Application of the MSAP Technique to Evaluate Epigenetic Changes in Plant Conservation.

Epigenetic variation, and particularly DNA methylation, is involved in plasticity and responses to changes in the environment. Conservation biology studies have focused on the measurement of this variation to establish demographic parameters, diversity levels and population structure to design the appropriate conservation strategies. However, in ex situ conservation approaches, the main objective is to guarantee the characteristics of the conserved material (phenotype and epi-genetic). We review the use of the Methylation Sensitive Amplified Polymorphism (MSAP) technique to detect changes in the DNA methylation patterns of plant material conserved by the main ex situ plant conservation methods: seed banks, in vitro slow growth and cryopreservation. Comparison of DNA methylation patterns before and after conservation is a useful tool to check the fidelity of the regenerated plants, and, at the same time, may be related with other genetic variations that might appear during the conservation process (i.e., somaclonal variation). Analyses of MSAP profiles can be useful in the management of ex situ plant conservation but differs in the approach used in the in situ conservation. Likewise, an easy-to-use methodology is necessary for a rapid interpretation of data, in order to be readily implemented by conservation managers.

Lipid Thermal Fingerprints of Long-term Stored Seeds of Brassicaceae.

Thermal fingerprints for seeds of 20 crop wild relatives of Brassicaceae stored for 8 to 44 years at the Plant Germplasm Bank-Universidad Politécnica de Madrid and the Royal Botanic Gardens, Kew's Millennium Seed Bank-were generated using differential scanning calorimetry (DSC) and analyzed in relation to storage stability. Relatively poor storing oily seeds at -20 °C tended to have lipids with crystallization and melting transitions spread over a wide temperature range (c. 40 °C) that spanned the storage temperature, plus a melting end temperature of around 15 °C. We postulated that in dry storage, the variable longevity in Brassicaceae seeds could be associated with the presence of a metastable lipid phase at the temperature at which they are being stored. Consistent with that, when high-quality seed samples of various species were assessed after banking at -5 to -10 °C for c. 40 years, melting end temperatures were observed to be much lower (c. 0 to -30 °C) and multiple lipid phases did not occur at the storage temperature. We conclude that multiple features of the seed lipid thermal fingerprint could be used as biophysical markers to predict potential poor performance of oily seeds during long-term, decadal storage.

Genetic and epigenetic stability of recovered mint apices after several steps of a cryopreservation protocol by encapsulation-dehydration. A new approach for epigenetic analysis.

The genetic and epigenetic stability (analysis of DNA methylation using MSAP markers) of mint (Mentha x piperita L.) apices was studied after each step of a cryopreservation protocol, by encapsulation-dehydration. The effect of the addition of an antioxidant (ascorbic acid) during one of the protocol steps was also evaluated. Eight-week old in vitro recovered shoots from apices after each step of the protocol were genetically stable when compared to control in vitro shoots, using RAPD and AFLP markers. The addition of ascorbic acid in the medium with the highest sucrose concentration did not improve recovery and did not have any effect on stability. Apices sampled immediately after each step showed increased epigenetic differences as the protocol advanced, compared to in vitro control apices, in particular related to de novo methylation events. However, after one-day in vitro recovery, methylation status was similar to control apices. To improve the quality of methylation data interpretation, a simple and fast method for MSAP markers analysis, based on R programming, has been developed which allows the statistical comparison of treatments to control samples and its graphical representation.

Masa pulmonar en paciente con infección por el virus de la inmunodeficiencia humana / Pulmonary mass in a patient with human immunodeficiency virus infection

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Cryopreservation of Thymus lotocephalus shoot tips and assessment of genetic stability.

BACKGROUND: Thymus lotocephalus is a rare endemic species from the Algarve, Portugal, and is legally protected by Portuguese and European legislation. OBJECTIVE: The aim is to develop a cryopreservation protocol for T. lotocephalus shoot tips, as an alternative approach for the long-term conservation of this species. METHODS: Several methods (droplet-vitrification, vitrification and encapsulation-dehydration) were tested. Conditions regarding the subculture period, cold-hardening and preculture were optimized. Cryopreserved shoot tips were also assessed for their genetic stability using RAPD markers. RESULTS: Droplet-vitrification presented the best results. The best regrowth of cryopreserved shoot tips obtained eight weeks after rewarming was 67%. This was accomplished with four weeks subculture period of in vitro-donor plants at 25 degree C, preculture of excised shoot tips for one day on MS medium containing 0.3 M sucrose, treatment in PVS2 for 60 min, and MS supplemented with 0.2 mg per L zeatin as recovery medium. The assessment using RAPD markers observed variation at a low frequency and shoots regenerated from cryopreserved apices showed normal development compared to the regular in vitro-grown shoots. CONCLUSION: Droplet-vitrification is thus a viable method for the cryopreservation of T. lotocephalus shoot tips.

Approaches for the cryopreservation of Plantago algarbiensis, a rare endemic species of the Algarve.

BACKGROUND: Plantago algarbiensis is an endangered endemic species from the Algarve, Portugal. OBJECTIVE: The main goal of this study was to investigate the viability of cryopreservation procedures in the conservation of seeds and nodal explants from this species. MATERIALS AND METHODS: Seeds were directly immersed in liquid nitrogen (LN) for 30 days. Two methods were tested for the cryopreservation of nodal explants, namely droplet-vitrification and encapsulation-dehydration. For both methods, nodal segments were precultured on Murashige and Skoog (MS) medium and recovered on MS supplemented with 0.2 mg l(-1) 6-benzyladenine (BA), after freezing. RESULTS: After 30 days in LN, the germination capacity of seeds was not affected. The regrowth percentages of cryopreserved nodal segments were approximately 60%. With the droplet-vitrification method, a regrowth percentage of 60.0+/-15.2% was obtained after 120 min exposure to PVS2 (plant vitrification solution 2) and with encapsulation-dehydration method the highest percentage, 63.3+/-9.6%, was achieved after 3 h desiccation. CONCLUSION: Seed cryopreservation and cryopreservation of nodal segments by droplet-vitrification and encapsulation-dehydration are therefore effective approaches for the conservation of P. algarbiensis.

Genetic stability analysis of chrysanthemum (Chrysanthemum x morifolium Ramat) after different stages of an encapsulation-dehydration cryopreservation protocol.

Genetic stability in chrysanthemum (cultivar 'Pasodoble') apices was studied at each step of an encapsulation-dehydration cryopreservation protocol: control shoots (A), nodal segments after cold treatment (N), apices after osmotic stress (0.3M sucrose) and cold treatment (P), encapsulation and culture in 0.8M sucrose (S), dehydration (D), and cryopreservation (Cr). Two different markers were employed: RAPDs and AFLPs. Throughout the process, the origin of the apices (in vitro shoot from which they were excised) was recorded. Eight complete lines (from which DNA could be amplified after all the steps considered) were studied. Two out of twelve arbitrary primers showed polymorphisms. Three RAPD markers were replaced by three new ones in the Cr sample in one line. Using a different primer, a 700bp fragment was absent from all samples from the 0.3M sucrose-culture step ('P') onwards, in all the lines studied. The sequences of these fragments were studied to find similarities with known sequences. Polymorphic AFLP fragments were also observed, and most of the differences appeared from step 'P' onwards, pointing out the possible effect of this process (preculture on 0.3M sucrose) in the DNA variation. These results show that genetic variation can appear throughout the cryopreservation process, and the low temperature itself is not the only stress risk of the technique. Therefore, genetic stability of the regenerants obtained after cryopreservation should be monitored.