Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification

This paper investigates the effect of the origin and size of the explants employed and of the preconditioning (cold acclimation, preculture) and loading treatments on survival and regeneration of cryopreserved garlic shoot apices using vitrification with the PVS3 vitrification solution. Both the origin and size of explants had a significant effect on regeneration of cryopreserved apices. Higher regeneration was generally observed with apices excised from bulbs and bulbils, followed by cloves, and those originated from larger propagules regrew more rapidly. Smaller apices (1.5 or 3.0 mm in diameter) displayed higher regeneration than large ones (4.5 mm in diameter). Cold acclimation at 5 degree C of apices before freezing had no positive effect on regeneration after cryopreservation. Preculture of apices at 10 or 23 degree C for more than 3 days had a detrimental effect on regeneration. The optimal sucrose concentration in the preculture medium was 0.3-0.5 M. Loading apices for 30 or 60 min at 23 degree C in medium containing 2 M glycerol + 0.4 M sucrose or 1 M glycerol + 0.8 M sucrose had no effect on regeneration after cryopreservation, in comparison with apices cryopreserved without loading treatment. Under optimal conditions, regeneration of cryopreserved apices sampled from large cloves was above 90 percent.     

Cryopreservation of potato cultivated varieties and wild species: critical factors in droplet vitrification

The applicability of cryopreservation protocols to a broad range of genotypes is a key issue for genebanks. We tried to identify the critical factors causing differences in survival of cryopreserved shoot tips using potato varieties coming from cultivated and wild species. The droplet-vitrification method, a combination of droplet-freezing and solution-based vitrification, was selected from several protocols. High survival after freezing was observed after dehydration with PVS2 for 20 min, cooling shoot tips placed in a droplet of PVS2 solution on aluminum foil strips by immersing the foil strips in liquid nitrogen, warming them by plunging the foil strips into a 0.8 M sucrose solution (at 40 degrees C) for 30 s and unloading in 0.8 M sucrose for 30 min. This optimized protocol was successfully applied to 12 accessions with survival ranging between 64.0 and 94.4%.     

Cryopreservation of shoot apices of in-vitro grown gentian plants: comparison of vitrification and encapsulation-vitrification protocols

Using vitrification and encapsulation-vitrification protocols, we successfully cryopreserved shoot apices from in-vitro plants of different Gentiana cultivars (lines). Although both protocols gave high survival percentages after storage in liquid nitrogen, the encapsulation-vitrification protocol had several distinct advantages over the vitrification protocol: (i) survival was higher under optimal conditions, (ii) the range of optimal exposure periods to the plant vitrification solution 2 (PVS2) was broader, and (iii) regrowth of cryopreserved shoot apices was apparently more vigorous and faster. Shoot apices from ten cultivars/lines of three Gentiana species (G. scabra, G. triflora, and G. pneumonanthe) were successfully cryopreserved using the two protocols with average survival of 49.0 percent and 73.7 percent for vitrification and encapsulation-vitrification, respectively. These results indicate that the two protocols optimized in the present study are promising for cryopreservation of a wide range of Gentiana genetic resources.     

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.     

An improved cryopreservation protocol for pineapple apices using encapsulation-vitrification

Several modifications to the cryogenic protocols previously described for pineapple apices were performed using vitrification and encapsulation-vitrification. Pregrowth of apices in sucrose-proline before loading significantly reduced the exposure duration to PVS2 and PVS3 required for successful cryopreservation. Encapsulation and treatments with PVS3 at 0 degree C gave the highest survival before and after cooling. Optimal conditions involved the encapsulation of pineapple apices in calcium alginate (3 percent) followed by a 2-d preculture in liquid medium with 0.16 M sucrose + 0.3 M proline for 24 h and then transfer to 0.3 M sucrose + 0.3 M proline for an additional 24 h. After preculture, samples were loaded in 0.75 M sucrose + 1 M glycerol solution at room temperature (25 min) and dehydrated with PVS3 at 0 degree C for 60 min before immersion into liquid nitrogen. Following this procedure 54 percent and 83 percent of apices from MD-2 and Puerto Rico varieties respectively survived.     

Cryopreservation of cultivated and wild potato varieties by droplet vitrification: effect of subculture of mother-plants and of preculture of shoot tips

In this paper, we studied the effect of subculture of mother-plants and of preculture of shoot tips of two potato varieties (Dejima, cultivated and STN13, wild) cryopreserved using the droplet-vitrification technique. The subculture conditions (light intensity, aeration and planting density) significantly affected survival of both non-cryopreserved and cryopreserved shoot-tips in both varieties. The subculture duration and the position of the shoot tips on the axis of the in vitro plantlets had a significant (P<0.0001) effect on survival of cryopreserved shoot tips. The optimal subculture duration was 7 and 5 weeks and the optimal size of shoot tips was 1.5-2.0 and 1.0-1.5 mm for var. Dejima and STN13, respectively. Survival of cryopreserved shoot tips was influenced by the sucrose concentration in the preculture medium and the preculture duration. The highest survival of cryopreserved shoot tips was observed after preculture with 0.3 M sucrose for 8 h followed by 0.7 M sucrose for 18 h. These results indicate that the parameters of the subculture of mother-plants and of preculture of shoot tips should be carefully optimized, especially in the case of wild species.     

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 garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.     

Cryobanking of Korean allium germplasm collections: results from a 10 year experience

This paper reviews a 10-year experience in establishing a cryopreserved Allium germplasm collection at the genebank of the National Agrobiodiversity Center, Republic of Korea. A systematic approach to Allium cryopreservation included: 1. revealing the most critical factors that affected regeneration after cryostorage; 2. understanding the mechanisms of cryoprotection by analyzing the thermal behavior of explants and cryoprotectant solutions using DSC and influx/efflux of cryoprotectants using HPLC; 3. assessing genetic stability of regenerants; and 4. revealing the efficiency of cryotherapy. Bulbil primordia, i.e. asexual bulbs formed on unripe inflorescences, proved to be the most suitable material for conservation of bolting varieties due to high post-cryopreservation regrowth and lower microbial infection level, followed by apical shoot apices from single bulbs and cloves. A total of 1,158 accessions of garlic as well as some Allium species have been cryopreserved during 2005-2010 using the droplet-vitrification technique with a mean regeneration percentage of 65.9 percent after cryostorage. These results open the door for large-scale implementation of cryostorage and for simplifying international exchange for clonal Allium germplasm.     

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 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%).     

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 Arachis species by vitrification of in vitro-grown shoot apices and genetic stability of recovered plants

A storage protocol at cryogenic temperature was established for shoot apices from in vitro plants of the cultivated groundnut (Arachis hypogaea) and wild Arachis species (A. retusa and A. burchellii) using a basic vitrification protocol with direct immersion in liquid nitrogen (LN). The effect of pre-treatments of donor-plants with ABA as well as of different supplements in the post-thaw culture medium was studied. After rapid warming at 40 C, the explants were cultured on MS medium devoid of growth regulators (MS0) or MS supplemented with 4.4(M benzylaminopurine (BAP) and 0.5(M naphthalene acetic acid (NAA) plus 5(M silver nitrate (AgNO3), 0.25% polyvinylpyrrolidone (PVP) or 0.2% activated charcoal. Non-frozen explants from the three species formed one shoot through meristematic amplification when cultured on MS0 medium. These explants also developed callus on MS supplemented with growth regulators (4.4(M BAP and 0.5(M NAA) alone or plus PVP or AgNO3. Callus formation was suppressed in the presence of activated charcoal. Post-thaw regeneration ocurred only through indirect organogenesis on media containing AgNO3 or PVP. Preculturing on medium supplemented with abscisic acid (ABA) improved regrowth rate in these media. Recovery failed to occur in the presence of activated charcoal. The genetic stability of shoots of A. burchellii originated from shoot apices was analyzed through Random Amplified Polymorphic DNA (RAPD) markers.     

Newly developed encapsulation-dehydration protocol for plantcryopreservation

A simplified and efficient encapsulation-dehydration protocol, which is a compromise between vitrification and encapsulation-dehydration, was presented for plant cryopreservation. In this protocol, during the encapsulation process, the apices precultured with 0.3 M sucrose for 16 h were simultaneously osmoprotected with a mixture of 02 M glycerol plus 0.4 M sucrose for 1 h. These encapsulated apices were directly dehydrated with dry silica gel prior to a plunge into LN without the pretreatment of 0.8M sucrose for 16 h. This protocol produced much higher rates of recovery growth in the three plant species tested (wasabi, chrysanthemum, and mint) than those cryopreserved by the conventional encapsulation-dehydration. This protocol also considerably reduced the time needed for the cryogenic procedure. Thus, this new protocol appears promising for cryopreservation of shoot apices and other explants.     

Pellet freezing of in vitro produced bovine embryos using dry ice

In vitro produced bovine embryos were frozen by pellet freezing or vitrification method. In the pellet freezing method, the embryos were cooled on the dry ice and then frozen as pellets. At warming, the pellets were immersed directly into 0.5 M sucrose. The survival rates of blastocysts frozen by the pellet freezing method were higher (P<0.01) in 40% ethylene glycol (EG) than those in the lower concentrations (20 and 30% EG). Higher survival rates of blastocysts frozen by the pellet freezing method were obtained but the development rates did not differ, as compared with those by the vitrification method. There were no significant differences between the pellet freezing and vitrification method in the frequencies of post-thaw survival of hatched blastocysts. These results demonstrate that the pellet freezing method using dry ice can be used successfully for the cryopreservation of blastocysts.     

Effect of preculture, pvs2 and vitamin C on survival of recalcitrant Nephelium ramboutan-ake shoot tips after cryopreservation by vitrification

This paper reports the cryopreservation of Nephelium ramboutan-ake shoot tips derived from in vitro shoot multiplication and in vitro seed germination using vitrification. Preculture with either 0.5 M sucrose for 2 days or a combination of 0.3 M sucrose and 0.5 M glycerol for 3 days enhanced dehydration tolerance and resulted in the highest survival of shoot tips; however, none of the shoot tips withstood liquid nitrogen (LN) exposure. The use of a lower temperature (0 degree C) during exposure to plant vitrification solution (PVS2) led to higher survival of shoot tips, compared to exposure at 25 degree C. The survival percentage of shoot tips exposed to PVS2 for up to 20 min at 0°C was 83.3 percent. It was only 53.3 percent when shoot tips were exposed to PVS2 at 25 degree C for 5 min. The importance of vitamin C for reducing oxidative stress in shoots tips was demonstrated. The addition of 0.28 mM vitamin C during critical steps of the vitrification process resulted in a high survival (96.7 percent) without LN exposure, compared to 73.3 percent for shoot tips not treated with vitamin C. Moreover, 3.3 percent shoot tips withstood LN exposure when vitamin C was added during the loading step. This result suggests that cryopreservation is possible for this tropical, recalcitrant seeded tree species.     

Gelled droplet vitrification improves recovery of cryopreserved potato germplasm

The droplet vitrification method was improved for maneuverability by embedding shoot tips in gelled droplets before osmoprotection. This newly modified cryopreserving method -gelled droplet vitrification - was compared with other PVS2-based cryopreservation methods using potato shoot tips. Survival rates of each cryogenic procedure held at 25 degree C were about 40 percent by cryotube-vitrification procedures (vitrification and encapsulation vitrification methods) and about 70 percent by PVS2-droplet procedures (droplet vitrification and gelled droplet vitrification methods). Much higher cooling rates of PVS2-droplet procedures than cryotube- vitrification procedures increased their survival rates. The gelled droplet vitrification method was applied to shoot tips of 26 potato cultivars and six wild potatoes. After a little modifications of the conditions for preculture, osmoprotection and dehydration, all cultivars and wild potatoes produced high enough survival rates to be of value to genebanks and all surviving shoot tips developed normal shoots within 3 weeks.     

Cryopreservation of in vitro-grown shoot tips of cassava by encapsulation-vitrification method

Shoot tips of cassava (Manihot esculenta Crantz) in vitro plantlets were successfully cryopreserved using the encapsulation-vitrification technique. Nodal cuttings of 5 mm length with one leaf were cultured on modified MS medium in Petri dishes (90 mm x 20 mm) for about 28 days. Excised shoot tips were precultured on sucrose enriched (0.3 M) medium for 16 h, encapsulated and osmoprotected with a mixture of 2 M glycerol and 0.6 M sucrose for 90 min at 25 degree c before dehydration with PVS2 at 0 degree C for 4 h, then plunged in liquid nitrogen. Successfully vitrified shoot tips resumed growth within 3 days, without intermediary callus formation, and developed shoots. Shoot tips sampled from 21 day-old plantlets produced the highest survival of 80 %. The percentage survival of vitrified shoot tips differed from 38 to 80 % depending on the day of excision. The protocol was successfully applied to four cultivars of cassava with about 80 % average percentage of survival.     

Density of Electronic States in the Conduction Band of Ultrathin Films of Naphthalenedicarboxylic Anhydride and Naphthalenetetracarboxylic Dianhydride on the Surface of Oxidized Silicon

The results of examination of the electronic structure of the conduction band of naphthalenedicarboxylic anhydride (NDCA) films in the process of their deposition on the surface of oxidized silicon are presented. These results were obtained using total current spectroscopy (TCS) in the energy range from 5 to 20 eV above the Fermi level. The energy position of the primary maxima of the density of unoccupied states (DOUS) of an NDCA film was determined based on the experimental TCS data and calculated data and compared with the position of the DOUS maxima of a naphthalenetetracarboxylic dianhydride (NTCDA) film. The theoretical analysis involved calculating the energies and the spatial distribution of orbitals of the molecules under study at the B3LYP/6-31G(d) DFT (density functional theory) level and correcting the obtained energies in accordance with the procedure that was proven effective in earlier studies of the conduction band of films of small conjugated organic molecules. It was found that the DOUS maxima of the NTCDA film in the studied energy interval from 5 to 20 eV above the Fermi level are shifted toward lower electron energies by 1–2 eV relative to the corresponding DOUS maxima of the NDCA film Subdivision of the Ufa Federal Research Centre of the.

     

Development of a shoot-tip vitrification protocol and comparison with encapsulation-based procedures for plum (Prunus domestica L.) cryopreservation

Cryopreservation of plum (Prunus domestica L.), cv Regina Claudia, was obtained by a vitrification/one-step cooling procedure of shoot tips from cold-hardened in vitro-grown plants. Best survival (57%) was obtained when the shoot tips were precultured at 4 degree C for 2 days on 0.09 M sucrose-containing Quoirin and Le Poivre medium, loaded for 30 min with a cryoprotectant (2 M glycerol and 0.4 M sucrose), incubated with the PVS2 solution at 0 C for 90 min, and directly plunged into liquid nitrogen. After re-warming in a waterbath at 40 degree C, the shoot tips were washed in a 1.2 M-sucrose MS solution for 20 min and finally plated on a regrowth medium. In comparison with the one-step cooling procedure, both the slow cooling (-0.5 degree C/min up to -45 degree C), and the two-step cooling (-160 degree C for 25 min, then -196 degree C) gave lower percentages of shoot-tip survival. Among the other cryogenic procedures tested, the performance of the encapsulation-vitrification method was similar to the vitrification protocol in terms of shoot-tip regrowth (47.5%), while encapsulation-dehydration was unsatisfactory.