A droplet vitrification cryopreservation protocol for conservation of hops (Humulus lupulus) genetic resources.

Hops (Humulus lupulus L.) is essentially used in the brewing industry as it contributes to flavor, and aroma of beer. However, the genetic diversity of hops is increasingly threatened by diseases, environmental changes, and urbanization. Cryopreservation has emerged as a pivotal strategy for safeguarding and maintaining the genetic diversity of hops. The present work presents a comprehensive study on the cryopreservation of hops, focusing on the development and optimization of a droplet vitrification based cryopreservation protocol. Shoot tips excised from one month old in vitro cultures were precultured on 0.3 M sucrose, dehydrated in a loading solution followed by treatment with PVS2 solution for different durations. Significant effect of PVS2 dehydration was observed on post-thaw survival and regeneration after cryoconservation with maximum 50% post-thaw regeneration observed in shoot tips dehydrated in PVS2 for 30 min. Genetic fidelity of the regenerated plants was confirmed using 30 ISSR markers. Reproducibility of the developed protocol was tested on seven other accessions and post thaw regeneration ranging from 43 to 70% was observed across the accessions. The present study reports a highly efficient protocol for conservation of hops germplasm. The results indicate that droplet vitrification can be used as a reliable and sustainable approach for hop genetic preservation, with high survival rates and minimal genetic alterations observed in cryopreserved samples. To the best of our knowledge, this is the first report on DV based cryopreservation of hops germplasm.

Validation of Blood Glucose and Lipid-Lowering Effect of Solvent Fractions of the Crinum Abyssinicum Shoot Tips in Streptozotocin-Induced Diabetic Mice.

Background: Diabetes mellitus prevalence has reached epidemic levels despite the existence of contemporary treatments. People thus started looking at the possible therapeutic value of natural therapies. Crushed shoot tips of Crinum abyssinicum (Amaryllidaceae) are mixed with water in Ethiopia to treat diabetes, yet this practice is not well supported by science. Objective: In this experiment, mice models were used to verify the blood sugar and lipid-lowering benefits of solvent fractions of C. abyssinicum shoot tips. Materials and Methods: In a single-dose treated Streptozotocin (STZ)-induced diabetic model, mice were randomly grouped into eleven categories which include diabetic negative control, diabetic positive control, and 9 diabetic treatment groups. In repeated daily doses treated STZ-induced model, Mice were divided into 6 groups which included normal and diabetic negative control (TW80), diabetic positive control (5 mg/kg glibenclamide), and three diabetic treatment groups 100, 200, and 400 mg/kg). Finally, blood glucose, lipid level, and body weight were examined. Results: In the single-dose treated diabetic model, there was a significant blood glucose reduction at 200 and 400 mg/kg doses of aqueous fraction and glibenclamide starting from the sixth-hour post-administration unlike ethyl acetate and chloroform fraction compared to baseline and negative control. In repeated daily dose-treated diabetic mice, all three doses (100, 200, and 400 mg/kg of aqueous fraction) resulted in a substantial reduction (P < .001) in blood glucose compared to baseline and negative control on the seventh day and 14th day. Besides the AQF shows improvement in lipid levels and body weight parameters. Conclusion: The results of the study demonstrated that C. abyssinicum shoot tip fractions have the greatest potential to lower blood sugar and lipid levels, supporting conventional claims for the treatment of diabetes.

Critical Role of Regrowth Conditions in Post-Cryopreservation of In Vitro Plant Germplasm.

Cryopreservation is an effective option for the long-term conservation of plant genetic resources, including vegetatively propagated crops and ornamental plants, elite tree genotypes, threatened plant species with non-orthodox seeds or limited seed availability, as well as cell and root cultures useful for biotechnology. With increasing success, an arsenal of cryopreservation methods has been developed and applied to many species and material types. However, severe damage to plant material accumulating during the multi-step cryopreservation procedure often causes reduced survival and low regrowth, even when the optimized protocol is applied. The conditions at the recovery stage play a vital role in supporting material regrowth after cryopreservation and, when optimized, may shift the life-and-death balance toward a positive outcome. In this contribution, we provide an overview of the five main strategies available at the recovery stage to improve post-cryopreservation survival of in vitro plant materials and their further proliferation and development. In particular, we discuss the modification of the recovery medium composition (iron- and ammonium-free), exogenous additives to cope with oxidative stress and absorb toxic chemicals, and the modulation of medium osmotic potential. Special attention is paid to plant growth regulators used at various steps of the recovery process to induce the desired morphological response in cryopreserved tissues. Given studies on electron transport and energy provision in rewarmed materials, we discuss the effects of light-and-dark conditions and light quality. We hope that this summary provides a helpful guideline and a set of references for choosing the recovery conditions for plant species that have not been cryopreserved. We also propose that step-wise recovery may be most effective for materials sensitive to cryopreservation-induced osmotic and chemical stresses.

Alternative Plant Vitrification Solution A3-80% and Initial Ammonium-Free Regrowth Medium Enable Cryobanking of Chrysanthemum Germplasm.

Cryopreservation, storing biological material in liquid nitrogen (LN, -196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocol is hampered by a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing a droplet-vitrification cryopreservation procedure for chrysanthemum shoot tips. The standard procedure includes two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 40 min, cryoprotection with alternative plant vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with 1 mg L-1 gibberellic acid (GA3) and 1 mg L-1 benzyl adenine (BA) followed by an ammonium-containing medium with and without growth regulators was essential for the development of normal plantlets from cryopreserved shoot tips. A pilot cryobanking of 154 accessions of chrysanthemum germplasm initiated with post-cryopreservation regeneration of 74.8%. This approach will facilitate the cryobanking of the largest Asteraceae family germplasm as a complementary long-term conservation method.

High regrowth of potato crop wild relative genotypes after cryogenic storage.

Potatoes are consumed by millions of people and are the survival food in several countries. Cultivated varieties of potato (Solanum tubersosum L.) are results of selection and crossing of many wild species. Only 8-13% of wild potato species used for food are preserved by either in situ or ex situ methods. The U.S. National Potato Germplasm Collection maintains over 5900 accessions, of which 75% are crop wild relatives (CWR). The objective of the study was to investigate regrowth of cryogenically stored clonal propagules (shoot tips) of selected CWR accessions maintained in the collection. Sixty-nine accessions from 30 Solanum species and six accessions that are not yet assigned to a species were cryopreserved by a droplet vitrification method at the NLGRP. The post cryopreservation regrowth varied from 40 to 100% (average 68%) but was not significantly different between the tested accessions. Regrowth of six accessions tested after 10 years of cryogenic storage was between 35 and 90% (average 66%) and was significantly different from their initial regrowth (average 87%); the largest viability loss was in S. condolleanum; but for the other five accessions the regrowth was between 45 and 90% (average 72%) and suggested at least 10 years of successful storage in LN was possible. Twelve potato wild species cryopreserved in this study were reported in literature as important for developing cultivated varieties for changed weather conditions.

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies.

Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L-1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20-100%). The most effective result (70-100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in "Dunluce" free of PVS having higher numbers of microtubers and in "V500' free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

Ascorbic acid addition during dehydration improves garlic shoot tip cryopreservation but does not affect viral load.

Cryopreservation is known be an effective method for virus elimination in garlic. However, oxidative damage during the cryopreservation seriously affects the survival of garlic after cryopreservation. Ascorbic acid (AsA) can reduce oxidative damage and improve regrowth following cryopreservation, and its effect may be influenced by the step during which it is added. In this study, AsA was added at the osmoprotection (O) and dehydration (DE) steps of cryopreservation. By observing the dynamic changes in cell viability and reactive oxygen species (ROS) components with different AsA treatments, AsA has been linked to the reduced accumulation of ROS in the shoot tips. Increased gene expression levels of antioxidant enzymes also explained the ROS scavenging effect of AsA. The correlation analysis between cell viability, ROS, membrane lipid peroxidation-related indicators and antioxidant-related indicators showed that membrane lipid peroxidation caused by excess ROS was the main factor affecting cell viability. Ascorbic acid added during dehydration minimized the accumulation of ROS from dehydration to dilution and alleviated the oxidative damage during cryopreservation. Thus, the survival and regrowth of the garlic was significantly improved after cryopreservation. Dehydration was found to be the suitable step for the addition of AsA during garlic cryopreservation. We further evaluated the virus elimination effect under optimal AsA treatment. However, there was no significant difference in virus content in regenerated plants when compared with the control.

Thermotherapy Followed by Shoot Tip Cryotherapy Eradicates Latent Viruses and Apple Hammerhead Viroid from In Vitro Apple Rootstocks.

Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple growing regions. In addition to these viruses, a new infectious agent named Apple hammerhead viroid (AHVd) has been identified. We investigated whether thermotherapy or cryotherapy alone or a combination of both could effectively eradicate ACLSV, ASGV, and AHVd from in vitro cultures of four apple rootstocks developed in the Cornell-Geneva apple rootstock breeding program (CG 2034, CG 4213, CG 5257, and CG 6006). For thermotherapy treatments, in vitro plants were treated for four weeks at 36 °C (day) and 32 °C (night). Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture in 2 M glycerol + 0.8 M sucrose for one day followed by exposure to PVS2 for 60 or 75 min at 22 °C, either without or with liquid nitrogen (LN, cryotherapy) exposure. Combinations of thermotherapy and PVS2/cryotherapy treatments were also performed. Following treatments, shoot tips were warmed, recovered on growth medium, transferred to the greenhouse, grown, placed in dormancy inducing conditions, and then grown again prior to sampling leaves for the presence of viruses and viroids. Overall, thermotherapy combined with cryotherapy treatment resulted in the highest percentage of virus- and viroid-free plants, suggesting great potential for producing virus- and viroid-free planting materials for the apple industry. Furthermore, it could also be a valuable tool to support the global exchange of apple germplasm.

In Vitro Technologies for American Chestnut (Castanea dentata (Marshall) Borkh) Conservation.

American chestnut (Castanea dentata), a native species of eastern North America, is an economically important deciduous hardwood tree that has been designated as endangered in Canada. The population of American chestnut trees has dwindled significantly across Southern Ontario due to chestnut blight and many of the surviving trees continue to show blight disease symptoms. American chestnut requires efficient strategies for propagation and preservation for species recovery. The objective of this study was to develop a long-term plant conservation program using micropropagation and cryopreservation protocols. An in vitro technology using a liquid-based temporary immersion system (TIS) was developed for micropropagation of American chestnut. The highest rate of shoot multiplication was observed in cultures grown in the DKW (Driver and Kuniyuki 1984) basal medium supplemented with 2.2 µM 6-benzylaminopurine and 1.0 µM gibberellic acid. More than 95% of proliferated microshoots, about 40-50 mm in size, developed roots after 30 days of culture within bioreactor vessels containing DKW basal medium supplemented with 15 µM 3-Indolebutyric acid. Rooted plantlets transplanted to the greenhouse had a survival efficiency of 82% after one month of growth. The cryopreservation protocol for germplasm preservation was developed through droplet vitrification of shoots. Optimal regeneration of shoot tips occurred from explants precultured on stepwise concentrations of sucrose and subsequent dehydration in PVS3 for 30 min. Cryopreserved shoot tips were regenerated to whole plants using pre-optimized conditions of micropropagation. This study confirms the potential of TIS for micropropagation in ex situ conservation and reintroduction of endangered American chestnuts and possibly other woody plant species.

Micropropagation and Cryopreservation of Yukon Draba (Draba yukonensis), a Special Concern Plant Species Endemic to Yukon Territory, Canada.

Yukon Draba (Draba yukonensis) is a small, short-lived perennial mustard species that is endemic to southwestern Yukon in Canada. This plant has been categorized as a species of Special Concern. It faces the threat of habitat loss due to natural and man-made causes and a population that is unevenly distributed to a few large and several small subpopulations in the area. It will therefore be judicious to undertake investigations on the conservation of this species to save it from further deterioration which may lead to its extinction. In this study, a protocol was developed for in vitro propagation and cryopreservation of Yukon Draba. The micropropagation protocol was optimized using shoot tips which enabled clonal propagation and in vitro storage of the species. Shoots grew best in the medium containing MS basal salts and had the highest multiplication with the addition of 2 µM 6-benzylaminopurine or 5 µM Kinetin with 3% sucrose. The addition of 10 µM Indole Butyric Acid (IBA) produced the highest number of adventitious roots on the shoots and the longest root length was observed at 2 µM IBA. The rooted plantlets were transferred to greenhouse and the highest survival (87.5%) was observed for the plantlets treated with a lower concentration of IBA (2 µM). Cryopreservation protocol was developed using the droplet-vitrification method for in vitro shoot tips. Two-week-old shoots had the highest survival and regrowth following exposure to plant vitrification solution 3 (PVS3) for 30 min, prior to direct immersion of the droplets into the liquid nitrogen. The optimized protocols for the micropropagation and cryopreservation may be useful for the long-term germplasm conservation and reintroduction of this species in its natural habitat.

Epigenetic and Genetic Integrity, Metabolic Stability, and Field Performance of Cryopreserved Plants.

Cryopreservation is considered an ideal strategy for the long-term preservation of plant genetic resources. Significant progress was achieved over the past several decades, resulting in the successful cryopreservation of the genetic resources of diverse plant species. Cryopreservation procedures often employ in vitro culture techniques and require the precise control of several steps, such as the excision of explants, preculture, osmo- and cryoprotection, dehydration, freeze-thaw cycle, unloading, and post-culture for the recovery of plants. These processes create a stressful environment and cause reactive oxygen species (ROS)-induced oxidative stress, which is detrimental to the growth and regeneration of tissues and plants from cryopreserved tissues. ROS-induced oxidative stresses were documented to induce (epi)genetic and somatic variations. Therefore, the development of true-to-type regenerants of the source germplasm is of primary concern in the application of plant cryopreservation technology. The present article provides a comprehensive assessment of epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants developed in the past decade. Potential areas and the directions of future research in plant cryopreservation are also proposed.

Cryopreservation of Woody Crops: The Avocado Case.

Recent development and implementation of crop cryopreservation protocols has increased the capacity to maintain recalcitrant seeded germplasm collections via cryopreserved in vitro material. To preserve the greatest possible plant genetic resources globally for future food security and breeding programs, it is essential to integrate in situ and ex situ conservation methods into a cohesive conservation plan. In vitro storage using tissue culture and cryopreservation techniques offers promising complementary tools that can be used to promote this approach. These techniques can be employed for crops difficult or impossible to maintain in seed banks for long-term conservation. This includes woody perennial plants, recalcitrant seed crops or crops with no seeds at all and vegetatively or clonally propagated crops where seeds are not true-to-type. Many of the world's most important crops for food, nutrition and livelihoods, are vegetatively propagated or have recalcitrant seeds. This review will look at ex situ conservation, namely field repositories and in vitro storage for some of these economically important crops, focusing on conservation strategies for avocado. To date, cultivar-specific multiplication protocols have been established for maintaining multiple avocado cultivars in tissue culture. Cryopreservation of avocado somatic embryos and somatic embryogenesis have been successful. In addition, a shoot-tip cryopreservation protocol has been developed for cryo-storage and regeneration of true-to-type clonal avocado plants.

Nitrogen Levels Regulate Sugar Metabolism and Transport in the Shoot Tips of Crabapple Plants.

To comprehensively understand the responses of carbohydrate metabolism and transport to different levels of nitrogen supply in growing shoot tips of crabapple (Malus hupehensis Rehd), enzyme activities and related genes involved in the sugar metabolism pathway were investigated. The nitrogen and chlorophyll content of plants increased with increasing nitrogen supply. High nitrogen application increased the net photosynthesis rate and the growth rate of shoot tips but decreased the synthesis capability of sucrose and sorbitol in mature leaves. However, the shoot tips of plants under high-nitrogen treatment had higher contents of sucrose and sorbitol than did those under low-nitrogen treatment, while the activity of sucrose phosphate synthase and aldose-6-phosphate was increased and the transporters MdSOT and MdSUT were up-regulated. Moreover, the activities of enzymes involved in sucrose and hexose metabolism (including sucrose synthase, fructokinase, and hexokinase) were enhanced in the shoot tips of plants under high-nitrogen conditions, and the expression levels of MdSUSY3 and MdHK1 were significantly up-regulated. These findings indicate that a high nitrogen supply increases the metabolic capacity of assimilatory substances in shoot tips, accelerates the efficiency of sugar utilization and eventually leads to a rapid increase in the growth of shoot tips. Our results highlight that high nitrogen increases the capacity of sugar unloading and metabolic utilization in growing shoot tissues.

Two Advanced Cryogenic Procedures for Improving Stevia rebaudiana (Bertoni) Cryopreservation.

Cryopreservation is a useful tool for the long-term storage of plant genetic resources, and different cryogenic procedures have recently been developed. The present study focused on the use of the Droplet-vitrification (DV) and V cryo-plate protocol for the cryopreservation of Stevia rebaudiana in vitro-derived apical shoot tips and axillary shoot tips. A preliminary test showed that 90 and 120 min PVS2 (Plant Vitrification Solution 2) treatment significantly reduced the regrowth of the explants before immersion in liquid nitrogen (LN). For both procedures tested, the best osmoprotective condition for obtaining a higher regrowth of cryopreserved explants occurred when explants were PVS2 treated for 60 min. After direct immersion in LN, thawing and plating, the highest regrowth recorded was 80% with DV and 93% with V cryo-plate. Moreover, shoot tips proved to be a more suitable material for Stevia cryopreservation. A satisfactory vegetative regrowth was observed in the subcultures following cryopreservation by DV and V cryo-plate cryogenic procedures.

Endemic Plant Species Conservation: Biotechnological Approaches.

Endemic plant species are usually more vulnerable to anthropogenic threats and natural changes and, therefore, hold a higher extinction risk. The preservation of these species is a major concern on a worldwide context and in situ protection alone will not guarantee their conservation. Ex situ conservation measures must be undertaken to support the conservation of these species, and seed banking is the more efficient and cost-effective method. However, when seed banking is not an option, alternative approaches should be considered. Biotechnological tools provide new and complementary options for plant conservation including short-, medium-, and long-term strategies, and their application for plant species conservation has increased considerably in the last years. This review provides information about the status of the use biotechnology-based techniques for the conservation of endemic plant species. Particular attention is given to cryopreservation, since is the only long-term ex situ conservation strategy that can complement and support the other conservation measures. The cryopreservation of plant genetic resources is, however, more focused on crop or economically important species and few studies are available for endemic plant species. The plant material used, the cryopreservation methods employed, and the assessment of cryogenic effects are reviewed. The reasons to explain the difficulties in cryopreserving these species are discussed and new strategies are proposed to facilitate and increase the interest on this matter. We expect that further studies on the conservation of endemic plant species will increase in a near future, thus contributing to maintain these valuable genetic resources.

Development, progress and future prospects in cryobiotechnology of Lilium spp.

Lilium is one of the most popular flower crops worldwide, and some species are also used as vegetables and medicines. The availability of and easy access to diverse Lilium genetic resources are essential for plant genetic improvements. Cryopreservation is currently considered as an ideal means for the long-term preservation of plant germplasm. Over the last two decades, great efforts have been exerted in studies of Lilium cryopreservation and progress has been made in the successful cryopreservation of pollen, seeds and shoot tips in Lilium. Genes that exist in Lilium, including those that regulate flower shape, color and size, and that are resistant to cold stress and diseases caused by fungi and viruses, provide a rich source of valuable genetic resources for breeding programs to create novel cultivars required by the global floriculture and ornamental markets. Successful cryopreservation of Lilium spp. is a way to preserve these valuable genes. The present study provides updated and comprehensive information about the development of techniques that have advanced Lilium cryopreservation. Further ideas are proposed to better direct future studies on Lilium cryobiotechnology.

In vitro shoot multiplication of Rumex vesicarius L., and quantification of ascorbic acid and major phenolics from its leaf derived callus.

An optimised method for enhanced in vitro shoot multiplication of Rumex vesicarius (Polygonaceae)-a branched succulent herb-was achieved. The in vitro seed pre-treatment with 4% urea was able to show 95% seed germination on MS medium within 2 weeks of culturing. In vitro shoot bud induction from shoot tip explants was best in the presence of 2 mg L-1 kinetin on Murashige and Skoog medium (MS medium), wherein 8.3 shoots/explants were observed. Shoot elongation was found to be high (3.75 ± 0.5 cm) in 2 mg L-1 BA comprising medium. The transfer of micro-shoots on to MS medium comprising 1.5 mg L-1 IBA and 1% activated charcoal (w/v) supported efficiently in vitro rooting (2.5-4.0 cm) in 3-4 weeks. Upon hardening, 70% rooted plants survived under greenhouse conditions. Though friable callus was produced on MS medium-containing 2 mg L-1 BA followed by 3 mg L-1 BA, no organogenesis was noticed. The ascorbic acid content of 78.62 ± 0.25 mg 100 g-1 FW was recorded in callus cultures grown on medium supplemented with 2 mg L-1 BA, and it is 1.74-fold more compared to normal ex vitro leaves of the same age. In vitro raised plant leaf showed 1.98-fold more ascorbic acid (89.42 ± 0.18 mg 100 g-1 FW) to that of ex vitro leaves. The total phenolic content was found to be 60 mg in callus as compared to 610 mg (per 100 g GAE FW) of ex vitro leaves. The major phenolic compounds quantified were synergic, chlorogenic, ferulic, and generic acids, respectively. This optimised protocol will facilitate to pursue scale-up studies for in vitro ascorbic acid production and also to further investigate the kinetics of biosynthetic pathway genes involved.

Cryobiotechnology: A Double-Edged Sword for Obligate Plant Pathogens.

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.

Long-term preservation of potato leafroll virus, potato virus S, and potato spindle tuber viroid in cryopreserved shoot tips.

Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips (58-60%) than in 0.5-mm-ones (30-38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRV-preserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation, similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.

Effects of Leucaena leucocephala (lamk. ) shoot tips plus young leaf extract containing mimosine on reproductive system of male rats / Efectos de los brotes de Leucaena leucocephala (lamk. ) y del extracto de hojas jóvenes que contiene mimosina en el sistema reproductivo de ratas macho

This study aimed to determine the mimosine level and examine the male reproductive toxicity effects of Leucaena leucocephala (LL) shoot tips plus young leaf extract. Mimosine level in LL extract was determined by thin layer chromatography before administration in animals. Male rats were divided into control and LL (1,500 mg/KgBW) groups (n = 6). After 60 days of experiment, serum sex hormones, sperm quality, and testicular histopathology were assayed and observed. Malondialdehyde (MDA) level and expressions of steroidogenic acute regulatory (StAR) and phosphorylated proteins in testicular lysate were examined by western blotting. Results showed that mimosine levels in LL extract was 17.35 ± 1.12 % of dry weight. LL significantly decreased FSH & LH levels, sperm qualities, and seminiferous tubule diameter compared to the control (p<0.05). Seminiferous tubular atrophies, germ cell sloughing, and degenerations were observed in LL group. In addition, testicular MDA level and StAR protein expression were significantly decreased in LL group. LL extract could increase the expression of a 50 kDa phohorylated protein in testicular lysate. In conclusion, LL extract has mimosine and reproductive toxicity effects on males.

Este trabajo tuvo como objetivo determinar el nivel de mimosina y examinar los efectos de la toxicidad reproductiva de los brotes de Leucaena leucocephala (LL), más el extracto de hojas jóvenes, en ratas macho. El nivel de mimosina en el extracto de LL se determinó mediante cromatografía en capa fina antes de la administración en animales. Las ratas se dividieron en grupos de control y LL (1,500 mg / kgBW) (n = 6). Después de 60 días, se analizaron y observaron las hormonas sexuales séricas, la calidad de los espermatozoides y la histopatología testicular. A través de Western Blot se examinaron el nivel de malondialdehído (MDA), las expresiones de reguladores agudos esteroidogénicos (StAR) y las proteínas fosforiladas en el lisado testicular. Los resultados mostraron que los niveles de mimosina en el extracto de LL fueron 17.35 ± 1.12 % del peso seco. LL disminuyó significativamente los niveles de FSH y LH, la calidad de los espermatozoides y el diámetro de los túbulos seminíferos en comparación con el control (p <0,05). Se observaron atrofias en los túbulos seminíferos, desprendimiento de células germinales y degeneraciones en el grupo LL. Además, el nivel de MDA testicular y la expresión de la proteína StAR se redujeron significativamente en el grupo LL. El extracto de LL podría aumentar la expresión de la proteína fosforilada de 50 kDa en el lisado testicular. En conclusión, el extracto de LL tiene mimosina y efectos de toxicidad reproductiva en los hombres.