CRYO 2019 San Diego

Dani Ballesteros, Christina Walters, Hugh W. Pritchard

Royal Botanic Gardens Kew, Wellcome Trust Millennium Building, United Kingdom, United States Department of Agriculture., United States

Often various plant propagules (e.g., seeds, pollen and fern spores) can be stored dry (or partially dry) at sub-zero temperatures for extended periods of time. Under such conditions the cell cytoplasm of these propagules solidifies and forms a glass, lethal water freezing does not occur, and metabolic reactions are inhibited. However, dry (or partially dry) plant propagules deteriorate and eventually die during sub- zero storage, including at liquid nitrogen temperatures (i.e. < -150°C). The rates of deterioration are diverse, depend on species and genotypes, and are often influenced by the dry architecture of the propagule’s cells. Probing life and death in the glassy state of cryopreserved materials is challenging, which makes the understanding of the ageing mechanisms of dry plant propagules difficult. However, new techniques in tomography, microscopy and biophysics have been adopted for the study of longevity of cryopreserved dry plant propagules from a structural point of view. Of importance among these tools is: Differential Scanning Calorimetry, Dynamic Mechanical Analysis, micro-Computer Tomography, and Cryo- Scanning Electron Microscopy. They are providing interesting clues about the deterioration mechanisms of seeds, fern spores and pollen during drying and sub-zero exposure and storage. Understanding these deterioration mechanisms will lead to the development of optimized storage protocols to maximize plant germplasm ex situ preservation.

Contributing Author(s): 
Date Recorded: 
Tuesday, July 23, 2019

Meera Das*, Reema Diengdoh, Seram Devika, Suman Kumaria, North-Eastern Hill University, Shillong, India, *Speaker

North-east India, being one of the global hot-spots, is considered the richest reservoir of diverse plant genetic resources. However, the vast utilities of plant species in medicines, ornamentals, fragrances, urbanization and illegitimate harvest of plant genetic resources have immense impact on establishment and multiplication in the natural habitat and many of them have already been listed in the threatened category of CITES. Hence, storage and conservation are the prime concern for plant conservationists to safeguard plant genetic resources in this region. In addition to micropropagation, cryopreservation plays an important role in conservation science due to its additional potential of storing genetic resources without altering its genetic makeup. Optimization of protocols desirable for each species is the key aspect in conservation science since species react differently to the formulations and conditions opted for experimentation. Studies were therefore carried out to developed protocols for long-term storage of some important plant species of North-east India such as Aquilaria malaccensis, Dendrobium chrysanthum, D. nobile, Paphiopedilum insigne, P. villosum etc. Different techniques such as dehydration, encapsulation-dehydration, vitrification and encapsulation-vitrification were applied to study the efficiency of each plant species for long-term storage. A protocol each was developed for all the species studied with a different level of effectiveness in the techniques applied. Also, a modification in the standard protocols of cryopreservation techniques, such as rehydration of explants prior to its transfer into regeneration medium in dehydration technique and incorporation of antioxidants in vitrification technique, improved media for post-cryo survival and regeneration in some of the species studied. A few contributions consequently towards conservation of plant genetic resources of North-east India through cryopreservation will be discussed in detail during the presentation.

Date Recorded: 
Thursday, July 25, 2019

Lourdes Delgado-Aceves, Maria T. Gonzalez- Arnao, Liberato Portillo, Raquel Folgado, CUCBA-University of Guadalajara, University of Veracruz, The Huntington Library, Art Collections and Botanical Gardens

Agave tequilana cv. chato is an important resource widely used to produce a very emblematic and popular Mexican beverage called mezcal; however, its wild populations are currently severely degraded due to unsustainable management, mainly overexploitation. The application of tissue culture and cryopreservation would contribute to the propagation and conservation of this less studied cultivar. This work reports the in vitro multiplication via indirect somatic embryogenesis(ISE) and cryopreservation of regenerants. For ISE induction, segments (1 cm) of young leaves were cultivated for 40 days on MS basal solid medium with 10mg·L-1 4- amino-3,5,6-trichloro-2-pyridinecarboxylicacid (PIC) and 0.75 mg·L-1benzylaminopurine (BA). Generated calli were transferred to basal MS médium with reduced (76%) concentration of NH4NO3 and supplemented with 500 mg·L-1 glutamine, 250 mg·L- 1 casein hydrolysate and solidified with 6 g·L-1 phytagel for ISE conversion. After 50 days of culture, an average of 30±5 somatic embryos per explant were regenerated from calli mass. Cryopreservation experiments were performed following the V- cryoplate method and using somatic embryos of 1-3 mm in length, which were precultured on MS solid medium with 0.3 M sucrose for 1 d in dark, encapsulated over the cryoplate with calcium alginate (2%) containing 0.4 M sucrose, loaded in solution with 1 M sucrose and 2 M glycerol (15 min), and exposed to the vitrification solutions PVS2 and PVS3 for 30 min prior to direct immersion in liquid nitrogen. Rewarming took place in liquid medium with 1.2 M sucrose and samples were transferred to solid conversion medium in darkness for 7 days. More than 90% of somatic embryos shown post- cryopreservation recovery after 15 days of reculture, irrespective of the PVS used. Samples treated with PVS2 presented faster regeneration detected by the elongation of coleoptile and radicle. This is the first report on successful cryopreservation of somatic embryos from Agave genus.

Date Recorded: 
Thursday, July 25, 2019

Jose Faria, Lisa Hill, Christina Walters, Tree Seed Laboratory, Federal University of Lavras, Brazil, USDA-ARS, National Center for Genetic Resources Preservation, United States

Quercus imbricaria is included in the red oak group (Lobatae) and is broadly distributed in the Midwestern US. The embryonic axes are about 1 mg dry mass and have 0.68 g H2O/g dw in the acorn. Their small size and considerable desiccation tolerance made them ideal to compare various approaches for cryopreservation. Drying over a stream of nitrogen gas for 120 min reduced axis water content to 0.15 g/g, with 55% survival following liquid nitrogen (LN) exposure. To test whether addition of cryoprotectants could improve survival, axes were bathed in Plant Vitrification Solution #2 (PVS2) for 5-60 min and tested for viability before and after LN exposure. Axes submitted to PVS2 treatments and exposed or not to LN attained survival of 100% (or close to) after two weeks of tissue culture. Differential scanning calorimetry (DSC) was used to infer water freezing and melting patterns. In axes dried without PVS2, water melted at about 178 J/g H2O and melting transitions were not observed in axes dried to less than 0.34 g/g when standard methods of 10oC/min scanning rates were used. Using similar DSC methods, the water melting signal was no longer apparent in axes soaked in PVS2 for 45 minutes. To evaluate water melting behavior during fast cooling and warming, axes were plunged into LN and scanned at 300oC/min, revealing broad endothermic events between -120 to -40oC in axes that received no cryoprotectant, which we interpreted as crystal growth during warming. The enthalpy for melting transitions was reduced to about 70 J/g H2O, but there was no effect on the water content at which the melting signal was not detected. From our collective results, we suggest that PVS2 alters the rate of lethal ice crystal formation during rapid cooling and warming allowing for greater survival of axes exposed to LN.

Date Recorded: 
Thursday, July 25, 2019

Josette Tin, Taylor La Val, Sean Lahmeyer, John Trager, Raquel Folgado*, The Huntington Library, Art Collections and Botanical Gardens, San Marino, CA., United States, *Speaker

Succulent plants are significant to the horticultural industry, and they are also sources for food, fibers, medicines, and cosmetics. The main threats for the wild population of these often emblematic plants are human activities, such as over-collection in the wild. The Huntington Desert Garden holds one of the largest ex-situ collections of succulent plants. Besides the traditional propagation methods for the field collections and the cryopreservation of seeds, in vitro repositories have been created to assure the preservation of the clonal type plants, which often have historic and botanical value. Experiments of droplet-vitrification based techniques have been used to cryopreserve clonal accessions of aloes and agaves. Apical shoot tips of 1 mm size from 5-week- old in vitro plantlets (aloe or agave) were exposed to loading solution for 20 min at room temperature, dehydrated with Plant Vitrification Solution 2 (PVS2) for different times (from 0 to 90 min) at 0 °C, transferred to aluminum foil strips and directly plunged into liquid nitrogen. For re-warming, aluminum strips were rinsed in unloading solution for 20 min at room temperature. Explants were transferred to regeneration media and kept in the dark for one week. In additional experiments, shoot-tips excised from donor plants pretreated onto a sucrose- enriched medium for two weeks were also submitted to cryoprocedure. The pretreatment with sucrose- supplemented medium improved the regeneration of both aloe and agave cryopreserved explants. The optimized protocols that have been developed for Aloe fievetii and Agave sobria spp frailensis (70 % and 90 % of plants recovered after cryopreservation, respectively) are being tested for other Aloe and Agave species. Regenerated plants were acclimated to ex vitro conditions.

Date Recorded: 
Thursday, July 25, 2019

Megan Philpott, Valerie PenceL*, Cincinnati Zoo & Botanical Garden, United States *Speaker

Threats to global plant biodiversity compel the need for ex situ collections of species worldwide. However, the subset of species known as exceptional plants are often overlooked. These species produce few or no seeds or produce recalcitrant seeds. The Center for Conservation and Research of Endangered Wildlife (CREW) at the Cincinnati Zoo & Botanical Garden maintains a liquid nitrogen bank of exceptional plant seeds and tissues known as the CryoBioBank®. CREW has partnered with the Lyon Arboretum in Honolulu to develop cryopreservation protocols for 21 endangered exceptional Hawaiian plant species and bank them in the CryoBioBank®. To date, micropropagation protocols to produce target tissues have been tested in 13 species, and cryopreservation protocols have been tested in 7 species. As an example, in Cyrtandra gracilis, micropropagation on medium containing 2mg/L of the auxin indole-3-acetic acid and 0.1mg/L of the cytokinin 6-benzylaminopurine induces shoot organogenesis on excised leaves. These leaves were dissected into leaf bud segments and compared with shoot tips for survival after liquid nitrogen storage. Cryopreservation of shoot tips using droplet vitrification (DV) with PVS2 yielded 0% survival, while leaf bud segments yielded 20% survival. DV using PVS3 increased survival in these leaf buds to 60%. Cryopreservation of leaf bud segments immediately after bud primordia formed yielded 25% survival compared to 50% survival if the buds were left to develop into shoots before cryopreservation. Shoot tips from another species, Melicope mucronulata, have been banked in the CryoBioBank® using the DV method following an experimental survival rate of 57% after 2 days of pre- culture on a 0.3M mannitol medium instead of one day on 0.3M mannitol and one day on 0.5M mannitol medium. This project will result in the long-term protection of many endangered exceptional Hawaiian species that would otherwise be unbankable using conventional methods. (Supported by IMLS grant #MG-30-17-0055-17).

Contributing Author(s): 
Date Recorded: 
Thursday, July 25, 2019

Manuela Nagel, Hans-Peter Mock, Markus Kuhlmann, Johanna Stock
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany

Plant cryopreservation is a back-up approach for vegetatively propagated genetic resources. The cryopreservation process represents a model for complex stress response mechanisms. During cryopreservation, meristematic tissues have to cope with several stresses including mechanical, osmotic and cold stress. The molecular regulation and response pathways in combination with changes in the DNA and/or RNA methylation pattern are poorly understood. Therefore, the presentation aims to summarize current results and ongoing projects to decipher detailed molecular processes during shoot tip cryopreservation of the model plant Arabidopsis thaliana L. Arabidopsis is beneficial because shoot tips can be obtained from homogeneous seeds and effects of pre-culture environment and endophytic contamination can be avoided. Furthermore, sophisticated protocols were developed and guarantee high regrowth percentages of shoot tips after cryo.
Transcriptome analysis (high throughput RNA sequencing) from Arabidopsis showed that members of the transcription factor families WRKY, MYB and AP2 EREBP, such as C-repeat binding factors (CBFs)/ dehydration-responsive element-binding proteins (DREBs) are involved in the stress response mechanisms. The transcription factor WRKY22 was identified as key regulator which is assumed to affect the expression of the Pathogenesis Related gene 5 (PR5) in response to shoot tip preparation. Furthermore, during the cryopreservation the CBFs transcription factors were identified as differential expressed genes (DEGs) and were shown to be upregulated. In principle, they can alter the expression of more than 100 cold-regulated (COR) genes. As significantly overrepresented GO term RNA methylation was found. The analysis of T-DNA insertion plants impaired for CBF1 or CBF3 gene function, resulted in a significant reduced regrowth after cryopreservation.
Understanding these signaling cascades involved in cryopreservation could be useful to improve and develop successful cryopreservation protocols for other plant genetic resources in
future and might reveal the yet unknown biological f unction of RNA methylation in plants.

Date Recorded: 
Thursday, July 25, 2019

Christina Walters, Jennifer Crane, Lisa Hill, United States Department of Agriculture., USDA-ARS

Seeds of papaya (Carica papaya) are tolerant to desiccation, but they are quickly damaged when stored at -5 to -20C. The seeds have a high lipid content that reflect “tropical oils,” which are high in saturated and mono-unsaturated fatty acids. Differential Scanning Calorimetry (DSC) experiments show that the lipids tend to crystallize into alpha and beta crystals and then re-crystallize to more stable crystalline beta prime structure as the seeds slowly pass through the -25 to +5C temperature range. A working hypothesis is that these transitions are associated with the poor storage response of papaya seeds. Cooling and warming sufficiently quickly (~ 50C/min) to liquid nitrogen temperatures allowed only the alpha-type crystals to form and slowed recrystallization. Seeds survived longer when stored at liquid nitrogen temperatures. However, after 15 years of storage, papaya seeds showed measurable degradation that may be associated with 1) multiple freeze-thaw cycles that occurred when retrieving samples for monitoring, 2) failure to warm seeds adequately before they were tested for viability, or 3) degradation processes that continue at liquid nitrogen temperatures. Papaya seeds serve as a convenient model to study a large class of seeds within the ‘intermediate’ storage category that originate from tropical or subtropical areas and cannot be stored using conventional seed banking methods. We show that storage at liquid nitrogen temperatures offers improved success, but may not provide long-term storage usually anticipated at such low temperatures.

Date Recorded: 
Tuesday, July 23, 2019

Daniela Impe, Daniel Ballesteros, Till Ischebeck, Claudia Köpnick, Hardy Rolletschek, Michael Melzer, Manuela Nagel, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Germany, Royal Botanic Gardens, Kew, United Kingdom, Department of Plant Biochemistry, Georg-August University Göttingen, Germany

Cryopreservation of pollen is used to preserve and exchange nuclear genes of plant genetic resources and to facilitate crosses in breeding programs. For many species, pollen preservation protocols have been established. In wheat breeding programs, long- term pollen preservation would be of high interest to improve the crossing management in hybrid production. However, favorable storage conditions for wheat pollen have not been studied yet and physiological, biochemical and genetic processes in pollen after shedding are hardly understood.

The present project aims to investigate factors that influence wheat pollen viability in order to extend its longevity by cryopreservation. Firstly, a viability test and evaluation scheme based on pollen tube growth in vitro was established and comparisons between in vivo germination, staining methods and impedance flow cytometry showed that more than one test is required to determine wheat pollen viability.

Secondly, storage of wheat pollen under four different non-cryogenic conditions showed that wheat pollen lose germination ability rapidly. At room temperature, viability was lost within one hour at both low and high relative humidity (RH). Under cold temperatures (6-8 °C, both RH) pollen survival was extended moderately. Sugar contents (fructose, glucose and sucrose) increased two- to threefold in pollen stored for one hour under all non-cryogenic conditions. Therefore, we speculate that sugars are actively mobilized during pollen storage. To gain a comprehensive overview of processes contributing to viability loss, a metabolomics and transcriptomic study has been initiated and is complimented by transmission electron microscopy that illustrates ultrastructural differences between fresh, stored and dead wheat pollen.

Finally, storage under ultra-low temperatures (- 196°C) is being investigated in detail. A self-made flash drier was used to control the pollen water content by fast drying. The effects of drying and cryogenic storage are investigated using differential scanning calorimetry and will indicate the feasibility of long-term pollen cryopreservation.

Contributing Author(s): 
Date Recorded: 
Tuesday, July 23, 2019

Karin Van Der Walt, Jayanthi Nadarajan, David J Burritt, Peter Kemp, Otari Native Botanic Garden - Wellington City Council & Massey University New Zealand, The New Zealand Institute for Plant and Food Research Limited, University of Otago, Massey University

The pandemic pathogen, Myrtle Rust(Austropuccinia psidii) was discovered in New Zealand in 2017 and has now become established through most of the climatically suitable areas. Of the approximately 30 native Myrtaceae species, three genera representing four species are of particular concern due to unknown seed storage behaviour. One of these, Syzygium maire (swamp maire) - New Zealand’s only Syzygium species, was found to be highly sensitive to desiccation with complete embryo viability loss following dehydration to 20% moisture content. Comparison of desiccation sensitivity profiles across populations and seasons is
being conducted to understand the role of genotype and environmental conditions on chilling sensitivity and desiccation tolerance. Preliminary studies investigated cryopreservation of zygotic
embryos using naked embryo desiccation, encapsulation-dehydration and Plant Vitrification Solution 2 (PVS2) techniques. No survival was recorded following naked embryo and PVS2 vitrification cryopreservation. Limited root formation, but no regeneration following encapsulation-dehydration was also
found. Optimization of cryopreservation steps are being investigated by using oxidative damage and antioxidant metabolism as biomarkers of stress, as well as embryo/seedling germination to build a better understanding of how stress caused by embryo excision, decontamination, desiccation rate (rapid vs slow) and rehydration are impacting embryo survival. In addition, embryo survival following exposure to PVS2 is studied to optimize duration (30-90minutes), temperature (0oC and 20oC) and method (conventional vitrification and vacuum infiltration vitrification). Further research is underway to refine cryopreservation protocols further through preculture and acclimatization and comparing embryo desiccation tolerance at three seed developmental stages.

Date Recorded: 
Tuesday, July 23, 2019