Encapsulation-vitrification Protocol Research Articles

Improved Conservation of Coffee (Coffea arabica L.) Germplasm via Micropropagation and Cryopreservation

Coffee (Coffea spp.) is an important tropical agricultural crop that has significant economic and social importance in the world. The ex situ conservation of plant genetic resources through seeds is not feasible due to the sensitivity of coffee seed to desiccation and low temperatures. The cryopreservation of zygotic embryos may allow for an efficient and long-term storage of coffee germplasm. This study describes the cryopreservation methods for conserving zygotic embryos of Coffea arabica L. for the long-term conservation of currently available germplasm. Zygotic embryos were successfully cryopreserved in liquid nitrogen at −196 °C under controlled environmental conditions with either droplet-vitrification or encapsulation–vitrification protocols without dehydration. Zygotic embryos had the highest regrowth (100%) following droplet-vitrification cryopreservation using the Plant Vitrification Solution 3 (PVS3) for 40 min at 23 °C. In the case of encapsulation–vitrification using PVS3 for 40 min at 23 °C, the embryo regeneration response was 78%. Plantlets were recovered following shoot multiplication using a temporary immersion system (TIS) and in vitro rooting. The prolific rooting of shoots was observed after 4 weeks of culture in the liquid medium with plugs made of the inert substrate Oasis® In vitro Express (IVE) compared to the semi-solid medium. The successful cryopreservation of coffee zygotic embryos using droplet vitrification and encapsulation–vitrification followed by micropropagation in temporary immersion culture system has not been reported earlier and together these technologies are anticipated to further facilitate the initiatives for the conservation and distribution of coffee germplasm.

Gold nanoparticles affect the cryopreservation efficiency of in vitro-derived shoot tips of bleeding heart

The popularity of nanoparticles (NPs) is continuously increasing. To date, however, there has been little research on the application of NPs in plant cryopreservation, i.e. storage of tissues in liquid nitrogen (LN). The aim of this study is to analyze the effect and evaluate the usefulness of gold nanoparticles (AuNPs) in regard to cryobiology studies. In vitro-derived shoot tips of Lamprocapnos spectabilis ‘Valentine’ were cryopreserved with the encapsulation-vitrification protocol. Gold nanoparticles (at 10–30 ppm concentration; 13 nm in size) were added either into the preculture medium; to the protective bead matrix during encapsulation; or to the recovery medium after rewarming of samples. The control plants were produced from cryopreserved explants non-treated with nanoparticles or treated with colloid dispersion medium without NPs. A non-LN-treated standard was also considered. The influence of AuNPs on the cryopreservation efficiency was determined by evaluating the recovery rate of explants and their morphogenic response; the membrane stability index (MSI); the concentration of pigments in shoots; and the antioxidant enzymes activity. The genetic stability of the plant material was evaluated using Start Codon Targeted Polymorphism (SCoT) markers. It was found that 10 ppm of AuNPs added into the alginate bead matrix improved the recovery level of LN-derived shoot tips (70.0%) compared to the non-NPs-treated cryopreserved control (50.5%). On the other hand, the presence of nanoparticles in the recovery medium had a deleterious effect on the survival of explants. AuNPs usually had no impact on the MSI (73.9–85.9%), except for those added into the recovery medium at the concentration of 30 ppm (decline to 55.8%). All LN-derived shoots were shorter and contained less chlorophyll and carotenoids than the untreated standard. Moreover, the application of AuNPs affected the enzymatic activity in L. spectabilis. Minor genetic variation was found in 8.6% of plants if AuNPs were added either into the preculture medium (at 10 and 20 ppm) or to the alginate matrix (at 30 ppm). In conclusion, AuNPs added at a lower concentration (10 ppm) into the protective bead matrix can significantly improve the cryopreservation efficiency in L. spectabilis with no alternation in the DNA sequence.

Shoot Tip Cryopreservation of Lamprocapnos spectabilis (L.) Fukuhara Using Different Approaches and Evaluation of Stability on the Molecular, Biochemical, and Plant Architecture Levels.

The aim of this study is to optimize and evaluate the effectiveness of vitrification, droplet-vitrification, and encapsulation-vitrification techniques in the cryopreservation of Lamprocapnos spectabilis (L.) Fukuhara ‘Gold Heart’, a popular medicinal and ornamental plant species. In vitro-derived shoot tips were used in the experiments. All three techniques were based on explant dehydration with plant vitrification solution 3 (PVS3; 50% glycerol and 50% sucrose) for 0, 30, 60, 90, 120, 150, or 180 min. The recovered microshoots were subjected to morphometric, biochemical, and molecular analyses (RAPD, ISSR, SCoT). The highest recovery level was reported with the encapsulation-vitrification protocol based on 150 min dehydration (73.1%), while the vitrification technique was the least effective (maximum 25.8% recovery). Explants cryopreserved with the encapsulation-vitrification technique produced the highest mean number of shoots (4.9); moreover, this technique was optimal in terms of rooting efficiency. The highest fresh weight of shoots, on the other hand, was found with the vitrification protocol based on a 30-min PVS3 treatment. The concentrations of chlorophyll a and b were lower in all cryopreservation-derived plants, compared to the untreated control. On the other hand, short dehydration and cryopreservation of non-encapsulated explants stimulated the synthesis of anthocyanins. A small genetic variation in 5% of all samples analyzed was detected by RAPD and ISSR marker systems. Only plants recovered from the encapsulation-vitrification protocol had no DNA sequence alternations.

Cryopreservation of in Vitro Grown Shoot Tips of Sweet Potato (Ipomoea batatas L.) by the Encapsulation-Vitrification Method

Sweet potato (Ipomoea batatas L.) shoot tips grown in vitro were successfully cryopreserved by encapsulation-vitrification. Encapsulated explants are very easily manipulated, due to the relatively large size of the alginate beads, and a large number of samples can be treated simultaneously. In this study, the effects of sucrose preculture, cryoprotectant preculture, and post-warm recovery media on regrowth, following liquid nitrogen (LN) exposure, were investigated to establish an efficient encapsulation-vitrification protocol for sweet potato. Shoot tips of plants grown in vitro were precultured in 0.3 M sucrose for 2 d before encapsulation. Encapsulated shoot tips were pre-incubated in liquid MS (Murashige and Skoog) medium containing 0.5 M sucrose for 16 h, before preculturing in sucrose-enriched medium (0.7 M sucrose) for 8 h. Shoot tips were osmoprotected with 35% plant vitrification solution 3 (PVS3) for 3 h, before being dehydrated with PVS3 for 2 h at <TEX>$25^{\circ}C$</TEX>. The encapsulated and dehydrated shoot tips were transferred to 2 mL cryotubes, suspended in 0.5 mL PVS3, and plunged directly into liquid N. High levels of shoot formation were obtained for the cv. Yeulmi (65.7%) and Yeonwhangmi (80.3%). The regrowth rates of cryopreserved samples in Yeulmi (78.9%) and Yeonwhangmi (91.3%), following culture on ammonium-free MS medium for 5 d, were much higher than those cultured on standard MS medium (65.7% and 80.3%, respectively). This encapsulation-vitrification is a promising method for the long-term preservation of sweet potato.

Open encapsulation-vitrification for cryopreservation of algae

Vitrification offers a cost-effective solution for the preservation and management of genetic resources with, low-cost international movement of selected genetic materials and for long-term maintenance of stable stocks of a wide variety of microorganisms. However, its success is limited by the wide range of algal species. Here, we report a simple open encapsulation-vitrification protocol of cryopreservation. Results showed that ∼58% and ∼27% of Oocystis sp. survived vitrification-warming after the open and closed system of vitrification respectively when compared to non-cryopreserved controls. The improved success in an open system of vitrification was also observed in Anabaena sp. Furthermore, with the addition of 2-mercaptoethanol or glutathione the post-warming viability of vitrified algae in both open and closed system of vitrification was significantly improved (p < 0.05). The present case study aimed to develop a vitrification-based cryopreservation protocol and confirms an improvement in survival percentage over conventional encapsulation-vitrification method.

C-2013: Cryopreservation of Passiflora pohlii Mast Nodal Segments and Assessment of Genetic Stability of Recovered Plants

Passiflora pohlii Mast. (Passifloraceae) is a wild species native to Brazil, with a potential agronomic interest due to its tolerance to soil-borne pathogens that cause damage to the passion fruit culture. Because this species occurs in impacted regions, the goal of this study was the development of an efficient cryopreservation strategy for nodal segments excised from axenic plants using vitrification-based methods. The genetic stability of the recovered plants was also evaluated by RAPD and ISSR. In the encapsulation–vitrification protocol, precultured explants on medium with high sucrose concentrations (0.3–0.7 M) were encapsulated in calcium alginate beads, followed by incubation in liquid 1/2MSM medium with 0.75 M sucrose for 24 h. After this, beads were exposed to PVS2 or PVS3 for different periods before immersion into LN. In the vitrification protocol, precultured explants were incubated in the cryoprotectant solutions and stored in LN using the same conditions adopted for encapsulation–vitrification. Frozen materials were rewarmed at 40 °C and transferred to the recovery medium (MSM supplemented with 30.8 μ M BA). Explants were kept in the dark for 30 or 60 days before transfer to the presence of light. Although plant recovery was achieved using both vitrification-based protocols, higher recovery rates were obtained with the vitrification technique, when explants were incubated in the dark. RAPD and ISSR analyses indicated that shoots derived from cryopreserved explants were genetically stable at the genomic regions tested. These results show that cryopreservation of nodal segments through the vitrification technique can be used for long-term storage of P. pohlii germplasm.

Cryopreservation of Rose Cultivars: Comparison of Two Encapsulation Protocols

800x600 Shoot tips excised from in vitro plants of Rosa x hybrida L., cvs. Ioana, Mariana and Vulcan were successfully cryopreserved using encapsulation-dehydration and encapsulation-vitrification methods. Shoot tips (2-3 mm in length) were excised, encapsulated in natrium alginate beads, precultured in sucrose (0.25, 0.5, 0.75 M) for 24 h and either desiccated in laminar air flow (up to 5 h) or dehydrated with a vitrification solution (up to 220 min) prior to direct immersion in liquid nitrogen. The highest shoot development (72% for cv. Vulcan, 69% for cv. Mariana and 66% for cv. Ioana) after storage in liquid nitrogen was obtained after using the encapsulation-vitrification protocol. Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4

Melatonin enhances the recovery of cryopreserved shoot tips of American elm (Ulmus americana L.)

Climate change and global migrations of people and goods have exposed trees to new diseases and abiotic challenges that threaten the survival of species. In vitro germplasm storage via cryopreservation is an effective tool to ensure conservation of tree species, but plant cells and tissues are exposed to multiple stresses during the cryopreservation process. The current study was designed to evaluate the potential of melatonin to improve survival through the process of cryopreservation. Shoot tips of in vitro-grown plantlets and dormant winter buds of American elm were successfully cryopreserved in liquid nitrogen (LN) at -196°C under controlled environmental conditions following melatonin treatment and cold acclimation with either vitrification or encapsulation–vitrification protocols. Explants had optimal regrowth following cryopreservation when treated with the plant vitrification solution#2 (PVS2) for 10 min. Supplementation of both preculture and regrowth media with melatonin significantly enhanced regrowth of frozen shoots compared with the untreated control (P < 0.05). Approximately 80–100% of shoot explants grew under optimized conditions using melatonin-enriched media. Shoot tips of dormant winter buds consistently produced nearly 100% regrowth with both techniques. The main steps of the optimized protocol are14-day cold-acclimated cultures exposed to preculture medium with 0.1–0.5 lM melatonin for 24 hr, application of PVS2 for 10 min, rapid cooling in LN, rapid rewarming, removal of cryoprotectants, and recovery on a medium supplemented with 0.1–0.5 lM melatonin. Our results demonstrate the usefulness of the antioxidant melatonin for long-term storage of naturally resistant elm germplasm.

23. Comparison of vitrification-based techniques in the efficacy of cryopreservation of Passiflora suberosa L. and P. foetida L. shoot tips

Passiflora are used as ornamentals and in popular medicine. Passiflora suberosa is commonly used to treat hypertension, diabetes, skin diseases and as a sedative, whereas P. foetida shows analgesic and antibacterial activities. The goal of this study was the development of in vitro conservation strategies for long-term storage for both species through cryopreservation using vitrification and encapsulation-vitrification techniques. The efficacy of vitrification and encapsulation-vitrification protocols was compared, and several parameters were evaluated, including pregrowth on medium with high sucrose concentrations, type of vitrification solution (PVS2 and PVS3), exposure time to vitrification solutions, and recovery conditions. Shoot tips (0.3 cm) excised from in vitro-grown plants were precultured for 24 h on solid 1/2 MSM medium supplemented with different sucrose concentrations (0.3, 0.5 or 0.7 M), at 25 °C. Explants precultured on medium supplemented with 0.3 M sucrose were incubated in the cryoprotectant solutions PVS2 or PVS3 for different periods (0–240 min) at room temperature, and immersed into liquid nitrogen (LN) for 72 h. In the encapsulation-vitrification protocol, precultured shoot tips were encapsulated in calcium alginate beads, followed by incubation in liquid 1/2 MSM medium containing 0.75 M sucrose for 24 h, on a rotary shaker (100 rpm) at 25 ± 2°C. After this period, beads were maintained in cryovials containing PVS2 or PVS3 for 30, 60, 120 or 180 min, before immersion into LN. The materials were rapidly thawed in a warm water bath (38-40 °C) for 2 minutes and transferred to the recovery medium (1/2 MSM or MSM supplemented with 0.44, 2.2 or 4.4 μM BA). Alternatively, explants were kept in the dark for 30 or 60 days before transfer to the presence of light. Plant recovery from the shoot tips before and after cooling in LN was evaluated by assessing the percentage of shoot formation. The shoots were cultured on 1/2 MSM medium for 60–90 days for root induction, and whole plants were transferred to ex vitro conditions. The highest recovery rates for both P. suberosa and P. foetida (28% and 60%) were obtained with the encapsulation-vitrification protocol, after a pre-treatment with 0.3 M sucrose, followed by exposure to PVS2 for 60 or 120 min, respectively, and post-freezing incubation in the absence of light for 30 days on MSM medium supplemented with 0.44 μM BA. These results suggest that alginate beads provided additional protection, reducing the toxicity of the cryoprotectant solution. In addition, they demonstrate that different species from genus Passiflora display distinct responses to cryopreservation treatments.

72. Cryopreservation of Passiflora pohlii – Vitrification and encapsulation-vitrification

Passiflora pohlii is a wild tropical species of the genus Passiflora , it is native to Brazil and found in Cerrado, Atlantic Forest and Pantanal. This species has a potential agronomic interest due to its tolerance to soil-borne pathogens of Phytophthora sp, which causes damage to passionfruit culture. P. pohlii occurs in regions that are impacted by human actions, with the possibility of disappearance of many genotypes that could be used in breeding. Considering these facts, the establishment of different conservation strategies for this species is of great importance. The goal of this work was to develop cryopreservation protocols for P. polhii using vitrification-based techniques. Nodal segments (0.3 cm) excised from in vitro -grown plants were precultured for 24 h on solid MSM (Monteiro et al. In vitro Cell Dev Biol – Plant 36, 527–531) medium supplemented with 0.7 M sucrose, at 25 °C. For vitrification, the precultured explants were placed into 2.0 ml cryovials and incubated in the cryoprotectant solution PVS2 for different periods (0, 30, 60 or 120 min) at room temperature. After this, the cryovials were plunged into liquid nitrogen (LN 2 ) and stored for 72 h. For encapsulation-vitrification, the precultured explants were suspended in calcium-free MS medium supplemented with 3% sodium alginate. The mixture, including the nodal segments, was dispensed with a sterile pipette into MS medium containing 100 mM CaCl 2 , and held for 20 min at 25 °C. Beads, each containing one explant, were precultured in liquid 1/2 MSM medium containing 0.75 M sucrose for 24 h on a rotary shaker (100 rpm) at 25° ± 2 °C, and then placed into 2 ml cryovials containing PVS2 for 0, 30, 60 or 120 min. After these periods, cryovials were immersed into liquid nitrogen (LN 2 ) and stored for 72 h. Thereafter, the cryovials were removed from LN 2 and rapidly re-warmed in a water bath (40 °C) for 2 min, and transferred to the MSM supplemented with 31 μM BA. The explants were maintained in the dark for 30 or 60 days in a growth chamber at 30° ± 2 °C. After these periods, the explants were exposed to total irradiance (46 μ mol m −2 s −1 ). When using the vitrification technique, the highest recovery rate (40%) was obtained after exposure to PVS2 for 60 or 120 min. On the other hand, a very low survival rate was attained when explants were submitted to the encapsulation-vitrification protocol. In summary, in the present study we established a protocol for long-term conservation of P. pohlii through cryopreservation. We demonstrated that the vitrification protocol resulted in higher recovery rates when compared to the results obtained with the encapsulation-vitrification technique. In addition, the high multiplication capacity of nodal segments was not affected by cryostorage.

Cold hardening and sucrose treatment improve cryopreservation of date palm meristems

Date palm (Phoenix dactylifera L.) cv. Khenizi caulogenic meristems were initiated from achlorophyllous leaves excised from in vitro shoot cultures and then proliferated on a specific culture medium supplemented with 70 g dm-3 sucrose. Regeneration rates obtained when using standard vitrification, droplet-vitrification, and encapsulation-vitrification protocols reached 26.7, 60.0, and 40.0 %, respectively. Only explants smaller than 3 mm in diameter were found to survive cryogenic treatments. Sucrose preculture, cold hardening and loading solution pretreatments showed significant effects on regeneration rates. Moreover, our results indicate that both sucrose preculture and cold acclimation of explants increased proline content. Cryopreservation of date palm tissue with high proliferation capacity can directly benefit large scale micropropagation projects.

153. Changes in alginate bead water status and dry weight content during encapsulation–dehydration and encapsulation–vitrification protocols

153. Changes in alginate bead water status and dry weight content during encapsulation–dehydration and encapsulation–vitrification protocols

Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification

Alginate coated meristems from in vitro-grown axillary buds of potato (Solanum tuberosum L.) were successfully cryopreserved by vitrification. Excised meristems were precultured on sucrose-enriched MS medium and then encapsulated. To induce dehydration tolerance (osmotolerance), encapsulated meristems were treated with a mixture of 2 M glycerol plus 0.6 M sucrose for 90 min. These encapsulated meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 hr at 0°C prior to a plunge into liquid nitrogen. Successfully vitrified meristems developed shoots within 3 weeks after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 70%. No difference was observed in RAPD analysis using 17 primers between cryopreserved and non-treated plantlets. The cryogenic protocol was successfully applied to 14 cultivars. It was also confirmed that the encapsulated vitrified meristems produced much greater shoot formation than the encapsulated dried meristems. Thus, the encapsulation vitrification protocol appears promising for cryopreservation of potato germplasm.

4335457 Method for semiconductor memory testing

Vitrification offers a cost-effective solution for the preservation and management of genetic resources with, low-cost international movement of selected genetic materials and for long-term maintenance of stable stocks of a wide variety of microorganisms. However, its success is limited by the wide range of algal species. Here, we report a simple open encapsulation-vitrification protocol of cryopreservation. Results showed that ∼58% and ∼27% of Oocystis sp. survived vitrification-warming after the open and closed system of vitrification respectively when compared to non-cryopreserved controls. The improved success in an open system of vitrification was also observed in Anabaena sp. Furthermore, with the addition of 2-mercaptoethanol or glutathione the post-warming viability of vitrified algae in both open and closed system of vitrification was significantly improved (p < 0.05). The present case study aimed to develop a vitrification-based cryopreservation protocol and confirms an improvement in survival percentage over conventional encapsulation-vitrification method.