cryopreservation

Maria Teresa Gonzalez-Arnao, Pedro Isidro-Adolfo, Maria F. NetzahualcoyotlMata, Lourdes G. Iglesias Andreu, Jaime Martínez-Castillo, University of Veracruz, INBIOTECA, CICY, 

In this study we used six selected ISSR primers (T05, T06, C07, UBC823, UBC825, UBC848) to evaluate the genetic stability during cryopreservation of vanilla (V. planifolia) shoot-tips subjected to a Droplet-Vitrification (D-V) protocol using PVS2 solution for dehydration. We studied the impact of tissues culture and cryopreservation, assessing the effect of multiplication cycles of donor plantlets, with few (less than 4) and multiples (more than 12) subcultures, from which were isolated the shoot-tips to be cryopreserved. It was also assessed the effect of treatments, which allowed growth recovery after immersion in liquid nitrogen. To apply D-V procedure, shoot-tips were dissected of motherplantlets with few or multiple subcultures, and then preconditioned for 1 day on MS semisolid medium supplemented with 0.3M trehalose, loaded in a solution of 0.4M sucrose + 2M Glycerol and exposed to PVS2 solution for 30 min at room temperature prior to the ultra-rapid cooling in liquid nitrogen placed on droplets of PVS solution over aluminium cryoplates. The analysis of electrophoretic profiles of in vivo plants and in vitro plantlets with different subculture numbers revealed a total of 153 bands and 21.2% of polymorphism. UPGMA dendrogram based on Nei’s genetic distance demonstrated that plants with multiple subcultures induced the greatest genetic variation but kept a high (0.90) similarity index. The electrophoretic profiles of all successive steps of D-V protocol revealed a total of 199 bands and 34.2% polymorphism. Treatment with PVS2 promoted the greatest variation and continued declining the level (0.816) of similarity compared to that of the plantlets with multiples subcultures. Plantlets regenerated after cryopreservation kept the same level (0.819) of those treated with PVS2, indicating that liquid nitrogen immersion did not induce a further genetic variation. Our studies revealed that both, subculturing number and the composition of vitrification solution influenced genetic variability.

Date Recorded: 
Tuesday, July 23, 2019

Chris O'Brien, Jayeni Hiti Bandaralage, Raquel Folgado, Sean Lahmeyer, Alice Hayward, Neena Mitter Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Australia The Huntington Library, Art Collections and Botanical Gardens, San Marino, CA., United States

Australia’s avocado industry is worth over ~$557 million and produces more than 77,000t. The genetic diversity of the avocado genus remains largely unexplored but a vast resource (approximately 90 species/>3000 varieties) for crop improvement. Persea spp. are traditionally restricted to ex-situ conservation due to high heterozygosity and recalcitrance nature of seeds. Field banks are vulnerable to abiotic and biotic stresses. Thus, size of the gene pool, replications and quality are restrained by environmental conditions, space and funding. Cryopreservation is a safe and cost-effective method of germplasm conservation. Preserving shoot-tips enables conservation of exact gene pool of interest. Cryo-protocols that use vitrification solutions usually cause high mortality. Avocado is highly susceptible to osmotic stresses upon vitrification. To date there are no reports of survival/regrowth of in vitro cryopreserved shoot tips. This study aimed to optimize sucrose pre-culture to sustain shoot tip survival/regrowth after cryopreservation. In vitro shoots of cultivar ‘Velvick’ were pre-cultured for 2 weeks on 0.3 M sucrose and 100 mg/L Ascorbic acid containing media to be compared with normal sucrose concentration of 0.09 M. For cryo-treatments shoots were dissected to obtain 1 x 1 mm shoot-tips. When treated with PVS2 and evaluated for survival, pre-cultured shoots displayed higher survival 83%, higher regrowth 73% with vigorous green cultures and appeared morphologically normal. In contrast non-precultured shoots-tips recorded 70% survival, 23% regrowth resulted in stunted yellow-brown and less vigorous cultures. Very interestingly, after liquid nitrogen treatment pre-cultured shoot-tips showed 60% survival while no sucrose treatment lead to 0% survival. The cryo-preserved shoots developed into green proliferating clumps after 8 weeks in culture. This result is the first report of successful survival and proliferation of cryopreserved shoots of avocado and further optimizations may lead to development of a high efficiency cryopreservation protocol of the species.

Date Recorded: 
Tuesday, July 23, 2019

Gayle Volk, USDA ARS National Laboratory for Genetic Resources Preservation, United States

The USDA-ARS National Plant Germplasm System has over 30,000 clonally maintained accessions within its field, screenhouse, greenhouse, and tissue culture collections. These fruit, nut, tuber, and bulb crop collections are usually not duplicated at secondary locations and are vulnerable to bioticabiotic, and climatic threats. Only about 15% of the clonally maintained accessions are currently secured in long-term storage at the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado. The labor required to cryopreserve the clonal collections at NLGRP exceeds that which is available, even when reliable, robust cryopreservation methods are available. We have sought to prioritize collection materials for cryopreservation and to identify methods that improve the efficiency of the shoot-tip cryopreservation procedure. In particular, we have used field-, screenhouse-, and growth-chamber harvested plant tissue as source material for shoot tip cryopreservation, rather than relying on in vitro grown cultures. This strategy has been particularly effective for garlic, citrus, and grape cryopreservation efforts. In addition, incorporation of antioxidants and shoot tip micrografting methods have made cryopreservation protocols widely applicable to diverse genetic resources for each crop.

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Date Recorded: 
Tuesday, July 23, 2019

Shin-Ichi Yamamoto, Genetic Resources Center, National Agriculture and Food Research Organization (NARO)

The National Agriculture and Food Research Organization (NARO) conducts Genebank Project for collecting, conserving, evaluating, multiplying and distributing plants (228,198 accessions), microorganisms (35,407 acc.), animals (1,971 acc.) and DNA materials (394,440 clones) related to agriculture since 1985. The Genetic Resources Center, NARO (NGRC) coordinates this activity in collaboration with a network of institutes (so-called sub-banks) throughout Japan. In the plant section of the project, orthodox seeds are conserved in a seed storage at -18 ℃ for long term and at -1 ℃ for medium term, respectively. Approximately 30,000 accessions of plant genetic resources are clonal crops. They are basically conserved in fields or greenhouses of sub-banks and very useful for immediate use. However, the construction of cryobank will contribute significantly to the cost-effective long term preservation of vegetatively propagated crops in a stable manner in safe and disease-free conditions as safe backup. In NGRC, winter dormant buds of 1,283 mulberry accessions have been cryopreserved. But this method cannot be available for the crops which do not form dormant buds. Although existing cryopreservation methods such as vitrification and droplet methods for in vitro shoots can be applicable for those crops, more systematical protocol is desirable for facilitating cryobank for them. Therefore, we developed efficient and simple cryopreservation procedures, V and D cryo-plate procedure using small hard aluminum plates with micro-wells. These methods have many advantages such as simple handling, high regrowth rate, very high cooling and warming rate, easy learning and so on. We already started to preserve clonal plant genetic resources such as potato (310 acc.), mat rush (181 acc.) and so on by these methods in NGRC liquid nitrogen tanks. We plan to increase accessions stored systematically and establish a cryobank project in Japan.

Date Recorded: 
Tuesday, July 23, 2019

Bart Panis, Bioversity International, Belgium

More than 800 million people are undernourished and 200 million children under five years of age are underweight. Moreover, the world’s population is expected to reach 10,500 million by 2050. Reliable and sustainable improvements in yield will thus be needed to meet the demands of this growing population. The availability of the largest possible crop diversity is central to food security. Crop collections encompass seed propagated as well as vegetatively propagated crops. Seed is classically stored at -20°C (and sometimes cryopreserved) while vegetatively propagated crops are maintained in the field, stored as in vitro collection under reduced growth conditions or through cryopreservation. Cryopreservation plays an essential role in the safe conservation of plant genetic resources of vegetatively propagated crops like bananas, cassava, potato, yams and sweet potato. Cryopreservation research on these crops already started in the 80ties but it was only with the development of vitrification protocols and more recently with the use of droplet vitrification that a significant portion of such collections are now stored in liquid nitrogen. The droplet vitrification protocol was established because it combines the application of highly concentrated vitrification solutions (often PVS2) with ultra-fast freezing and thawing rates both leading to a lower chance of lethal ice crystal formation. Currently, over 10,000 accessions starting from in vitro cultures are safely preserved for the long term through cryopreservation. More than 80% of these belong to 5 crops; potato, cassava, bananas, mulberry and garlic. Other important cryopreserved collections representing thousands of accessions are those of dormant apple buds. One of the recommendations of an expert group to apply cryopreservation to a wider diversity of vegetatively propagated crops was to establish a collaborative effort among researchers and genebanks that is focused on the specific technical and practical issues.

Date Recorded: 
Monday, July 22, 2019

Dr. Oliver Ryder, Barbara Durrant, Marlys L. Houck, Marisa Korody, Cynthia Steiner, Institute for Conservation Research, San Diego Zoo Global

Cryobanking of viable tissue culture cells at the San Diego Zoo, named the “Frozen Zoo® by its founder, Kurt Benirschke, has contributed knowledge advancing conservation science and species recovery from its beginnings in 1975. This diverse collection of cryopreserved diploid fibroblast cell cultures contributes to the legacy his endeavor. Banked gametes and cryopreserved reproductive tissues expanded the possibility of conservation applications. Identification of chromosomal errors affecting fertility of mammalian species as diverse as tigers, dik-diks, and gorillas has been enabled by its collections. Key studies delimiting species boundaries, evolutionary relationships, phylogeny and systematics of mammals and birds have utilized samples from the Frozen Zoo and its extended network of samples. Major contributions to the advancement of comparative vertebrate genomics have been facilitated by the collections of the Frozen Zoo. A crucial contributor to the 200 Mammals project, Genome10K, and the Vertebrate genome project, intact cells and high molecular weight DNA extracts from the Frozen Zoo have facilitated whole genome sequencing efforts and, notably, high quality de novo genome assemblies. Cryobanking of viable early passage diploid fibroblasts has allowed investigation of somatic cell nuclear transfer cloning technology to be pursued in the conservation context. Live offspring of two endangered species of wild cattle, the gaur, and the Javan banteng have been produced with cells banked for decades in the Frozen Zoo. Fibroblast cells from the Frozen Zoo were used for the first reported cellular reprogramming of endangered species to produce induced pluripotent stem cells (iPSC), including an African primate, the drill, and the critically endangered northern white rhinoceros were successfully reprogrammed. These studies have been extended and recently, using nonintegrating methods, iPSC have from two southern white and eight northern white rhinoceroses have been produced and characterized, an effort crucial to preventing its otherwise-certain extinction.

Date Recorded: 
Monday, July 22, 2019

Oliver A. Ryder, Director, Conservation Genetics, Kleberg Endowed Chair, San Diego Zoo Institute for Conservation Research

In January, 1975 genetics studies commenced at the San Diego with group of researchers who would become the core of CRES, the Center for Reproduction of Endangered Species – now the San Diego Zoo Institute for Conservation Research. Founded by Kurt Benirschke, MD, cell culture and chromosomal analyses were first established. In June of that year, the first postdoctoral fellow joined the team, Oliver Ryder. By the time that Dr. Benirschke left the employ of the Zoo (to join its Board of Trustees), the research disciplines had expanded to include reproductive sciences, endocrinology, behavior and virology. Ryder oversaw the continuing development of the Frozen Zoo® and projects in molecular evolution, systematics, hybridization, speciation, kinship analyses, and population genetics, each area evolving as new technologies became available. An historical overview, the continuing development of conservation genetics at ICR in the era of genomics, and the incorporation of advanced cellular technologies for genetic rescue into our Conservation Genetics toolkit will be presented.

Date Recorded: 
Tuesday, September 10, 2019

Plant Cryopreservation Post Doc - San Diego Zoo Institute for Conservation Research

The Plant Conservation team at the Institute for Conservation Research and the Horticulture Department at San Diego Zoo seek a Plant Cryobiology Postdoctoral Associate to investigate and refine protocols for cryopreservation of IUCN endangered Quercus dumosa shoot tips, embryos, and pollen.  In the course o

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CREW’s CryoBioBank, where samples, such as those from Hawaii, will be stored.
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Photo Credit: 
Cincinnati Zoo and Botanical Garden

Valerie Pence and Megan Philpott, Cincinnati Zoo & Botanical Garden

Crotalaria avonensis is a Florida endemic found in three populations and characterized by low seed production. In the late 1990s, CREW developed protocols for tissue culture propagation from field collected shoot cuttings as well as cryopreservation methods. In order to develop a genetically representative collection for conservation, in vitro lines were established from shoots collected at all three populations from 2008-2012. Plants were produced and sent to Bok Tower Garden for further growth and for use in an outplanting by Archbold Biological Station. The resulting collection of genotypes in culture at CREW provides an example of the challenges of a genetically diverse collection of an exceptional species. C. avonensis cultures require maintenance subculturing every 2-3 months. Only a low number of replicates could be maintained for each genotype, resulting in some loss of genotypes over time. Cryopreservation offered a solution to this challenge and over the course of 16 years, a number of lines were cryopreserved. In a study of lines stored for 5.5 Ð 16 years in liquid nitrogen, there was no change in average viability of the collection in storage, although specific survival differed by genotype. A cost estimation indicated that cryopreservation could decrease the cost of maintaining the collection over 20 years by at least 1/3. Genetic analysis of the collection and the wild populations is also underway in order to determine the genetic representation of the collection.

Contributing Author(s): 
Date Recorded: 
Friday, May 3, 2019