Cryopreservation of 'Nabali' olive (Olea europea l.) somatic embryos by encapsulation-dehydration and encapsulation-vitrification

Olive (Olea europea L.) somatic embryos were successfully cryopreserved using encapsulation-dehydration and encapsulation-vitrification. In the encapsulation-dehydration procedure, a maximum of 48% embryo survival was obtained when bead moisture content was decreased to 21.1% after 4 h dehydration. Preculture of embryos for 4 d in medium containing 0.75 to 1.25 M sucrose produced higher (40 to 34 %, respectively) regrowth after cryopreservation using encapsulation-dehydration procedure. Dehydration of beads for 3 h in PVS2 ensured higher survival (64%) of encapsulated-vitrified and cryopreserved (EVN) somatic embryos. Thermal treatment of embryogenic callus for 1 d at 30 degree C was very effective to increase survival of encapsulated-dehydrated and cryopreserved (EDN) (58%) and EVN (68%) embryos. Plantlets produced from control and cryopreserved embryos were phenotypically similar.     

Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-dehydration technique

Cryopreservation of ovules and somatic embryos from several genotypes of citrus was achieved using the encapsulation-dehydration technique. Survival of cryopreserved ovules was occasional and erratic after different pregrowth conditions in liquid medium with 0.75M, 1M or up to 1.25M sucrose. An efficient cryopreservation protocol was established for somatic embryos derived from two embryogenic sources (ovules and cut thin layer explants from stigma, style and ovaries). High survival rates (75-100%) were consistently obtained after 1 day pregrowth in 0.75M sucrose, desiccation down to 20-25% moisture content in the beads and direct immersion in liquid nitrogen. The histological study showed that embryos subjected to the encapsulation-dehydration, accumulated high sucrose levels which appear to ensure the recovery of the whole embryo after cryopreservation.     

Cryopreservation of sugarcane somatic embryos

In this paper, we compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos. Viability of somatic embryos was evaluated by measuring electrolyte leakage and by regrowth on recovery medium. Droplet-vitrification was the most efficient technique. Optimal conditions included loading with a solution containing 1.5 M glycerol and 0.3 M sucrose for 30 min at 25 degree C, treatment with the PVS2 solution for 20-40 min at 0 degree C followed by rapid immersion in liquid nitrogen of clumps of somatic embryos placed in microdroplets of cryoprotectant solution. Under such conditions, viability of cryopreserved somatic embryos reached 55 percent.     

Cryopreservation of Vanda coerulea protocorms by encapsulation-dehydration

Protocorms of Vanda coerulea were successfully cryopreserved by encapsulation-dehydration in combination with a loading solution. Protocorms were selected 70 days after sowing seeds harvested from 7-month-old fruits. After encapsulation in an alginate matrix composed of 2 percent Na-alginate, 2 M glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.7 M sucrose on a shaker (110 rpm) at 25 +/- 3 degree C for 20 h. Encapsulated protocorms were then dehydrated in a sterile air-flow in a laminar air-flow cabinet at 25 +/- 3 degree C for 0-10 h and then directly plunged into liquid nitrogen for 1 d. After thawing at 40 degree C for 2 min, cryopreserved beads were cultured on modified VW agar medium for regrowth. The highest regrowth of 40 percent was observed with cryopreserved beads with 35 percent water content after 8 h dehydration. No morphological variation was detected between non-cryopreserved and cryopreserved plantlets, and ploidy level was unchanged as a result of cryopreservation.     

Cryopreservation of somatic embryos of paradise tree (Melia azedarach L.)

In paradise tree (Melia azedarach L.), immature zygotic embryos sampled from immature fruits are the starting material for the production of somatic embryos. These somatic embryos are employed for freezing experiments. Immature fruits could be stored at 25 degrees C for up to 80 days without impairing the embryogenic potential of zygotic embryos, which represents a four-fold increase in immature fruit storage duration, compared with previous studies. Among the three cryopreservation techniques tested for freezing paradise tree somatic embryos, namely desiccation, encapsulation-dehydration and pregrowth-dehydration, only encapsulation-dehydration and pregrowth-dehydration led to successful results. The optimal protocol was the following: i) somatic embryos (encapsulated or not) pretreated in liquid Murashige & Skoog medium with daily increasing sucrose concentration (0.5 M/0.75 M/1.0 M); ii) dehydrated with silica gel to 21 - 26% moisture content (fresh weight basis), for encapsulation-dehydration, or to 19% moisture content, for pregrowth-dehydration; iii) frozen at 1 degree C/min from 20 degrees C to -30 degrees C with a programmable freezing apparatus; iv) rapid immersion in liquid nitrogen. The highest recovery achieved was 36% with encapsulation-dehydration and 30% with pregrowth-dehydration. Regrowth of frozen embryos was direct in most cases, as secondary embryogenesis originating from the root pole was observed on only around 10% of cryopreserved somatic embryos. Plants recovered from cryopreserved embryos presented the same phenotypic traits as non-frozen control plants.     

Cryopreservation of Citrus madurensis embryonic axes encapsulation-dehydration

In this paper, we demonstrate that C. madurensis embryonic axes can withstand cryopreservation using the encapsulation-dehydration technique. Up to 57.5 % survival was achieved using a standard encapsulation-dehydration protocol, which included pregrowth of encapsulated axes for 16 h in medium containing 0.8 M sucrose + 1 M glycerol, desiccation of beads to around 30 % moisture content (fresh weight basis) followed by rapid freezing. A slightly higher survival percentage (65 %) was obtained using a modified encapsulation-dehydration protocol, which included pretreatment of axes with 2 M glycerol + 0.6 M sucrose for 1 h, concomitantly with their encapsulation in 3 % calcium alginate beads, followed by desiccation of the beads to around 30 % moisture content.     

Cryopreservation of seeds and in vitro-cultured protocorms of Oncidium bifolium sims. (Orchidaceae) by encapsulation-dehydration

Encapsulation-dehydration was employed for cryopreserving seeds and in vitro-cultured protocorms of Oncidium bifolium. Freshly harvested seeds, 120 days after pollination, were encapsulated in beads containing 1/2 MS medium with 3% sucrose and 3% calcium alginate and subsequently pretreated in agitated (80 rpm) liquid medium supplemented with 0.15 M sucrose (24 h) followed by 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h). The beads with seeds were dehydrated with silica gel for 5 h to 19.2% moisture content and immersed in liquid nitrogen for 1 h, thawed at 30 degrees C for 2 min, post-treated using the same series of liquid media [0.5 M sucrose (24 h), 0.25 M sucrose (48 h), 0.15 M sucrose (24 h)], and recultured on 1/2 MS medium with 0.1M sucrose and 0.7% percent agar. As much as 4.8% of the cryopreserved seeds produced complete plants. In-vitro cultured protocorms were successfully cryopreserved following the same procedure, allowing 11.3% of them to produce plants.     

Cryopreservation of shoot tips of blackberry and raspberry by encapsulation-dehydration and vitrification

Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm. Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to c. 20 percent moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5 percent dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25 degree C , followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60 to 100 percent using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71 percent average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.     

Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut

This work describes experiments demonstrating the feasibility of long-term conservation of Castanea sativa germplasm through cryopreservation of embryonic axes or somatic embryo clumps. Between 93 % and 100 % of excised embryonic axes of recalcitrant chestnut seeds survived storage in liquid nitrogen (LN) following desiccation in a laminar flow cabinet to moisture contents of 20-24 % (on a fresh weight basis), and some 63 % subsequently developed as whole plants. Desiccation to moisture contents less than 19 % produced damage resulting in loss of organized plant development after cryostorage, allowing only root growth. When 6-8 mg clumps of globular or heart-shaped somatic embryos were precultured for 7 days on high-sucrose medium and then desiccated to a moisture content of 25 % before storage in LN, the embryogenesis resumption level after thawing was 33 %. When the embryo clumps were precultured for 3 days on high-sucrose medium followed by 60 min application of PVS2 vitrification solution before cryostorage, the post-storage embryogenesis resumption level was 68 %.     

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.     

Cryopreservation of plant germplasm using the encapsulation-dehydration technique: review and case study on sugarcane

Encapsulation-dehydration is a cryopreservation technique based on the technology developed for producing synthetic seeds, i.e. the encapsulation of explants in calcium alginate beads. Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before freezing. Encapsulating the explants allows the subsequent application of very drastic treatments including preculture with high sucrose concentrations and desiccation to low moisture contents which would be highly damaging or lethal to non-encapsulated samples. An encapsulation-dehydration protocol comprises the following steps: pretreatment, encapsulation, preculture, desiccation, freezing and storage, thawing and regrowth. Encapsulation-dehydration has been applied to around 40 different plant species. The optimization of the successive steps of the encapsulation-dehydration protocol is illustrated for sugarcane apices.     

Cryopreservation of the late stage embryos of Spodoptera exigua (Lepidoptera: Noctuidae)

Genetic devolution, genetic drift and contamination are all threats to maintain germplasm stability during mass rearing of many insects. Cryopreservation of beet armworm (Spodoptera exigua) embryos was studied to provide information to improve mass rearing. A series of experiments was conducted on late-stage embryos (45-48 h at 27 degree C) of the beet armyworm, which included evaluation of cryoprotectants (CPAs), their toxicity and glass-forming tendency and optimization of experimental procedures. The results showed that ethylene glycol (EG) was the best CPA with comparatively low toxicity compared to the other six CPAs tested (methanol, 1,3-propanediol, glycerol, 2-amino-1-ethanol, 3-amino-1-propanol 3-methoxy-1 and 2-propanediol). The highest hatching rate of 8.8 degree was attained after freezing with a 3-step loading procedure and a 1-step unloading procedure, but the hatched larvae from frozen-thawed embryos did not actively feed and could not develop to a later stage. This was attributed to injuries from freezing in late stage embryos of S. exigua which had formed midguts.     

Cryopreservation of peach palm zygotic embryos

Cryopreservation is a safe and cost-effective option for long-term germplasm conservation of non-orthodox seed species, such as peach palm (Bactris gasipaes). The objective of the present study was to establish a cryopreservation protocol for peach palm zygotic embryos based on the encapsulation-dehydration technique. After excision, zygotic embryos were encapsulated with 3 percent sodium alginate plus 2 M glycerol and 0.4 M sucrose, and pre-treated or not with 1 M sucrose during 24 h, followed by air-drying. Fresh weight water contents of beads decreased from 83 percent and 87 percent to 18 percent and 20 percent for pre-treated or non-pretreated beads, respectively, after 4 h of dehydration. Sucrose pre-treatment at 1 M caused lower zygotic embryo germination and plantlet height in contrast to non-treated beads. All the variables were statistically influenced by dehydration time. Optimal conditions for recovery of cryopreserved zygotic embryos include encapsulation and dehydration for 4 h in a forced air cabinet to 20 percent water content, followed by rapid freezing in liquid nitrogen (-196 degree C) and rapid thawing at 45 degree C. In these conditions 29 percent of the zygotic embryos germinated in vitro. However, plantlets obtained from dehydrated zygotic embryos had stunted haustoria and lower heights. Histological analysis showed that haustorium cells were large, vacuolated, with few protein bodies. In contrast, small cells with high nucleus:cytoplasm ratio formed the shoot apical meristem of the embryos, which were the cell types with favorable characteristics for survival after exposure to liquid nitrogen. Plantlets were successfully acclimatized and showed 41+/-9 percent and 88+/-4 percent survival levels after 12 weeks of acclimatization from cryopreserved and non-cryopreserved treatments, respectively.     

Cryopreservation of Mediterranean fruit fly embryos

In this paper we present a procedure to cryopreserve the embryos of a tephritid, the Mediterranean fruit fly (Ceratitis capitata), by vitrification. Developmental stages between 24 and 32 hours after oviposition were examined for tolerance to cryopreservation. Embryos, 27-hr-old and incubated at 29 C, were found to be at the most suitable stage for treatment. Effects of the previtrification steps of our protocol, dechorionation, permeabilization, cryoprotectant loading, and dehydration, on survival to hatching were also assessed. Dechorionation did not affect viability, while isopropanol and a hexane treatment used in the permeabilization step of the protocol reduced hatching by about 15%. This reduction was dependent on the amount of isopropyl alcohol carried over into the hexane rinse. The remaining previtrification steps reduced hatching by an additional 10%. After optimization of the procedure, normalized hatching was 44% after vitrification in liquid nitrogen vapor followed by storage under liquid nitrogen for a test period of 7 days. Post cryopreservation larval diets containing wheat bran, corncob grits, or agar as the base were examined for survival to pupation and emergence. A yield of 34% egg to adult emergence was obtained when the agar-based diet was used for rearing larvae that had experienced cryopreservation during the embryonic stage.     

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei: Istiophoridae)

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation, mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).     

Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification

Cryopreservation of fish oocytes is challenging because these oocytes have low membrane permeability to water and cryoprotectant and are highly chilling sensitive. Vitrification is considered to be a promising approach for their cryopreservation as it involves rapid freezing and thawing of the oocytes and therefore minimising the chilling injury. In the present study, vitrification properties and the toxicity of a range of vitrification solutions containing different concentrations of Me2SO, methanol, propylene glycol and ethylene glycol were investigated. Two different base media and vitrification methods were compared. The effect of different post-thaw dilution solutions together with incubation periods on oocyte viability were also investigated. Stage III zebrafish oocytes were equilibrated in increasing concentrations of cryoprotectants for 30 min in 3 steps. Oocytes were thawed rapidly in a water bath and cryoprotectants were removed in 4 steps. Oocyte viability was assessed using trypan blue staining. The results showed that vitrification solutions V3 and V4 in KCl buffer had low toxicity and vitrified well. The survivals of oocytes after stepwise dilution using solutions containing permeable cryoprotectants were significant higher than those diluted in 0.5M glucose, and the use of CVA65 vitrification system improved oocyte survival when compared with plastic straws after 30 min at 22 degrees C post-thawing. Cryopreservation of zebrafish oocytes by vitrification is reported here for the first time, although oocyte survivals after cryopreservation assessed by trypan blue staining were relatively high shortly after thawing, they became swollen and translucent after incubation in KCl buffer. Further studies are needed to optimise the post-thaw culturing conditions.     

Cryopreservation of black chokeberry in vitro shoot apices

In vitro shoot apices of black chokeberry (Aronia melanocarpa Elliott) were successfully cryopreserved utilizing three methods, namely vitrification, encapsulation-vitrification and encapsulation-dehydration. Encapsulation-dehydration and encapsulation-vitrification, however, seem preferable to vitrification, since the highest respective survival levels of apices (71.1 +/- 2.2 percent and 77.8 +/- 4.4 percent) by both methods were higher than that (60.0 +/- 3.9 percent) by vitrification. In encapsulation-dehydration, the highest survival was achieved when the moisture content of beads was reduced to 19 percent by drying with silica gel for 6 h. In the present study, it was shown that adding 1.0 M glycerol to beads and loading solution during encapsulation-dehydration resulted in high survival (91.7 - 95.0 percent) regardless of lines and polyploids of black chokeberry.     

Developing cryopreservation for Picea sitchensis (sitka spruce) somatic embryos: a comparison of vitrification protocols

Two vitrification-based cryopreservation protocols, encapsulation/dehydration and PVS2 were applied to Stage 2 (globular) and Stage 4 (torpedo) somatic embryos (SE) from Picea sitchensis. Two recovery responses: partially differentiated embryogenic suspensor masses (ESM) and dedifferentiated non-embryogenic masses (NEM) were observed following exposure to LN. All genotypes tested, proliferated NEM, approximately 10 to 100% of the total SE cryopreserved. A General Linear Model applied to NEM recovery data demonstrated several different factors (developmental state and genotype, treatment, culture age) interacted at a significant level (P less than 0.05) to influence proliferation. One genotype was capable of proliferating ESM after cryopreservation using encapsulation-dehydration, this response was achieved for Stage 4 embryos derived from the youngest ESM tissue.     

Cryopreservation of the australian species Macropidia fuliginosa (Haemodoraceae) by vitrification

Somatic embryos were used to develop a cryopreservation protocol for Macropidia fuliginosa, a commercially-important species endemic to the south-west of Western Australia. Somatic embryos were allowed to develop from embryogenic callus for three weeks on an kinetin medium prior to processing. These were transferred and cultured on a agar solidified basal medium supplemented with 0 to 0.6 M sorbitol for 2 d prior to incubation in Plant Vitrification Solution Two (PVS2). Following this, embryos were then washed in 1 M sucrose solution (treated controls) or cooled in liquid nitrogen (LN). Cooled embryos were then warmed and washed in sucrose solution. Highest survival for cooled treatments (67.3%) was achieved by preculture with 0.4 M sorbitol, then incubation in PVS2. Further experimentation varying pre-culture duration (2 or 3 d) and incubation on either glycerol (0.8 M) or sorbitol (0.4 M) indicated that very high survival (90.6%) of embryos was achievable by adopting a 2 d preculture period on 0.8 M glycerol. The phenotype and growth rates of plants obtained using this protocol were similar to those of parent plants. This optimised procedure was then applied to tissue culture-derived shoot apices of the same clone also resulting in a high survival rate (84.4%).     

Apex cryopreservation of several strawberry genotypes by two encapsulation-dehydration methods

This paper presents results from a study to develop cryopreservation procedures for apices of several strawberry genotypes. Five Fragaria x ananassa Duch. cultivars and two wild species (F. chiloensis and F. virginiana) have been screened using the encapsulation-dehydration method and/or a protocol which compromises vitrification and encapsulation-dehydration. Apices were encapsulated in an alginate gel, precultured on media containing high levels of sucrose (0.8 M, conventional protocol), or a combination of 0.4 M sucrose and 2 M glycerol. Recovery rates varied among genotypes (23-63%). The latter method reduced considerably the time needed for the cryogenic procedure by eliminating the pre-treatment with 0.8 M sucrose for 19 h prior to dehydration, as required by the conventional procedure.