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.

Transcriptome assessment in 'Red Globe' grapevine zygotic embryos during the cooling and warming phase of the cryopreservation procedure.

Cryopreservation has the potential for long-term germplasm storage. The metabolic pathways and gene regulation involved in cryopreservation procedures are still not well documented. Hence, the genetic expression profile was evaluated using RNA-Seq in zygotic embryos of grapevines subjected to cryopreservation by vitrification. Sequencing was performed on the Illumina NextSeq 500. The average alignment of reads was 96% against the reference genome. The expression profiles showed 229 genes differentially expressed (186 repressed and 46 induced). The main biological processes showing upregulated enrichment were related to nucleosome assembly, while downregulated processes were related to organ growth. The most highly repressed processes were associated with the organization of the cell wall and membrane components. The unnamed protein product and 17.3 kDa class II heat shock protein (HSP) were highly induced, while ATPase subunit 1 and expansin-A1 were repressed. The response to the cooling and warming process during cryopreservation probably indicates that the changes occurring in transcription may be related to epigenetics. In addition, the cell exhibits an increase in the reserve of nutrients while seeking to survive modestly using available energy and pausing the plant's development. Additionally, energy containment occurred to cope with the stress caused by the treatment where deactivation of components of the cell membrane was observed, possibly due to changes in fluidity caused by alterations in temperature.

The world's largest potato cryobank at the International Potato Center (CIP) - Status quo, protocol improvement through large-scale experiments and long-term viability monitoring.

Long-term conservation of Plant Genetic Resources (PGR) is a key priority for guaranteeing food security and sustainability of agricultural systems for current and future generations. The need for the secure conservation of genetic resources collections ex situ is critical, due to rapid and extreme climatic changes which are threatening and reducing biodiversity in their natural environments. The International Potato Center (CIP) conserves one of the most complete and diverse genetic resources collections of potato, with more than 7500 accessions composed of 4900 cultivated potato and 2600 potato wild relative accessions. The clonal conservation of cultivated potato, principally landraces, through in vitro or field collections is indispensable to maintain fixed allelic states, yet it is costly and labor-intensive. Cryopreservation, the conservation of biological samples in liquid nitrogen (-196°C), is considered the most reliable and cost-efficient long-term ex-situ conservation method for clonal crops. Over the last decade, CIP has built one of the largest potato cryobanks worldwide, cyopreserving more than 4000 cultivated potato accessions which represents 84% of the total cultivated potato collection currently conserved at CIP. In approximately, four years the entire potato collection will be cryopreserved. The development of an applied, robust cryopreservation protocol for potato, serves as a model for other clonally maintained crop collections. The CIP cryobank designs experiments with a high number of genetically diverse genotypes (70-100 accessions, seven cultivated species), to obtain reliable results that can be extrapolated over the collection as genotypes can often respond variably to the same applied conditions. Unlike most published reports on cryopreservation of plants, these large-scale experiments on potato are unique as they examine the acclimatization process of in vitro plants prior to, as well as during cryopreservation on up to ten times the number of genotypes conventionally reported in the published literature. As a result, an operational cryopreservation protocol for potato has advanced that works well across diverse potato accessions, not only with reduced processing time and costs, but also with an increased average full-plant recovery rate from 58% to 73% (+LN) for routine cryopreservation. The present article describes the composition of CIP's cryobank, the cryopreservation protocol, methodology for the dynamic improvement of the operational protocol, as well as data collected on regeneration from long term cryopreserved potatoes.

Cryopreservation of Potato Shoot Tips for Long-Term Storage.

Cryopreservation is currently the only method which allows long-term conservation of living clonal plant material in the vapor or liquid phase of nitrogen (at -140 to -196 °C) allowing tissue to be viable for decades or perhaps centuries. Specifically, for species with recalcitrant seeds or requiring constant vegetative propagation, it is the method of choice for the long-term conservation of its genetic resources. The protocol described here is a modification of a previously developed plant vitrification solution 2 (PVS2)-droplet vitrification method of potato shoot tips, adapted from Musa species. Utilizing this protocol, the International Potato Center (CIP) has successfully stored in the cryobank more than 3000 cultivated potato accessions, belonging to seven species and nine different taxa [16], originating principally from ten countries in South and Central America. As part of CIP's quality management system, all vegetative material placed in cryo is routinely subsampled, thawed, and assessed to confirm that whole plantlets can be produced after storage in liquid nitrogen. Complete plant recovery rates of thawed shoot tips range from 20% to 100% (average rate: 60%). This chapter describes the complete set of steps from the routine procedure of cryopreserving potato shoot tips for long-term conservation.

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.

Cryopreservation of carnation (Dianthus caryophyllus L.) and other Dianthus species.

MAIN CONCLUSION: This paper reviews the cryopreservation of the ornamental, carnation (Dianthus caryophyllus L.), as an important method for the long-term preservation of this plant's germplasm. Carnation (Dianthus caryophyllus L.) is an important ornamental plant that is used as a potted plant as well as a cut flower. Important Dianthus germplasm would benefit from long-term strategies such as cryopreservation. Unlike the in vitro tissue culture literature of this ornamental, which has been studied in considerable detail, and with several genetic transformation protocols, surprisingly, the literature on its cryopreservation is still fairly scant, with barely two dozen or so studies, mostly having employed shoot tips. Early (< 2007) and more recent (2007-2020) cryopreservation techniques for carnation, including ultra-rapid cooling, encapsulation-vitrification, and encapsulation-dehydration, efficiently replaced programmed slow cooling processes used in early studies in the 1980s. Two large gaps (1997-2006, and 2016-2020) in which no carnation cryopreservation studies were published, requires future studies to cover new knowledge to fill gaps in information. Carnation cryopreservation research would benefit from testing a wide range of in vitro explants, new techniques such as the cryo-mesh, improved regeneration protocols for post-cryopreserved material, and the use of low-temperature storage as a mid- to long-term complementary germplasm storage strategy. This mini-review provides details of what has been achieved thus far and future objectives that could fortify cryopreservation research of this ornamental, as well as provide a robust long-term germplasm repository.

Cryopreservation of Brazilian green dwarf coconut plumules by droplet-vitrification / Criopreservação de coqueiro anão verde do Brasil de Jiqui por vitrificação em gotas

ABSTRACT: This study evaluated the effect of vitrification solutions and exposure time on the cryopreservation of Brazilian green dwarf coconut plumules (BGD) using the droplet vitrification technique. Explants were excised from BGD mature fruits from the Active Germplasm Bank of Embrapa Tabuleiros Costeiros, Sergipe, Brazil. Firstly, embryos were disinfected, and after excision, plumules were pre-cultivated for 72 hours in Y3 + 0.6 M sucrose + 2.2 g L-1 Gelrite® culture medium. Plumules were exposed to PVS2 and PVS3 solutions for 15 and 30 minutes and rapidly immersed in liquid nitrogen (-196 ºC). After cryopreservation, they were thawed in culture medium solution (Y3 + 1.2 M sucrose) and cultured in regeneration medium. The experimental design was completely randomized in a 2x2 factorial scheme (vitrification solutions per exposure times), with five replicates per treatment. Data were compared by the Tukey's test at 5% probability. Significant differences were observed in the callogenesis percentage for the solutions x exposure time interaction for non-cryopreserved cultures (-NL) and for exposure time after cryopreservation (+NL). PVS2 and PVS3 combined with 15 minutes of exposure promoted the highest callus formation (70 and 100%, respectively) in control cultures. The exposure time of 30 min, regardless of vitrification solution, resulted in 30% embryogenic callus formation after cryopreservation. These results contributed to the long-term conservation of coconut palm.

RESUMO: O objetivo desse estudo foi avaliar o efeito das soluções de vitrificação e do tempo de exposição na criopreservação de plúmulas de coqueiro anão verde do Brasil de Jiqui (BGD), pela técnica de vitrificação em gotas. Os explantes foram excisados de frutos maduros oriundos do Banco de Germoplasma Ativo de Embrapa Tabuleiros Costeiros, Sergipe, Brasil. Os embriões foram desinfestados e as plúmulas, após a excisão, pré-cultivadas durante 72 horas em meio de cultura Y3 suplementado com sacarose 0,6 e 2,2 g L-1 Gelrite®. As plúmulas foram expostas em soluções de PVS2 e PVS3 durante 15 e 30 minutos, e rapidamente imersas em nitrogênio líquido (-196 ºC). Após a criopreservação, foram descongeladas na solução de meio de cultura Y3 com 1,2 M de sacarose, e cultivadas em meio de regeneração. O delineamento experimental foi inteiramente casualizado em esquema fatorial 2x2 (soluções de vitrificação x tempos de exposição), com cinco repetições por tratamento. Os dados foram comparados pelo teste de Tukey à probabilidade de 5%. Observaram-se diferenças significativas na porcentagem de calogênese para a interação entre soluções e tempo de exposição para as culturas não criopreservadas (-NL), e para o tempo de exposição após a criopreservação (+NL). O PVS2 e o PVS3 combinados com 15 minutos promoveram a maior formação de calo (70 e 100%, respectivamente) nas culturas de controle. O tempo de exposição de 30 min, independente da solução de vitrificação, promoveu 30% da formação de calos embriogênicos após a criopreservação. Estes resultados contribuem para a conservação em longo prazo do coqueiro.

Anti-freezing-protein type III strongly influences the expression of relevant genes in cryopreserved potato shoot tips.

KEY MESSAGE: AFP improved cryopreservation efficiency of potato (Solanum tuberosum cv. Superior) by regulating transcript levels of CBF1 and DHN1. However, the optimal AFP concentration required for strong induction of the genes was dependent on the type of vitrification solution to which the AFP was added: This finding suggests that AFP increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Despite the availability of many studies reporting the benefits of anti-freeze protein III (AFP III) as a cryoprotectant, the role of AFP III in this process has not been well demonstrated using molecular analysis. In addition, AFP III has not been exploited in the cryopreservation of potato thus far. Therefore, we studied the effects of AFP III on the cryopreservation of potato (Solanum tuberosum cv. Superior). We found that CBF1 and DHN1 genes are low temperature-inducible in potato leaves (S. tuberosum cv. Superior). Transcript levels of these genes expressed in shoot tips cryopreserved with AFP III were higher than those of the controls. However, the optimal AFP III concentration required for strong induction of the genes was dependent on the type of cryoprotection solution to which the AFP III was added: 500 ng/mL worked best for PVS2, while 1500 ng/mL was optimal for LS. Interestingly, the involvement of AFP III in the cryoprotection solutions improved cryopreservation efficiency as compared to the control, and expression levels of the detected genes were associated with cryopreservation efficiency. This finding suggests that AFP III increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Therefore, we expect that our findings will lead to the successful application of AFP III as a potent cryoprotectant in the cryopreservation of rare and commercially important plant germplasms.

Ascorbate Peroxidase Activity of Aranda Broga Blue Orchid Protocorm-like Bodies (PLBs) In Response to PVS2 Cryopreservation Method.

Throughout the cryopreservation process, plants were exposed to a series of abiotic stresses such as desiccation and osmotic pressure due to highly concentrated vitrification solution. Abiotic stress stimulates the production of reactive oxygen species (ROS) which include hydrogen peroxide, superoxide radicals, and singlet oxygen. Higher production of ROS may lead to oxidative stress which contributes to the major injuries in cryopreserved explants. Antioxidant enzymes in plant such as ascorbate peroxidase (APX) can protect plants from cell damage by scavenging the free radicals. This study was determined based on APX enzyme activity of Aranda Broga Blue orchid's protocorm-like bodies (PLBs) in response to PVS2 (Plant Vitrification Solution 2) cryopreservation treatments at different stages. PLBs that were precultured at 0.25 M sucrose for 3 days were subjected to vitrification cryopreservation method. Results obtained showed that the highest APX activity was achieved at PVS2 cryoprotectant treatment prior liquid nitrogen (LN) storage. This phenomenon indicating that accumulation of osmotic and dehydrating stress throughout the cryopreservation treatment resulted in oxidative burst which in turn leads to higher APX activity in order to control the excess production of ROS. To conclude, PVS2 treatment was revealed as the most detrimental step throughout cryopreservation treatment. Thus, this research also suggested that exogenous antioxidant such as ascorbic acid can be added throughout cryopreservation procedure especially at PVS2 treatment in the future experiments to aid in regrowth of cryopreserved explants by reducing oxidative stress.

Cryopreservation the seeds of a Taiwanese terrestrial orchid, Bletilla formosana (Hayata) Schltr. by vitrification.

BACKGROUND: The cryopreservation of orchid seeds is an important conservation method, studies of the effects of cryopreservation on the seeds of wild orchids are scant. This investigation was to establish a method for the vitrification and cryopreservation of seeds of B. formosana that may be suitable for the long term storage of Taiwan native orchid germplasm for conservation purposes. RESULTS: The germination rate and morphological stability of seeds from spontaneous-dehiscent capsules of Bletilla formosana (Hayata) Schltr. were evaluated after cryopreservation by vitrification. The germination rates of cryopreserved seeds varied according to immersion time and the vitrification method used. Seeds that were dehydrated by immersion in loading solution (LS; 2.0 M glycerol, 0.4 M sucrose) for 10 min to 30 min then transferred to plant vitrification solution 2 (PVS2) for 30 min prior to freezing in liquid nitrogen (LN) showed significantly higher germination rates than seeds immersed in PVS2 only. The optimal immersion times were 10 min for LS and 30 min for PVS2, resulting in an in vitro germination rate of 91%. Germination was not observed for cryopreserved seeds that were dehydrated by immersion in LS only. Seed viabilities and germination rates did not vary significantly for cryostorage times from 10 minutes to 1 year. CONCLUSIONS: This study improve, an efficient protocol was established that maintained seed viability and enhanced the germination rates of seeds, compared with previously described cryopreservation methods, and the germinated seeds showed normal morphology of both vegetative and reproductive organs.

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique.

Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10-50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized.