Germplasm Storage Research Articles

Encapsulation of Rauvolfia tetraphylla microshoots as artificial seeds and evaluation of genetic fidelity using RAPD and ISSR markers

Synseeds of Rauvolfia tetraphylla were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Woody Plant Medium (WPM) (Lloyd and McCown, 1981) and 100 mM calcium chloride. Effect of different medium on in vitromorphogenic response of synseed was evaluated. The maximum frequency (90.3%) of conversion of encapsulated beads into plantlets was achieved on WPM containing 7.5 μM 6-benzyladenine (BA) and 2.5 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. Encapsulated nodal segments stored at 4°C for 1 to 8 weeks also showed successful conversion, followed by development into complete plantlets when returned to regeneration medium. Healthy root formation was achieved in plantlets following 4 weeks of their transfer to Murashige and Skoog medium containing 0.5 μM indole-3-butyric acid (IBA). Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. Genetic stability of synseed-derived plantlets was assessed and compared with mother plant using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analysis. No changes in RAPD and ISSR profiles were found among the regenerated plantlets. This synseed protocol could be useful for conservation, short-term storage and exchange of germplasm of this endangered medicinal plant between national as well as international laboratories. Key words: Encapsulation, germplasm storage, regeneration, synthetic seeds, random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR).

Crioconservación: herramienta para la conservación ex situ de material vegetal

Nowadays different in vitro techniques are employed in order to preserve plant species. Among these, cryopreservation (the conservation of plant material at ultralow temperature, usually at -196 oC, which is the temperature of liquid nitrogen) is widely employed for long-term storage of plant germplasm. These techniques could complement other ex situ strategies like seed banks or field collections, and are the unique reliable way for long-term preservation of species that are vegetatively propagated or have recalcitrant seeds. Recently, cryopreservation protocols in Tetraclinis articulata and Thymus moroderi explants have been developed.

华山新麦草光合特性对干旱胁迫的响应

以濒危植物华山新麦草为材料进行盆栽试验,设置3个水分梯度,研究生长指标、气体交换、叶绿素荧光参数、光合色素含量、光响应特征及丙二醛(MDA)含量。结果表明:随干旱胁迫的加剧,株高、叶宽和光合色素含量降低,根长和丙二醛(MDA)含量增加;水分胁迫导致净光合速率(<em>P</em>_N)、气孔导度(<em>g</em>_s)、蒸腾速率(<em>E</em>)、最大荧光(<em>F</em>_m)、光合电子传递速率(ETR)和光化学猝灭系数(<em>q</em>_P)降低,胞间CO₂浓度(<em>C</em>_i)、瞬时水分利用效率(WUE)、初始荧光(<em>F</em>_o)和非光化学淬灭系数(NPQ)升高;光系统Ⅱ最大光化学效率(<em>F</em>_v/<em>F</em>_m)不变;干旱胁迫下光响应曲线拟合结果显示,干旱胁迫造成最大光合速率和光能利用效率下降。综上表明,干旱对光系统Ⅱ的伤害是制约华山新麦草光合作用的主要原因。;<em>Psathyrostachys huashanica </em>Keng is a perennial herb belongs to Gramineae and only distributes in Huashan area of Qinling Mountain, Shaanxi Province, China. At present, the amount of the population is much less than the past and suffers the risk of extinction. Owing to distributing limitation and importance as breeding material for germplasm storage, it has been listed into the first class of rare and endangered species of the nation and imperatively protected wild species in relation to crops. Drought is the major environmental stress limiting growth and yield of plants worldwide. Mean global temperature and evaporation rate are expected to rise over the next few decades and as a consequence the crop demand for water will increase. It is well known that inhibition of photosynthesis is one of the primary physiological consequences of drought stress. For this reason, the study of photosynthetic characteristics with related to drought stress is worthwhile in exploration of the acclimation and conservation of this endangered species. In the present paper, the response of photosynthetic characteristics to drought stress in <em>P. huashanica </em>Keng was studied by measuring growth parameters, gas-exchange, photosynthetic pigment contents, modulated chlorophyll fluorescence and malonaldehyde (MDA) contents under 3 different drought stresses in a pot experiment. The result exhibited that, when increasing drought stress, plant height, the width of leaf and photosynthetic pigment contents decreased and root length and MDA contents enhanced. Drought stress induced the decrease of net photosynthesis (<em>P</em>_N), stomatal conductance (<em>g</em>_s), ratio of transpiration (<em>E</em>), the maximum fluorescence (<em>F</em>_m), the electron transport rate (ETR) and photochemical quenching (<em>q</em>_P) and an increase of ratio of intercellular CO₂, intrinsic water use efficiency (WUE), the minimal fluorescence (<em>F</em>_o) and non-photochemical quenching (NPQ). Meanwhile, the maximum quantum yield of photo-system II (PSII) (<em>F</em>_v/<em>F</em>_m) kept invariable under drought stress. Besides, the fitted indexes from photo-response curves of <em>P</em>_N showed that photosynthesis and photosynthetic capacity were progressively reduced under drought conditions. As a consequence, lower light intensity is required to saturate photosynthesis. Under these conditions, plants cannot use all the excess of light energy for photosynthesis, increasing the susceptibility of plants to photoinhibition. In this experiment, photosystem II was injuried by severe drought, the quantum yield of photosystem II are significantly reduced by nonphysiologically low relative water contents when photoinhibition occurs, which may be the main cause restraining factors of photosynthesis. Electron transport is very resistant to drought and can remain unchanged during mild drought while it decreased when suffering severe drought stress. Summing up and obtains a conclusion, Gas-exchange data showed that the closure of stomata during drought stress, stomatal closure is one of the first responses to soil drying in <em>Psathyrostachys huashanica </em>Keng, and when drought stress increased, the photosynthetic apparatus was destroyed irreversible (Chlorophyll fluorescence parametres exhibited the functioning of the photosynthetic apparatus), and non-stomatal limitations may be the main inhibition of photosynthesis. Since <em>Psathyrostachys huashanica </em>Keng was distributed narrowly and disconnectedly and it's habitat is often on roadside or cliff or rock hollow (water deficit), namely quite rigorous, drought stress may be one of the main cause of the species endangered.

CRIOCONSERVACIÓN DE ÁPICES DE CRISANTEMO (Dendranthema grandiflorum Kitam) POR ENCAPSULACIÓN-DESHIDRATACIÓN Y POR VITRIFICACIÓN

The development of biotechnological methods of cryopreservation allows safe, long-term germplasm storage of valuable species such as chrysanthemum, which is one of the most important ornamental plants in Mexico and around the world. This study used the cryogenic methods of encapsulation-dehydration and vitrification to conserve chrysanthemum cv. Indianapolis White plants grown in vitro. Results showed similar behavior with both procedures during tissue recovery. The highest survival rates after cryopreservation were 60 % for encapsulation-dehydration and 74 % with vitrification. However, regeneration in both cases passed through a primary phase of callus formation and rates of obtention of new plants were low, 12 and 10 %, respectively. Therefore, further research is needed to improve cryoprotectective conditions in order to increase rate of recovery of chrysantemum plants.

CONSERVATION OF POTATO GERMPLASM IN THE CZECH REPUBLIC

In this study we tested a new cryopreservation method for conservation of old potato cultivars of the Czech origin. Explants recovered after cryopreservation were introduced into ex vitro conditions and tests of genotype stability were evaluated by molecular markers, morphological traits and tuber proteins. Fifty old potato cultivars of the Czech origin were cryopreserved and successfully recovered. Genetic stability of cryopreserved cultivars was evaluated in 29 cultivars by molecular markers, 14 cultivars by morphological traits and 8 cultivars by tuber protein analyses. Plants regenerated after cryopreservation were compared with plants from in vitro bank. No significant differences were found between the samples before and after cryopreservation. The results showed the possibility of using the cryopreservation method for long-term storage of potato germplasm.

Genetic studies of seed longevity in hexaploid wheat using segregation and association mapping approaches

Genebanks are entrusted with the storage, preservation and distribution of crop germplasm. Seed longevity is an important character in this context, but little is known regarding its genetic basis, largely because it is so strongly influenced by non-genetic factors. Here we present the outcome of a genetic dissection of seed longevity in bread wheat. We applied both a standard quantitative trait locus analysis based on segregation from a biparental cross, and an association analysis using a germplasm panel to detect marker trait associations. The latter revealed more loci than the former. Some of the genomic regions identified are known to contain genes determining spike architecture or aspects of biotic and abiotic stress responses. The results open perspectives for identification of favourable longevity alleles and the more accurate prediction of seed longevity in cereal germplasm collections.

Agroforestry Tree Seed Production and Supply Systems in Malawi

A sustainable agroforestry tree germplasm supply system is vital to resource-constrained smallholder farmers who depend on agroforestry to improve the productivity of their farm enterprises. Successful adoption of agroforestry hinges on the development of a sustainable agroforestry tree germplasm supply system. This paper reviews the agroforestry tree seed supply system in Malawi, with a view to determining its sustainability and quality. Currently, more than 90% of the documented agroforestry tree seed distributed to farmers is produced by smallholder farmers collected mainly from scattered farmland trees, the remainder being produced from seed orchards and seed stands owned or controlled by research organizations. Three organizations—namely the Land Resources Centre (LRC), National Tree Seed Centre (NTSC) of the Forestry Research Institute of Malawi (FRIM) and the World Agroforestry Centre (ICRAF)—were identified as major procurers of agroforestry tree seed produced by smallholder farmers. Agroforestry germplasm is distributed to farmers by non-governmental organizations (NGOs) and government agricultural and forestry extension departments. The procurement and distribution of germplasm to farmers is in general effective. The major challenge to sustainability of agroforestry tree germplasm distribution in Malawi is dependence on donor funding. The agroforestry tree seed system is, to some extent, sustainable with regards to production, although the genetic quality of the germplasm is low. Germplasm storage facilities at national level are available and possibly adequate, but knowledge and information on effective low-cost tree germplasm storage systems at household level are limited. Sustainability could be enhanced by strengthening of grass-root organizations involved in tree seed production to institutionalize the distribution through farmer–farmer exchange. There is also a need to support the development, promotion and adoption of low-cost tree germplasm storage facilities by smallholder farmers.

Is Australia ready for assisted colonization? Policy changes required to facilitate translocations under climate change.

Assisted Colonization (AC) has been proposed as one method of aiding species to adapt to the impacts of climate change. AC is a form of translocation and translocation protocols for threatened species, mostly for reintroduction, are well established in Australia. We evaluate the information available from implementation of translocations to understand how existing policies and guidelines should be varied to plan, review and regulate AC. While the risks associated with AC are potentially greater than those of reintroductions, AC is likely to be the only available method, other than germplasm storage and establishment of captive populations, of conserving many taxa under future climate change. AC may also be necessary to maintain ecosystem services, particularly where keystone species are affected. Current policies and procedures for the preparation of Translocation Proposals will require modification and expansion to deal with Assisted Colonization, particularly in relation to risk management, genetic management, success criteria, moving associated species and community consultation. Further development of risk assessment processes, particularly for invasiveness, and guidelines for genetic management to maintain evolutionary potential are particularly important in the context of changing climate. Success criteria will need to respond to population establishment in the context of new and evolving ecosystems, and to reflect requirements for any co-establishment of interdependent species. Translocation Proposals should always be subjected to independent peer review before being considered by regulators. We conclude that consistent approaches by regulators and multilateral agreements between jurisdictions are required to minimize duplication, to ensure the risk of AC is adequately assessed and to ensure the potential benefits of AC are realized.

Importance of in vitro technology to future conservation programmes worldwide

The latest IUCN statistics show that of over 12,000 plant species, 70% are threatened, 19% are critically endangered and 28 species are extinct in the wild. Target 8 of the Global Strategy for Plant Conservation (GSPC) highlights the importance of ex situ conservation of critically endangered plants. Long-term germplasm storage for species with recalcitrant seeds needs alternative measures. In vitro methods complement seed banking and other ex situ measures and are vital for long-term conservation. Conservation Biotechnology at RBG Kew is currently working on a number of rare and threatened recalcitrant species from biodiversity-rich areas of the world to develop good quality in vitro propagules for cryopreservation, recovery and restoration projects. The importance of successful in vitro propagation methods, transplantation technologies, cryopreservation and international networking for the integrated conservation of these species are discussed in detail.

Application of Cryopreservation Technique by Encapsulation-dehydration Pretreatment for Long-term Storage of Germplasm in Seeds of Lily and Embryonic Axes of Persian Lilac and Tea

This study focused on increasing the freezing rate in cell vitrification cryopreservation by using a cryopreservation container possessing rigid mechanical properties and high heat-transfer efficiency. Applying a fast freezing rate in vitrification cryopreservation causes a rapid temperature change in the cryopreservation container and has a substantial impact on mechanical properties; therefore, a highly rigid cryopreservation container that possesses a fast freezing rate must be developed. To produce a highly rigid cryopreservation container possessing superior heat transfer efficiency, this study applies an electrochemical machining (ECM) method to an ANSI 316L stainless steel tube to treat the surface material by polishing and roughening, thereby increasing the freezing rate and reducing the probability of ice crystal formation. The results indicated that the ECM method provided high-quality surface treatment of the stainless steel tube. This method can reduce internal surface roughness in the stainless steel tube, thereby reducing the probability of ice crystal formation, and increase external surface roughness, consequently raising convection heat-transfer efficiency. In addition, by thinning the stainless steel tube, this method reduces heat capacity and thermal resistance, thereby increasing the freezing rate. The freezing rate (3399 ± 197 °C/min) of a stainless steel tube after interior and exterior polishing and exterior etching by applying ECM compared with the freezing rate (1818 ± 54 °C/min) of an original stainless steel tube was increased by 87%, which also exceeds the freezing rate (2015 ± 49 °C/min) of an original quartz tube that has a 20% lower heat capacity. However, the results indicated that increasing heat-transferring surface areas and reducing heat capacities cannot effectively increase the freezing rate of a stainless steel tube if only one method is applied; instead, both techniques must be implemented concurrently to improve the freezing rate.

Cryopreservation of fish sperm: applications and perspectives

Summary Cryopreservation is of interest not only for fish farming but also for the conservation and genetic improvement of resources. This technique has been well established in some freshwater fish species mainly, salmonid, sturgeons and carps, however, only in the last decade research was focused in marine fish species. The benefits of sperm cryopreservation include: (i) synchronization of gamete availability of both sexes, (ii) sperm economy; (iii) simplification of broodstock management, (iv) transport of gametes from different fish farms, and (v) germplasm storage for genetic selection programs or conservation of species. These issues would certainly benefit the aquaculture industry. The tremendous impact that biotechnology is having in aquaculture has been particularly obvious in recent years. Several species are being used as research models not only for aquaculture development applications but also for medical research. Sperm cryopreservation can give an important contribution in the germ storage of all transgenic lines. However, in all applications in fish sperm, cryopreservation needs to overcome a lack in standardization of methodologies and procedures, a correct assay of seminal quality and the development of tools to characterize cryoinjury. Many efforts have recently been made in the study of DNA using different approaches such as the comet assay (single cell gel electrophoresis), TUNEL (terminal deoxynucleotidyl transferase-nick-end-labelling), SCSA (sperm chromatin structure assay) and the analysis of specific DNA sequences using RT-PCR, since DNA damage may impair fertility or embryo development. Cryopreservation of gametes would certainly benefit from a higher concern on male improvement, basically through nutrition or selection of resistant stocks (e.g. stress resistant individuals or highly adapted to captivity) producing gametes of higher quality. There is a huge window of opportunities for improve the resistance of cells to cryopreservation through diet supplementation of certain compounds such as amino acids (taurine and hypotaurine), vitamins (Vit. E and C) and lipids or through a direct supplementation of the extender media. An equilibrium of those compounds will improve spermatozoa and seminal plasma composition protecting cells against oxidative stress (lipid peroxidation, protein oxidation, DNA fragmentation, enzyme protection) that is gaining each day more importance in cryodamage research.

In vitro proliferation and minimum growth storage of fraser photinia: Influences of different medium, sugar combinations and culture vessels

Protocols for the in vitro proliferation and storage of fraser photinia were developed by comparing 6-benzyladenine (BA) concentrations (0.5–4 mg/L) together with different media formulations [Murashige and Skoog (MS) media and Quoirin and Lepoivre (QL) media], sugar combinations (sucrose and mannitol), culture vessels (baby food jars and vitrovents) and methods (synthetic seed technology and slow growth storage). The best responses in terms of both proliferation percentage and multiple shoot formation were obtained in QL medium containing 1 mg/L BA. Synthetic seed production was optimized by encapsulating shoot apices in 3% sodium alginate. Encapsulated shoot apices could be maintained up to 6 months at 4 °C in dark with 91.6% sprouting in MS medium. Microshoots were stored at 4 °C up to 15 months on sucrose and mannitol containing QL medium in both baby food jars and vitrovents without subculture. The stored material could be recovered and multiplied normally in 1 mg/L BA supplemented QL medium. Both in vitro propagated and conserved microshoots were rooted (∼75%) on QL medium with 1 mg/L indole butyric acid (IBA). Optimized synthetic seed and slow growth storage system can be used for short and medium-term storage of fraser photinia germplasm.

Encapsulation for in vitro short-term storage and exchange of ginger ( Zingiber officinale Rosc.) germplasm

Synseeds of ginger ( Zingiber officinale) were produced using aseptically proliferated 2-week old encapsulating explants (microshoots) upon complexation of 4% sodium alginate prepared in Murashige and Skoog (1962) medium (MS) and 100 mM calcium chloride. Conversion of synseeds into plantlets (conversion) was recorded as 66% and 53% on MS (3% sucrose) and on MS (3% sucrose) + 2.5 mg/l BA media, respectively. However, shoots/synseed were significantly higher on MS (3% sucrose) + 2.5 mg/l BA medium. For short-term storage of germplasm, sucrose-dehydrated synseeds were found better than air-dehydrated or fresh synseeds. Synseeds dehydrated in 0.25 M sucrose liquid medium for 16 h and stored in cryovials (with out medium) at 25 °C for 8 weeks and 12 weeks exhibited 53% and 13% conversion, respectively, on MS (3% sucrose) + 2.5 mg/l BA medium. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plants. This synseed protocol could be useful for short-term storage and exchange of germplasm of ginger between national as well as international laboratories.

Effect of liquid nitrogen storage time on the survival and regeneration of somatic embryos of cocoa

Investigations were undertaken on the effect of liquid nitrogen (LN) storage time on survival and regeneration of somatic embryos of cocoa (Theobroma cacao l.). Somatic embryos from different cocoa genotypes (AMAZ 3-2, AMAZ 10-1, AMAZ 12, SIAL 93, and IMC 14) at 15.45% moisture content were cryopreserved in LN for one h, four and eight weeks. Somatic embryos of the genotypes emerged from the alginate beads at different periods 4 to 12 weeks post-cryopreservation. Individual genotypes subjected to low temperature storage timedid not show significant differences in post-cryopreservation survival, although different genotypes responded differently with AMAZ 12 and IMC 14 recording the highest and lowest mean survival rates of 58% and 35%, respectively. Plantlets originating from five genotypes had been weaned and developed normally comparable to non-cryopreserved somatic embryo-derived plantlets in the glasshouse. The effects of low temperature storage time on survival and genetic stability of somatic embryos using genotypes from the same parental and distinct lines may contribute to long-term storage of cocoa germplasm.

Morphological and physiological seed heterogeneity in the Mediterranean annual plant Tuberaria macrosepala (Cistaceae)

The morphometric features, germination requirements (under controlled conditions of light and temperature, and after different pre-sowing treatments) and cryopreservation tolerance of Tuberaria macrosepala (Cistaceae) seeds were studied. Constant (5, 10, 15, 20, 25, 30°C) and alternating temperature regimes (25/15°C, 30/20°C) with 16 h/8 h light/dark photoperiod and constant darkness conditions were used. Mechanical scarification, dry heat, boiling water, soaking in distilled water and soaking in gibberellic acid solution were used as pre-sowing treatments to enhance germination. Tuberaria macrosepala seeds were separated into three groups (light brown, medium brown and dark brown) according to the color of their seed coat. These three types of seeds also differed in size, weight and germinability. Dark brown seeds were found to be inferior to both medium and light brown seeds in size, final germination percentage and germination rate. No significant differences were found in seed weight between the three seed color types. Most seeds of the studied population were light brown. The final germination percentages of this type of seed ranged from 12 to 69%, depending on the temperature and light conditions used. Mechanical scarification significantly improved the final germination percentages of the three seed color types (86–96%). Therefore, seed coat impermeability to water (physical dormancy) appears to be the main cause of seed dormancy present in this species and seed color may be an indicator of hardseededness. Cryopreservation did not significantly decrease the final germination percentages. Therefore, cryopreservation could provide a method for long-term storage of germplasm of this species.

In vitro germplasm conservation of Podophyllum peltatum L. under slow growth conditions

Germplasm conservation of Podophyllum peltatum L. was attempted by using synthetic seed technology and media supplemented with osmotic agents. Excised buds from in vitro cultures were encapsulated in calcium alginate beads and cultured on different substrates then stored at 5, 10, and 25°C for up to 8 mo. Survival and vigor in re-growth were the parameters used to evaluate the germplasm storage conditions. Vigor in re-growth was measured by number of buds induced after storage, which was achieved on a substrate containing water solidified with 1% w/v agar under 10°C. In vitro storage of shoot cultures was also evaluated by supplementing osmotic agents, mannitol, or sorbitol to the media. Such treatment had a negative impact on post-storage re-growth (at 25°C), even though the inclusion of 2% w/v sorbitol and mannitol each to the media increased plantlet survival during 10°C storage treatment. A deleterious effect was noticed among cultures in re-growth when higher concentrations of these supplements were added to the media. Genetic stability was assessed following 8 mo of storage using a PCR-based multilocus DNA fingerprinting technique, amplified fragment-length polymorphism. No differences in the DNA fragment patterns were observed using eight primer combinations in stored clones. However, a polymorphic band was noticed in the accession that served as explant source, suggesting that the mutation has occurred prior to this study perhaps during the 9 years of in vitro cultivation.

Germination requirements and cryopreservation tolerance of seeds of the endangered species Tuberaria major

Summary The germination requirements and the cryopreservation tolerance of Tuberaria major (Cistaceae) seeds, an endangered plant species endemic from the Algarve region (South of Portugal) were studied. The final germination percentage of untreated seeds was very low (less than 2%) and was significantly improved by heat treatments (maximum of 54%). No significant differences were observed among the germination percentages reached by seeds submitted to boiling water or dry heat (100°C for different periods: 15, 30 and 60 min). Moreover, the temperature regime did not affect the final germination percentage. Cryopreservation did not affect the germination rate in comparison with the control, although the heat treatment was still necessary after cryopreservation. In conclusion, it was observed that heat stimulated germination of T. major seeds and cryopreservation could provide a method for long-term storage of germplasm of this endangered species. Experimental and discussion

Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation

AbstractCryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra‐low temperature, usually that of liquid nitrogen (−196°C), and is considered as an ideal means for long‐term storage of plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature‐limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo‐treatment and regeneration of healthy shoots from the surviving pathogen‐free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen‐free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana (Musa spp.), Citrus spp., grapevine (Vitis vinifera), Prunus spp., raspberry (Rubus idaeus), potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing ‘citrus greening’. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy.

Medium, container and genotype all influence in vitro cold storage of apple germplasm

The goal of this study was to evaluate the in vitro storage of apple germplasm by screening a range of genotypes followed by more comprehensive testing of multiple parameters on two genotypes of differing species, Malus domestica cultivar Grushovka Vernenskaya and wild Malus sieversii selection TM-6. Stored plants were rated on a 6 point scale (0 low to 5 high) for plant appearance at 3 month intervals after storage at 4°C. Combinations of carbon source (sucrose and/or mannitol), nitrate nitrogen content (25, 50 or 100%) and plant growth regulators (ABA, BAP, IBA) were studied in three types of containers (tissue culture bags, test tubes or jars). An initial screen of 16 genotypes stored in tissue culture bags indicated that plantlets could be stored at 4°C for 9–14 months without subculture on standard 3% sucrose Murashige and Skoog (1962) (MS) medium with no plant growth regulators (PGRs). In subsequent in-depth studies on the two genotypes, ANOVA indicated highly significant interactions of medium, container and genotype. ‘Grushovka Vernenskaya’ shoots with no PGRs and 3% sucrose remained viable (ratings of ≥1) for 21 months of storage in bags. Storage on reduced nitrogen (MS with 25% nitrogen), PGRs, and 3% sucrose kept ‘Grushovka Vernenskaya’ shoot condition rated >2 at 21 months. Addition of 0.5 or 1 mg−1 abscisic acid (ABA) also improved plant ratings at 21 months. The longest storage for ‘Grushovka Vernenskaya’ was 33–39 months with PGRs and 3% sucrose in either tubes or jars. Addition of abscisic acid (ABA) to the medium did not improve storage of plantlets in jars and tubes at 15 months. TM-6 stored best in tubes on 3% sucrose with PGRs or in jars on 2% mannitol and 2% sucrose. Overall it appears that cold storage of apple shoot cultures can be successful for 21 months in tissue culture bags with 25% MS nitrate nitrogen, 3% sucrose, and no PGRs or for 33 months in jars or tubes on MS with 3% sucrose and PGRs. Preliminary RAPD analysis found no significant differences between plants stored for 39 months and non-stored controls.

How bryophytes came out of the cold: successful cryopreservation of threatened species

The use of ex situ techniques for the conservation of threatened plants has been increasing over the past decades. Cryopreservation is often used for the long-term storage of plant germplasm where conventional methods (i.e. seedbanking) are inappropriate. Simple encapsulation-dehydration protocols were developed for the cryopreservation of bryophytes at The Royal Botanic Gardens, Kew, as part of an ex situ project for the conservation of UK threatened species. The applicability of these methods was tested on 22 species with a broad range of ecological requirements and found to be highly successful across the board. Regeneration rates from frozen material were >68% for all species tested and half had regeneration rates of 100%. The high regeneration rate and broad applicability of the protocols across a range of species was attributed to a combination of the inherent totipotency of bryophytes and the in-built recovery periods in the pre-treatment protocol. In conclusion, bryophytes are well suited to cryopreservation and such techniques would be applicable for the long-term storage of similar conservation collections across the globe.