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The objective of this study was to evaluate the efficiency of cryoprotective solution (PVS2) combined with phloroglucinol for the cryopreservation of seeds of two orchid species, Encyclia cordigera and Epidendrum ciliare. Seeds of Encyclia cordigera had 91.03% initial viability and 91.99% germination. The treatment of the seeds with PVS2 at 0 °C with 1% phloroglucinol for 60 min returned 93.79% viability and 91.01% germination after recovery from LN, consequently resulting in faster development of protocorms. For Epidendrum ciliare, seed viability was 85.65% and germination was 85.90%. Seed exposure to the PVS2 at 0 °C with 1% phloroglucinol for 180 min showed viability of 39.23% and germination of 37.88%. Despite lower germination, 78.90% of the protocorms reached stage P3 of development, when evaluated 45 days after sowing, not significantly different from the control 1, and showed normal development. These results indicate that PVS2 cryoprotective solution is efficient when combined with phloroglucinol for the cryopreservation and successful recovery of seeds of Encyclia cordigera and Epidendrum ciliare. The present study also indicates that response to cryopreservation and success of recovery after cold storage is species-specific and requires adjustments in exposure time to PVS2 at 0 °C prior to immersion in LN. Resumo O objetivo deste estudo foi avaliar a eficiência da solução crioprotetora (PVS2) combinada com floroglucinol para a criopreservação de sementes de duas espécies de orquídeas, Encyclia cordigera e Epidendrum ciliare. Sementes de Encyclia cordigera apresentaram viabilidade inicial de 91,03% e germinação de 91,99%. O tratamento das sementes com PVS2 a 0 °C com 1% de floroglucinol por 60 min promoveu a recuperação de 93,79% da viabilidade e 91,01% da germinação após a recuperação do nitrogênio líquido, resultando consequentemente no desenvolvimento mais rápido dos protocormos. Para Epidendrum ciliare, a viabilidade inicial das sementes foi de 85,65% e germinação de 85,90%. E quando suas sementes foram tratadas com solução PVS2 a 0 °C com 1% floroglucinol por 180 min atingiram viabilidade de 39,23% e germinação de 37,88%. Apesar de observada menor porcentagem de germinação para o referido tratamento, 78,90% dos protocormos atingiram estágio de desenvolvimento P3, quando avaliados 45 dias após a semeadura, não apresentando diferença significativa comparada ao controle 1, e apresentando protocormos com desenvolvimento normal. Esses resultados indicam que a utilização da solução crioprotetora de PVS2 combinado ao floroglucinol 1% foi eficiente na recuperação de sementes criopreservadas de Encyclia cordigera e Epidendrum ciliare. O presente estudo também indica que a resposta à criopreservação após o armazenamento a frio é específica da espécie e requer ajustes no tempo de exposição ao PVS2 a 0 °C antes da imersão em nitrogênio líquido.

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 by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots.

EnglishThis 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. portuguesO objetivo desse estudo foi avaliar o efeito das solucoes de vitrificacao e do tempo de exposicao na criopreservacao de plumulas de coqueiro anao verde do Brasil de Jiqui (BGD), pela tecnica de vitrificacao em gotas. Os explantes foram excisados de frutos maduros oriundos do Banco de Germoplasma Ativo de Embrapa Tabuleiros Costeiros, Sergipe, Brasil. Os embrioes foram desinfestados e as plumulas, apos a excisao, pre-cultivadas durante 72 horas em meio de cultura Y3 suplementado com sacarose 0,6 e 2,2 g L-1 Gelrite®. As plumulas foram expostas em solucoes de PVS2 e PVS3 durante 15 e 30 minutos, e rapidamente imersas em nitrogenio liquido (-196 oC). Apos a criopreservacao, foram descongeladas na solucao de meio de cultura Y3 com 1,2 M de sacarose, e cultivadas em meio de regeneracao. O delineamento experimental foi inteiramente casualizado em esquema fatorial 2x2 (solucoes de vitrificacao x tempos de exposicao), com cinco repeticoes por tratamento. Os dados foram comparados pelo teste de Tukey a probabilidade de 5%. Observaram-se diferencas significativas na porcentagem de calogenese para a interacao entre solucoes e tempo de exposicao para as culturas nao criopreservadas (-NL), e para o tempo de exposicao apos a criopreservacao (+NL). O PVS2 e o PVS3 combinados com 15 minutos promoveram a maior formacao de calo (70 e 100%, respectivamente) nas culturas de controle. O tempo de exposicao de 30 min, independente da solucao de vitrificacao, promoveu 30% da formacao de calos embriogenicos apos a criopreservacao. Estes resultados contribuem para a conservacao em longo prazo do coqueiro.

Common cypress (Cupressus sempervirens L.) is one of the most widespread species in the Mediterranean area. It has been traditionally cultivated for its ornamental value, becoming a typical feature of urban and rural landscapes, and high timber quality. In the last 30 years, cypress has been subjected to important breeding programmes, aimed to select clones tolerant to the widespread canker, caused by the pathogenic fungus Seiridium cardinale, leading to various patented varieties today available on the market, as well as for genotypes producing null or low amount of allergenic pollen. Somatic embryogenesis is a suitable in vitro regeneration method for fast cloning of conifer trees, and the cryopreservation of embryogenic callus is a significant tool for the safe long-term conservation of valuable cell lines. Recently, a complete protocol for the production of cypress plants from somatic embryogenesis was developed for the patented clone ‘Mediterraneo’. Here, the coupling of somatic embryogenesis and cryopreservation may offer a superior tool to propagate and maintain selected genotypes of cypress by overcoming repetitive subculturing of selected embryogenic callus lines. For the above, this study aimed to compare different cryopreservation techniques (PVS2-based vitrification and slow cooling) with the ‘Mediterraneo’ embryogenic callus line. Best results were obtained after the optimization of a slow cooling procedure, based on the 30-min treatment of embryogenic masses with a cryoprotective solution containing 180 g l-1 sucrose and 7.5% DMSO, followed by the reduction of the temperature at a rate of -1 °C min-1 up to -40 °C and the subsequent immersion in liquid nitrogen (“two-step freezing”).

Dammar (Agathis damara) seeds categorized as an intermediate seeds since their viability tend to decrease when subjected to storage more than 2 weeks conventionally. Storage period of seeds could be prolonged when the seeds cryopreserved in liquid nitrogen since the metabolism of the cells could be minimized without loss of viability. The objective of the study was to identify suitable vitritification method for dammar storage seeds. Seed water content was decreased gradually from initial water content (28.48% as a control) using desiccators and vacuum method. Vitrification solution (PVS2) containing glycerol 30%, ethylene glycol 15% and dimethylsulfoxide (DMSO) 15% in 0.4M sucrose solution was used as a cryoprotectant of peeled or unpeeled dammar seeds during freezing process. The samples were soaked in PVS2 for 1 hour followed by exposure to liquid nitrogen in cryotube for 1 hour. The samples were then thawed in water bath at 28°C for 1 hour prior to germination in IPB-78 germinator with UDK and UKdDp germination methods. Results showed that the highest viability of cryopreserved dammar seeds (22.32% moisture content) was 100% obtained from those germinated with UKdDp method. A negative effect of cryoprotectant was occurred in both peeled and unpeeled seeds cryopreserved for 1 hour. However, it was effective for seeds cryopreserved for 4 weeks which indicate the possibility to preserve for a longer period in the future. © 2006 Jurusan Biologi FMIPA UNS SurakartaKey words: dammar, Agathis damara, seeds, PVS2, crypreservation.

Axillary buds from greenhouse-grown plants of Rosa chinensis 'Old Blush' were successfully used to establish cryopreservation protocols using both droplet-vitrification and encapsulation-dehydration methods. In droplet vitrification, regrowth occurred after exposure to liquid nitrogen even without pre-culture in the loading solution (LS) before immersion in the plant vitrification solution 2 (PVS2). However, a 20-80 min LS step followed by a short immersion in PVS2 for 3 or 15 min, at 0 A degrees C gave the best regrowth rates (82-86 %). In encapsulation dehydration, the level of dehydration significantly influenced shoot regrowth. The best regrowth rate, 60 %, was obtained at a bead water content of 0.35 g water per g dry weight. These results demonstrate the possibility of using greenhouse plants of rose for cryopreservation by droplet vitrification and encapsulation dehydration.

The objective of this work was to evaluate the phytotoxicity of a plant vitrification solution (PVS2), and the survival of shoot tips of the sugarcane variety SP716949, after cryopreservation by droplet-vitrification. Shoot tips were precultured for 24 hours in MS medium containing 0.3 mol L-1 sucrose, and exposed to PVS2 for 0, 20 or 30 min. Shoot tips were then immersed in liquid nitrogen. Thawing was fast in concentrated sucrose solution (1.2 mol L-1). PVS2 is a nontoxic to shoot tips, which in turn are sensitive to liquid nitrogen. The best results occurred when shoot tips were maintained for up to 20 min in PVS2 solution, before freezing, with 20% survival.

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.

In order to guarantee safe, long-term conservation of the Agrocampus Ouest - INHP Pelargonium collection, apex cryopreservation studies have been undertaken. An encapsulation-dehydration procedure has been first developed. More recently, studies were undertaken in order to adapt the droplet-vitrification procedure to this genus. For both procedures, protocols were optimized using P. × peltatum 'Balcon Lilas' as model accession. Survival and plant regeneration were obtained with the two newly determined protocols. However, droplet-vitrification gave higher and more reproducible regeneration results. To confirm these results, we carried out experiments in order to compare both protocols applying them to four accessions. Droplet-vitrification gave always better results than encapsulation-dehydration, and permitted to obtain shoot regrowth for each accession. To compare the two protocols, a histo-cytological study was performed on P. × peltatum 'Balcon Lilas' apices. Samples were fixed after each step of both protocols in order to observe the modifications at the cellular level. Important differences were noted concerning starch accumulation and nucleus aspect. The droplet-vitrification was then tested on 28 genotypes representative of the diversity of the genus. Apex survival was obtained for each accession out of the 28 tested. An average of 65% survival rate was obtained ranging from 14.8% for P. × fragans to 90% for P. capitatum and P. × hortorum 'Neurot'. Plants were regenerated for all genotypes, except P. × peltatum 'Papa Crousse'.

Advanced cryopreservation techniques can provide cheap and safe back-ups to traditional ex situ conservation approaches of fruit germplasm, including Citrus species. At the ultra-low temperature of liquid nitrogen (-196°C), plant cells, tissues and organs can be safely stored for theoretically unlimited time. Among the innovative cryoprocedures, storage of synthetic seeds (i.e., encapsulated in vitroderived propagules) in liquid nitrogen has repeatedly proven to be one of the most effective. The technique is a multi-step process, including (i) pre-culturing of the encapsulated explants (generally shoot tips or nodal segments) in sucrose-containing medium, (ii) removal of the freezable water from the cells by desiccation over silica gel or by treatment with highly-concentrated cryoprotectant solutions (such as PVS2), (iii) direct immersion of synthetic seeds in liquid nitrogen, (iv) thawing and (v) conversion of synthetic seeds into plantlets. As a preliminary step towards the optimization of a cryopreservation protocol for 'Carrizo' citrange [Citrus sinensis (L.) Osbeck x Poncirus trifoliata L. Raf.] synthetic seeds, this study investigated the effect on moisture content and regrowth rate of different pre-freezing treatments, i.e., (i) the exposure to increasing sucrose concentrations (0.1, 0.3 and 0.7 M, each one for 24 h), (ii) the desiccation (from one to 7 h), and (iii) the treatment with PVS2 (90 or 180 min, at 0°C). Best result was achieved when sucrose preculture was followed by a 5-hour exposure to silica gel; this treatment induced a consistent reduction of synthetic seed moisture content (MC) (from the initial 82 up to 23%), while the regrowth rate was affected to more limited extent (from 62 to 48%).