Search

Micrografting, which was developed almost 50 years ago, has long been used for virus eradication, micropropagation, regeneration, rejuvenation, and graft compatibility. Recently, micrografting has been used for studies of long-distance trafficking and signaling of molecules between scions and rootstocks. The graft transmissiveness of obligate plant pathogens, such as viruses, viroids, and phytoplasmas, facilitated the use of micrografting to study biological indexing and pathogen transmission, pathogen-induced graft incompatibility, and screening for the pathogen resistance during the past 20 years. The present study provides comprehensive information on the latter subjects. Finally, prospects are proposed to direct further studies.

Fruit and vegetable species and varieties, their wild relatives, and pollinators and other associated organisms underpin diverse food production systems and contribute to worldwide health and nutrition. This biodiversity, however, is threatened, remains poorly conserved, and is largely undocumented. Its loss leads to a narrowing of new food options, reduced variation for breeding, and yield gaps due to pollinator decline. This constrains the supply of climate-resilient and nutritious foods to the global human population and limits long-term progress towards the 2030 Sustainable Development Goals and any future goals set thereafter. It will require that awareness be raised globally to safeguard and sustainably use fruit and vegetable biodiversity and that a global rescue plan for reducing and reversing the decline in this biodiversity be devised. Success will depend on a global partnership of custodians and users of fruit and vegetable biodiversity and requires an investment of at least 250 million USD over ten years.

The National Historic Preservation Act of 1966 mandates that historic resources, including orchards, on federal lands be evaluated for potential significance. Evaluations include assessments and documentation of an orchard’s layout and location, as well as the architecture, ages, conditions, and cultivars of trees. These factors may reveal an orchard’s history and uniqueness and determine an orchard’s historic importance. Fruit tree cultivar identities are a key piece of this puzzle. They reveal information about an orchard’s use, origin, and uniqueness. In the past, historic preservationists relied on pomologists to identify cultivars based primarily on the phenotypic traits of the tree. DNA fingerprinting technologies, in combination with high-quality reference cultivar collections, have dramatically improved the accuracy of heritage cultivar identification. We demonstrate the utility of using DNA technologies for cultivar identifications on federal lands using examples from trees in orchards of Redwood National and State Parks and the Eldorado National Forest. A total of 87 historic apple trees from these locations were genotyped and cultivar names were determined for 35 trees, using a reference set of 1319 apple cultivars from the USDA National Plant Germplasm System, Washington State University, Seed Savers Exchange, and Temperate Orchard Conservancy. Some of the trees that were not identified may be cultivars that are not included in the reference sets and others may be unique trees derived from seeds. The information derived from genetic fingerprinting analyses will aid in the determination of the historic significance of orchards on public lands.

Societal Impact Statement Yosemite National Park, California, USA has hundreds of apple trees in orchards that were planted in the 1800s by local settlers. These orchards played a cultural role in the early history of the land that was to become a national park. Most of Yosemite’s orchards are well over one hundred years old and have reached the end of their lifespan. Genetic data has facilitated the assignment of cultivar names to historic apple trees within Yosemite and this information will be used to develop an Orchard Management Plan. Yosemite will use this Plan to balance orchard conservation with other necessities such as reducing human‐wildlife conflict from bears that enter developed areas to consume fruit from the trees. Summary Yosemite National Park, California, USA has hundreds of historic apple trees that were planted within orchards in the 1800s, prior to the establishment of the park. These apple trees have been a concern within Yosemite for decades because their fruit attracts bears and other wildlife into visitor recreation areas, causing undesirable wildlife–human interactions. Herein, 361 of Yosemite's apple trees were genotyped to determine cultivar identities. The cultivar names of 117 apple trees were identified by matching genotypic data with genetic data available for an apple cultivar reference dataset for the United States Department of Agriculture‐Agricultural Research Service (USDA‐ARS) National Plant Germplasm System as well as collections maintained by other sources. An additional 92 trees were determined to be likely cultivars because they matched genotypes in a reference set of trees whose identities are not known. A total of 152 trees had unique genotypes that did not match reference datasets. Cultivar information, made possible by having digital genetic information available, will allow for the preparation of a more comprehensive Orchard Management Plan. As such, park managers will be able use the Plan to more effectively make decisions about balancing orchard conservation with other park necessities, including eliminating the human–wildlife conflict that results from bears entering developed areas to feed on the fruit trees.

Cryopreservation of clonal plant germplasm is a reliable way to preserve important agronomic traits and protect against loss of crop genetic diversity of many horticultural species. Dormant bud cryopreservation techniques present an efficient alternative to the labor-intensive shoot tip cryopreservation process and may allow a single technician to preserve large quantities of germplasm in a season. This method of cryopreservation takes advantage of the natural dormancy in cold hardy crops, making it a viable technique for only deciduous trees and shrubs. Many factors must be considered when attempting to perform dormant bud research methods to applied-level germplasm preservation efforts. This process is necessarily a seasonal endeavor, which puts strain on labor and facilities particularly in winter. Integration of methods and procedures using different crop species or new equipment provides additional challenges that must be tested in advance. By identifying variables of dormant bud processing in cryopreservation literature, options emerge that allow for the modification of reported methods to work within the confines of institutional resources. Infrastructure, pre-processing, and recovery stages are discussed in terms of necessity and available alternatives to allow informed decision making in establishing an applied dormant budwood genebank. This review aims to provide information about the DB cryopreservation method with a focus on its many variables and permutations reported in literature to allow genebanks and germplasm managers to make informed decisions regarding the designation of critical resources.

Plant cryopreservation technologies are used within gene banks for the long-term preservation of vegetatively propagated collections. Surface-sterilized plant tissues grown in the field, greenhouse/screenhouse, growth chamber, or in vitro are the source of shoot tips subjected to vitrification-based cryopreservation methods. Here, we describe the methods used to minimize microbial contamination during the tissue culture initiation process. We also discuss the occurrence and possible elimination of endophytes after extended in vitro culture and during recovery after liquid nitrogen exposure. We describe two case studies in which bacterial endophytes were observed in

The availability of and easy access to diverse Vitis species are prerequisites for advances in breeding programs. Plant genebanks usually maintain collections of Vitis taxa as field collections that are vulnerable to biotic and abiotic stresses. Cryopreservation has been considered an ideal method of preserving these collections as safety back-ups in a cost-effective manner. We report a droplet vitrification method used to cryopreserve 12 Vitis species (Vitis vinifera cvs. Chardonnay and ‘Riesling, V. actinifolia, V. aestivalis, V. jacquemontii, V. flexuosa, V. palmata, V. riparia, V. rupestris, V. sylvestris, V. ficifolia, V. treleasi, and V. ×novae angeliae) using shoot tips excised from plants grown in vitro. Our results demonstrated wide applicability of this technique, with regrowth levels at least 43% for 13 genotypes representing 12 Vitis species. We demonstrated that the droplet vitrification procedure can be successfully replicated by technical staff, thus suggesting that this method is ready for implementation.

Plant genetic resource collections are national treasures that are critical to the success of breeding programs and the long-term resiliency of agriculture in the United States and worldwide. The USDA National Plant Germplasm System (NPGS) is a coordinated network of 19 genebank locations throughout the United States that conserve and protect nearly 600,000 accessions representing ∼16,000 plant species. The expertise of current curators must be captured, and training materials must be developed to educate the next generation of those who maintain and use plant genetic resources. A group of experts convened in April 2018 to discuss the needs, pedagogical approaches, educational content, delivery platforms, and mechanisms for sustaining a possible future plant genetic resources management training program. A three-component approach was envisioned to achieve the task of educating current and future genebank managers, as well as those who use genetic resources in their research and breeding programs. The proposed training program will include the development of online Resource Libraries, online courses, and workshops. Resource Libraries, hosted by GRIN-Global, will make learning objects, downloadable information, and links to other online sources publicly available. These Resource Libraries will be available for use in existing classes, as well as for the development of new workshops and online courses. Development of, and public access to, training resources will capture key information about genebanking, make it more widely available, and secure its long-term viability.

In apple, virally transmitted diseases cause economic losses as a result of their direct effect on production and yield and by causing an increased susceptibility to other phytopathogenic agents. This study evaluated the effectiveness of a droplet-vitrification cryopreservation technique in eradicating latent viruses: Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) from in vitro axillary shoot tips excised from ‘SC417 Monalisa’ apple cultivar shoot tips. In vitro stock cultures, four weeks after subculture, infected with ASGV, ASPV and ACLSV were used as source material. Axillary shoot tips were excised from stock cultures, precultured in MS + 2 M glycerol + 0.8 M sucrose, and then exposed to plant vitrification solution 2 (PVS2) for between 0 and 80 min (six treatments) at 0 °C or room temperature prior to liquid nitrogen (LN) exposure. Shoot tips were then warmed and recovered on growth medium. The survival and regrowth of cryopreserved (+LN) and non-cryopreserved (-LN) 'Monalisa' shoot tips were evaluated and the efficiency of virus eradication was determined using RT-PCR after recovered plants were grown in a greenhouse for 6 months. With this protocol, the highest degree of regrowth (45%) were obtained after 20 to 40 min PVS2 exposure at room temperature. After 6 months of growth in a greenhouse, all of the plants regenerated after cryopreservation were free of ASPV, 95% were free of ACLSV, and 35% were free of ASGV. This promising cryotherapy procedure may facilitate the production of virus-free plants.

Apples are important from both a cultural and nutritional standpoint. The fruit produced from the cultivated apple, Malus × domestica Borkh., is grown in temperate regions throughout the world. Ornamental crabapple trees represent other Malus species and are generally valued for their landscape properties rather than their fruits. Genetic analyses of apple cultivars and species have revealed domestication pathways, pedigree relationships, as well as data that guide genebank collection management practices. Molecular information is also critical for breeding programs that are using new techniques to identify novel genetic combinations with enhanced biotic and abiotic stress resistance as well as desirable fruit quality and production traits. This chapter includes information about wild apple species, origins of cultivated apples, genetic assessments, as well as some basic information about tree phenology, architecture, and propagation methods.

Diverse apple cultivars and wild Malus species offer necessary allelic diversity to breeding and research programs. Wild apple species are found in native landscapes of countries throughout the Northern Hemisphere; however, some of these species are at risk of being lost, particularly if trees are unable to thrive under changing climatic conditions, disease/pathogen outbreaks, and human encroachment. Apple genebanks have been established to ensure that user communities have current and future access to these genetic resources. Herein, we describe necessary components of apple genebanks, including collection maintenance forms (trees in orchards, in vitro cultures, low-temperature medium-term storage, and cryopreservation), acquisition of genebank accessions, documentation and data management, distribution of collection materials, as well as characterization and evaluation (phenotypic evaluations and genetic tools for collection management). We conclude with a summary of international apple genebank holdings and provide some examples of how collaborations among genebanks would benefit the global community.

The USDA-ARS National Plant Germplasm System (NPGS) maintains a citrus genetic resource collection of 1522 accessions of Citrus cultivars and crop wild relatives. Of these, 540 are maintained as pathogen-free duplicate clones in a screenhouse at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) in Riverside, California. An additional 815 accessions of citrus and citrus relatives are maintained in greenhouses or orchards in Riverside until sanitation for pathogens can be completed. This paper describes progress in backing up the 540 pathogen-tested accessions using cryopreservation techniques and methods to accelerate processing and viability testing. Vegetative budwood was harvested from trees in the screenhouse and sent to NLGRP. At least 170 shoot tips from each accession were excised from surface-sterilized budwood, and then cryopreserved using a PVS2 droplet-vitrification technique. Viability was assessed by micrografting 10 thawed shoot tips onto in vitro grown 'Carrizo' seedling rootstock. The remaining shoot tips are held in long-term liquid nitrogen storage. A total of 451 of the pathogen-tested citrus accessions were cryoprocessed: 354 accessions had regrowth levels of 40% or greater, 17 had 0% regrowth, 47 had viability levels between 10 and 30%, and 33 had endogenous contaminants. Technical staff familiar with shoot-tip cryoprocessing could be fully trained for specific applications of citrus in about 2 months and thereafter processed a single accession of at least 170 shoot tips in about 16 h. This large-scale effort has revealed that shoot tip cryopreservation can be successfully scaled-up to secure the NPGS Citrus collection.

The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticulture Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases, and other threats. We sought to identify a method whereby clonally propagated sugarcane accessions could be successfully introduced into tissue culture, multiplied, and then cryopreserved as shoot tips for long-term preservation at the National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado. For many accessions, 70% isopropyl alcohol and 20% commercial bleach treatments, followed by three rinses of sterile water were sufficient to remove microbial contaminants during the introduction process. However, in some cases, cefotaxime was particularly effective for removing bacterial contamination. We found that antioxidant treatments of glutathione, glycine betaine, and ascorbic acid did not improve regrowth after liquid nitrogen exposure using either PVS2 or PVS3 as cryoprotectants in droplet vitrification cryopreservation methods. Exposure durations of PVS2 and PVS3 were optimized, with and without exposure to liquid nitrogen (LN), and shoot tip regrowth levels ranged from 0 to 37% after LN exposure. Published methods for encapsulation-dehydration and V-plate vitrification cryopreservation procedures were tested to determine if acceptable results could be obtained. Using these methods, shoot tip regrowth levels ranged from 0 to 50% after LN exposure. We conclude that the sugarcane cryopreservation methods that we tested are not yet ready for implementation in the NPGS.

Thousands of apple trees were planted in Wyoming’s orchards and homesteads in the 1800s, many of which are still alive today. Unfortunately, cultivar identity of these trees has mostly been lost or obscured. The purpose of this research was to identify heritage apple cultivars in Wyoming using genetic fingerprinting (microsatellite) techniques and to use this information to make recommendations on candidate cold-hardy cultivars for specialty crop and breeding programs. Leaf samples were collected from 510 heritage apple trees from 91 sites in 19 locales across Wyoming. Known cultivars from the U.S. Department of Agriculture (USDA)–National Plant Germplasm System, Seed Savers Exchange, and Washington State University apple collections were used as standards to determine cultivar identities. Overall, 328 (64%) of the previously unidentified apples trees were identified to 47 known cultivars. Fifteen of these known cultivars comprised more than 80% of the samples that were identified, with all 15 of those cultivars developed in states and countries with average temperatures or winter conditions similar to Wyoming. Seventy-one of the heritage trees were identified as the Wealthy cultivar. Other commonly identified cultivars were Haralson, Patten’s Greening, Yellow Transparent, Northwestern Greening, and McMahon. It is likely that a combination of popularity and cultivar origin affected the choice of cultivars that were grown in Wyoming. Although most original Wyoming heritage apple trees are reaching the end of their life span, many surviving trees continue to produce fruit. This strongly suggests that despite lower resistance to certain pathogens than many modern cultivars, these heritage trees should be considered for use today. The results provide insights into possible cultivars that could be grown in Wyoming and also in other states with similar harsh growing conditions.

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.

Apple (Malus × domestica Borkh.) trees, either abandoned or cared for, are common on the North American landscape. These trees can live for decades, and therefore represent a record of large- and small-scale agricultural practices through time. Here, we assessed the genetic diversity and identity of 330 unknown apple trees in northern Minnesota with 9 simple sequence repeat (SSR) markers. The unknown (not identified by cultivar name) trees were compared to > 1000 named cultivars in the U.S. Department of Agriculture Plant Genetic Resources Unit Malus collection and also to each other to identify repeated genotypes. Overall, the 330 unknown trees had high diversity (average He = 0.75), and consisted of 264 unique genotypes. A total of 76 of the unknown trees were matched to 20 different named cultivars, and these cultivars were mainly derived from either the local breeding program at the University of Minnesota, or were Russian cultivars imported for horticulture in the northern US. This study demonstrates the importance of local breeding programs, and also the challenges associated with identifying clones in a genetically diverse crop like apple.

Cryopreservation provides valuable genes for further breeding of elite cultivars, and cryotherapy improves the production of virus-free plants in Malus spp., thus assisting the sustainable development of the apple industry. Apple (Malus spp.) is one of the most economically important temperate fruit crops. Wild Malus genetic resources and existing cultivars provide valuable genes for breeding new elite cultivars and rootstocks through traditional and biotechnological breeding programs. These valuable genes include those resistant to abiotic factors such as drought and salinity, and to biotic factors such as fungi, bacteria and aphids. Over the last three decades, great progress has been made in apple cryobiology, making Malus one of the most extensively studied plant genera with respect to cryopreservation. Explants such as pollen, seeds, in vivo dormant buds, and in vitro shoot tips have all been successfully cryopreserved, and large Malus cryobanks have been established. Cryotherapy has been used for virus eradication, to obtain virus-free apple plants. Cryopreservation provided valuable genes for further breeding of elite cultivars, and cryotherapy improved the production of virus-free plants in Malus spp., thus assisting the sustainable development of the apple industry. This review provides updated and comprehensive information on the development and progress of apple cryopreservation and cryotherapy. Future research will reveal new applications and uses for apple cryopreservation and cryotherapy.

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.

Citrus germplasm is conserved in genebanks at sites around the world to provide genetic resources for breeding and research programs. The value of genebank collections is particularly evident as diseases and climate change threaten citrus production areas. We provide historical, inventory, and maintenance information about national citrus collections in Russia and in the United States. The Russian Research Institute of Floriculture and Subtropical Crops (RRIFSC) in Sochi, Russia maintains a collection of 132 citrus accessions representing 50 taxa. Southern Russia is one of the most northerly citrus growing areas in the world and many accessions in the RRIFSC collection were specifically selected for cold tolerance to facilitate citrus production in this region. Tree response data taken during the frequent severe winters in Sochi has provided an understanding of the relative cold tolerance of many RRIFSC accessions. Particularly noteworthy is the array of cold-tolerant lemon accessions maintained at the RRIFSC. The Aurantioideae collections at the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) and at the University of California Citrus Variety Collection in Riverside, California, maintain 1328 accessions of citrus cultivars and wild relatives. Because of federal and state quarantine regulations, accessions at this facility are tested for graft-transmissible pathogens and undergo therapy to eliminate known pathogens. In contrast to Sochi, desert-adapted lemon and grapefruit varieties are a major interest of the California-based collection. The collection of citrus and citrus relatives is being screened for resistance and/or tolerance genes to diseases such as huanglongbing which is currently threatening the US citrus industry. Through collaborative exchange efforts, the diversity in the two genebanks could be expanded to increase availability of desirable genetic resources to breeding and research communities throughout the world.

Genetic improvements in plant breeding are dependent upon having access to novel plant genetic resources that are available in plant genebanks. Many crops that are vegetatively-propagated are maintained as plants in the field or greenhouse, making them vulnerable to biotic and abiotic threats. Increasingly, plant genebanks are using cryopreservation technologies to secure vegetatively propagated collections at secondary locations. Droplet vitrification and cryo-plate cryopreservation methods have been used to successfully cryopreserve the shoot tips of many plant species. New propagule types, including small leaf square-bearing adventitious buds, stem disc-bearing adventitious buds, microtubers and rhizome buds are alternative explants for use in cryopreservation. This review describes new technologies for in-vitro based cryopreservation systems that have advanced the field of plant cryopreservation. Future advances will allow even more diverse germplasm to be successfully preserved in cryobanks. New technologies for in-vitro based cryopreservation systems have advanced the field of plant cryopreservation since the twenty first century. Further advances will certainly facilitate even more diverse germplasm to be successfully preserved in cryobanks.

The cultivated pear is a major fruit crop in Eurasia that underpins many local economies. However, its origin and domestication history, as well as the diversity of wild pears in natural ecosystems, are at the early stages of exploration. In this chapter, we provide an overview of the described diversity and genetic relationships among wild and cultivated Pyrus species. Non-discriminatory morphological characters, poor diagnostic genetic tools, and lack of access to samples scattered throughout worldwide genebank collections make it difficult to definitively elucidate relationships of pear species and more generally Pyrus diversification and domestication. High-throughput sequencing is providing advancements in our understanding of the domestication process of the pear, and of biogeography, taxonomy, and ecology of wild pears. This knowledge will be crucial for future breeding programs focused on improving quality and production traits.

Pathogen-free stock plants are required as propagation materials in nurseries and healthy materials are needed in germplasm exchange between countries or regions through quarantine programs. In addition, plant gene banks also prefer to maintain pathogen-free germplasm collections. Shoot tip cryotherapy is a novel biotechnology method whereby cryopreservation methods are used to eradicate obligate pathogens from vegetatively propagated plants. Long-term preservation of pathogens is necessary in all types of virus-related basic research and applications such as antigen preparation for virus detection by immunology-based methods, production of plant-based vaccines, genetic transformation to produce virus-derived resistant transgenic plants, and bionanotechnology to produce nano drugs. Obligate plant pathogens such as viruses and viroids are intracellular parasites that colonize only living cells of the hosts. Therefore, their long-term preservation is difficult. Cryotreatments cannot completely eradicate the obligate pathogens that do not infect meristematic cells and certain proportions of plants recovered from cryotreatments are still pathogen-infected. Furthermore, cryotreatments often fail to eradicate the obligate pathogens that infect meristematic cells. Cryopreservation can be used for the long-term cryopreservation of the obligate plant pathogens. Thus, cryobiotechnology functions as a double-edged sword for plant pathogen eradication and cryopreservation. This review provides updated a synthesis of advances in cryopreservation techniques for eradication and cryopreservation of obligate plant pathogens.

Transcripts related to abiotic stress, oxidation, and wounding were differentially expressed in Arabidopsis shoot tips in response to cryoprotectant and liquid nitrogen treatment. Cryopreservation methods have been implemented in genebanks as a strategy to back-up plant genetic resource collections that are vegetatively propagated. Cryopreservation is frequently performed using vitrification methods, whereby shoot tips are treated with cryoprotectant solutions, such as Plant Vitrification Solution 2 (PVS2) or Plant Vitrification Solution 3 (PVS3); these solutions remove and/or replace freezable water within the meristem cells. We used the model system Arabidopsis thaliana to identify suites of transcripts that are up- or downregulated in response to PVS2 and PVS3 treatment and liquid nitrogen (LN) exposure. Our results suggest that there are many changes in transcript expression in shoot tips as a result of cryoprotection and that these changes exceed the number detected as a result of LN exposure. In total, 180 transcripts showed significant changes in expression level unique to treatment with either the cryoprotectant or cryopreservation followed by recovery. Of these 180 transcripts, 67 were related to stress, defense, wounding, lipid, carbohydrate, abscisic acid, oxidation, temperature (cold/heat), or osmoregulation. The responses of five transcripts were confirmed using qPCR methods. The transcripts responding to PVS2 + LN suggest an oxidative response to this treatment, whereas the PVS3 + LN treatment invoked a more general metabolic response. This work shows that the choice of cryoprotectant can have a major influence on the patterns of transcript expression, presumably due to the level and extent of stress experienced by the shoot tip. As a result, there may be divergent responses of study systems to PVS2 and PVS3 treatments.

Vegetatively propagated plant collections within genebanks are vulnerable to threats when they are maintained in field or greenhouse conditions. International genebanking standards recommend that these collections be securely backed-up at either secondary locations or by using in vitro culture or cryopreservation. A series of tools and tables were developed to assist genebank managers in determining the predicted number of viable explants after cryopreservation procedures have been employed based on the number of samples processed and the viability for a range of confidence levels (0.5, 0.75, 0.90, 0.95, and 0.99). In addition, critical viabilities were estimated which represent the minimum viability required to have at least one viable explant for a given number of explants stored per cryovial or tube. The plant cryobanking literature was surveyed to summarize the sample processing practices that have been implemented within genebanks. The minimum number of explants predicted to be viable for accessions within these cryobanked collections was calculated based on the published minimum viability levels and numbers of explants processed. Implementation of the proposed tools and tables will allow genebank managers to determine if shifts in their current methods could result in more efficient back-up procedures.

Many species in genebank collections are maintained vegetatively either as in vitro cultures or as plants in the field or greenhouse. In these cases, individual genotypes (cultivars) are the focus of conservation. In crop wild relatives, where the value of an accession is in the heterogeneity of segregating genotypes, seeds may be the most representative form of conservation. We used two sets of field-planted Malus sieversii apple trees that originated from seeds collected from two wild populations in Kazakhstan as a model system to determine if seeds from hand-pollinated crosses could be used to represent the allelic diversity of the parent trees in a genebank orchard. A crossing design was developed that used each of the selected trees from the two Kazakhstan collection sites as mothers and also as fathers in the form of bulked pollen pools. The sets of offspring from these crosses were genotyped using microsatellite markers. Paternity analyses revealed the percentage of offspring that was derived from each of the parent trees. We also determined the contribution of each parent to the fertilization events from the pollen pools. Although there were some unintended pollination events (fertilization by trees in unexpected pollen pools) and unequal contribution of pollen parents within pollen pools, we demonstrated that the seedlings effectively captured the allelic frequency and diversity of the parents. A seed collection strategy is the most efficient way to conserve the diversity of crop wild relatives even in collections typically conserved as individual clones.

Apples (Malus Mill.) have been economically and socially important throughout the centuries in North America. Apple cultivar (Malus ×domestica Borkh.) collections that include historic cultivars are valued for their unique diversity, historical significance, and also as a resource to identify unknown trees; however, not all of the historically significant apple cultivars are currently included in these collections. We used historic books, publications, and nursery catalogs to develop an inventory of apple cultivars that were propagated and grown in the United States before 1908. We collected synonym, introduction date, and original source country information for 891 historic apple cultivars. Most of the historic American cultivars originated as seedlings first planted in the United States. Some cultivars were brought to the United States from the United Kingdom, France, Russia, Germany, and other European countries. We classified historic apple cultivars based on their availability over time and popularity in nursery catalogs. Ninety percent of the most popular historic apple cultivars in the United States were available in 2015 in the U.S. and U.K. national collections and within several commercial and private collections. This work identified high priority historic cultivars that are not currently protected within genebanks that could be added to genebank collections in the future.