Plant Science 161 (2001) 1099 1106 www.elsevier.

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Genetic delity and viability of Anigozanthos 6iridis following tissue culture, cold storage and cryopreservation
Shane Turner a,b,*, Siegfried L. Krauss a,c, Eric Bunn a, Tissa Senaratna a, Kingsley Dixon a, Beng Tan b, Darren Touchell d
a

Botanic Gardens and Parks Authority, Kings Park and Botanic Garden, Research Laboratory, West Perth, WA, 6005, Australia b Curtin Uni6ersity of Technology, Bentley, WA, 6102, Australia c Department of Plant Science, Faculty of Agriculture, Uni6ersity of Western Australia, Nedlands, WA, 6009, Australia d School of Forestry and Wood Products, Michigan Technological Uni6ersity, Townsend Dri6e, Houghton, Michigan, USA Received 14 June 2001; accepted 26 July 2001

Abstract The effects of long-term storage conditions on the viability and genetic delity of plant somatic tissues are poorly known. In this study, the effects of three storage methods (tissue culture, cold storage and cryostorage) on genetic delity and shoot apex viability were evaluated for Anigozanthos 6iridis subspp. terraspectans (Haemodoraceae), a threatened plant from south west Australia. Genetic delity was assessed following 12 months of storage using the PCR-based multi-locus DNA ngerprinting technique Amplied Fragment Length Polymorphism (AFLP). Shoot apex viability was evaluated at 0, 3, 6 and 12 months for cryogenically stored material. The AFLP technique generated a total of 95 fragments for three primer pairs, and no differences were detected across treatments. Post-cryostorage viability was high (mean = 85%) and not signicantly different across storage times. These results show that genetic delity and shoot apex viability (for cryopreserved material) were maintained following tissue culture, cold storage and cryostorage of A. 6iridis subspp. terraspectans for up to 12 months. 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Anigozanthos 6iridis ssp terraspectans; AFLP; Genetics; Conservation; Cryopreservation; Vitrication

1. Introduction It is estimated that currently worldwide, there are 33 418 species or over 12% of the total world ora classied as threatened [1]. In Australia the situation is no better with 2245 species considered threatened and in need of conservation action [1]. The ex situ storage of representative genotypes is an important strategy for the conservation of these species. Techniques such as seed storage (at room temperature, 5, 20 or 196 C) and container, in vitro or arboretum collections are being used to maintain genetic diversity [2,3]. However, clonal collections of endangered species are advantageous because they can facil-

* Corresponding author. Tel.: + 61-8-9480-3647; fax: +61-8-94803641. E-mail address: turnersr@pop.ses.curtin.edu.au (S. Turner).

itate the conservation of targeted clonal lines from genetically representative samples. This may be preferable to the use of seed, which can be highly variable and contain unt genotypes [4]. Seed may also have complex dormancy mechanisms and be hard to germinate, short lived or simply not produced in sufcient quantities [5,6]. In a modern conservation agency, the efcient preservation of large clonal collections is achieved through techniques such as tissue culture, slow growth (including cold storage) or cryostorage. These techniques are highly space efcient, minimise disease and pest problems and allow for the manipulation and control of all external variables, which may cause irreplaceable loss of important mother plants when collections are maintained outdoors [2]. In particular, the technique of cryostorage optimises these advantages, has very low maintenance costs and, theoreti-

0168-9452/01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 5 1 9 - 2

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cally, material can be maintained under cryogenic conditions indenitely with virtually no maintenance [7]. However, for in vitro and cryogenically stored cultures, there may be risks to the maintenance of genetic delity associated with maintaining plant material under these conditions for extended periods of time [8,9,4]. This may be due to the extended duration that these cultures have been maintained under, as well as the use of various chemicals including plant growth regulators or cryoprotectants [10,11]. Reports of somaclonal variation in tissue culturederived plant material have been described for many species including, horseradish, pecan and alfalfa [12 14]. However, these reports concern plantlets derived from callus tissues. Nevertheless, there are still concerns about the genetic stability of shoot cultures over extended periods of time [9,4]. The exact causes of somaclonal variation in tissue culture are not known, although it is believed that medium to high concentrations of various plant growth regulators such as NAA, 2,4-D and BAP may contribute signicantly [15]. Variation can also occur when plants are placed under different culture conditions, which may induce stress like responses. These include media with high sugar concentrations and temperature reduction [15]. For example, Harding [16,17] found genetic changes in ribosomal RNA genes were generated after storage under slow growth conditions (6 months on 6% mannitol). Nevertheless, the genetic consequences of culture incubation under such stress conditions for long periods of time is still poorly known. Few studies have assessed somaclonal variation of plants derived from cryogenically stored material. Most studies that have been undertaken including those by Harding and Benson [18] on potato, and Haggman et al. [19] on Scots pine have found no genetic differences in regenerated plants following LN immersion. Though, Vannini and Poli [20] have stated that the commonly used cryoprotectant DMSO, has been shown to cause genetic alterations under some conditions. Additionally, the number of studies completed on the effects of extended LN storage are also limited, though several studies on agriculturally important species such as potato, cassava and sugarcane have found no differences in shoot apex viability after 4 years (potato and cassava) and 12 months storage (sugarcane), respectively, [21,22]. Anigozanthos 6iridis ssp terraspectans (Haemodoraceae), commonly known as the dwarf green kangaroo paw, is a small herbaceous perennial found growing in winter wet depressions, in a restricted part of the Western Australian wheatbelt. It is currently known from four populations and is classied as rare and endangered due to extensive clearing and its limited distribution [23,24]. As part of an integrated approach to its

conservation, we are establishing a cryostored population of representative genotypes. The aims of this study were to rstly determine if immersion in LN causes a decline in shoot apex viability over a 12 month period, and to secondly assess genetic delity over the same duration, when somatic germplasm is stored under standard tissue culture, cold storage and cryogenic conditions. Genetic delity was assessed using the powerful PCR-based multi-locus DNA ngerprinting technique AFLP [25]. AFLP, when used with uorescent markers, automated sequencer and dedicated software, is the most stringent, reproducible and powerful PCR-based multilocus technique for genotyping and ngerprinting currently available [26].

2. Materials and methods

2.1. Standard culture conditions for Anigozanthos viridis

Shoot cultures of Anigozanthos 6iridis ssp terraspectans, Hopper used during this study were obtained from in vitro stocks derived from eld collected shoots and maintained at the Plant Science Laboratory, Kings Park and Botanic Garden, Perth, Western Australia. To ensure genetic uniformity, a single plantlet was used as the starting point to build a single clonal line. This was established 6 months before the initiation of this experiment. In vitro explants were sub-cultured every 3 weeks onto basal medium (BM) which consisted of half strength MS [27] salts, 3.0 mM thiamine hydrochloride, 2.5 mM pyridoxine hydrochloride, 4.0 mM niacin, 0.5 mM MES buffer, 0.5 mM myo-inositol, 60 mM sucrose and solidied with 0.8% w/v agar. This was supplemented with 0.5 mM 6-benzylaminopurine (BAP). The pH of all media was adjusted to 6.0 prior to the addition of agar, then autoclaved at 121 C and 107 KPa for 20 min. Plant cultures were maintained at 2224 C and illuminated with 16 h light with PPFD of 30 mM m 2s 1.

2.2. Modications to storage conditions

About 3 weeks after the last sub-culture and when sufcient numbers of plantlets were achieved, they were divided into three groups; tissue culture plantlets, cold storage plantlets and cryopreservation plantlets. However, before initiation of the experiment 20 samples of parent plants were randomly collected from each group for future AFLP analysis. This material was maintained in the vapour phase of liquid nitrogen ( 130 C) until required.

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2.2.1. Tissue culture plantlets Plantlets were maintained under standard tissue culture conditions (see above) and a sub-sample was randomly selected at each sub-culture interval (every 3 weeks) for further sub-culturing. For AFLP analysis shoot apices were extracted at 0 and 12 months, and then cultured under standard cryogenic recovery conditions (see below) for three months to obtain actively growing plantlets. 2.2.2. Cold storage plantlets About 3 weeks after the last sub-culture, plantlets were placed under cold storage conditions for 12 months (with no change in the culture medium, at 4 C and illuminated with 16 h light with PPFD of 30 mM m 2 s 1). For AFLP analysis shoot apices were extracted at 0 and 12 months and cultured under standard cryogenic recovery conditions (see below) for 3 months to obtain actively growing plantlets. 2.2.3. Cryogenic plantlets Shoot apices were harvested and subjected to the cryopreservation protocol [28,29] described below. At 0, 3, 6 and 12 months three vials were warmed and the shoot apices within, recovered under standard cryogenic recovery conditions (see below). Survival was recorded over 28 days. Surviving shoot apices were then cultured for three months to obtain actively growing plantlets. 2.2.3.1. Preculture. Shoot apices 1 1.5 mm long, were excised from 21-day-old plantlets and precultured on BM supplemented with 0.8 M glycerol for 72 h. 2.2.3.2. Loading. Following preculture, shoot apices were placed in 2 ml of loading solution consisting of 2 M glycerol, in liquid BM supplemented with 0.4 M sucrose. Shoot apices were incubated in this medium for 20 min at room temperature before placing in modied PVS2 vitrication solution. 2.2.3.3. Vitrication and cooling. PVS2 (plant vitrication solution 2) of Sakai et al. [30] was modied by Turner et al. [29] and comprised 30% w/v glycerol, 15% w/v ethylene glycol and 7.5% w/v dimethyl sufoxide (DMSO) and 7.5% propylene glycol in liquid BM supplemented with 0.4 M sucrose was used as the cryoprotective medium. The medium was lter-sterilised using a 0.2 mM Acrodisc 32 lter prior to use. Following preculture and loading, excised apices were incubated at 0 C for 25 min in 1.0 ml NUNC cryovials containing modied PVS2. Shoot apices were then plunged into LN. 2.2.3.4. Warming and washing. Shoot apices were stored in LN for at least 30 min (0 months) or 3, 6 or 12

months prior to warming in a 40 C water bath for 1 min. After warming, apices were removed from the modied PVS2 medium and rinsed three times for 4 min per rinse in liquid BM supplemented with 1.0 M sucrose.

2.2.3.5. Reco6ery and post reco6ery. Washed shoot apices were placed onto recovery medium consisting of BM solidied with 0.8% w/v agar and supplemented with 2 mM choline chloride (cc) for 7 days, and incubated in darkness for this duration [31]. Shoot apices were then removed and placed under standard light conditions, on BM solidied with 0.8% w/v agar and supplemented with 1 mM zeatin for a further 21 days. After this time shoot apices were placed on BM solidied with 0.8% w/v agar and supplemented with 0.5 mM kinetin/0.5 mM GA3 medium for 21 days. Finally after this time developing plantlets were placed onto 2.5 mM kinetin/0.25 mM BAP until harvesting. 2.2.3.6. Scoring of sur6i6al and regeneration. For the cryogenic treatment three replicates consisting of 15 shoot apices each were used. Survival was determined as detectable growth of all or part of apical tissues 128 days after treatment. Data were analysed by an Analysis of Variance (ANOVA). The original data expressed as a percentage were transformed (arcsin
) to conform to ANOVA assumptions. 2.3. Genetic analysis
High molecular weight genomic DNA was extracted from plantlets weighing between 20 and 100 mg using plant DNAzol reagent according to the manufacturers instructions (Life Technologies) and stored in Tris EDTA (TE) buffer. Five replicate plantlets for each of nine treatments were genotyped using the AFLP technique [25]. AFLP procedures were modied from Krauss [32] and Krauss and Peakall [33] and involved the following steps. 1. Restriction of the DNA: For each sample, approximately 300 ng of DNA was digested with 1.25 U of EcoRI/MseI restriction enzyme and 2.5 ml 5 reaction buffer in a reaction volume of 12.5 ml and incubated at 37 C for 2 h. Samples were then transferred to a 70 C bath for 15 min, before briey cooling on ice. 2. Ligation of adapters: About 12 ml of adapter ligation solution and 0.5 U of T4 DNA ligase were added to the digested DNA, incubated at 20 C for 2 h, then diluted 1:10 with TE buffer. 3. Preselective amplication by PCR: About 1.25 ml of the diluted ligation mix was combined with 10 ml of pre-amplication primer mix I, 1.25 ml 10 PCR

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buffer, 1.5 mM MgCl2, and 0.3 U Taq DNA polymerase and PCR was performed for 20 cycles of 94 C for 30 s, 56 C for 60 s, and 72 C for 60 s on a Perkin-Elmer 9700 thermocycler in 9600 emulation mode. Subsequently, the pre-amplication mixture was diluted 1:50 with TE buffer. 4. Selective amplication by PCR: About 2.5 ml of the diluted pre-selective PCR product was combined with 15 ng MseI-CAC primer, 7.5 ng of each of three uorescently labelled primers (EcoRI-ACT (FAM), EcoRI-AGG (JOE) and EcoRI-ACC (TAMRA)), 0.2 mM of each of four dNTPs, 1.0 ml 10 PCR buffer, 1.5 mM MgCl2, 0.25 U Taq DNA polymerase and distilled water to a nal volume of 10 ml. A touchdown PCR reaction commenced with one cycle of 94 C for 30 s, 70 C for 2 min, and 72 C for 2 min. In subsequent cycles, the annealing temperature was reduced in 1 C steps to 61 C, followed by 23 cycles at 61 C. A single step of 60 C for 30 min followed before holding at 4 C. All solutions were purchased from Life Technologies except for Taq and uorescently labelled EcoRI primers, which were purchased from Applied Biosystems. The uorescently labelled amplied fragments were analysed by gel electrophoresis (5% acrylamide gels) using an ABI Prism 377 Automated Genetic Analysis System (AGAS). The inclusion of internal size standards (ROX-500) in each lane enabled the accurate sizing and scoring (presence/absence of DNA fragments) of multi-locus DNA ngerprint fragments between 60 and 500 base pairs using ABI GeneScan software. All 45 samples were electrophoresed on the one gel, which was replicated once.

4. Discussion We found no decline in the viability of shoot apices of A. 6iridis following storage in LN for a period of up to 12 months. Our results agree with the ndings of Haskins and Kartha [34] and Kartha et al. [35], who found no loss in viability after 2 and 10 years storage in LN for pea and strawberry shoot apices, respectively. Consequently, storage in LN does not appear to affect the ability of shoot apices to recover and regenerate plantlets. Nevertheless, as shoot apices in this study were only stored in LN for 12 months (a duration still considered short term), our experiments are being continued for a much longer duration to determine if LN storage has an ultimately deleterious effect on long term viability. However, as there is virtually no biophysical processes or water movement below 139 C [36], theoretically there should be no decline in viability over extended periods of storage. The only stresses experienced by appropriately maintained cryogenically stored plant tissues are during the critical cooling and warming phases, so material maintained correctly should maintain viability. There are however, currently no studies to determine if stability of cultures in LN varies among the different cryopreservation procedures e.g. encapsulation/dehydration, slow cooling or vitrication [37]. In addition, during extended cryostorage, samples may also be unintentionally exposed to warming and cooling cycles as other samples are added or removed to the same storage vessel [37]. Over extended periods of time these small changes may cause cellular damage and eventually lead to a decline in viability [37]. These are issues requiring further study.

3. Results

3.1. Shoot apex 6iability o6er 12 months.

Shoot apices warmed after different lengths of time in LN showed no signicant differences (P \ 0.05) in survival regardless of LN immersion time (Fig. 1). Survival ranged from 84.7 to 88.6%.

3.2. Genetic delity

Three AFLP primer pairs generated a multilocus DNA ngerprint with a total of 95 DNA fragments scored between 60 and 500 base pairs in length. No qualitative (presence/absence) differences were detected among the 45 DNA ngerprints (six of which are shown in Fig. 2). Some lane to lane variation was observed in peak height, but replicate runs conrmed that these were due to subtle PCR effects and/or gel loading artefacts.

Fig. 1. Post LN survival after 28 days recovery for A. 6iridis after immersion for 0, 3, 6 or 12 months. (bars = 2 standard error (S.E.)).

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Fig. 2. Part of AFLP DNA ngerprints for six of 45 samples of an Angozanthus 6irdis genotype subjected to three storage treatments (tissue culture, cold storage, and cryopreservation) for upto 12 months. Shown are ngerprints between 100 and 300 bp base pairs. No qualitative differences were seen in any ngerprints. The arrow indicates a region where some fragments were poorly amplied, but replicate runs (not shown) conrmed these fragments to be present.

The genetic delity of A. 6iridis was apparently maintained following 12 months of storage, as no detectable differences were found with AFLP among storage treatments. Our result agrees with that of Wilkinson et al. [38], who found no genetic differences for Cosmos atrosanguineus, between material initiated into tissue culture and material that had been cryopreserved for 12 months. AFLP has also been used recently to evaluate somaclonal variation in somatic embryo cultures of oak and pecan [39,13]. No evidence of somaclonal variation was found in oak plantlets derived from the same clonal line, but for pecan a higher level of polymorphism was detected for some somatic embryos derived from the same callus lines. These differences in the pecan study were thought to be more related to the genotype of the cell line rather than to the age of the culture. Using other genetic markers, such as RFLP or RAPDs, prolonged maintenance of cultures under stan-

dard conditions (such as conventional tissue culture) or under slow growth conditions (cold storage or the use of growth retardants) has generated mixed results for genetic delity [9]. For example, Harding [16,17] found changes in ribosomal DNA of potato after cultures were maintained for extended duration under slow growth conditions, with the use 6% mannitol acting as a growth retardant. These differences were attributed to hypermethylation of genomic DNA and ribosomal genes, and were thought to serve as an adaptive response to conditions of high osmotic stress. Culture for extended periods under such conditions has been shown to cause physical changes such as hyperhydricity, and reduced internodal length and leaf size in shoot cultures [9], while the genetic consequences, on cellular and developmental pathways is still poorly understood [9]. On the other hand, Goto et al. [40], found no changes in 36 micropropagated plantlets of Pinus thunbergii, maintained for over 10 years in conventional

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tissue culture, while no mutations were detected in strawberry cultures maintained under cold storage conditions using RAPDs [41]. In contrast, Rani et al. [42] detected 13 polymorphisms from six micropropagated plants maintained under standard tissue culture conditions, out of 23 plants screened from the one clone of Populus deltoides. This is perhaps surprising given that shoot cultures (as used in this study) are normally highly genetically stable, when compared with other tissue types such as, callus or adventitiously derived shoots [10], although under conditions of long-term maintenance the chances of somaclonal mutations may be increased. Many laboratories limit the number of subcultures that can be made from an explant and generally replace the source material after about 1 year with fresh cultures derived from the original mother plant [10]. Additionally, problems with the RAPD technique, as used in the Goto et al. [40], Kumbar et al. [41] and Rani et al. [42] studies are also a potential source of these contrasting results, that are addressed by AFLP due to the higher specicity and reproducibility of this technique. For cryogenically derived material, most studies have found no differences following cryoprotectant exposure and/or cryopreservation. Studies on potato by Harding [16], Harding and Benson [18], Ward et al. [43] and Benson et al. [44] have found no differences between cryopreserved material and other in vitro manipulations. These studies applied a variety of genetic techniques including ow-cytometric assessments of ploidy stability, and analysis of nuclear and chloroplast DNA using RFLPs. However, Harding [45] did nd some differences in ribosomal RNA proles in some regenerated plants after cryoprotection with 10% DMSO and cryopreservation. These differences were attributed to DNA methylation occurring at specic target sites. Additionally, in several other studies on cryopreserved cultures of spruce and Scots pine, no genetic differences that could be attributed to LN immersion were detected [46,47,19]. This suggests that most plant material maintains genetic delity following LN immersion and that the Harding [45] study was more likely the exception rather than the rule. Further, the unexpected polymorphism found by Harding [45] was not directly attributed to cryopreservation per se but was more likely the result from the whole process (i.e. tissue culture, pre-growth, cryoprotection, freezing, thawing, recovery and plant regeneration). However, in another study on embryogenic cultures of Abies cephalonica, genetic changes were detected in DMSO treated cultures, but not in cryostored samples (also pretreated with DMSO) [48]. DMSO treated cultures were kept on ice for 2 hours and then returned to culture. At this temperature, slow metabolic activity and interactions with the DMSO are still possible. DMSO may act by changing metabolic function of cells

and interact with chromatin and nucleic acids, potentially causing numerous DNA changes [48]. As cryopreserved cells did not express this level of genetic variability, it was suggested that cryostorage of cultures treated with DMSO probably eliminated a high proportion of cells bearing DMSO induced genetic changes, as only small meristematic cells can survive LN immersion [48]. A confounding effect in Aronen et al. [48] is that cultures were also derived from open-pollinated material that were assumed to be inter-specic hybrids. These may often contain genetic incompatibilities or imbalances which may make them more susceptible to external mutagenic effects. Further, when DMSO was used in a cryoprotectant mixture labelled PGD (consisting of polyethylene glycol, glucose and DMSO), the level of mutation was no more than the background level, suggesting that cryoprotectant mixtures may minimise the detrimental effects of DMSO alone [48]. Therefore, the use of vitrication solutions as used in our study, containing combinations of DMSO, ethylene glycol, propylene glycol, glycerol and sucrose may minimise the deleterious effects of DMSO exposure. In addition, the much shorter exposure duration in our study (25 min compared with 2 h) may have also reduced the possible mutagenic effects of DMSO. A potential limitation in the present study is that only one clone was assessed, which may have been unusually stable under the conditions evaluated. Skirvin et al. [10] states that the level of somaclonal variation that should be expected in any in vitro cultures is about 13%, but this depends on many factors, including age, tissue type culture environment, the species and clone. Previous studies on other Haemodoraceae species with AFLP [Krauss unpublished results, [49]] have detected an extremely high level of variation among individuals. Consequently, AFLP is an extremely powerful technique, and a sensitive marker for the detection of genetic changes, especially in the Haemodoraceae. Nevertheless, we have still only looked at a very small proportion of the genome (but more than previously possible). As a consequence, although this study has not detected genetic changes, it is possible some changes may have occurred that were not detected. Although AFLP does appear to sample the genome evenly and widely, there is the possibility of point mutations outside of the priming sites that may go undetected. A more thorough test would include, (a) sequencing analysis, particularly of more variable regions; and/or (b) a greater sampling of the genome with AFLP. AFLP lends itself to increased sampling because it is more straightforward to use than other primer combinations and generates many more ngerprints. In conclusion, this study did not detect an effect on genetic delity and viability as a result of storage protocols used in our laboratory, and we now have a

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degree of condence that the storage techniques described, do not cause major genetic aberrations to critically important somatic germplasm. Nevertheless, there remains limitations to our study, which are being addressed by on-going research extended to other species, involving longer periods of storage and increased genome sampling.

[13]

[14]

[15]

Acknowledgements We wish to thank Robyn Taylor for her generous help and direction in sample preparation and processing.

[16]

[17]

[18]

References
[19] [1] K.S. Walter, H.J. Gillett, 1997 IUCN Red List of Threatened Plants. IUCN, Gland, Switzerland/Cambridge, UK, 1998 pp. 1862. [2] D.H. Touchell, M. Richardson, K.W. Dixon (Eds.), Germplasm Conservation Guidelines for Australia. Australian Network for Plant Conservation Canberra, Australia, 1997, pp.1 40. [3] F. Engelmann, Importance of cryopreservation for the conservation of plant genetic resources, In: F. Engelmann, H. Takagi (Eds.), Cryopreservation of Tropical Germplasm. Japanese International Research Center for Agricultural Sciences, Tsukuba, Japan/International Plant Genetic Resources Institute, Rome, Italy, 2000, pp 8 20. [4] D.H. Touchell, K.W. Dixon, In vitro preservation, in: B.G. Bowes (Ed.), A Colour Atlas of Plant Conservation and Propagation, Manson Publishing Ltd, London, 1999, pp. 108 118. [5] J.M. Iriondo, C. Perez, Propagation from seeds and seed preservation, in: B.G. Bowes (Ed.), A Colour Atlas of Plant Conservation and Propagation, Manson Publishing Ltd, London, 1999, pp. 46 57. [6] M.C. Munoz, In Vitro culture (IVC) and plant conservation, in: B.G. Bowes (Ed.), A Colour Atlas of Plant Conservation and Propagation, Manson Publishing Ltd, London, 1999, pp. 77 86. [7] Y.P.S. Bajaj, Cryopreservation of plant cell, Tissue, organ culture for the conservation of germplasm and biodiversity, in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry 32. Cryopreservation of Plant germplasm I, Springer-Verlag, Berlin, 1995, pp. 3 28. [8] R.L. Jarret, N. Gawel, Chemical and environmental growth regulation of sweet potato (Ipomoea batatas (L) Lam) in vitro, Plant Cell Tis. Org. Cult. 24 (1991) 13 18. [9] K. Harding, Approaches to assess the genetic stability of plants recovered from in vitro culture, in: M.N. Normah, M.K. Narimah, M.M. Clyde, (Eds.), Proceedings of the International Workshop on In vitro Conservation of Plant Genetic Resources. Plant Biotechnology Laboratory University Kebangsaan, Kuala Lumpur Malaysia, 1996, pp 135 168. [10] R.M. Skirvin, K.D. McPheeters, M. Norton, Sources and frequency of somaclonal variation, Hortscience 29 (1994) 1232 1235. [11] B.P. Finkle, M.E. Zavala, J.M. Ulrich, Cryoprotective compounds in the viable freezing of plant tissues, in: K.K. Kartha (Ed.), Cryopreservation of Plant Cells and Organs, CRC Press, Boca Raton, FL, 1985, pp. 75 113. [12] O. Rostiana, M. Niwa, W. Marubashi, Efciency of inter-simple sequence repeat PCR for detecting somaclonal variation among

[20]

[21]

[22]

[23]

[24]

[25]

[26] [27]

[28]

[29]

[30]

[31]

leaf-culture-regenerated plants of horseradish, Br. Sci. 49 (1999) 245 250. W.A. Vendrame, G. Kochert, H.Y. Wetzstein, AFLP analysis of variation in pecan somatic embryos, Plant Cell Rep. 18 (1999) 853 857. E. Piccioni, G. Barcaccia, M. Falcinelli, A. Standardi, Estimating alfalfa somaclonal variation in axillary branching propagation and indirect somatic embryogenesis by rapid ngerprinting, Int. J. Plant Sci. 158 (1997) 556 562. E.F. George, Plant Propagation by Tissue Culture. Part 1 The Technology, Second ed., Exegetics Ltd, London, 1993, pp. 1 574. K. Harding, Molecular stability of the ribosomal RNA genes in Solanum tuberosum plants recovered from slow growth and cryopreservation, Euphytica 55 (1991) 141 146. K. Harding, The methylation status of DNA derived from potato plants recovered from slow growth, Plant Cell Tis. Org. Cult. 37 (1994) 31 38. K. Harding, E.E. Benson, Analysis of nuclear and chloroplast DNA in plants regenerated from cryopreserved shoot-tips of potato, Cryo-Letters 21 (2000) 279 289. H.M. Haggman, L.A. Ryynanen, T.S. Aronen, J. Krajnakova, Cryopreservation of embryogenic cultures of scots pine, Plant Cell Tis. Org. Cult. 54 (1998) 45 53. G.L Vannini, F. Poli, Binucleation and abnormal chromosome distribution in Euglena gracilis cells treated with dimethylsulphoxide, Proto. 114 (1983) 62 66. Y.P.S. Bajaj, Cryopreservation of germplasm of potato (Solanum tuberosum L.) and cassava (Manihot esculenta Crantz), in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and forestry 32. Cryopreservation of Plant Germplasm I, Springer-Verlag, Berlin, 1995, pp. 398 416. M.T. Gonzales-Arnao, C. Urra, F. Engelmann, R. Ortiz, C de la Fe, Cryopreservation of encapsulated sugarcane apices: effects of storage temperature and storage duration, Cryo-Letters 20 (1999) 347 352. S.D. Hopper, S. Van leeuwen, A. Brown, S. Patrick, (Eds) Western Australias Threatened Flora, Department of Conservation and Land Management. Perth, Australia, 1990, pp 1 140. S.D. Hopper, Kangaroo paws and cats paws, Department of Conservation and Land Management. Perth, Australia, 1993, pp 1 144. P. Vos, R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, M. Zabeau, A new technique for DNA ngerprinting, Nuc. Acid Res. 23 (1995) 4407 4414. U.G. Mueller, L.L. Wolfenbarger, AFLP genotyping and ngerprinting, Tr. Ecol. Sys. 14 (1999) 389 394. T. Murashige, F. Skoog, A revised medium for rapid growth and bio-assay with tobacco tissue cultures, Physiol. Plant 15 (1962) 473 497. S.R. Turner, D.H. Touchell, K. Dixon, B. Tan, Cryopreservation of Anigozanthos 6iridis ssp viridis and related taxa from the south west of Western Australia, Aust. J. Bot. 48 (2000) 739 744. S.R. Turner, T. Senaratna, E. Bunn, B. Tan, K.W. Dixon, D.H. Touchell, Cryopreservation of shoot tips from six endangered Australian species using a modied vitrication protocol, Ann. Bot. 87 (2001) 371 378. A. Sakai, S. Kobayashi, I. Oiyama, Cryopreservation of nucellar cells of naval orange (Citrus sinesis Osb. var. brasiliensis Tanaka) by vitrication, Plant Cell Rep. 9 (1990) 30 33. D.H. Touchell, S.R. Turner, E. Bunn, K.W. Dixon, Cryostorage of somatic tissues of endangered Australian species, In: Y.P.S. Bajaj, (Ed.) Biotechnology in Agriculture and Forestry 32. Cryopreservation of Plant Germplasm II. Berlin: Springer-Verlag, in press.

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S. Turner et al. / Plant Science 161 (2001) 10991106 [42] V. Rani, A. Parida, S.N. Raina, Random amplied polymorphic DNA (RAPD) markers for genetic analysis in micropropagated plants of Populus deltoides Marsh, Plant Cell Rep. 14 (1995) 459 462. [43] A.C.W. Ward, E.E. Benson, N.W. Blackhall, S. Cooper-Bland, W. Powell, J.B. Power, M.R. Davey, Flow-cytometric assessments of ploidy stability in cryopreserved dihaploid Solanum tuberosum and wild Solanum species, Cryo-Letters 14 (1993) 145 152. [44] E.E. Benson, M. Wilkinson, A. Todd, Developmental competence and ploidy stability in plants regenerated from cryopreserved potato shoot tips, Cryo-Letters 17 (1996) 119 128. [45] K. Harding, Stability of the ribosomal genes in Solanum tuberosum L. plants recovered from cryopreservation, Cryo-Letters 18 (1997) 217 230. [46] D.R. Cyr, W.R. Lazaroff, S.M.A. Grimes, G. Quan, T.D. Bethune, D.J. Dunstan, D.R. Roberts, Cryopreservation of interior spruce (Picea glauca Engelmanni complex) embryogenic cultures, Plant Cell Rep. 13 (1994) 574 577. [47] L.L. DeVerno, Y.S. Park, J.M. Bonga, J.D. Barrett, Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.], Plant Cell Rep. 18 (1999) 948 953. [48] T.S. Aronen, J. Krajnakova, H.M. Haggman, L.A. Ryynanen, Genetic stability of cryopreserved embryogenic cultures of open pollinated Abies cephalonica, Plant Sci. 14 (1999) 163 172. [49] S.L. Krauss, S.D. Hopper, From Dampier to DNA: the 300 year old mystery of the identity and proposed allopolyploid origin of Conostylis stylidioides (Haemodoraceae). Aust. J. Bot. in press.

[32] S.L. Krauss, Complete exclusion of non-sires in an analysis of paternity in a natural population using AFLP, Mol. Ecol. 8 (1999) 217 226. [33] S.L. Krauss, R. Peakall, An evaluation of the AFLP ngerprinting technique for the analysis of paternity in natural populations of Persoonia mollis (Proteaceae), Aust. J. Bot. 46 (1999) 533 546. [34] R.H. Haskins, K.K. Kartha, Freeze preservation of pea meristems: cell survival, Can. J. Bot. 58 (1980) 833 840. [35] K.K. Kartha, N.L. Leung, K. Pahl, Cryopreservation of strawberry meristems and mass propagation of plantlets, J. Am. Soc. Hort. Sci. 105 (1980) 481 484. [36] G.J. Morris, Cryopreservation: An Introduction to Cryopreservation in Culture Collections, Cambridge, Institute of Terrestrial Ecology, Cambridge, UK, 1981, pp. 1 27. [37] B.M. Reed, Implementing cryogenic storage of clonally propagated plants, Cryo-Letters 22 (2001) 97 104. [38] T. Wilkinson, A. Wetten, M.F. Fay, Cryopreservation of rare and endangered species, Cryo-Letters 22 (2001) 87. [39] E. Wilhelm, Somatic embryogenesis in oak (Quercus spp.), In Vit. Cell Dev. Biol. 36 (2000) 349 357. [40] S. Goto, R.C. Thakur, K. Ishii, Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. Using RAPD markers, Plant Cell Rep. 18 (1998) 193 197. [41] M.B. Kumbar, R.E. Barker, B.M. Reed, Morphological and molecular analysis of genetic stability in micropropagated Fragaria x ananassa cv. Pocahontas, In Vitro Cell Dev Biol. 35 (1999) 254 258.