Gene Banks in vitro recovery environment The embryos of more than 50 species have been successfully cryopreserved Other parts of the plant can also be used to establish ex situ gene banks for species with nonbankable seeds, as described in the following section Vegetative Parts of Plants Vegetative tissues of both pteridophytes and bryophytes can be banked for germplasm preservation (Pence, 2000) Gametophytes of many bryophyte species are naturally adapted to desiccation stress and can be cryopreserved after sufficient drying Gametophytes of pteridophytes and some desiccationintolerant bryophytes can also be frozen when provided with some cryoprotection, such as encapsulation in alginate beads followed by dehydration or the use of abscisic acid and the amino acid, proline, as a pre-treatment Shoot tip freezing of sporophytes of pteridophytes has also been demonstrated It is estimated that fewer than 200 taxa of bryophytes and pteridophytes combined are currently banked worldwide using vegetative tissues, primarily at the Cincinnati Zoo and Botanical Garden and the University of Kansas in the United States, but there is significant potential for increasing this number The National Seed Storage Laboratory of the U.S Department of Agriculture (USDA) also cryopreserves about 1700 apple lines using dormant scion sections, which are retrieved by grafting (i.e., no culture of meristems) The lines are mainly from Malus x domestica, plus 10 to 15 other apple species Some pear and cherry species (Towill and Forsline, 1999) are also banked in this way Apical shoot tips and other meristems/buds are the most popular vegetative materials for cryopreservation Lines from about 50 species are now routinely cryopreserved Initial studies used shoot tips from cold hardy, temperate zone species (apple, pear), but cryopreservation methods have been extended to tropical zone species (banana, pineapple) Successful cryopreservation depends on defining the physiological adaptation of the stock plant, the explant size and type, and its water content, the steps in the preservation process (cryoprotectant concentrations and rates of addition/removal; cooling/warming rates), and the recovery system Two-step cooling procedures have been useful for some species, but vitrification procedures (solution-based systems and encapsulation/dehydration systems) are more favored because of the technical simplicity (Sakai, 1993) All methods are designed to reduce ice crystal growth in the specimen Other vegetative material that has been cryopreserved using similar methodological approaches include cell suspensions and callus (more than 40 species), protoplasts (more than 10 species), and root cultures (5 species) It should be noted that cryopreservation of vegetative germplasm overcomes the problem of genetic instability during storage as all cellular divisions and metabolic processes are stopped In contrast, two other methods of in vitro preservation, normal and slow growth techniques, run the risk of genetic changes (somaclonal variation) in the conserved germplasm that may result in the loss of distinct genotypes Species are stored under normal growth conditions (e.g., Coffea at 27 1C) for short-term purposes only The explant 651 (usually meristem or nodal cutting) is frequently transferred (subcultured) to fresh nutrient medium with the risk of microbial contamination, or loss through human error To retard growth and hence extend the subculture interval, temperature and light intensity are reduced For example, to 1C and 1000 Lux are generally used for cold tolerant species, and 15 to 22 1C and reduced light intensity for tropical species Alternatively, growth can be slowed down by the addition to the medium of chemicals to induce mild osmotic stress (e.g., mannitol) or hormonal retardants (paclobutrazol, abscisic acid) Also, maintenance of tissue under reduced oxygen (e.g., under mineral oil or liquid medium) slows growth Under the appropriate conditions subculture intervals can be extended to one year or longer The slow growth technique is now routinely used for the medium-term conservation of a number of species such as banana, potato, yam, cassava, and strawberry Although in vitro culture without cryopreservation poses considerable threat of genetic drift, the propagation of plant material in an aseptic environment ensures the production of disease-free stock material, which is readily accessible internationally because it satisfies most country quarantine requirements Undoubtedly, in vitro culture is a valuable complementary approach to field conservation and is particularly useful when applied to species that are predominately propagated vegetatively (banana, potato, and pear), produce non-bankable or highly heterozygous seeds, and have a particular gene combination (i.e., elite genotypes; see Ashmore, 1997) The impact of these positive features of the technique is such that FAO estimates that 37,600 accessions of plant material (vegetative and embryos) are conserved in vitro (including cryopreservation) worldwide Animal Germplasm Samples A majority of ex situ animal germplasm is maintained in zoological gardens and institutes around the world The NIAR in Japan holds 621 accessions of animal germplasm, including silk worms in the living state More than 100 of these accessions, mostly sperm, are cryopreserved (for a general methodology, see the discussion presented later) Similarly, the main gene bank methodology for ova is cryopreservation At present, however, procedures for sperm and ova preservation are not well developed for wild species, even though a number of reported successes with artificial insemination and frozen semen can be found in the literature, especially for ungulates such as deer and antelopes The concept of gamete rescue from tissues has considerable value for spermatozoa, where epididymal spermatozoa are readily obtainable post mortem and can be frozen using glycerol as a reasonably standard cryoprotectant Oocyte cryopreservation has only been achieved in the hamster, rat, rabbit, and cow and is therefore not a practical proposition at present Interest in freezing ovarian tissue, and then culturing follicles and oocytes by various methods after thawing, has recently been resurrected and progress has included the birth of a lamb originating from ovarian tissue autotransplanted into the donor-recipient after freezing and thawing In another recent study, isolated rat spematogenic cells were transferred to a mouse testis, where they displayed the ability to develop into