava (Manihot esculentum), Roca (1978) reported that nodal cultures were sensitive to storage temperature of 15° C and, at 20° C, there was one-tenth reduction in growth rate compared to that at 30° C day/ 25° C night temperature regime. However, in recent years, Roca et al. (1983) have successfully shown that nodal cuttings from meristem derived plantlets could be maintained for two years on a medium with low osmotic concentration and activated charcoal.

The rapid in-vitro multiplication of banana has been achieved by culturing explant in MS semisolid basal medium supplemented with low concentration of indole-3-acetic acid (IAA) and a fairly high level of 6-benzylaminopurine (BAP). The genomic constitution of the cultivar as well as the concentration of BAP seem to control the number of shoot buds (Banerjee, 1989). In banana and plantain cultivars of different genomic constitutions, it has been shown that some genotypes displayed 92 percent viability following 17 months storage at 15° C whereas others exhibited upto 50 percent within 13 months of storage (Banerjee and de Langhe, 1985). At temperatures below 15° C, none survived more than three months. Shoot tip cultures of banana could be successfully stored for over two years without being transferred to fresh medium at 15° C and 1000 lux.

In strawberry, cultures have been successfully stored for six years (Mullin and Schlegel, 1976), the meristem plantlets being kept viable at 4° C in dark with renewal of fresh medium, occasionally. Experimenting with 19 strawberry cultivars, Damiano (1979) demonstrated that in cultures stored at 2° C for 13 months, survival was 100 percent with one cultivar whereas it was 50 percent for two cultivars stored for 27 months.

In-vitro storage of apple shoots (Lundergan and Janick, 1979) at -17° C, 1° C, 4° C and 26° C in the light revealed that shoots could not survive at -17° C. Following storage for 12 months at 1°C, 100 percent cultures survived whereas at 4° C, only 70 percent exhibited survival. According to Marino et al. (1985), Prunus root stocks could be stored for six months at 3 to 4° C.

Among forage legumes and grasses, i.e., Trifolium repens, T. pratense and Medicago sativa, cultures could be stored for 15 to 18 months at 4 to 6° C in dark or at 2 to 4° C in 8-hour photoperiod (Cheyne and Dale, 1980). Cultured shoot tips of T. repens, stored in dark at 5° C exhibited 100 percent survival and rapid multiplication following 10 months of storage (Bhojwani, 1981). In Lolium multiflorum, Dactylis spp. and Festuca spp., meristem tip cultures could be maintained at 2 to 4° C for over one year in the former and over two years in the latter two species (Dale, 1978, 1980).

In Capsicum annuum, callus could be stored at 4° C for 70 days following pretreatment with ethanolamine (Withers, 1980).

Among other techniques, desiccation and mineral oil overlay have been employed as a tool for in-vitro storage as in Daucus carota (Caplin, 1959; Jones, 1974), while nutrient depletion from the culture medium has been found beneficial for storage of cultures in coffee (Kartha et al., 1981). In Saccharum spp., Sreenivasan and Sreenivasan (1988) reported that plantlets generated from shoot apices could be stored in White's basal medium for more than a year at 25 ± 1° C. By reducing temperature from 25 to 15° C, subculture period could be extended from 26-30 weeks to 52 weeks. Recovery was found to be 60 to 70 percent.

Cryopreservation of in-vitro cultures

Examples of successful cryopreservation
Preservation of embryos
Clonal plantlets and seedlings
Mature plant pieces
Freeze preservation of pollen

Cryopreservation or storage in liquid nitrogen at a temperature of -196° C, at which all the cells are in a state of suspended animation, is the most promising method of in-vitro germplasm storage (Stushnoff and Fear, 1985). Cryopreservation has been successfully applied to callus, protoplast, pollen, meristems, zygotic and somatic embryos and suspension cultures of around 42 crop species. The first report of successful Cryopreservation of plant cell suspension (Quatrano, 1968) and regeneration of somatic embryos from cryopreserved cells (Nag and Street, 1973), led to numerous studies on Cryopreservation of plant system (Finkle et al, 1985; Kartha, 1985a; Steponkus, 1985; Withers, 1985a, b).

Various stages in a freeze preservation protocol can be categorized under pregrowth, cryoprotection, freezing, storage, thawing and regrowth.

1. Pre-growth is prior to storage at ultra-low temperature. Pre-growth manipulations involve cold hardening by exposure to low temperature.

2. Cryoprotective pretreatment procedure involves reduction in net water content of the cells prior to freezing (Withers, 1985a). Cryoprotectants can be used either alone or in combination, as no single protectant or combination seems to be the best (Ulrich, 1985). Amongst various cryoprotectants used, mannitol, proline, hydroxyproline, betaine aldehyde, sucrose, sorbitol, methanol, ethylene glycol, dimethyl sulphoxide, glyceraldehyde, glucose and glycerol have been in frequent use. According to some research workers (Ulrich, 1985; Withers, 1985a; Finkle et al., 1985), cryoprotectants reduce the amount of ice at any temperature during freezing and moderate the rise in concentration of solutes, thereby maintaining cell viability.

3. Freezing can be categorised as slow, rapid or droplet freezing; and is accomplished using a programmable controlled freezing apparatus to give linear slow freezing rates or stepwise freezing to one or more intermediate temperature.

Various factors like cooling rate, pretreatment, cryoprotection, type and physiological state of the material and the temperature prior to immersion in liquid nitrogen, determine the success of slow freezing (Finkle et al., 1985). Most commonly, slow cooling is regulated at a constant rate of 0.5 to 2° C min-1 to terminal temperature between - 30 and - 40° C, and subsequent storage in liquid nitrogen. In rapid freezing, cryoprotected specimens were directly immersed into liquid nitrogen, the rate of temperature reduction being several hundred degrees per minute. Droplet freezing technique has been applied to cassava meristems (Kartha et al., 1982); cryoprotected specimen were kept in 2-3 µl droplets on a aluminum foil contained in a petridish and then frozen by slow cooling (0.5°C min-1) to a temperature between -20 and -40°C prior to immersion in liquid nitrogen.
4. Storage, thawing and regrowth: Storage of material is best carried out in liquid nitrogen immersion phase (-196° C) or in its vapour phase (-150° C). Rapid thawing found suitable for most of the cryopreservation protocols is accomplished by removing the specimen from liquid nitrogen and immersing it in a warm (34-40° C) water bath for about 1-2 min or until the phase change occurs and ice is transformed into water. Regrowth of cryopreserved specimen is the most accurate criterion for assessing viability as compared to other methods such as vital staining or triphenyl tetrazolium chloride test (Kartha, 1985b; Withers, 1985 a, b). Various factors, such as, handling of specimen, incorporation of certain additives in regrowth medium and physical environment during early regrowth are important.

Examples of successful cryopreservation

Initial attempts were made with plant systems where specimens were subjected to temperatures far below 0° C but not as low as -196° C (Latta, 1971). In subsequent years, Sakai and Sugawara (1973) reported freezing of callus of Populus euramericana down to -196° C, whereas Nag and Street (1973) reported regeneration of somatic embryos from cell cultures stored in liquid nitrogen. Since then, encouraging progress has been made with different culture systems as summarized below.

1. Suspension culture: Cryopreservation has attained the highest degree of refinement with suspension cultures (Kartha, 1985b). The selection of the appropriate growth stage is important for the survival of suspension cultures. Use of many compounds, such as proline (upto 10 percent), mannitol (6 percent), DMSO (5 or 10 percent), sucrose or glycerol, either as media components or as pre-growth additives has considerably improved the viability of suspension cultures following LN treatment. Important examples include Daucus carota (Latta, 1971), Saccharum sp. (Chen et al., 1979), Nicotiana tabacum (Hauptman and Widholm, 1982). Brassica napus, Datura innoxia and Glycine max (Weber et al., 1983).
In recent years, Kartha (1985-86) reported that cell lines of Brassica napus usually maintained at 13 percent sucrose have the potentiality to form somatic embryos. One to seven days old cultures cryoprotected with sorbitol plus DMSO were frozen and stored in liquid nitrogen. Recovered frozen cells grown on solid media formed somatic embryos and some specimens also regenerated plantlets.
2. Callus culture: Callus culture being a heterogenous system, poses problems during cryopreservation as rapid freezing does not ensure even cooling and also cryoprotectant penetration may not be uniform (Ulrich, 1985). This system, though always protected by various chemicals before freezing, yet in an interesting example, Sakai and Sugawara (1973) employed omission of cryoprotectant stage in Populus euramericana. Callus hardened to low tempertaure regimes (0° C, -20° C, -120° C) was subsequently stored at -196° C. Upon thawing at 0° C, recovery growth of callus could be observed within two weeks. A noteworthy example of successful cryopreservation has been with callus of Saccharum sp. (Ulrich et al., 1979). Calli cryoprotected with a mixture of DMSO, glycerol and polyethylene glycol were frozen slowly first to - 40° C and then exposed to - 80° C for 5 min and later plunged in liquid nitrogen. Following rapid thawing, regrowth of callus and also rootlet formation could be observed. Some of the important species in which calli have been stored for a period varying from one week (Saccharum sp.) to over one to three years include Malus domestica, Medicago sativa, Oryza sativa and Phoenix dactylifera (Ulrich, 1985).

3. Meristem, shoot tip and bud cultures: Meristem cultures have assumed a significant place in the domain of plant tissue culture due to its role in clonal propagation as well as in the production of virus-free plants. Moreover, apical meristems are less differentiated and genetically more stable which makes this an ideal system for germplasm preservation (D'Amato, 1975; Kartha, 1985b; Sakai, 1985).

Following successful cryopreservation of shoot tips of Dianthus caryophyllus (Seibert, 1976) and tomato (Grout et al., 1978), researchers explored the possibilities of cryopreserving meristems of potato, a system that had been used extensively to provide virus-free plants for international exchange. In Solanum goniocalyx, a diploid species known to be particularly amenable to shoot tip culture, Grout and Henshaw (1980) reported 20.5 and 62.6 percent survival (plantlet formation from frozen shoot tips) using 5 percent and 10 percent DMSO, respectively. In these experiments, shoot tips carried on hypodermic needles were thawed directly by plunging needles into culture medium at 35° C.

In another species of this genus, S. tuberosum subsp. andigena, frozen meristems exhibited 36 percent survival whereas S. tuberosum subsp. tuberosum showed between 2 and 32 percent survival following recovery (Henshaw et al., 1979). Using improved procedures, Towill (1983) reported high survival (71 percent average) and regeneration of larger number of multiple plantlets following LN storage.

Successful cryopreservation of meristems has also been reported in several other species, such as Manihot esculenta, Pisum sativum, Fragaria ananassa (Kartha et al., 1979, 1980, 1982), Cicer arietinum (Bajaj 1979; Kartha 1985b) and Arachis hypogoea (Bajaj, 1983). In most of these cases, DMSO, sucrose or glycerol, either alone or in combination have been used as cryoprotectants and percent survival of cultures varied from 16 to 80.

Manipulation of post-thaw conditions such as low light level during the recovery of meristems of S. goniocalyx has been found beneficial (Grout and Henshaw, 1978). Addition of NAA (0.5 mg l-1) and GA3 (0.2 mg l-1) to the recovery medium increased the survival percentage of meristems of S. tuberosum subsp. tuberosum (Henshaw et al., 1979).

Plants growing under extremely cold conditions undergo hardening, a phenomenon associated with biochemical changes within the plant and can be induced in some tissues by gradual reduction of temperature. Buds on hardy fruit trees become dormant with the onset of winter. It has been shown in apple (Malus domestica Borkh.) by Sakai and Nishiyama (1978) that 77 percent buds, from apple shoots immersed in LN for two hours after prefreezing to - 40° C, could be successfully grafted onto two-year old seedlings. These buds developed normally and continued their growth. Interestingly, apple foliar buds immersed in LN for 23 months still remained alive. Other hardy fruit trees whose buds could survive LN treatment after prefreezing to 40° C and slow rewarming included pear, raspberry, gooseberry and currant (Sakai, 1985).

Preservation of embryos

The data available in literature regarding survival of embryos and their subsequent regeneration into plants, points that their most important use could be for conservation of: (a) recalcitrant species, (b) species in which less number of seeds are produced, (c) hybrid embryos produced out of incompatible crosses, and (d) haploid germplasm.

For cryopreservation, four types of embryos have been used, i.e., zygotic, somatic, nucellar and the pollen embryos obtained from androgenic anthers. In Daucus carota, somatic embryos at a younger stage of development could be successfully cryopreserved using 5 to 10 percent DMSO (Bajaj, 1976; Withers, 1979).

Freeze preservation of zygotic embryos has been attempted in rice, wheat, barley, mustard and coconut (Bajaj, 1985). In these, rapid freezing followed by thawing at 35 to 40° C was employed. Recovery percentage for these systems varied from 15 to 100. In Citrus spp., nucellar embryos were subjected to rapid freezing in LN in presence of DMSO and sucrose. On a medium supplemented with 500 mg l-1 casein hydrolysate, the retrieved material formed pseudobulbils and also polyembryonic axes and shoots (Bajaj, 1985).

Freeze preservation of pollen embryos assumes an important role upon realizing the instability of haploid cultures as also their importance in mutation biology and biochemical genetics (Ulrich, 1985). Bajaj (1983) reported successful storage of pollen embryos of several species for a period of one year in LN with varying percent survival in Arachis hypogoea (29), A. villosa (38), Brassica campestris (31), B. napus (44) and Triticum aestivum cv. Kalyansona (19). For B. campestris, thawed pollen embryos elongated and formed plantlets or callused after a lag of five weeks in culture. For wheat, retrieved cultures exhibited a lag phase of four to six weeks before anther segments proliferated and callus differentiated into malformed shoots and plants. With excised rice anthers, frozen and stored at -196° C for seven days, 70 percent survival was observed.

Clonal plantlets and seedlings

Review of literature indicates that cryopreservation of seedlings has been attempted in Pisum sativum (Sun, 1958), Lycopersicon esculentum (Grout et al, 1978) and Dactylis sp. (Withers, 1980). In most of these cases, freezing has been rapid and cryopreservation has been done with DMSO. Percent survival varied between 8 and 45.

Mature plant pieces

According to Withers (1980), relatively mature plant pieces can provide basic information on interaction of freezing and thawing rates, effects of ultra-low temperature on storage of tissues and cold-hardening in relation to low temperature tolerance. Substantial information has been provided by Sakai (1985) on fundamental aspects of cryobiology. Very small pieces (20 to 30 µm thick) of hardened stem tissue of Morus species could survive rapid freezing and thawing as tested by vital staining and plasmolysis tests (Sakai and Yoshida, 1967). Pre-freezing has been found beneficial for less hardy species as also cryoprotection with 1 M glucose (Sakai and Otsuka, 1972). Sakai (1965) reported that twigs of Salix spp., Populus spp., Betula spp. and Rosa pendulina could survive rapid freezing treatment.

Freeze preservation of pollen

Pollen storage can be of considerable value in supplementing germplasm conservation strategies (Roberts, 1975). It not only facilitates hybridization between plants with different flowering times but can also be of immense value for crops with short lived pollen e.g. Graminae. Prolonged storage is required for species which flower erratically. The ease of transport of pollen is also helpful in its exchange to many countries for its use in crop improvement and germplasm collection programmes. In case of woody trees, which take longer time to flower, pollen distribution can speed up crossing of desired strains (Towill, 1985). Additionaly, pollen can be cultured to regenerate haploid and homozygous diploid (dihaploids) tissues and plants.

Pollen from many species can be desiccated before it can be preserved at very low temperature. Reports on successful freeze-preservation of pollen have appeared in literature since long (Roberts, 1975; Towill 1985; Ulrich 1985; Withers, 1980). The data provided therein necessitates the desirability of pollen banks in germplasm conservation. A pollen bank of 60 peach cultivars has been established in Japan using freeze-drying, referigeration or storage in LN (Omura and Akihama, 1980).

The method of cryostoring pollen is usually done by enclosing pollen in a vial, direct immersion in LN (cooling rates are often not specified) and storage therein followed by thawing in a warm water bath (Towill, 1985; Withers, 1980). Viability is tested by in-vitro germination, pollen staining/fluorescence and fruit-setting.

Towill (1981) reported that exposure of pollen to -196° C enhanced their percent germination in 25 tuber bearing Solanum species. Ichikawa and Shidei (1972) reported that pollen from 33 species, stored at -196° C, have been shown to retain germination ability.

Papaya pollen has been shown to lose viability, germinability and fertilization capacity following storage for 4 to 66 months under low temperature and humidity (Ganeshan, 1986). However, it was further reported that viability of papaya pollen cryopreserved in liquid nitrogen for 485 days, remained as much as that of fresh pollen, when germinated in-vitro. Pollen stored for 300 days, could bring about normal fruit and seed-setting.

Other species in which cryopreservation of pollen has been successful are Prunus spp. (Parfitt and Almehdi, 1983), Humulus lupulus (Haunold and Stanwood, 1985) and Solanum species (Towill, 1981).

Ganeshan (1985) has reported that grape pollen stored in LN for 64 weeks maintained as much viability as the fresh pollen. In this study, it was also indicated that pollen stored at room temperature completely lost its capacity to germinate within four weeks. In a recent report by Alexander and Ganeshan (1988), successful cryogenic preservation of pollen from several genera has been demonstrated. In grapes (Vitis vinifera), cryogenic storage of pollen has been achieved for four years without any decline in germination capacity. Successful seed-setting could also be induced with stored pollen. In tomato, pollen stored at -196° C for three years could induce normal plants and also seed-set, though number of seeds per fruit was very low.

Pollen from corn, sorghum, rye, oats and wheat exhibited positive staining for tetrazolium bromide, when subjected to -180° C (Collins et al., 1973). Of the above systems, only rye pollen could effectively bring about seed-setting. Maize pollen, dried to less than 25 percent moisture content, showed good germination and seed set (Barnabas and Rajki, 1976; 1981). Onion pollen has been found to lose viability completely by the sixth day of anthesis (Alexander and Ganeshan, 1988). It has been demonstrated that onion pollen cryopreserved at -196° C for 720 days showed viability equal to that of fresh pollen. Pollinations carried out with the pollen stored for 225, 360 and 720 days produced fruit and seed-setting on male sterile flowers. Even in brinjal (Solarium melongena), pollen stored for 300 days in liquid nitrogen could effect fruit and seed-setting.

Cryopreservation of Seeds

Orthodox seeds
Recalcitrant seeds

Physiological deterioration contributes significantly towards reducing the storage life of seed, thereby resulting in a loss of precious genetic material (Stanwood, 1985). Storage temperatures down to -20° C have been effective in prolonging storage life of a seed sample, yet cryopreservation technology (LN storage at -196° C) offers the potentiality for 'indefinite' storage of seed germplasm (Roberts, 1975).

Of the two major categories of seeds, those referred to as desiccation tolerant (orthodox) can be dried to 1-3 percent moisture without suffering viability loss whereas desiccation sensitive ones (recalcitrant) get killed on being subjected to drying below a critical value, generally between 12 and 35 percent moisture content (Roberts, 1972).

Orthodox seeds

Numerous reports have appeared in literature wherein seeds of common agricultural and horticultural species have been shown to be tolerant to desiccation and exposure to LN (Stanwood, 1985). About 155 species and 455 accessions have been included in a list prepared by Stanwood (1985) which strongly support that LN preservation of such seeds is a feasible proposition.

Lipman and Lewis (1934) demonstrated that seeds of sugarcane, spinach, cucumber, sugarbeet, buckwheat, barley, oat, onion, mustard and Melilotus spp. could be stored for 30 days and those of pea, corn, squash, alfalfa and sunflower for 60 days at -196° C without any decline in their germinability or vigour. Both laboratory and greenhouse evaluation confirmed this. Later, Lipman (1936) reported that seeds of wheat, barley, tobacco, flax, buckwheat, spinach, milo, maize and Melilotus spp. subjected to -272° C for several hours showed no difference in germinability when compared with the controls.

According to Sakai and Noshiro (1975), seeds of rice, winter wheat, soybean, alfalfa and Italian ryegrass having less than 8 percent moisture content were not damaged by liquid nitrogen.

Seeds of 14 vegetable and two flower species survived liquid nitrogen storage at -196° C for 180 days without any loss in germination percentage (Stanwood and Roos, 1979). Vigour tests with seeds of Phaseolus vulgaris, Lactuca sativa and Pisum sativum also pointed towards no decline in germination or vigour after exposure to liquid nitrogen.

For several tuber bearing Solanum species, Towill (1982) reported that seeds exposed to liquid nitrogen exhibited no difference in germination percentage and rate of germination when compared with the controls.

Seeds of many fruit and nut crops, such as Prunus sp., Juglans sp., Corylus sp. and Coffea sp. can be dried to moisture contents lower than 10 percent but cannot tolerate temperatures lower than - 40° C (Stanwood, 1985), and in many cases get killed. High amount (60 to 70 percent) of storage lipids have been expected to play a role in seed sensitivity towards LN though it is not clearly established. Thorough research is needed to characterize the behaviour of such seeds during LN cooling/ rewarming cycle. The studies on cryopreservation of such species are extremely important as these species are being propagated and preserved currently through vegetative propagules.

Recalcitrant seeds

The past fifty years have witnessed many different methods of storage for long-term conservation of recalcitrant seeds but none has resulted in storage for more than a year (Chin, 1988). This group includes many large-seeded tree species, such as coconut, rubber, cacao, jackfruit and also oil palm (Grout et al., 1983). According to Bajaj (1987), an effective way to conserve recalcitrant species could be through cryopreservation of excised embryos. Induction of somatic embryos in such species can be greatly useful for their use in cryopreservation (Chin, 1988).

Manipulation of seed moisture content is important in cryogenic storage of recalcitrant seeds (Stanwood, 1985) as there is threshold of seed moisture above which a decrease in viability of seed sample occurs during cooling to a temperature of LN (Chin, 1988). It has been suggested that, for storage purposes, embryos of some recalcitrant seeds can be dried to a value below their critical moisture level and later subjected to LN treatment. Alternatively, 'colligative cryoprotection' can also be used for conservation of recalcitrant species. In this, cell water is replaced with a cryoprotectant, such as glycerol, DMSO or proline (Merryman and Williams, 1981).

There are reports that excised embryos of Artocarpus heterophyllus dried to 11 percent moisture content could survive in culture (Chin, 1988). Pritchard and Prendergast (1986) also observed that there was survival of tissue from desiccated and cryopreserved embryos of Araucaria hunsteinii. Partial desiccation of embryos prior to LN exposure has been employed with rubber seeds. In this species, Normah et al. (1986) observed that following LN exposure for 24 h, 20 to 69 percent of excised embryos formed seedlings with normal roots and shoots when cultured in-vitro. These excised embryos had moisture content between 14 and 20 percent (desiccation for 2-5 h).

Interestingly, certain species e.g. Citrus and oil palm once designated as recalcitrant, now have been shown to be orthodox in behaviour following removal of seed coat. In the former, i.e., Citrus limon, Mumford and Grout (1979) observed that seeds without seed coat could be dried to a low moisture content and subjected to LN treatment without significant loss of viability. In oil palm, plantlets could be raised from excised embryos desiccated to about 10.4 percent moisture content and stored at -196° C for about 8 months (Grout et al., 1983).

New conservation strategies - Indian scenario

To meet the conservation requirements of the vegetatively propagated plant species, NBPGR established a National Facility for Plant Tissue Culture Repository (NFPTCR), a project funded by Department of Biotechnology. This project started operating in 1986 with the objectives of using tissue culture (in-vitro) and cryopreservation technology for medium and long-term conservation of germplasm. The in-vitro methods involve use of meristems, shoot tips, axillary buds, embryos, pollen and roots whereas freeze preservation would include seeds, pollen and in-vitro culture systems. The associated aspects, such as monitoring of genetic stability through morphological and biochemical characterization (isozyme polymorphism) and cytological techniques involving Q and Giemsa banding also receive adequate emphasis in this national programme. For in-vitro conservation, major thrust has been assigned to tuberous and bulbous crops, spices, plantation and new industrial crop plants, fruit crops as well as medicinal, aromatic and endangered plant species. The schematic representation of the NFPTCR activities is given in the flow chart (Fig. 1).

The research carried out during the past four years (1987-90) have led to significant achievements (Table 1). Among tuberous crops, high priority has been assigned to sweet potato and yams. Using MS medium with growth regulators, 56 promising strains of sweet potato have been cultured using nodal segments as explants. Cultures have been maintained with 3 percent mannitol upto 16 months at 25° C. In-vitro culture work on Dioscorea alata and D. esculenta has also made substantial progress. In bulbous group, emphasis has been given to garlic (Allium sativum) and other wild Allium species. The research using shoot bud explants have led to the conservation of 38 accessions of A. sativum at 10° C for nearly 16 months. In-vitro conservation experiments are being continued to conserve Allium germplasm at 4° C. In-vitro bulbing has also been observed. Further research work has been intensified to induce bulbingin other species of Allium. Successful establishment of plantlets in the field has been accomplished.

In-vitro multiplication of ginger (Zingiber officinale) and three species of turmeric (Curcuma domestica, C. feruginosa and C. longa var. koova) have been achieved with adequate multiplication rate. Field establishment and survival among above two important spices has been 100 percent. In-vitro storage experiments using mannitol and low temperature are in progress. Replacement of cotton plugs with polypropylene caps has resulted in prolonging the sub-culture cycle upto 7 months (Balachandran et al., 1990).

Among fruit crops, high priority has been accorded to banana, citrus and grapes due to the problems of pathogens. In-vitro cultures of 95 promising cultivars of banana of Indian origin have been established. These belong to both diploid and triploid forms of Musa acuminata and M. balbisiana as well as their inter-specific hybrids. In-vitro multiplied plantlets have been directly transferred and established in the field. For in-vitro conservation, low temperature (15° C) incubation and mannitol have been successfully prolonging storage for full 2 years. Isozyme analyses involving six enzyme systems have been accomplished.

In lime (Citrus aurantifolia), plantlets have been regenerated from root cultures. Shoot buds were found to originate de novo from roots. Plantlets have been established in the field. Histological studies have confirmed the origin of shoots from pericycle tissue.

Fig. 1. Schematic representation of in-vitro conservation and cryopreservation of germplasm in National Plant Tissue Culture Repository, NBPGR

Table 1. Status of in-vitro conservation programme at NFPTCR


No. of spp./accessions in culture

Field establishment

Storage temp. °C

Media modification

** Storage period (months) M

Cytological analysis

Isoenzymes analysed

Allium sativum






Five enzyme systems

Allium tuberosum





Allium spp. (Wild)





Ipomoea batatas






Five enzyme systems

Dioscorea alata



D. esculenta



Zingiber officinale





Eight enzyme systems

Curcuma spp.





Three enzyme systems

Musa spp.






Six enzyme systems



Citrus aurantifolia and other spp.






Rauvolfia serpentina





Coleus forskohlii




Tylophora indica




Sausurrea lappa




* C. aurantifolia (lime) in-vitro established plants showed no chromosomal variation
** Experiments contd.
Several medicinal and aromatic plants and endangered plant species of Indian origin have also been studied. Prominent among these are Rauvolfia serpentina, Tylophora indica, Saussurea lappa, Pogostemon patchouli, Gentiana kurroo and Coleus forskohlii. Using nodal segments, high multiplication rate and field establishment with 100 percent survival has been accomplished. The cultures have been continuously maintained for well over six months at 15° C.

Cryopreservation has been attempted for several orthodox seed species e.g. amaranth, sunflower, Brassica, sesame, minor millets, pearl millet and several vegetable crops. Seeds have maintained their viability and germination potential for well over 3 years (Chaudhary et al, 1989; Chandel and Chaudhary, 1990) following storage in liquid nitrogen (-196° C). Freeze preservation of cardamom seeds in liquid nitrogen has proved successful. Preservation of pollen was undertaken on Brassica species, sesame and primitive landraces of maize and its wild related genera and species from Central America.

Biochemical characterization using seed protein polymorphism, gel electrophoresis and isozyme analysis has been undertaken on several in-vitro generated cultures as well as seeds and pollen. Cytological studies were also being undertaken for monitoring of the genetic stability.

Future perspectives

The past two decades have witnessed the development of new techniques for plant germplasm conservation which offer new options and permit conservation of diversity in the form of seeds, pollen, embryos and in-vitro cultures (Chandel et al., 1988). In-vitro conservation using meristem and shoot tip cultures under minimal media, low temperatures and low light intensity provides adequate methods for germplasm conservation. Cryopreservation of seeds, pollen and in-vitro cultures offer a unique method for preservation and use of germplasm. These can be especially rewarding for vegetatively propagated crops, plant species with recalcitrant seeds, wild and endangered species and also wild relatives of crop plants. The rapid strides made in the past few years with regard to these novel approaches have enhanced the value of genebanks and clonal repositories.

The phenomenon of somaclonal variation associated with certain culture systems, in any in-vitro conservation programme needs to be looked into carefully. The instability has been documented to occur at the karyotypic, morphological and biochemical levels. It involves polyploid and aneuploid changes, structural changes in chromosome morphology and mitotic aberrations. The majority of these changes occur during culture. For example, callus lines which were derived from isolated single cells have been shown to contain diploid, tetraploid and aneuploid cells. Relatively, cryopreservation offers a potential method as evident by the fact that more than 50 species have been successfully freeze preserved. About 30 species can be cryostored in the form of cell cultures. Despite substantial data, it is difficult to recommend universally applicable storage protocols. This necessitates concerted research efforts on the study of behaviour of cells and cell constituents at all the stages of cryopreservation. If cryopreservation ever becomes a routine method, it could be used in the storage of plant tissue cultures with important biochemical and morphological characters and the long-term storage of tissue culture collections in any repository. There is need to establish strong linkages between institutions and centres of excellence involved in research on different aspects of genetic conservation. Gene cloning technology has led in recent years to fundamental advances in many areas of biology. The use of cloned fragments of chromosomal DNA as genetic markers, usually termed 'RFLPs' (Restriction Fragments Length Polymorphism) has acquired significant importance. This new technology promises to revolutionize some areas of plant genetics, crop breeding and genetic conservation. It is evident that isozyme characterization and RFLP markers can be directly used as probes for the presence or absence of certain chromosome segments. Thus, early research on RFLP analysis will be of immense value and research efforts have to be initiated right now to detect genetic variations in repositories. Eventually, various approaches discussed in this account will serve the useful purpose of creating awareness among scientific community and would contribute significantly in establishing conservation programmes, particularly in the gene rich countries of the third world, in the right perspective.


Recent years have witnessed significant advancement in researches demonstrating increasing applications of in-vitro techniques and cryopreservation technology in the genetic conservation of germplasm particularly of vegetatively propagated crop plants and other difficult materials. In-vitro techniques now provide suitable approaches which can lead to the safe conservation of germplasm employing slow growth procedures through the use of minimal media, osmotica, growth retardants or subjecting cultures to the reduction of light/temperatures. Long-term preservation of meristems, shoot tips, embryos (both zygotic and somatic), embryonic axes, protoplast and cell suspension culture is possible. These can be successfully freeze preserved in liquid nitrogen (-196°C) and plantlets could be established at ease. Cryopreservation of seeds, pollen, excised embryos/embryonic axes and buds has also proved feasible and practical in many cases. However, genetic stability in such programme is of utmost importance which is simultaneously monitored using morphological, cytological and biochemical parameters. Molecular techniques, such as isozymes and RFLP offer strong tools in the characterization and genome mapping and help in detecting genetic variants. National Plant Tissue Culture Repository at NBPGR has made significant progress in the field of in-vitro conservation technology on several vegetatively propagated species and their wild relatives. Similarly, cryopreservation work has also received adequate emphasis. The chapter briefly discusses these recent approaches in genetic conservation of valuable plant resources and highlights its important role.


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