AOBPreview originally published online on November 13, 2002
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Annals of Botany 91: 65-74, 2003
© 2003 Annals of Botany Company
Suitability of Cryopreservation for the Long-term Storage of Rare and Endangered Plant Species: a Case History for Cosmos atrosanguineus
1 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK and 2 School of Plant Sciences, University of Reading, Department of Agricultural Botany, Whiteknights, PO Box 221, Reading RG6 6AS, UK
* For correspondence. Fax +44 20 8332 5310, e-mail m.fay{at}rbgkew.org.uk
Received: 3 January 2002; Returned for revision: 19 March 2002; Accepted: 6 October 2002 Published electronically: 13 November 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The suitability of cryopreservation for the secure, long-term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non-frozen controls.
Key words: Cosmos atrosanguineus, cryopreservation, shoot tips, alginate encapsulation, somaclonal variation, AFLP, TEM.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cryopreservation of plant germplasm has obvious advantages over in vitro storage in terms of space saving and improved phytosanitation (Towill, 1991; Engelmann, 1997). However, in the context of germplasm conservation it is a fundamental requirement that the species in question can be maintained so that regeneration is true to type. The potential for spontaneous genetic alteration in the form of somaclonal variation in long-term tissue culture storage is well known (Scowcroft, 1984), and examples of variation have been reported in Musa spp. (Vuylsteke and Swennen, 1990), Solanum tuberosum L. (Harding, 1991), Vitis vinifera L. (Harding et al., 1996) and others. The risk of genetic instability may be minimized through the selection and optimal use of organized tissues such as meristems or shoot tips (Kartha, 1982). Further problems that arise with in vitro germplasm storage are the different responses of genotypes to standard culture conditions. This can seriously affect the representative gene pool in the collection by loss of those accessions that do not respond well to the culture conditions. The effect of this has been reported following low-temperature storage of Ribes germplasm collection (Brennan et al., 1990) and more recently by Dussert et al. (1997) in an in vitro collection of Coffea spp.
In the cryopreservation procedure developed for the endangered species Cosmos atrosanguineus (Hook.) Voss in Vilm. (Wilkinson et al., 1998), the necessity for cellular dehydration prior to freezing was demonstrated. The removal of available water is necessary to reduce the damage caused to the cells by ice formation (Grout and Henshaw, 1980; Benson, 1999). Under favourable conditions, rapid cooling of the plant tissues in this state will allow vitrification to occur and will prevent ice crystal formation and cellular damage. However, the degree of dehydration required to achieve this may itself cause considerable damage through excessive plasmolysis and osmotic shock. This damage may be responsible for the non-ideal regeneration observed from most shoot tips of C. atrosanguineus, in which callus formation and possible indirect shoot formation were observed. Callus formation was a feature only in post-thaw regeneration, and was not observed during the original shoot culture initiation (Wilkinson et al., 1998). It has been suggested that indirect regeneration of this type may be due to damage incurred during cryopreservation, e.g. by freeze fracture (Reed, 2001), although there will also be a base mutation rate resulting from free radical activity or atmospheric radiation (Benson, 1990). Following the cryopreservation procedure it is important to determine whether regeneration is direct and to assess the regions of cellular survival. There is increasing evidence that providing the cryopreservation technique applied ensures the greatest possible maintenance of the integrity of the stored specimen, there will be no modification at the phenotypic, biochemical, chromosomal or molecular level due to cryopreservation (Engelmann, 1997).
For the cryopreservation of rare and endangered species the stability of the samples is especially important. Although it has been suggested that somaclonal variation may constitute a source of novel variation in endangered species where genetic bottlenecks appear to be a problem (Bramwell, 1990), other authors (e.g. Towill, 1991; Fay, 1992) have taken a more conservative view, given the high percentage of mutations that are deleterious even in nature. In addition, somaclonal variation can include cytological and chromosomal abnormalities in addition to point mutations (Harding, 1999). Where population size may be extremely low, any further loss of material due to genetic degradation would be unacceptable. In the development of a cryopreservation protocol for the storage of shoot tips, in vitro cultures must be established first to allow the multiplication of sterile shoots. The shoots produced must show uniform growth characteristics to be used for developing a cryopreservation protocol and, ideally, the shoots recovered from these trials should show exactly the same characteristics.
Since somaclonal variation can arise with in vitro culture, the importance of using a reliable technique to detect any novel variation in plant tissues that have been stored through cryopreservation is clear. Characterization of plant material after cryopreservation has been achieved using a variety of techniques including (1) morphological markers and agronomic traits; (2) cytological markers including karyotype description at the chromosome and sub-chromosome level; (3) biochemical markers, including isozyme analysis, protein electrophoresis and secondary products; and (4) DNA markers (Harding, 1999). The use of DNA to assess variation has the advantage that while phenotypic changes to in vitro or cryopreserved specimens may be a reversible response to stress, any genotypic change could have a permanent and heritable effect on the species. Therefore, genetic fingerprinting studies have been carried out to look for any variation that may have arisen during the period of tissue culture of C. atrosanguineus, the cryopreservation process or the long-term storage of material in liquid nitrogen (LN).
The dependence of polymerase chain reaction (PCR)-based procedures on relatively small quantities of DNA makes them more applicable to rare and threatened species than restriction fragment length polymorphism (RFLP)-based genetic fingerprinting techniques (Chase and Fay, 1997) and means they are also suitable for in vitro material that is normally only maintained in relatively small quantities. The PCR-based technique of randomly amplified polymorphic DNAs (RAPDs) has been widely used to determine variation in cultivated and wild species and has been successfully utilized for the identification of somaclonal variation in embryogenic cultures of Picea glauca (De Verno, 1999). However, the technique can suffer from a lack of reproducibility, and scoring of the resultant profiles is often difficult (Edwards, 1998; Jones et al., 1998b). Another PCR-based procedure, amplified fragment length polymorphisms (AFLPTM), was developed by Keygene Inc. (Wageningen, The Netherlands) (Zabeau and Vos, 1993; Vos et al., 1995), and is the most sensitive multilocus fingerprinting technique currently available. It has been demonstrated to sample DNA widely from the nuclear genome, an attribute that is essential where the detection of small random variations within clonal material is required (Vuylsteke et al., 1999). This technique has now been automated, making it fast and allowing a highly accurate assessment of band homology. In addition to requiring relatively small quantities of DNA, it provides 10–100-times more markers and is thus more sensitive than most other fingerprinting techniques (e.g. RAPDs; Matthes et al., 1998), and is reproducible between laboratories (Jones et al., 1998a). Furthermore, the recent proven application of the technique for the identification of clonal material of rare species (e.g. Fay et al., 1999) made it the procedure of choice for the assessment of variation of C. atrosanguineus through tissue culture and cryogenic storage.
To establish whether the protocol developed for the cryopreservation of C. atrosanguineus is suitable for the long-term storage of this species, the quality of regeneration, temporal stability and genetic stability of stored cultures were assessed. This paper presents work from an ultrastructural study of the cellular effects of freezing and a morphological and molecular assessment of C. atrosanguineus material regenerated after 12 months storage in LN.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cryopreservation
The methods of in vitro stock plant maintenance and shoot tip cryopreservation have been reported previously (Wilkinson et al., 1998). Briefly, shoot cultures of C. atrosanguineus were transferred to new growth media every 4 weeks to maintain healthy, active growth. Shoots (1 cm long) were transferred to new medium to give optimal growth 10 d before removal of shoot tips for encapsulation. Shoot tips (0·4–0·6 mm long) with two or three leaf primordia were dissected and embedded in an alginate-coated filter paper strip (five tips per strip) that was then encapsulated by repeating the coating procedure for each strip.
To assess the effect of storage duration on recovery, sets of 40 strips (i.e. 200 shoot tips per set) were produced in a staggered manner and cryopreserved for 1, 3 and 12 months prior to simultaneous thawing and recovery. Samples were taken for genetic studies at each stage (see below). After a two-stage sucrose media pre-treatment (0·3 M for 72 h followed by 0·75 M for 96 h), strips were dehydrated in a laminar airflow hood for 7 h. Alginate strips were then placed in 1·75 ml cryovials and rapidly frozen by direct immersion in LN for 30 min followed by rapid transfer to a 35-l, narrow-neck LN storage dewar. While this process may induce vitrification of intracellular water rather than freezing, we have no direct evidence that this occurred. For this reason, we refer to the process as ‘freeze/thaw’ from here on. After the appropriate storage periods, strips were warmed rapidly in a 40 °C water bath for 2 min, removed from the cryovials and plated on recovery medium. Control strips were removed from each set after sucrose pre-treatment, dehydration and 30 min LN treatment and recovered as above.
Following pre-treatment or pre-treatment and cryopreservation, a shoot tip was considered to have survived if active growth of part or the entire shoot tip could be observed after 2 weeks on recovery medium. The quality of regeneration was noted and distinction was made between the production of single shoots, multiple shoots, and shoot tips that survived but did not produce shoot regeneration within 8 weeks. Five shoot tips that were removed from the 12-month cryopreserved set were transferred to rooting medium [full Murashige and Skoog (1962) medium (MS) with no growth regulators]. Rooted shoots were potted in a 1 : 1 : 1 mixture of compost : FullasorbTM : PerliteTM. Plantlets were grown to maturity and compared with non-frozen controls.
Comparisons were made in terms of survival and regeneration between three storage periods (1, 3 and 12 months) to assess (a) the variation between the sample sets and any correlation with the number of previous transfers the stock material had been exposed to, and (b) any effect of storage duration on recovery and shoot regeneration. ANOVA and standard deviation of the mean were used to assess levels of significance of the results.
Microscopy
To assess the damage caused by the pre-treatment and freezing process, sample shoot tips were taken at four stages during the cryopreservation protocol: (1) from stock material grown for 10 d after transfer: shoot tips were dissected and transferred to filter paper rafts on C1 medium for 24 h to allow some recovery before processing; (2) from encapsulated, sucrose-pre-treated and dehydrated material which was then cut to give individual tips in 2 mm3 blocks of dried alginate; (3) from material treated as in (2) but exposed to LN for 30 min and thawed prior to processing; and (4) from material treated as in (3) but transferred, after thawing, to 0·3 M sucrose recovery medium in the dark for 7 d and then moved to C1 medium for a further 48 h. Shoot tips were then removed from their alginate coat before being processed for microscopy.
All shoot tips were then fixed in Karnovsky’s solution (Glauert, 1975) at room temperature before being washed in 0·05 M Sörensen phosphate buffer pH 7·2 (Dawson et al., 1969). Osmium tetroxide (1 % w/v) was used as a secondary fixative and stain prior to a second wash in phosphate buffer. Samples were progressively dehydrated through ethanol solutions to a final concentration of 100 % and then embedded in acrylic resin for sectioning.
Specimens were transferred into supported gelatine capsule halves filled with fresh acrylic LR White Resin® and placed into a vacuum oven (58–60 °C and 440 mbar) for 18–24 h to polymerize the resin. Blocks were allowed to cool for at least 4 h prior to sectioning using an ultra microtome (Reichert–Jung Ultracut). Sections (0·5 µm) were cut, mounted and stained with 0·5 % w/v solution of Toluidine Blue-O in 0·1 M phosphate buffer (pH 7·0) and viewed and photographed under a light microscope (Leica DMLB).
For transmission electron microscopy, silver and silver/gold sections 80–160 nm thick were cut and mounted on Formvar grids made using a 0·5 % w/v Formvar solution. Sections were stained with uranyl acetate followed by lead citrate using an LKB Bromma 2168 ultrostainer. After air-drying, sections were viewed and photographed using a JEOL JEM-1210 TEM operating at 100 kV.
AFLP analysis
Clonal shoot material was removed from culture prior to each set of shoot tips being removed for dissection and alginate encapsulation. This material was then transferred to silica gel (Chase and Hills, 1991) and stored for the duration of the experiment so that a comparison could be made between the stored material and transfer stock material. For each subsequent control or storage treatment, the first shoot regenerating from each shoot tip was removed as soon as it reached a size suitable for transfer to standard growth medium. The individual shoot tip was placed on multiplication medium and allowed to grow for 4 weeks before being divided into nodal explants. These explants were then allowed to grow on C1 multiplication medium for another 4 weeks. The material obtained from each single regenerant shoot after 8 weeks of growth was transferred to silica gel for later DNA extraction.
DNA was extracted from 16 samples of 0·1–0·3 g of silica gel-dried leaves using the 2X CTAB method of Doyle and Doyle (1987). Genetic studies were then carried out on these samples and on DNA that had been extracted 4 years previously from C. atrosanguineus cultures after initiation into tissue culture. This DNA sample had been purified on caesium chloride/ethidium bromide gradients (1·55 g ml–1 density) and stored in a –80 °C DNA bank.
AFLPs were conducted according to the AFLPTM Plant Mapping Protocol of PE Applied Biosystems Inc. (ABI), involving four main steps:
Restriction–ligation.
The template DNA fragments were generated by restricting 0·5 µg genomic DNA with the restriction enzymes EcoRI (a relatively rare six-base cutter) and MseI (a frequent four-base cutter). EcoRI and MseI adapters were then ligated onto the ends of the restriction sites, generating primer binding sites.
Preselective PCR.
Preselective primers based on these binding sites with the addition of a single nucleotide were used to amplify a subset of restriction fragments having the matching nucleotide downstream from the restriction sites, resulting in an approx. 16-fold reduction (4 x 4) in the number of amplified fragments.
Selective PCR.
The preselective products were amplified with primers having two additional selective bases (three in total). The EcoRI-based primers were also labelled with fluorescent dyes. The first base was the same as that used in the preselective amplification. Only that subset of fragments with matching nucleotides at all three positions was amplified, a further reduction in the number of fragments by approx. 256-fold (16 x 16). Twenty-nine primer combinations were tested to select those combinations that gave the highest number of clear peaks that could be used to compare the 17 samples with as high a definition as possible. Seven combinations of selective bases were used in this study: (ACG + CTT, ACT + CTG, AAC + CAA, AAC + CTG, ACG + CTA, ACC + CAA and ACT + CAT, with the first triplet being attached to the EcoRI-based sequence and the second to the MseI-based sequence).
Fragment analysis.
The fragments were sized by running dye-labelled size standards in each lane. These were then separated and visualized using an ABI 377 automated sequencer. Gel analysis to obtain the AFLP profile was carried out using Genescan 2.0.2 and Genotyper Version 1.1 (PE Applied Biosystems Inc.). Only amplified fragments with sizes ranging from 50–500 base pairs (bp) were scored since bands outside this size range cannot be sized accurately.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Effects of pre-treatment and storage duration on shoot tip survival
The effects of pre-treatment, freezing and storage on shoot tip survival were dependent on the sample set. For the 12-month storage set there was no significant difference in survival at the 1 % level, irrespective of treatment. Shoot tips that had been exposed to a freeze/thaw step did show a significant (P < 0·05) drop in survival compared with those subjected to the sucrose control treatment. However, there was no significant difference between those tips that were frozen and thawed as a control and those frozen and stored for 12 months. No significant difference was observed in survival of shoot tips for the 3- and 1-month sets, irrespective of pre-treatment or duration of exposure to LN.
Effects of pre-treatment and storage duration on shoot tip regeneration
When shoot tip regeneration of the frozen/stored shoot tips was compared with that of the frozen/thawed controls, the 12-, 3- and 1-month sets showed a large and highly significant (P < 0·001, P < 0·01 and P < 0·001, respectively) decrease in the ability to regenerate into shoots compared with the frozen/thawed controls. No increase in callus or abnormal development was noted between these sets during their post-thaw recovery.
In the majority of cases, shoot regeneration occurred in the form of multiple shoot development, apparently from the region associated with the basal area of the shoot primordia. Shoot tips recovered from the 1-month set produced noticeably more callus on regrowth than those from all other sets. Shoot regeneration, when it occurred, was slow in comparison, and resultant shoots exhibited high levels of hyperhydricity. This decreased after transfer from recovery media to multiplication media, although some shoots that developed after the second transfer remained prone to hyperhydric growth.
Those shoots that grew initially as a single shoot were removed and the material bulked up by two transfers. In all cases the shoot tip meristem/primordia region produced multiple shoots from the area surrounding the base of the first shoot subsequent to the removal of this initial shoot. This rapid development of multiple shoots was observed in all regenerating shoot tips regardless of whether the initial regeneration was of a single shoot or directly into multiple shoots. The removal of the first shoot had no significant effect on the number of shoots produced thereafter.
Shoot tips that were removed from the final sample for rooting, weaning and returning to glasshouse conditions showed no significant variation to those from unfrozen controls in rooting ability after three transfers on basal MS medium. Shoots removed from the 1-month samples were slightly hyperhydric initially but a normal growth habit returned after the second transfer. No significant variation was noted in weaning ability, and all rooted shoots developed into plants with normal growth characteristics under glasshouse conditions.
Recovery and weaning of cryogenically stored shoot tips
Plants recovered and weaned from LN-stored shoot tips were morphologically similar to those from non-treated control shoot tips. As with all weaned and recovered shoot tips of C. atrosanguineus, no flower production occurred during the first flowering period after weaning but flower development in the following season did not differ from that of control plants.
Structural analysis
Figure 1A illustrates a section of a frozen/recovered shoot tip viewed under a light microscope (LM) depicting the four zones (A–D) that were compared for tissue damage. Figure 1B and C depicts three of these regions in a control shoot tip. Figure 1B shows cells located in the meristematic region (zone B) that are generally in good condition with abundant mitochondria and containing numerous small vacuoles and occasional starch grains. Nuclei and nuclear membranes are well defined. Figure 1C shows procambium and parenchyma cells located within the lower primordial region (zone C) which, like the meristematic cells, contain dense cytoplasm with numerous small vacuoles. Starch grains are more abundant and occupy a larger proportion of the cell. Figure 1D illustrates larger cortical parenchyma cells from the basal zone (A), which exhibit a greater vacuole : cytoplasm ratio.
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Frozen/recovered cells are illustrated in Fig. 2 and appear in marked contrast to the control cells of Fig. 1. Some of the samples exhibit regions of the meristematic zone where recovery from the freezing process has been rapid. Figure 2A shows such a region where the cells have made an almost complete recovery and are rich in mitochondria and virtually indistinguishable from the unfrozen controls. The cytoplasm, although sparser than in the control cells, is still dense and well-stained with small vacuoles. Cells from the lower primordial zone (C) generally appear in good condition with discernible organelles including mitochondria. The size of the vacuoles in these cells appears to have increased and multiple small vesicles have formed. Figure 2C shows a region of the outer leaf primordial cells. In all samples this region suffered massive damage with most cells showing complete disruption of all cell and organelle membranes. Cell walls also show signs of rupture, and cells in this region are considered necrotic.
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Sections obtained from shoot tips fixed and embedded in a dehydrated state did not show good preservation. Figure 2D shows a section taken from a dehydrated but unfrozen specimen that had not been allowed to re-hydrate prior to fixation. Folding of the plasmalemma and the formation of multiple small vesicles is observed between the cell wall and the plasmalemma. The cytoplasm in all such sections is granular and no organelles are identifiable.
Assessment of genetic stability
The number of bands obtained per AFLP primer combination ranged from 36 to 121, giving 433 bands in total. All bands were present in all individuals examined, indicating that, within the limits of resolution of the technique, the samples obtained from C. atrosanguineus were identical. Not only were all the bands present, but the relative shape and size of the peaks in the electropherograms were highly reproducible. Representative traces from regenerated material, transfer stock material and the original DNA extracted from C. atrosanguineus post-initiation into tissue culture are shown in Fig. 3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The cellular damage attributed to tissue pre-treatment, freezing and thawing has been well documented in a range of species. The nature and location of cell damage appears to be dependent on the pre-growth cryoprotectant, freezing protocol and species under study. In shoot tips of Dianthus, most of the subapical tissue appeared damaged after thawing while most surviving cells were located in the apical meristem and primordial leaf tissue (Kartha, 1985). In frozen shoot-tip apices of Pisum sativum L. (Haskins and Kartha, 1980), undifferentiated cells in the dome area were found to be less likely to survive freezing than cells in the primordial leaf tissue and areas of the dome. Grout and Henshaw (1980) observed the same results for shoot apices of S. tuberosum. Fukai (1995) determined more accurately from which cells the majority of regeneration was occurring by using a periclinally chimeric cultivar of Chrysanthemum; 70 % of regenerating shoot tips had lost their chimeric form indicating regeneration originating from the L1 layer and not the entire original meristem as hoped. Assessment of regeneration suggested that groups of cells survived in areas of the meristem and shoot primordia, whereas cells in other regions showed either complete necrosis or considerable disruption. Using the encapsulation technique for potato shoot tips, Benson et al. (1996) noted that primordia growth occurred more quickly during recovery and that the primordial region gave the best cell survival.
In this study of C. atrosanguineus, complete survival of the entire shoot tip during the freezing process was not achieved. Observation of recovering shoot tips clearly showed a lag phase before growth was resumed, indicating that cell recovery is required before growth can resume. In many cases, when shoot regeneration occurs it can be seen to come from the primordial base/meristem region. Shoot regeneration is usually in the form of multiple shoots. The ultrastructural investigation described here suggests why this is the case and allows some explanation for the type of regeneration observed. Sections through the different regions of the shoot tip show pockets of cells that survive the freezing process. These pockets exist within the meristematic dome and leaf primordia, whereas other areas show complete disruption of the cells. The ultrastructural state of the surviving and damaged regions suggests why the recovery is rarely direct, but assessment of when the damage to the cells occurs is vital if improvements are to be made to the protocol.
The introduction to and subsequent thawing of material from the cryogen are unlikely to be the only factors responsible for the damage observed. Cryoprotection and especially prolonged periods of dehydration, though not lethal in themselves, can act synergistically and lethally with freezing stress (Berjak et al., 1999). During loading of osmotically active cryoprotectants and desiccation, ultrastructural studies have shown many similarities in cell response. The cytoplasm is often retracted from the cell wall and the vacuole : cytoplasm ratio is increased (Pritchard et al., 1986; González Arnao et al., 1993; Isaacs and Mycock, 1999). The organization of the nucleolus and disposition of heterochromatin that are normally encountered are lost, and the nuclear shape is often aberrant (Wesley-Smith et al., 1995; Berjak et al., 1999). Spatial arrangement of the organelles is often disorganized, possibly as a result of damage to the cytoskeleton (Kioko et al., 1998), and the occurrence of internalized vesiculation of the plasmolemma, and exocytotic protrusions and vesicle formation from the plasmalemma is observed (Singh, 1981; Gordon-Kamm and Steponkus, 1983; Gnanapragasam and Vasil, 1992;). With regards to this latter phenomenon the final authors suggest that internalized vesiculations represent irreversible membrane loss during the contraction phase of plasmolysis, leading to lysis on subsequent re-expansion. The protrusions and folding of the plasmalemma and attached vesicles, however, represent areas that may contain subducted lipid material, or from which lipid could be withdrawn during contraction or re-expansion, respectively. The ultrastructure of C. atrosanguineus shoot tips showed signs of all of these features. It should be noted, however, that a problem with attempting to explain the phenomena observed during dehydration and immediately after freezing is the use of an aqueous fixative that has been shown to be inaccurate and inadequate in the preservation of dried material (Thomas and Platt, 1997). This may have led to the production of artefacts that misled interpretation.1
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Despite this, the overall condition of the cells could be broadly assessed on the severity of membrane disruption and the general state of the cytoplasm and organelles. It is difficult, however, to assign the cause of damage, and no clear examples of ice crystal damage were seen in any of the sections viewed. Formation of microscopic ice crystals may have been responsible for some of the cytoplasmic granulation, but further work utilizing freeze-fracture techniques would be required to assess this. From the present study, cells in the area of the meristematic zone were no more likely to withstand the freezing process than were cells found in the lower primordial leaf tissue. In all but the control samples damage to the larger basal parenchyma cells was extensive, indicating that the pre-treatment does not affect all cells to the same extent. Regeneration observed from regions of the meristematic tissue and the fact that multiple regeneration can occur from a single meristem suggest that the dome does not need to survive in its entirety to allow regeneration of shoots. Meristematic cells can be assumed to have the ability of meristematic regeneration to produce a new dome area, and the regenerative capacity of the procambium cells can be assumed to facilitate the adventitious regeneration of shoots.
The stability of regeneration over storage duration shows that although variation in regenerative ability is observed among samples, there was no significant decrease to survival or shoot regeneration between 1 and 12 months of storage in LN. The variation that was observed among samples has also been reported in S. tuberosum (Benson et al., 1996), Fragaria (Kartha, 1985) and Malus (Zhao et al., 1999). In all cases this fluctuation has been attributed to variation of the physiological state of the donor plant at the time of shoot tip removal, and there is no evidence to suggest that the variation observed with C. atrosanguineus is any different. Despite the type of shoot regeneration, the use of AFLPs gave no indication of any variation among the samples tested. By using seven primer sets, a comparison of 433 fragments for each sample provides strong evidence of clonal uniformity and thus no variation in the samples tested. DNA that was originally purified and banked shortly after the initiation of C. atrosanguineus into in vitro culture was indistinguishable from DNA that had been extracted from shoots subcultured nearly 50 times over a 4-year period. It is unwise to assume that no somaclonal variation is to be found among the tissue culture material but of the specimens tested, the stability of C. atrosanguineus in tissue culture appears good. This compares favourably to the decline in culture quality of Vitis vinifera over time that may be related to methylation of the genomic DNA (Harding et al., 1996).
Although the quality of regeneration is not ideal, the temporal stability of frozen shoot tips has been shown to be good. Any growth abnormality observed directly after regeneration such as hyperhydric growth was not carried through to later growth stages and so it may be concluded that the method of strip encapsulation, sucrose pre-treatment, dehydration and rapid freezing is an effective system for the long-term cryogenic storage of C. atrosanguineus shoot tips. The accuracy and reproducibility of the AFLP procedure also allowed a detailed assessment of the genetic stability of stored vegetative material. However, the importance of stability cannot be underestimated and, wherever possible, a combination of techniques should be used to ensure the stability of cryopreserved material.
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ACKNOWLEDGEMENTS |
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T.W. thanks the Royal Botanic Gardens, Kew for financial support towards his PhD. We thank Paula Rudall for useful comments on an earlier draft of this manuscript and Robyn Cowan and Gloria Beltran for assistance with the AFLP study.
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LITERATURE CITED |
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