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Daniel Baskaran Krishnapillay
Forest Research Institute Malaysia, Kepong, Malaysia

A large proportion of plant species produce seeds that can be dried to a sufficiently low moisture content that permits them to be stored at low temperatures. These seeds are termed orthodox (Roberts 1973). There is another category of seeds that are termed as recalcitrant. A number of tropical fruit and timber species fall into this category. In Malaysia, which houses 6% of the world's flowering plant species, a majority of these flowering species produce recalcitrant seeds. These recalcitrant seeds cannot tolerate desiccation to low moisture contents and remain viable only for a short time ranging from a few days to a few weeks. Another category of seed as described by Ellis et al. (1990) comprises seeds that can be desiccated to fairly low moisture contents but which do not withstand exposure to low temperature. Even though the storage of these seeds can be prolonged from a few months to some years, their long-term conservation as seeds is still not possible.

Traditionally, the field genebank has been the ex situ method of choice for those species which produce recalcitrant seeds or that are propagated vegetatively. This method of conservation, however, presents certain drawbacks which limit its efficiency and threaten its security. Genetic resources in field genebanks remain exposed to pests, diseases and natural calamities such as droughts, fire and flood. Furthermore, cost of maintaining these germplasm as field collections are very prohibitive in terms of land and costs.

In Malaysia, the following methods are/have been tested and/or devised as protocols for the mid to long term storage of recalcitrant seeded v. forest tree species:

In vitro propagation for germplasm conservation
Tissue culture techniques provide the opportunity for very high rapid multiplication rates in aseptic environment of those desired germplasm. The in vitro propagation of mature forest trees is faced with a number of difficulties at various stages of the propagation process, including notably high levels of contamination in initial explants, high secretion of polyphenols and tannins which inhibit the development of the explants and often cause necrosis, vitrification and low-rooting ability.

Juvenile tissues have been seen to be the most responsive in culture. Marcotts from selected trees have been taken and raised in the nursery and ex-plants from these have been used as starting materials for initiating cultures. Successful examples have been for Acacia mangium, Acacia auriculiformis, Dyera costulata, Tectona grandis, Azadirachta excelsa, Aqualaria malaccense, Calamus manan (Aziah et al. 1992, 1994, 1999; Fadillah et al. 1999). Clonal materials propagated in this manner can be safely stored, planted out or used in the germplasm exchange programmes. For storage in vitro, the culture medium and the physical environmental growth conditions are modified to reduce the growth rate of the plantlets.

Classical cryopreservation procedures comprise a pre-treatment with cryoprotective substances followed by slow controlled freezing. Such procedures have been successful with culture systems consisting of small units of uniform morphology such as in protoplast culture, actively dividing cell suspension culture and fragmented callus cultures (Withers and Engelmann 1995). However, this method gives erratic results for culture systems that consist of large units comprising a mixture of cell sizes and types, such as shoot-tips, zygotic embryos or relatively mature somatic embryos (Krishnapillay and Englemann 1996; Krishnapillay 1999). Currently, reproducible and efficient methods are available such as encapsulation,/dehydration, vitrification, desiccation and pre-growth desiccation (Englemann 1999).

The encapsulation-dehydration technique is based on the technology developed for the production of synthetic seeds (Redenbaugh 1993). For cryopreservation, apices, somatic or small zygotic embryos are encapsulated in a bead of alginate and pregrown for various duration in liquid medium with high sucrose concentrations. Beads are then partially dehydrated under the air current of a laminar flow cabinet or using silica gel, down to a water content of about 20%. Freezing is usually rapid, by direct immersion of the samples in liquid nitrogen. For recovery, samples are usually placed directly under standard culture conditions. Growth recovery of the cryopreserved material is generally rapid and direct, without callus formation. This technique has been successfully used for the zygotic embryos of Swietenia macrophylla (mahogany) (Marzalina et al. 1994). Reports show that successful extension of this protocol for conservation has been performed routinely on 11 varieties of pear, 9 varieties of apple and 14 varieties of sugarcane.

Vitrification consists in placing samples for pretreatment in extremely concentrated cryoprotective solutions and freezing them ultra-rapidly. In these conditions, the intracellular solutes vitrify i.e. form an amorphous glassy structure, thus avoiding the formation of intercellular ice crystals, detrimental for cell survival. Vitrification procedures have been developed for cell suspensions, somatic embryos and apices of various species (Sakai 1993; Takagi et al. 1997; Thinh et al. 1999). Recently this technique has been successfully used for the cryopreservation of zygotic embryos of Arthocarpus heterophyllus and Naphelium lappaceum ( Wong 1999 unpubl.; Tammasiri 1999; Ginibun 1999 unpubl.).

Cryopreservation using a desiccation procedure is the simplest approach. It consists of dehydrating the plant material, then freezing it rapidly by the direct immersion in liquid nitrogen. A number of tropical forest trees such as Dipterocarpus alatus, D. intricatus and Pterocarpus indicus and palms like Veitchia merrillii and Howea fosteriana have been successfully cryopreserved using this technique (Chin et al. 1988; Krishnapillay et al. 1992, 1994).

Seedling storage under low light conditions
On a commercial scale for the continuous supply of planting materials of recalcitrant seeds, it is necessary to develop complementary methods to cryopreservation that are easily executed by nurserymen. It is well established that dipterocarp seedlings usually have low survival and slow growth rates over a period of several months when grown under subdued light. The idea of using this phenomenon was first proposed by Hawkes (1980). Two methods outlined below have been tested.

These are (a) storage of germinated seeds in a controlled chamber and (b) storage of germinating seeds on the forest floor under subdued light conditions.

Seedling Chamber
With this method freshly collected seeds are surface treated with a fungicide (0.1% Benlate/Tiram mixture) and allowed to germinate under ambient conditions in containers kept at high humidity with moistened tissue paper. After radicle emergence, germinated seeds are packed loosely in polythene bags, trays or boxes lined with moist tissue paper and stored in a specially constructed seedling chamber in which the temperature, humidity and light are controlled. The temperature is 16° C, the relative humidity 80% and the photoperiod 4 hours. Light is supplied from a fluorescent source, giving 800-1000 lux.

Development of the germinated seeds into seedlings occurs slowly in the chamber. Seventeen dipterocarp species have been tested to date and the storage period ranges 4-12 months (Krishnapillay and Tompsett 1998).

Seedlings developed slowly in the chamber, barely attaining heights of 20-25 cm over the storage periods tested. Seedlings which were transferred to the nursery and grown in polythene bags needed to be weaned in at least 70% shade for a period of 2-3 weeks before they could be placed under direct sunlight. Survival percentage was between 60-80%, dependent on the species.

Forest Floor
The second approach for storage of seedling is on the forest floor under subdued light. Areas are cleared of undergrowth and freshly collected seeds are sown. Seedlings develop very slowly and so can remain within manageable height for long periods of time.

Seedlings of Hopea odorata did not grow to a height greater than 10 cm under these conditions over a period of three years. Seedlings transferred to the nursery and grown in polythene bags began to increase in size rapidly. Weaning in 70% shade for two weeks before transfer to direct sunlight was however necessary. Survival was approximately 80-90% depending on the species. Approximately 8 species have been tested to date.

The constraints with this method are as follows: in the early stages after sowing, unprotected seeds are likely to be predated by squirrels, birds and wild boars. Fencing the area with barbed wire and covering the seed with a plastic sheet is thus necessary. The plastic sheet can be removed when the seedlings have emerged when damage by birds and squirrels is unlikely.

Cryopreservation offers interesting technical possibilities for the conservation of valuable germplasm compared to conventional preservation systems. For the tropical recalcitrant forest tree species, there are several prerequisites which have to be fulfilled before long-term in vitro conservation/storage is to be considered.

With proper reduction in moisture content and controlled desiccation, seed-derived material can be stored in liquid nitrogen. Over issues relating to continuous supply of planting materials of recalcitrant seeded species, the slow growth strategy of storing germinated seeds either in special chambers or on the forest floor under subdued light although does not offer a long term storage solution but nevertheless, provides an option for mid term storage of such species.

1Received July 2000. Original language: English


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Chin, H.F., B. Krishnapillay and Z.C. Alang (1988). Cryopreservation of Veitchia and Howea palmembryos: non-development of the haustorium. Cryoletters 9: 372-379.

Darus H.A. and M.Y. Aziah (1993) Mass propagation of Dyera costulata for forest plantations. Paper presented at the 2nd Symposium on Trends in Biotechnology: Meeting the challenge of the 21st Century. 30th Nov. - 2nd December, 1993. Universiti Pertanian Malaysia, Serdang, Malaysia.

Ellis, R.H., T.D. Hong and E.H. Roberts (1990). An intermediate category of seed storage behaviour? J. Exp. Bot. 41:1167-1174

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Krishnapillay D.B, and P.B. Tompsett (1998). Seed Handling. In: Appanah S. and J.M. Turnbull (Eds.). A Review of Dipterocarps-Taxanomy, Ecology and Silviculture. Centre for International Forestry Research (CIFOR), Bogor, Indonesia.

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Krishnapillay, D.B. (1999) Towards the Use of Cryopreservation as a technique for Conservation of Tropical Recalcitrant Seeded Species. Pp. 137-163 In: Razdan M.K. and E.C. Cocking (Eds.). Conservation of Plant Genetic Resources in vitro. Vol 2: Applications and Limitations. Science Publishers, Inc. U.S.A.

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Marzalina M., M.N. Normah and B. Krishnapillay (1994) Artificial seeds of Swietenia macrophylla. Pp. 132-134. In: Krishnapillay B., M. Haris, M.N. Normah and L.G. Lim (Eds.). Proceedings of the 2nd National Seed Symposium. Cawangan Pembangunan Komoditi, Jabatan Pertanian Malaysia, Kuala Lumpur, Malaysia.

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Sakai, A. (1993). Cryogenic Strategy for survival of plant cultured cells and meristems cooled to -196°C. Cryopreservation of Plant Genetic Resources: Technical Assistance Activities for Genetic Resources Project. Japan International Cooperation Agency.

Takagi, H., N.Tien Thinh, O.M. Islam, T. Senboku and A. Sakai (1997). Cryopreservation of in vitro grown shoot tips of taro (Colocasia esculenta Scott) by vitrification. 1. Investigation of Basic condition of the vitrification procedure. Plant Cell Rep. 16: 594-599.

Thammasiri, K. (1999) Cryopreservation of embryo axes of Jackfruit. Cryoletters 20: 21-28

Thinh, N.T., H. Takagi and S. Yashima (1999) Cryopreservation of in vitro grown shoot tips of banana (Musa spp.) by vitrification method. Cryoletters 20: 163-174.

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