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 chayote (Sechium edule JACQ. SW.) zygotic embryos and shoot-tips from in vitro plantlets

This paper presents the development of cryopreservation protocols for zygotic embryos and apices of chayote (Sechium edule Jacq. Sw.), a tropical plant species with recalcitrant seeds. Zygotic embryos of two cultivars, Ccocro negro (CN) and Claudio (Cl) could withstand cryopreservation, with survival percentages of 10 and 30 %, after desiccation to 23 and 19 % moisture content (fresh weight basis), respectively. Apices sampled on in vitro plantlets of cultivars Cl, 13 and JM were successfully cryopreserved using a vitrification technique. Optimal conditions included the culture of mother-plants for 22 days on medium containing 0.3 M sucrose, culture of excised apices on the same medium for 1 day, loading of apices for 20 min with 2M glycerol + 0.4M glycerol, treatment with a series of diluted PVS2 solution (60 % PVS2 followed by 80 % PVS2 solution for 15 min (cultivar Cocoro Blanco [CB]) or 30 min (cultivars CN and Cl) at each concentration), rapid freezing and thawing, washing of shoot-tips with a 1.2 M sucrose solution, followed by recovery on media with progressively decreasing sucrose concentrations until the standard concentration of 0.1 M was reached. The highest survival percentages achieved ranged between 17 and 38 %, depending on the cultivar.     

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 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 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 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 black iris (Iris nigricans) somatic embryos by encapsulation-dehydration

Somatic embryos of black iris (Iris nigricans) were cryopreserved using encapsulation-dehydration. Embryos size of 2-4 mm gave the highest survival after cryopreservation. Preculturing embryos on medium containing 0.75 M sucrose for 3 d at 22 degree C, then at 30 degree C for 1 d ensured maximum survival (60%). Viable embryos were brown in color after cryopreservation and during early recovery while dead ones where light brown or creamy. The first sign of regrowth was noted after 15 d. The final regrowth percentage of living embryo was 90% after 35 d. Only 10% of the embryos in all treatments showed secondary embryogenesis. Direct sowing in vivo of cryopreserved embryos was not successful in achieving germination.     

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 '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 chick islets

The avian endocrine pancreas shares some similarities with those of mammals. Previously we have reported a technique of isolation of B islets from chick pancreas and also demonstrated their possible use for hypoglycemic drug screening as efficiently as mammalian islets. Here we describe a novel cryopreservative medium for the cryopreservation of chick islets. Isolated chick islets were suspended in a cryo medium consisting of Dulbecco's modified Minimum Essential Medium: Ham's F12 (1:1), Fetal Bovine Serum (20%), Dimethylsulfoxide (10%) with different concentrations (50 microg/ml to 500 microg/ml) of riboflavin or nicotinamide. The viability of revived islets after three and half months was checked by trypan blue dye exclusion and functionality was assessed by insulin secretion in response to glucose challenge. The maximum recovery of viable islets and insulin secretion was obtained in response to glucose challenge at 250 microg/ml concentration of Riboflavin or Nicotinamide. This is a first report on cryopreservation of chick islets exhibiting cryoprotective role of water soluble vitamins without vitamin C.     

Cryopreservation of Pelargonium apices by droplet-vitrification

The droplet-vitrification method was adapted to Pelargonium apices by optimizing the duration of the loading solution (LS) as well as the plant vitrification solution 2 (PVS2). The excised apices were dehydrated in two steps (20 min in LS and 15 min in PVS2) and then immersed directly in liquid nitrogen (LN). After thawing and unloading in the recovery solution at room temperature for 15 min, apices were plated onto semi-solid Murashige and Skoog medium. This simple protocol without any pretreatment was successfully applied to eight cultivars with a survival level ranging between 55.6 - 96.2 percent and a regrowth level between 9.1 and 70.6 percent. These results prove the feasibility of the long-term storage of Pelargonium germplasm through cryopreservation.     

Cryopreservation of Arabidopsis thaliana shoot tips

Development of a successful shoot tip cryopreservation method for Arabidopsis thaliana L. will enable researchers to use molecular tools to study processes important for successful cryopreservation in this model organism. We demonstrate that Arabidopsis can be successfully cryopreserved using either plant vitrification solution 2 (PVS2) or plant vitrification solution 3 (PVS3) as cryoprotectants prior to rapidly cooling shoot tips in liquid nitrogen (LN). Shoot tip regrowth after PVS2 cryoprotectant treatment was improved after cold acclimation treatments of 8 or 18 days. All of the shoots tips regrew after LN exposure when cryoprotected with PVS3 for 60 min at 22 degree C. In addition, shoot tips could be cryopreserved using a two-step cooling procedure with PGD (polyethylene glycol-glucose-dimethyl sulfoxide) as a cryoprotectant. The high levels of shoot formation after LN exposure of Arabidopsis shoot tips makes this a desirable system in which molecular tools can be used to examine how alterations in biochemical, metabolic and developmental processes affect regrowth after cryoprotective treatments.     

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 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.     

Curvelet-based palm vein biometric recognition

<正>A novel personal recognition system utilizing palm vein patterns and a novel technique to analyze these vein patterns is presented.The technique utilizes the curvelet transform to extract features from vein patterns to facilitate recognition.This technique provides optimally sparse representations of objects along the edges.Principal component analysis(PCA) is applied on curvelet-decomposed images for dimensionality reduction.A simple distance-based classifier,such as the nearest-neighbor(NN) classifier,is employed. The experiments are performed using our palm vein database.Experimental results show that the algorithm reaches a recognition accuracy of 99.6%on the database of 500 distinct subjects.     

Cryopreservation of embryonic axes of selected amaryllid species

A study on cryopreservation of excised embryonic axes of fifteen species of the amaryllidaceae is reported. Embryonic axes that after flash-drying had a water content in the range 0.4 to 0.1 g/g and survival greater than 60% were selected for cryopreservation procedures. The highest post-thaw viabilities (roots and shoots produced) across all species were recorded for embryonic axes subjected to rapid rather than slow cooling. With rapid cooling and no cryoprotection, the highest post-thaw viabilities for the fifteen species investigated was 0% in one species; ranged between 10 and 35% for nine species; and between 45 and 55% for five species. With cryoprotection and rapid cooling the highest post-thaw viabilities for these fifteen species was 0% for one species; ranged between 15 and 35% for six species; and between 40 and 75% for eight species. The highest post-thaw survival in ten out of fifteen species was obtained for axes dried to between 0.24 +/- 0.06 and 0.14 +/- 0.08 g/g(-1) (and rapidly cooled). With only one exception (Strumaria discifera; 45%), post-thaw survival after slow cooling ranged between 10 and 30%. Survival after vitrification plus slow cooling was achieved for seven species but was never higher than post-thaw survival in non-cryoprotected, rapidly cooled axes. The results suggest that species within the same family can exhibit commonalities in terms of amenability to cryopreservation techniques but for maximum success, axis water content and cooling rate particularly, must be optimised for each species in the family independently.     

Cryopreservation of Prunus avium L. embryogenic tissues

Embryogenic tissues from wild cherry (Prunus avium L.) were successfully cryopreserved by using a one-step freezing procedure. Cryoprotection consisted of a pretreatment on solid medium with increasing sucrose concentrations (0.25 M for 1 day, 0.5 M for 1 day, 0.75 M for 2 days, and 1.0 M for 3 days), followed by air desiccation to about 20 percent moisture content (fresh weight basis). This method was compared with a pretreatment on solid medium containing 5 percent DMSO and 2 percent proline, followed by immersion in a modified PVS2 cryoprotective solution. Pretreatment on solid medium with increasing concentrations of sucrose led to regrowth of frozen embryogenic tissues, and after 6 weeks of culture, growth was comparable to that of non-dehydrated and non-frozen tissues. By contrast, no regrowth was observed when embryogenic tissues were submitted to the solid/liquid pretreatment with DMSO/proline and a modified PVS2 solution.     

Cryopreservation of Quercus robur L. embryogenic calli

Embryogenic calli of pedunculate oak (Quercus robur L.) were cryopreserved using direct immersion in liquid nitrogen. The pretreatment consisted of culture on a solid medium with increasing sucrose concentrations (0.25 M for 1 day, 0.5 M for 1 day, 0.75 M for 2 days, and 1.0 M for 3 days), followed by air desiccation of embryogenic calli to 17.3 percent (fresh weight basis). This method of cryoprotection was compared to a liquid cryoprotection treatment using high concentrations of sucrose solutions, followed by glycerol solutions. Regrowth of frozen tissue pretreated on a solid medium was significantly higher than those pretreated in the liquid solutions.