Cryopreservation of Tropical Plant Germplasm

539 Pages·2012·2.59 MB·English

iv Cryopreservation of Tropical Plant Germplasm
Foreword
Cryopreservation, i.e. the storage of biological material at ultra-low temperature,
usually that of liquid nitrogen (–196°C), is the only method currently available to
ensure the safe and cost-effective long-term conservation of genetic resources of
species that have recalcitrant seeds or are vegetatively propagated.
Dramatic progress has been made over the last 10 years in this area with the
development of cryopreservation techniques for well over 100 plant species.
Cryopreservation protocols are increasingly becoming available for routine
application in genebanks. However, much of the work to date has been done on
temperate species, with research on tropical and subtropical species lagging
behind. This is of particular concern given the large number of tropical species
that are either vegetatively propagated or that produce recalcitrant seeds.
Both JIRCAS and IPGRI are heavily involved in cryopreservation research. In
the framework of its Visiting Fellowship Programme, JIRCAS has carried out a
project specifically to develop techniques for the long-term preservation of
vegetatively propagated crop germplasm. During the project, visiting scientists
from developing countries have developed cryopreservation protocols for
selected tropical crops. For more than 15 years, IPGRI and its predecessor IBPGR
has supported cryopreservation research in collaboration with partners in Asia,
the Pacific and Oceania, Africa, the Americas and Europe.
As a result of their experience in the field of cryopreservation, JIRCAS and
IPGRI, in October 1998, jointly organized an international workshop to assess the
current state of the science, to explore cryopreservation applications and to examine
outstanding problems. The focus of the workshop was on the use of
cryopreservation to conserve the germplasm of tropical plant species. An additional
objective was to identify priority areas for collaborative research, technology
development, transfer and application. The workshop was attended by a large
number of cryopreservation experts from both developing and developed countries
who presented their latest research results and contributed to the discussions.
This publication of the proceedings of the workshop thus presents a
comprehensive overview of current knowledge concerning the biological and
physical mechanisms involved in cryopreservation, and the status of the
development of protocols for new species and their application in genebanks. We
trust that it will help to stimulate further collaborative research and thus
contribute to the wider application of cryopreservation for the safe long-term and
cost-effective conservation of genetic resources of tropical species.
Nobuyoshi Maeno, Director General Geoff Hawtin, Director General
Japan International Research Center for International Plant Genetic
Agricultural Sciences (JIRCAS) Resources Institute (IPGRI)
Tsukuba, Japan Rome, Italy
v
Acknowledgements
The editors hereby acknowledge the invaluable contributions made by Profs.
Akira Sakai and H.F. Chin to the development of cryopreservation techniques
and their application to the long-term conservation of tropical crop germplasm,
and to the planning and implementation of this Workshop. They also wish to
thank JIRCAS, SGRP and IPGRI for financial contributions for the publication of
these proceedings.
Keynote presentations 1
Keynote presentations
Development of cryopreservation techniques
Akira Sakai
Sapporo, 001-0045 Japan
Recent advances in cryogenic procedures
For successful cryopreservation, it is essential to avoid lethal intracellular
freezing which occurs during rapid cooling in liquid nitrogen (Sakai and Yoshida
1967; Sakai 1985, 1995). Thus, specimens to be preserved have to be sufficiently
dehydrated to avoid intracellular freezing and thus vitrify upon rapid cooling in
liquid nitrogen. Vitrification may be the only freeze-avoidance mechanism that
enables hydrated cells, tissues and organs to survive at the temperature of liquid
nitrogen (Sakai 1960, 1965, 1995), as suggested by Luyet as early as 1937.
Vitrification, a physical process, can be defined as the phase transition of an
aqueous solution from a liquid into an amorphous glassy solid, or glass, at the
glass transition temperature (Tg), while avoiding ice crystallization.
A glass fills spaces in a tissue, and during dehydration may contribute to
preventing additional tissue collapse, solute concentration and pH alterations.
Operationally, a glass is expected to exhibit a lower water vapour pressure than
the corresponding crystalline solid and to thereby prevent further dehydration.
As glass is exceedingly viscous and stops all chemical reactions that require
molecular diffusion, its formation may lead to dormancy and stability over time
(Burke 1986).
Fahy et al. (1984) presented a highly concentrated vitrification solution for
successful cryopreservation of animal embryos. This solution easily supercools
down to a temperature below –100°C and finally solidifies into a metastable glass
at the glass transition temperature (Tg: about –110°C) at a practical cooling rate.
For plant cryopreservation, some simplified and valuable cryogenic
procedures such as vitrification (Langis et al. 1990; Sakai et al. 1990, 1991; Towill
1990), air-drying (Uragami et al. 1990) and the encapsulation-dehydration
technique (Fabre and Dereuddre 1990) have been presented, and the number of
species to be cryopreserved has increased dramatically over the last 10 years or
so (Engelmann 1997; Sakai 1997). At present, potentially valuable cryogenic
procedures for cryopreserving apical meristems and somatic embryos include
vitrification with or without encapsulation, and the encapsulation-dehydration
technique. These alternative dehydration procedures, referred to as “vitrification-
based techniques” (Engelmann 1997), offer practical advantages compared with
classical prefreezing protocols. They are more appropriate for freezing complex
organs (shoot-tips and somatic embryos) that contain a variety of cell types, each
with unique requirements under conditions of freeze-induced dehydration
(Withers 1979). When the vitrification technique is employed under well-
optimized conditions, the whole or most of the apical meristems remain alive
2 Cryopreservation of Tropical Plant Germplasm
(Yamada et al. 1991; Matsumoto et al. 1994), thus allowing direct, organized
regrowth. By contrast, classical prefreezing procedures can lead to the
destruction of large zones of apical domes, and callusing only or transitory
callusing is often observed before organized regrowth starts (Haskins and Kartha
1980; Touchell and Dixon 1996). By precluding ice formation in the system,
vitrification-based procedures simplify the cryogenic procedure and eliminate
concerns for the potentially damaging effects of intra- and extracellular
crystallization, producing high levels of post-cryopreservation regrowth, and
have greater potential for broad applicability, requiring only minor modification
for different cell types (Engelmann 1997; Sakai 1997).
Vitrification protocol for apical meristems
Vitrification can be achieved by direct immersion of samples in liquid nitrogen
without a freeze-induced dehydration step, by osmotically dehydrating the cells
and meristems in a highly concentrated vitrification solution. Such a technique is
referred to as vitrification (complete vitrification: both cytosol and suspending
solution are vitrified), distinct from conventional prefreezing methods (partial
vitrification, only the cytosol is vitrified). The vitrification procedure requires a
highly concentrated vitrification solution, which sufficiently dehydrates cytosols
without causing injury so that they turn into a stable glass when plunged in
liquid nitrogen. We have developed a glycerol-based, low-toxicity vitrification
solution designated PVS2 (Sakai et al. 1990, 1991), which does not permeate into
the cytosol during the dehydration process. This solution easily supercools below
–100°C at practical cooling rate and finally solidifies at –115°C. More recently, we
have found that a new vitrification solution (PVS4) which contains 35% glycerol
(w/v) and 20% EG (w/v), in the basal medium containing 0.6M sucrose (pH 5.8)
produced nearly the same recovery growth as PVS2.
The acquisition of tolerance to dehydration achieved by exposure to PVS2 and
the mitigation of injurious effects during the dehydration process are essential for
the successful cryopreservation of excised shoot-tips by vitrification. To induce
dehydration tolerance, excised shoot-tips from in vitro grown plantlets are
precultured on medium with high sucrose concentrations (0.3–0.6M) for 16 h and
then treated with a mixture of 2M glycerol plus 0.4M sucrose (LD solution) for 20
min before dehydration with PVS2. In our vitrification method, the injurious
effects caused by the dehydration process with PVS2 are reduced or eliminated by
optimizing the duration of exposure to PVS2, and by dehydrating shoot-tips
gradually in two steps: LD solution at 25°C, followed by PVS2 solution at 0°C.
Table 1 lists the successful reports of cryopreservation of shoot-tips from in
vitro grown plants using vitrification with PVS2. These results clearly demonstrate
that the vitrification protocol has a wide applicability, both in terms of species
coverage, since the protocol has been successfully established for hairy roots,
tubers, fruits, ornamentals and plantation crops of both temperate and tropical
origins, and in terms of number of genotypes/varieties within a given species.
Keynote presentations 3
Table 1. Successful cryopreservation of meristems cooled to –196°C by vitrification using
PVS2 vitrification solution
†
Plant Pretreatment References
Woody plants
Apple (5 spp., cvs.) CH, PC Niino et al. 1992a
Cherry (8 cvs.) CH, PC Tashiro et al. 1995
Grape (4 cvs.) PC, LD Matsumoto et al. 1998b
‡
Grevillea scapigera PC Touchell and Dixon 1996
‡
Grevillea cirsiifolia PC Touchell and Dixon 1996
Mulberry (13 spp., cvs.) CH, PC Niino et al. 1992b
Pear (5 cvs.) CH, PC Niino et al. 1992a
Tea plant CH, PC Kuranuki and Sakai 1995
Herbaceous plants
§
Garlic (12 cvs.) None Niwata 1995
Lily (4 cvs.), Lilium japonicum CH, PC, LD Matsumoto et al. 1995b
Mint (3 cvs.) CH, LD Hirai et al., unpubl.
Strawberry (4 cvs.) CH, LD Hirai et al. 1998
Wasabi (4 cvs.) PC, LD Matsumoto et al. 1994
White clover (3 spp.) PC Yamada et al. 1991
Hairy roots
Armoracia rusticana PC Phunchindawan et al. 1997
Panax ginseng PC, LD Yoshimatsu et al. 1996
Tropical plants
Banana (6 cvs.) PG, PC, LD Thinh 1997
Cassava (2 cvs.) PC, LD Charoensub et al., unpubl.
Orchid
Cymbidium (2 cvs.) PC, LD
Dendrodium (protocorm) ABA Thinh 1997
Pineapple PC, LD Wang et al. 1998
Tannia (2 spp.) PC, LD Thinh 1997
Taro Thinh 1997
Colocasia var. antiquorum (2 cvs.) PG, PC, LD Takagi et al. 1997; Thinh 1997
Colocasia var. esculenta (4 cvs.) PG, PC, LD Thinh 1997
Yam
Dioscorea rotundata (3 cvs.) PC Kyesmu et al. 1997
†
ABA: abscisic acid; CH: cold-hardening; LD: loading treatment with 2M glycerol plus
0.4M sucrose; PC: preculture; PG: pregrowth of meristem-donor plants on sucrose-
enriched medium for 1 month; cv: cultivar, sp: species.
‡
Australian endangered woody plants (6 other species).
§
Post-dormant bulbs.
Comparative study of different cryogenic procedures
Shoot formation from apical meristems of various plants or cultivars cooled to
-196°C was compared for different cryogenic protocols. With most of the plants
tested, the vitrification method with or without encapsulation produced much
higher levels of shoot formation than the encapsulation-dehydration technique
under optimal conditions (Matsumoto and Sakai 1995; Hirai et al. 1998, unpubl.).
The same results were observed with endangered Australian plants (Touchell
4 Cryopreservation of Tropical Plant Germplasm
1995; Touchell and Dixon 1996). In addition, growth recovery was much quicker
with vitrified meristems than with encapsulated meristems (Matsumoto and
Sakai 1995; Hirai et al. 1998). Thus, the vitrification method certainly offers
considerable advantages over the encapsulation-dehydration technique for the
cryopreservation of apical meristems. These results suggest that the induction of
dehydration tolerance by sucrose alone (0.8M sucrose for 16 h) may be insufficient
for many meristems (Mandal et al. 1996). Thus, we presented a revised technique
using a mixture of sucrose and glycerol (Matsumoto and Sakai 1995;
Phunchindawan et al. 1997). More recently, we have found that treating apices with
a mixture of 2M glycerol plus 0.6M sucrose for 90 min, followed by air-drying for
3–4 h, produced higher levels of growth recovery (94% with wasabi) than the
original encapsulation-dehydration procedure (about 60%) (Sakai et al., unpubl.).
Key problems for successful cryopreservation by vitrification
The key for successful cryopreservation by vitrification is to induce tolerance of
specimens to dehydration with a highly concentrated vitrification solution. In the
vitrification method, cells and excised meristems are usually precultured on
sucrose- or sorbitol-enriched medium for 1 or 2 days to induce dehydration
tolerance (Yamada et al. 1991; Matsumoto et al. 1994; Touchell 1995; Reinhoud
1996; Thinh 1997). A high level of sugar or sorbitol accumulated during
preculture has been reported to be very important in improving the survival of
cryopreserved cells and meristems (Uragami et al. 1990; Dereuddre et al. 1991;
Reinhoud 1996; Matsumoto et al. 1998a). The accumulation of sugars increases
the stability of membranes under conditions of severe dehydration (Crowe et al.
1989). Reinhoud (1996) clearly demonstrated that the development of tolerance of
cultured tobacco cells to PVS2 during preculture with 0.3M mannitol solution for
1 day appeared to be a combined result of mannitol uptake and the cellular
response to mild osmotic stress caused by the preculture: production of ABA,
proline and certain proteins including late embryogenesis abundant ones (LEAs).
With apical meristems of numerous species, preculture with sugar or sorbitol
did not lead to substantial increases in growth recovery after vitrification.
However, treatment with LD solution for 20 min following preculture with 0.3M
sucrose for 16 h was very effective in increasing the growth recovery of vitrified
wasabi shoot-tips (Matsumoto et al. 1994). The same results were observed in about
20 tropical monocotyledons (Thinh 1997). Thus, the treatment with LD solution
seems to be an important step in the vitrification procedure for some plants.
The LD solution was reported to be very effective in inducing dehydration
tolerance to freeze-dehydration (Nishizawa et al. 1992) or to PVS2 (Nishizawa et
al. 1993; Matsumoto et al. 1995a, 1995b). During treatment with the LD solution,
the cells are considerably dehydrated and plasmolyzed. However, little or no
permeation of glycerol into the cytosol was observed after a 20-min incubation.
Thus, the protective effect of a brief incubation with LD solution might be a result
of the concentration of cytosolic cryoprotectants accumulated during the
preculture with sucrose, and to the protective effect of plasmolysis. The presence
of LD solution in the periprotoplasmic space of plasmolyzed cells may mitigate
Keynote presentations 5
mechanical stress caused by severe dehydration (Tao et al. 1983; Jitsuyama et al.
1997) and give some protective action to minimize the injurious membrane
changes during severe dehydration (Steponkus et al. 1992), though the
mechanism of action is not well understood.
Successful cryopreservation is not only determined by the cryopreservation
procedure itself, but also by the condition of plant cells (exponential phase) or
apical meristems (age of growth). Thus, for successful cryopreservation, excised
apices must be in a physiological state suitable for the acquisition of
osmotolerance and the production of vigorous growth recovery. This should be
decided empirically and species- or culture-specifically.
Prospects for cryopreservation of tropical plants
Thinh (1997) succeeded in cryopreserving tropical monocotyledons such as taro,
banana, pineapple and orchids, totaling about 20 species or cultivars, using
vitrification with only slight modifications of the technique. More recently, yam
(Kyesmu et al. 1997), cassava (Charoensub, unpublished) and protocorms of
Dendrobium (Wang et al. 1998) have been successfully cryopreserved by
vitrification.
In view of the wide range of efficient and simple vitrification-based techniques
available, many tropical plant species or cultivars could be amenable to
cryopreservation, provided that the tissue culture protocols, such as apical
meristem and somatic embryo culture, are sufficiently operational for the species.
For further development of cryopreservation of tropical plants, studies on
preconditioning for the induction of dehydration tolerance appear to be most
important.
References
Burke, M.J. 1986. The glassy state and survival of anhydrous biological systems. Pp. 358-
363 in Membranes, Metabolism, and Dry Organisms. A.C. Leopold, ed. Comstock, Cornell
Univ. Press, Ithaca and London.
Crowe, J.H., L.M. Crowe, J.F. Carpenter, A.S. Rudolph, C.A. Wistrom, B.J. Spargo and T.J.
Anchordoguy. 1989. Interactions of sugars with membranes. Biochimica et Biophysica
Acta 947: 367–384.
Dereuddre, J., S. Blandin and N. Hassen. 1991. Resistance of alginate-coated somatic
embryos of carrot (Daucus carota L.) to desiccation and freezing in liquid nitrogen. 1:
Effect of preculture. Cryo–Letters 12: 125–134.
Engelmann, F. 1997. Importance of desiccation for cryopreservation of recalcitrant seed
and vegetatively propagated apices. Plant Genetic Resources Newsletter 112: 9–18.
Fabre, J. and J. Dereuddre. 1990. Encapsulation-dehydration: A new approach to
cryopreservation of Solanum shoot tips. Cryo–Letters 11:413–426.
Fahy, G.M., D.R. MacFarlane, C.A. Angell and H.T. Meryman. 1984. Vitrification as an
approach to cryopreservation. Cryobiology 21: 407–426.
Haskins, R.H. and K.K. Kartha. 1980. Freeze-preservation of pea meristems: cell survival.
Canadian Journal of Botany 58: 833–840.
Hirai, D., K. Shirai, S. Shirai and A. Sakai. 1998. Cryopreservation of in vitro-grown
meristems of strawberry (Fragaria x ananassa Duch.) by encapsulation vitrification.
Euphytica 101:109–115.
6 Cryopreservation of Tropical Plant Germplasm
Jitsuyama, Y., T. Suzuki, T. Harada and S. Fujikawa. 1997. Ultrastructural study of
mechanism of increased freezing tolerance to extracellular glucose in cabbage leaf
cells. Cryo–Letters 18: 33–44.
Kuranuki, Y. and A. Sakai. 1995. Cryopreservation of in vitro-grown shoot tips of tea
(Camellia sinensis) by vitrification. Cryo–Letters 16: 345–352.
Kyesmu, P.M., H. Takagi and S. Yashima. 1997. Cryopreservation of white yam (Dioscorea
rotundata ) shoot apices by vitrification. P. 162 in Proceedings of Annual Meeting of Japan
Molecular Biology, 20–12 July 1997, Kumamoto University, Kumamoto, Japan.
Langis, R., B. Schnabel–Preikstas, B.J. Earle and P.L. Steponkus. 1990. Cryopreservation of
carnation shoot tips by vitrification. Cryobiology 276: 658–659.
Luyet, B.J. 1937. The vitrification of organic colloids and protoplasm. Biodynamica 1: 1–14.
Mandal, B.B., K.P.S. Chandel and S. Dwivedi. 1997. Cryopreservation of yam (Dioscorea
spp.) shoot apices by encapsulation-dehydration. Cryo–Letters 17: 165–174.
Matsumoto, T. and A. Sakai. 1995. An approach to enhance dehydration tolerance of
alginate-coated dried meristems cooled to –196°C. Cryo–Letters 16: 299–306.
Matsumoto, T., A. Sakai and K. Yamada. 1994. Cryopreservation of in vitro-grown apical
meristems of wasabi (Wasabia japonica ) by vitrification and subsequent high plant
regeneration. Plant Cell Reports 13:442–446.
Matsumoto, T., A. Sakai, C. Takahashi and K. Yamada. 1995a. Cryopreservation in vitro-
grown apical meristems of wasabi (Wasabi japonica) by encapsulation-vitrification
method. Cryo–Letters 16: 189–206.
Matsumoto, T., A. Sakai and K. Yamada. 1995b. Cryopreservation in vitro-grown apical
meristems of lily by vitrification. Plant Cell, Tissue and Organ Culture 41: 237–241.
Matsumoto, T., A. Sakai and Y. Nako. 1998a. A novel preculturing for enhancing the
survival of in vitro-grown meristems of wasabi (Wasabia japonica) cooled to –196°C by
vitrification. Cryo–Letters 19: 27–36.
Matsumoto, T., A. Sakai and Y. Nako. 1998b. Cryopreservation of in vitro cultured
axillary shoot tips of grape (Vitis vinifera) by vitrification. Supplement, Journal of the
Japanese Society of Horticultural Science 67: 78.
Niino, T., A. Sakai and K. Nojiri. 1992a. Cryopreservation of in vitro-grown shoot tips of
apple and pear by vitrification. Plant Cell, Tissue and Organ Culture 28: 261–266.
Niino, T., A. Sakai, S. Enomoto and S. Kato. 1992b. Cryopreservation in vitro-grown shoot
tips of mulberry by vitrification. Cryo–Letters 13: 303–312.
Nishizawa, S., A. Sakai, Y. Amano and T. Matsuzawa. 1992. Cryopreservation of
asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant
regeneration by a simple freezing method. Cryo–Letters 13: 379–388.
Nishizawa, S., A. Sakai, Y. Amano and T. Matsuzawa. 1993. Cryopreservation of
asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant
regeneration by vitrification. Plant Science 91: 67-73.
Niwata, E. 1995. Cryopreservation of apical meristems of garlic (Allium sativum L.) and
high subsequent plant regeneration. Cryo–Letters 16: 102–107.
Phunchindawan, M., K. Hirata, A. Sakai and K. Miyamoto. 1997. Cryopreservation of
encapsulated shoot primordia induced in horse radish (Armoracia rusticana) hairy root
cultures. Plant Cell Reports 16: 469–473.
Reinhoud, P.J. 1996. Cryopreservation of tobacco suspension cells by vitrification. Doctoral Thesis,
Leiden University, Institute of Molecular Plant Sciences, Leiden, the Netherlands..
Sakai, A. 1960. Survival of the twig of woody plants at –196°C. Nature 185: 393–394.
Sakai, A. 1965. Determining the degree of frost-hardiness in highly hardy plants. Nature
206: 1064–1065.
Keynote presentations 7
Sakai, A. 1985. Cryopreservation of shoot tips of fruit trees and herbaceous plants. Pp.
135–158 in Cryopreservation of Plant Cells and Organs (K.K. Kartha, ed.). CRC Press,
Boca Raton, Florida.
Sakai, A. 1995. Cryopreservation of germplasm of woody plants. Pp. 53–69 in
Biotechnology in Agriculture and Forestry Vol. 32: Cryopreservation of Plant Germplasm.
1 (Y.P.S. Bajaj, ed.). Springer–Verlag, Heidelberg.
Sakai, A. 1997. Potentially valuable cryogenic procedures for cryopreservation of cultured plant
meristems. Pp. 53–66 in Conservation of Plant Genetic Resources In Vitro. Volume 1: General
Aspects. M.K. Razdan and E.C. Cocking (eds.). Science Publishers Inc., Enfield, USA.
Sakai, A. and S. Yoshida. 1967. Survival of plant tissue at super-low temperatures. VI.
Effects of cooling and rewarming rates on survival. Plant Physiology 42: 1695–1701.
Sakai, A., S. Kobayashi and I. Oiyama. 1990. Cryopreservation of nucellar cells of navel
orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Reports
9: 30–33.
Sakai, A., S. Kobayashi and I. Oiyama. 1991. Survival by vitrification of nucellar cells of
navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) cooled to –196°C. Journal of
Plant Physiology 137: 465–470.
Steponkus, P.L., R. Langis and S. Fujikawa. 1992. Cryopreservation of plant tissues by
vitrification. Pp. 1–61 in Advances in Low-Temperature Biology, Vol. 1. P.L. Steponkus
(ed.). JAI Press Ltd., Hampton Mill, UK.
Takagi, H., N.T. Thinh, O.M. Islam, T. Sendai and A. Sakai. 1997. Cryopreservation of in
vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1.
Investigation of basic conditions of the vitrification procedures. Plant Cell Reports 16:
594–599.
Tao, D., P.H. Li and J.V. Carter. 1983. Role of cell wall in freezing tolerance of cultured
potato cells and their protoplasts. Physiologia Plantarum 58: 527–532.
Tashiro, H., T. Niino, J. Magoshi and T. Akihama. 1995. Cryopreservation of in vitro–
grown meristems of cherry by vitrification. In Japanese Society of Plant Tissue Culture
th
14 Annual Meeting Report, p.120.
Thinh, N.T. 1997. Cryopreservation of germplasm of vegetatively propagated tropical monocots
by vitrification. Doctoral Papers of Kobe University, Department of Agronomy, Japan.
Touchell, D.H. 1995. Principles of cryobiology for conservation of threatened Australian plants.
Doctoral Thesis of the University of Western Australia, Botany Division, Australia.
Touchell, D.H. and K.W. Dixon. 1996. Cryopreservation for conservation of Australian
endangered plants. Pp. 169–180 in In Vitro Conservation of Plant Genetic Resources
(M.N. Normah, M.K. Narimah and M.M. Clyde, eds.). Plant Biotechnology
Laboratory, Faculty of Life Sciences, University Kebangsaan, Malaysia.
Towill, L.E. 1990. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell
Reports 9: 178–180.
Uragami, A., A. Sakai and M. Nagai. 1990. Cryopreservation of dried axially buds from
plantlets of Asparagus officinalis L. in vitro. Plant Cell Reports 9: 328–331.
Wang, J.H., J.G. Ge, F. Liu, H.W. Bian and C.N. Huang. 1998. Cryopreservation of seeds
and protocorms of Dendrobium candidum. Cryo–Letters 19: 123–128.
Withers, L.A. 1979. Freeze preservation of somatic embryos and clonal plantlets of carrot
(Daucus carota ). Plant Physiology 63: 460–467.
Yamada, T., A. Sakai, T. Matsumura and S. Higuchi. 1991. Cryopreservation of apical
meristems of white clover (Trifolium repens L.) by vitrification. Plant Science 78: 81–87.
Yoshimatsu, K., H. Yamaguchi and K. Shimomura. 1996. Traits of Panax ginseng hairy
roots after cold storage and cryopreservation. Plant Cell Reports 15: 555–560.