Search

Root and tuber crops (RTCs) are the second-most important carbohydrate commodity after cereals. Many species of the RTCs are vegetatively propagated, making their shoot tips the preferred material to be conserved for future uses. Shoot tip cryopreservation provides an important tool to support the long-term conservation of plant genetic resources. Over the past four decades, significant efforts have been undertaken to move shoot tip cryopreservation of RTCs from research projects to full-scale implementation in cryobanks. This comprehensive review focuses on the history of cryopreservation protocols developed in RTCs. The encapsulation and vitrification solution-based cryopreservation techniques followed by ultra-rapid freezing and thawing have been highly successful. Additionally, different strategies for improving the cryotolerance of shoot tips have been introduced to further increase post-cryopreservation recovery. Finally, the research conducted to explain the mechanism underlying cryoprotection and differential cryotolerance including the use of histological studies are highlighted.

Abstract 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.

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.

Root and tuber crops (RTCs) are the second-most important carbohydrate commodity after cereals. Many species of the RTCs are vegetatively propagated, making their shoot tips the preferred material to be conserved for future uses. Shoot tip cryopreservation provides an important tool to support the long-term conservation of plant genetic resources. Over the past four decades, significant efforts have been undertaken to move shoot tip cryopreservation of RTCs from research projects to full-scale implementation in cryobanks. This comprehensive review focuses on the history of cryopreservation protocols developed in RTCs. The encapsulation and vitrification solution-based cryopreservation techniques followed by ultra-rapid freezing and thawing have been highly successful. Additionally, different strategies for improving the cryotolerance of shoot tips have been introduced to further increase post-cryopreservation recovery. Finally, the research conducted to explain the mechanism underlying cryoprotection and differential cryotolerance including the use of histological studies are highlighted. [ABSTRACT FROM AUTHOR]

Diseases caused by plant pathogens such as viruses, viroids and phytoplasmas cause huge economic losses of agricultural production and limit the safe movement of plant materials across borders. The use of pathogen‐free planting materials provides a strategy for efficient management of these diseases and facilitates the global exchange of genetic resources. Shoot tip cryotherapy is a novel biotechnology method that uses cryogenic procedures to eradicate plant pathogens from the diseased plants. Combining thermotherapy or chemotherapy with shoot tip cryotherapy has further enhanced pathogen eradication efficiency. This review provides updated and comprehensive information on shoot tip cryotherapy and the combination of thermotherapy or chemotherapy with shoot tip cryotherapy for pathogen eradication. Prospects are proposed for future studies. [ABSTRACT FROM AUTHOR]

Key message: 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.Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • We could successfully cryopreserve two-viroid infected chrysanthemum. • We discover that stock hygiene significantly affects cryopreservation success. • We could provide explanations as to why cryopreservation failed to eradicate viroids. Abstract Chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) are the two viroids most frequently infecting chrysanthemum. This study attempted to cryopreserve in vitro shoot tips of Chrysanthemum morifolium 'Piato' infected with high or low titers of CSVd, and co-infected with CSVd and CChMVd. By optimizing several key factors including the time and light regimes during cold-hardening of stock shoots, and the size of shoot tips, an encapsulation-vitrification procedure was established for cryopreservation of viroid-infected shoot tips. Viroid-infected stock shoots were cold-hardened in vitro at 4 °C in a 16-h photoperiod for 6 weeks. Shoot tips (1.5 mm in size) containing 3-4 LPs were excised from the cold-hardened stock shoots and subjected to encapsulation-vitrification cryopreservation. With this protocol, about 65%, 45% and 42% of shoot regrowth levels were obtained in cryopreserved shoot tips derived from in vitro stock shoots infected with low or high titers of CSVd, and co-infected with CSVd and CChMVd, respectively. All regenerants from cryopreservation maintained their viroid-infected status, identical to those of their in vitro stock shoots. Histological observations and in situ hybridization elucidated why cryopreservation could maintain viroids in cryopreserved shoot tips. Cryopreservation of viroid-infected plant materials has potential applications to all types of viroid-related basic and applied studies. [ABSTRACT FROM AUTHOR]

We report a droplet-vitrification for efficient cryopreservation of valuable germplasm of blueberry. In this protocol, in vitro stock shoots were cold-hardened in the dark at 4 °C for 3 weeks. Shoot tips (1.5–2.0 mm in length) including 4–5 leaf primordia were excised from the cold-hardened stock shoots and precultured with 0.3 M sucrose for 1 day. Precultured shoot tips were treated with a loading solution containing 1.0 M sucrose and 2 M glycerol for 30 min, followed by exposure to PVS2 for 40 min at 0 °C. Dehydrated shoot tips were then transferred onto 3.5 μL PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen foils with shoot tips were re-warmed in 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for a recovery medium containing 0.3 mg L −1 zeatin for shoot regrowth. With optimized parameters, shoot regrowth levels were 46.7% for ‘O’ Neal’ and 91.7% for ‘Misty’, with a mean shoot regrowth level of 66.5% obtained across the five valuable blueberry genotypes tested. Histological observations showed that many cells in the upper part of apical dome and some in leaf primordia 1–3 survived, while those in lower part of apical dome and leaf primordia 4–5 were seriously damaged, following cryopreservation. No polymorphic bands were detected using inter-simple sequence repeat markers (ISSR) and random amplified polymorphic DNA (RAPD) in regenerants of ‘O’ Neal’ and ‘Misty’ recovered from cryopreservation. To the best our knowledge, this is the first report on blueberry cryopreservation by droplet-vitrification, which would provide a technical platform for establishment of cryo-banks of valuable blueberry germplasm. [ABSTRACT FROM AUTHOR]

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94–95%) in the apical dome (AD) were damaged or killed and only some cells (30–32%) in the leaf primordia (LPs) survived. In category 2, only a few cells (18–20%) in the AD and some cells (30–31%) in the LPs survived. In category 3, majority of the cells (60–62%) in the AD and some cells (30–33%) in the LPs survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips. [ABSTRACT FROM AUTHOR]

We reported here an efficient, widely applicable droplet-vitrification cryopreservation for shoot tips of Chrysanthemum morifolium . Nodal segments, each being 0.5 cm in length and containing one bud positioned on nodes 3–7, were taken from 6 weeks old stock shoots and cultured on a shoot maintenance medium (SMM) for 12 days to promote bud elongation. Shoot tips (2.0 mm in size) containing 5–6 leaf primordia were excised from elongated buds and precultured on Murashige and Skoog medium (MS) containing 0.5 M sucrose for 1 day. Precultured shoot tips were loaded, dehydrated with PVS2 for 30 min at 0 °C and then transferred onto droplets containing 2.5 μl PVS2 on aluminum foils (2 cm × 0.8 cm), prior to a direct immersion in liquid nitrogen (LN) for 1 h. Thawed shoot tips were post-cultured on shoot recovery medium containing MS supplemented with 0.05 mg L −1 GA 3 in the dark for 3 days and then transferred on the same medium under standard culture conditions for shoot recovery. The droplet-vitrification procedure resulted in the highest (83%) and lowest (43%) shoot regrowth rates for C. morifolium ‘Japanese Red’ and ‘Xizi Qiuzhuang’, with an average rate of 68% in six C. morifolium genotypes tested. Histological observations showed that the pattern and percentage of surviving cells were similar in cryopreserved shoot tips of these two genotypes. No polymorphic bands were detected by simple sequence repeats (SSR) and ploidy levels analyzed by flow cytometry (FCM) were maintained in plantlets regenerated from cryopreserved shoot tips of the two genotypes. [ABSTRACT FROM AUTHOR]

We previously reported successful cryopreservation of shoot tips of potato 'Zihuabai' by three vitrification-based protocols. In the present study, cryo-injury to shoot tips and genetic stability in regenerants recovered from cryopreserved shoot tips by the three vitrification-based protocols were further investigated. The results showed that sucrose preculture caused no obviously different injuries, while dehydration with plant vitrification solution 2 (PVS2) was the step causing major damage to cells of shoot tips, regardless of the cryogenic procedures. Compared with droplet-vitrification and encapsulation--vitrification, vitrification caused the most severe injury to cells of the shoot tips, thus resulting in much longer time duration for shoot recovery and much lower shoot regrowth rate. Cells in apical dome and the youngest leaf primordia were able to survive and subsequently some of them regrew into shoots following all three vitrification-based cryopreservation procedures. Analyses using inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers in shoots regrown from all three vitrification-based protocols did not find any polymorphic bands. The results reported here suggest that vitrification-based cryo-procedures can be considered promising methods for long-term preservation of potato genetic resources. [ABSTRACT FROM AUTHOR]