Genetic stability of cryopreserved shoot tips of <Emphasis Type="Italic">Rubus</Emphasis> germplasm

Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study examined the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for more than 12 yr, then rewarmed and regrown. We analyzed the genetic stability of Rubus grabowskii, two blackberry cultivars (“Hillemeyer” and ‘Silvan’), and one raspberry cultivar (“Mandarin”) as in vitro shoots and as field-grown plants. No morphological differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants appeared similar but were more vigorous than mother plants, with larger leaves, fruit, and seeds. Single sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for 7 mo before analysis. Ten SSR primers developed from “Marion” and “Meeker” microsatellite-enriched libraries amplified one to 15 alleles per locus, with an average of seven alleles and a total of 70 alleles in the four genotypes tested. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants regardless of subculture. Although no polymorphisms were detected in shoots analyzed immediately after recovery from cryopreservation, AFLP polymorphisms were detected in three of the four Rubus genotypes after they were subcultured for 7 mo. Field-grown plants from the polymorphic shoot tips of R. grabowskii and ‘Silvan’ displayed the same AFLP fingerprints as their corresponding mother plants. Only long-cultured in vitro shoot tips displayed polymorphisms in vitro, and they were no longer detected when the plants were grown ex vitro. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability.     

Survival and genetic stability of shoot tips of <Emphasis Type="Italic">Hedeoma todsenii</Emphasis> R.S.Irving after long-term cryostorage

Endangered and rare species for which seed banking is not possible require alternative methods of ex situ conservation for long-term preservation. These methods depend primarily on cryopreservation methods, such as shoot tip cryopreservation, but there are few datasets with information on the long-term survival of shoot tips stored in liquid nitrogen. In this study, survival and genetic stability of shoot tips of the endangered species, Hedeoma todsenii, banked over multiple years were examined. In vitro cultures cryopreserved with both the encapsulation dehydration and the encapsulation vitrification methods showed good average survival after up to 13 yr of storage in liquid nitrogen. The application of droplet vitrification to this species increased survival significantly, with an average of 72%, compared with 24–45% survival obtained with other methods. As measured with microsatellite and sequence-related amplified polymorphism (SRAP) markers, the genetic stability of the same genotypes stored over different periods of time typically did not change. However, there was an average of 10.4% band loss between replicate samples that did indicate a potential change in DNA composition. These results demonstrate the use of shoot tip cryopreservation as an effective ex situ conservation tool for this species, but genetic stability of the cryopreserved tissues should be closely monitored.     

Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.     

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings

The present study aimed at exploring the fidelity of coconut (Cocos nucifera L.) plants recovered from cryopreservation. Zygotic embryos from various different cultivars were cryopreserved following four successive steps, namely: rapid dehydration, rapid freezing, rapid thawing and in vitro recovery followed by acclimatization. At the end of the acclimatization period, the seedlings were compared to counterparts of the same age, which were produced from non-cryopreserved embryos. Both series were submitted to morphological, cytological and molecular comparisons. No significant differences in terms of growth rates could be measured. In addition, no morphological variation could be detected through the measurement of shoot elongation rates, production of opened leaves, and the number and total length of primary roots. Karyotype analysis revealed the same chromosome number (2n = 32) in all studied cultivars independently of cryopreservation. No significant differences could be observed between control and cryopreserved material concerning the type of chromosomes, the length of the long and short arms, the arm length ratio and the centromeric index. However, idiogram analysis did show a greater number of black banding on chromosomes isolated from cryopreserved material. Genetic and epigenetic fidelity was assessed through microsatellite (SSR) analysis and global DNA methylation rates; no significant differences would be observed between genomic DNAs isolated from seedlings originating from cryopreserved embryos and respective controls. In conclusion, our results suggest that the method of cryopreservation under study did not induce gross morphological, genetic or epigenetic changes, thus suggesting that it is an appropriate method to efficiently preserve coconut germplasm.     

Evaluation of genetic stability in cryopreserved Prunus

Summary The evaluation of the genetic stability of Prunus Ferlenain plants regenerated from cryopreserved apices was investigated. The analysis of plants recovered from frozen material was performed at the phenotypic (developmental competence), cytological (chromosomal preparations) and molecular level [random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP)]. No genetic change was detected among the plantlets regenerated from frozen apices in comparison to the non-frozen material, including leaves of the mother tree kept in an orchard and vitroplantlets regenerated from non-frozen apices. This result suggested that the procedure used for Prunus cryopreservation could be used for long-term conservation. The relevance of each strategy for the genetic stability evaluation of the cryopreserved material is discussed.     

Assessment of genetic and epigenetic changes following cryopreservation in papaya

A vitrification based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of such clonal material following cryopreservation. Shoot tips of 12 genotypes were cryopreserved, recovery rates were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was explored using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were recovered following cryopreservation in all but one genotype and recovery rates of 61-73% were obtained from six genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) of detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occur following cryopreservation which may result in somaclonal variation.     

Cryopreservation and long-term storage of pear germplasm

Summary Germplasm collections of vegetatively propagated crops are usually maintained as plants in fields or potted in greenhouses or screened enclosures. Safety duplication of these collections, as duplicate plants or separate collections, is costly and requires large amounts of space. Cryopreservation techniques which were recently developed for long-term storage of pear germalasm may offer an efficient alternative to conventional germplasm collection maintenance. Pear (Pyrus L.) germplasm may now be stored as seeds (species), dormant buds or pollen from field-grown trees, or shoot tips fromin vitro-grown plants (cultivars). Pear germplasm may now be cryopreserved and stored for long periods (> 100 yr) utilizing slow-freezing or vitrification ofin vitro-grown shoot-tips. Dormant bud freezing, pollen, and seed cryopreservation of other lines are being developed to complete the base collection forPyrus. This cryopreserved collection provides base (long-term) storage for the field-grown pear germplasm collection at the National Clonal Germplasm Repository, Corvallis, Oregon. Based on a presentation at the 1997 Congress on In Vitro Biology held in Washington, D.C., June 14–18, 1997.     

Phenotypic and molecular studies for genetic stability assessment of cryopreserved banana meristems derived from field and in vitro explant sources

Explants used for cryopreservation of banana (Musa L. spp.) are mainly sourced from tissue culture. Here, we demonstrate the successful use of sucker meristems (SM) obtained from field-raised plants for cryopreservation of Indian Musa ABB cv. ‘Karpura Chakkarakeli’. In addition, the genetic stability of plants recovered from cryopreserved and regenerated meristems after hardening and transfer to field conditions was studied using 11 phenotypic (biometric) characters and 21 simple sequence repeat (SSR) markers. The regenerative potential of cryopreserved SM was compared with two types of routinely employed explants of banana germplasm: in vitro-raised single-shoot meristems (IVM) and proliferating meristems (PM). The regeneration frequency of SM was high (60.0?±?11.5%) and statistically comparable to PM (68.3?±?4.4%) and IVM (55.6?±?11.1%) after using the droplet vitrification cryopreservation technique. The total time required for cryopreserving plants from SM (～2 mo) was substantially less than that for PM (14 mo) and IVM (8 mo). The SSR profiles of plants recovered from cryopreserved PM, IVM, and SM and compared with control plants had a similarity coefficient of 0.92. Data on phenotypic traits revealed that cryopreserved plants were statistically comparable to the mother plants raised from suckers for all important growth and yield parameters. This study broadens the possibilities to cryopreserve Musa germplasm, by applying the droplet vitrification method to a new type of explant, the SM. The results presented in this paper show that Musa meristems can be effectively cryopreserved for storage and regeneration of true-to-type plants.     

Genome fidelity during short- and long-term tissue culture and differentially cryostored meristems of silver birch (<Emphasis Type="Italic">Betula pendula</Emphasis>)

Clonal trueness of micropropagated or cryopreserved material is essential, especially with long-living tree species. In this study, the growth rate and morphology of regenerated silver birch (Betula pendula Roth) plants growing in the nursery were evaluated after different treatments: short-term (14 months) and long-term (70 months) tissue culture periods, cryostorage of in vivo buds and cryopreservation of in vitro shoot apices using four different slow cooling cryopreservation protocols with PGD (10% PEG, 10% glucose, 10% DMSO) as cryoprotectant. Genetic fidelity of the regenerated plants compared to the original donor trees was evaluated using RAPD assays together with chromosome analysis. The regenerated plants showed no genetic or phenotypic changes, and can thus be considered as reliable material for any research, breeding or silvicultural activities.     

Survival and genetic stability of Dendranthema grandiflora Tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration

The aim of this study was to compare the genetic stability of chrysanthemum (cv. Pasodoble) apices cryopreserved using two different methods: encapsulation-dehydration and vitrification. The assessment of the genetic stability was developed using RAPDs markers. Assessment of stability was evaluated in pot-cultivated mother plants (from which buds were excised for micropropagation), in shoots (leave tissue) from which apices were extracted for cryopreservation, and in shoots regenerated from cryopreserved apices 30 days after recovery and after further 3 months in culture. Throughout the process the origin of the apices (in vitro-shoot from which they were excised) was recorded. Twenty one regenerants cryopreserved by vitrification and 25 by encapsulation-dehydration were assessed. Only one cryopreserved regenerant from the encapsulation-dehydration method showed a different band pattern. These results support the necessity of monitoring the genetic stability of the regenerants obtained after cryopreservation, as this is a very useful technique for the conservation of plant genetic resources.     

Adaptive plasticity and epigenetic variation in response to warming in an Alpine plant

Environmentally induced phenotypic plasticity may be a critical component of response to changing environments. We examined local differentiation and adaptive phenotypic plasticity in response to elevated temperature in half‐sib lines collected across an elevation gradient for the alpine herb, Wahlenbergia ceracea. Using Amplified Fragment Length Polymorphism (AFLP), we found low but significant genetic differentiation between low‐ and high‐elevation seedlings, and seedlings originating from low elevations grew faster and showed stronger temperature responses (more plasticity) than those from medium and high elevations. Furthermore, plasticity was more often adaptive for plants of low‐elevation origin and maladaptive for plants of high elevation. With methylation sensitive‐AFLP (MS‐AFLP), we revealed an increase in epigenetic variation in response to temperature in low‐elevation seedlings. Although we did not find significant direct correlations between MS‐AFLP loci and phenotypes, our results demonstrate that adaptive plasticity in temperature response to warming varies over fine spatial scales and suggest the involvement of epigenetic mechanisms in this response.     

Genetic and biochemical stability assessment of plants regenerated from cryopreserved shoot tips of a commercially valuable medicinal herb <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Wettst

Shoot tips from four accessions (IC249250, IC 426442, IC 375976, and IC468878) of Bacopa monnieri (L.) Wettst., a commercially valuable memory revitalizing medicinal plant, were cryopreserved using a vitrification technique. Depending on the genotype, 0 to 20% plant regeneration without intermediary callus was achieved from cryopreserved shoot tips. Genetic stability of plants derived from cryopreserved shoot tips was assessed using biochemical and molecular markers. The regenerated plants from non-frozen controls and cryopreserved shoot tips exhibited morphological similarity to respective parental material when transferred to soil. On the basis of ten random amplified polymorphic DNA (RAPD) and bacoside A content using HPLC analysis, no significant reproducible variation was observed between the controls and in vitro-cryopreserved plants. Thus, after cryopreservation treatment, the regenerated plants exhibited molecular and biochemical genetic stability.     

Epigenetic Variation in Mangrove Plants Occurring in Contrasting Natural Environment

Background

Epigenetic modifications, such as cytosine methylation, are inherited in plant species and may occur in response to biotic or abiotic stress, affecting gene expression without changing genome sequence. Laguncularia racemosa, a mangrove species, occurs in naturally contrasting habitats where it is subjected daily to salinity and nutrient variations leading to morphological differences. This work aims at unraveling how CpG-methylation variation is distributed among individuals from two nearby habitats, at a riverside (RS) or near a salt marsh (SM), with different environmental pressures and how this variation is correlated with the observed morphological variation.

Principal Findings

Significant differences were observed in morphological traits such as tree height, tree diameter, leaf width and leaf area between plants from RS and SM locations, resulting in smaller plants and smaller leaf size in SM plants. Methyl-Sensitive Amplified Polymorphism (MSAP) was used to assess genetic and epigenetic (CpG-methylation) variation in L. racemosa genomes from these populations. SM plants were hypomethylated (14.6% of loci had methylated samples) in comparison to RS (32.1% of loci had methylated samples). Within-population diversity was significantly greater for epigenetic than genetic data in both locations, but SM also had less epigenetic diversity than RS. Frequency-based (G_ST) and multivariate (β_ST) methods that estimate population structure showed significantly greater differentiation among locations for epigenetic than genetic data. Co-Inertia analysis, exploring jointly the genetic and epigenetic data, showed that individuals with similar genetic profiles presented divergent epigenetic profiles that were characteristic of the population in a particular environment, suggesting that CpG-methylation changes may be associated with environmental heterogeneity.

Conclusions

In spite of significant morphological dissimilarities, individuals of L. racemosa from salt marsh and riverside presented little genetic but abundant DNA methylation differentiation, suggesting that epigenetic variation in natural plant populations has an important role in helping individuals to cope with different environments.     

Microsatellite primers for Texas wild rice (<Emphasis Type="Italic">Zizania texana</Emphasis>), and a preliminary test of the impact of cryogenic storage on allele frequency at these loci

Seed collections in gene banks are useful for the preservation of wild germplasm, providing inexpensive insurance for species that survive in conventional cold storage (–18 °C). Seeds that cannot survive these conditions must be pretreated with cryoprotectants and stored at liquid nitrogen temperatures, which presents unique technical and methodological challenges. Implicit in this approach is the assumption that these added manipulations do not change the genetic diversity of the preserved collections. We used polymorphic microsatellite markers for an endangered aquatic grass, Texas wild rice (Zizania texana), to conduct a preliminary evaluation of the effects of cryogenic preservation of mature embryos on genetic diversity. Using several statistical approaches, we show that allele frequencies did not change in collections of seeds that underwent cryopreservation (cryoprotected) compared to those samples that was not exposed to cryopreservation (control). The retention of the allelic diversity at the five loci examined suggests that there were no significant changes in genetic diversity due to treatments and that these protocols may be appropriate for ex situ conservation of genetically diverse wild germplasm.     

Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: influence of abscisic acid and cold acclimation

It has been previously shown that shoot tips of in vitro plantlets of sugar beet (Beta vulgaris L. clone SES1) can be cryopreserved using the encapsulation-dehydration technique (survival rate of 37% after freezing). This article reports the influence of abscisic acid (ABA) and cold acclimation on survival after cryopreservation. When ABA was added to the multiplication medium of the plants, the survival rate of shoot tips after cryopreservation was not increased (45%). After cold acclimation of the plants, their growth pattern differed (plants became apically dominant) and the survival rate of the shoot tips after cryopreservation clearly increased (70% survival and 50% plant regeneration after freezing). This improved protocol was successfully applied to three other clones. Received: 28 October 1996 / Revision received: 28 January 1997 / Accepted: 15 March 1997     

Biodiversity conservation and conservation biotechnology tools

This special issue is dedicated to the in vitro tools and methods used to conserve the genetic diversity of rare and threatened plant species from around the world. Species that are on the brink of extinction because of the rapid loss of genetic diversity and habitat come mainly from resource-poor areas of the world and from global biodiversity hotspots and island countries. These species are unique because they are endemic, and only a few small populations or sometimes only a few individuals remain in the wild. Therefore, the challenges to support conservation by in vitro measures are many and varied. The editors of this invited issue solicited papers from experts from Asia, Africa, Europe, Australia, and North and South America. This compilation of articles describes the efforts in these diverse regions toward saving plants from extinction, and details the direct application of in vitro and cryopreservation methods. In addition, these contributions provide guidance on propagation of rare plants, including techniques for large-scale propagation, storage, and reintroduction. The in vitro techniques for conserving plant biodiversity include shoot apical or axillary-meristem-based micropropagation, somatic embryogenesis, cell culture technologies and embryo rescue techniques, as well as a range of in vitro cold storage and cryopreservation protocols, and they are discussed in depth in this issue.     

Variation in cytosine methylation patterns during ploidy level conversions in Eragrostis curvula

In many species polyploidization involves rearrangements of the progenitor genomes, at both genetic and epigenetic levels. We analyzed the cytosine methylation status in a ‘tetraploid-diploid-tetraploid’ series of Eragrostis curvula with a common genetic background by using the MSAP (Methylation-sensitive Amplified Polymorphism) technique. Considerable levels of polymorphisms were detected during ploidy conversions. The total level of methylation observed was lower in the diploid genotype compared to the tetraploid ones. A significant proportion of the epigenetic modifications occurring during the tetraploid–diploid conversion reverted during the diploid–tetraploid one. Genetic and expression data from previous work were used to analyze correlation with methylation variation. All genetic, epigenetic and gene expression variation data correlated significantly when compared by pairs in simple Mantel tests. Dendrograms reflecting genetic, epigenetic and expression distances as well as principal coordinate analysis suggested that plants of identical ploidy levels present similar sets of data. Twelve (12) different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated, sequenced and characterized.     

Epigenetic Variability in the Genetically Uniform Forest Tree Species Pinus pinea L

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.     

Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae

In this study we established reliable methods for conservation of seeds of Phaius tankervilleae as an orchid genetic resource. The seeds, which were dehydrated to 5% water content and preserved at 4°C, showed no decrease in viability and germinability after three months. After storage for six months, however, the seeds showed a drastic decrease in germinability, even though survival rate was high. For long-term preservation of seeds of P. tankervilleae, cryopreservation is applied to the freshly harvested seeds. When the seeds were cryopreserved by the vitrification method for up to 12 months there was no apparent deterioration effect of storage time. These results indicate that cryopreservation by the vitrification method is useful for long-term conservation of P. tankervilleae seeds, which are difficult to preserve for more than three months under dry and low-temperature conditions.