Vitrification Protocol Research Articles

Effect of cryopreservation on human sperm messenger RNAs crucial for fertilization and early embryo development

During recent years, several studies have pointed out the importance of key paternal transcripts in early embryo development. Sperm cryopreservation is commonly applied in assisted reproductive technologies (ARTs) and it is important to know if it produces any relevant effect at this level. In this study, using normozoospermic donors, we present a candidate transcript approach in which we quantify the presence of sperm mRNAs considered as markers for male fertility and pregnancy success. Analyses were done using quantitative PCR. Our results demonstrated that the used cryopreservation protocol, which is routinely employed in clinical practice, alter transcripts considered as spermatozoa quality markers and markers for pregnancy success. Most of the studied transcripts considered as male quality markers (PRM1, PRM2, and PEG1/MEST) and one of studied mRNAs reported as markers of pregnancy success (ADD1) were reduced after cryopreservation. In order to check if vitrification protocols could reduce this alteration, another assay was carried out analyzing those transcripts with higher differences in the first study (PRM1 and PRM2). The results showed the same tendency. Although maternal mRNAs can compensate these deficiencies, these results could make advisable the optimization of freezing/thawing procedures.

Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue

Can a vitrification protocol using an ethylene glycol/dimethyl sulphoxide-based solution and a cryopin successfully cryopreserve baboon ovarian tissue? Our results show that baboon ovarian tissue can be successfully cryopreserved with our vitrification protocol. Non-human primates have already been used as an animal model to test vitrification protocols for human ovarian tissue cryopreservation. Ovarian biopsies from five adult baboons were vitrified, warmed and autografted for 5 months. After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. The influence of the vitrification procedure on the cooling rate was evaluated by a computer model. After vitrification, warming and long-term grafting, follicles were able to grow and maintain their function, as illustrated by Ki67, anti-Müllerian hormone (AMH) and growth differentiation factor-9 (GDF-9) immunostaining. Corpora lutea were also observed, evidencing successful ovulation in all the animals. Stromal tissue quality did not appear to be negatively affected by our cryopreservation procedure, as demonstrated by vascularization and proportions of fibrotic areas, which were similar to those found in fresh ungrafted ovarian tissue. Despite our promising findings, before applying this technique in a clinical setting, we need to validate it by achieving pregnancies. In addition to encouraging results obtained with our vitrification procedure for non-human ovarian tissue, this study also showed, for the first time, expression of AMH and GDF-9 in ovarian follicles. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4507.10, grant 3.4.590.08 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, and Department of Mechanical Engineering at Louisiana State University (support to Ram Devireddy), and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors has any competing interests to declare.

Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions

What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? Contrary to common beliefs, it was observed that the ICCP in vitrified zygotes is lower than after SLF, although the solutions used in VIT contain higher concentrations of cryoprotectants (CPs). To reduce the likelihood of intracellular ice crystal formation, which has detrimental effects on cell organelles and membranes, VIT was introduced as an alternative to SLF to cryopreserve embryos and gametes. Combined with high cooling and warming rates, the use of high concentrations of CPs favours an intracellular environment that supports and maintains the transition from a liquid to a solid glass-like state devoid of crystals. Although the up-to-date publications are reassuring in terms of obstetric and perinatal outcomes after VIT, a fear about exposing gametes and embryos to high amounts of CPs that exceed 3-4-fold those found in SLF was central to a debate initiated by advocates of SLF procedures. Two experimental set-ups were applied. The objective of a first study was to determine the ICCP at the end of the exposure steps to the CP solutions with our VIT protocol (n = 31). The goal of the second investigation was to compare the ICCP between VIT (n = 30) and SLF (n = 30). All experiments were performed in triplicates using mouse zygotes. The study took place at the GIGA-Research Institute of the University of Liège. Cell volume is modified by changes in extracellular osmolarity. Hence, we estimated the final ICCP after the incubation steps in the VIT solutions by exposing the cells to sucrose (SUC) solutions with defined molarities. The ICCP was calculated from the SUC concentration that produced no change in cell volume, i.e. when intra- and extracellular osmolarities were equivalent. Cell volume was monitored by microscopic cinematography. ICCP was compared between SLF and VIT based on the principle that a high ICCP lowers the probability of (re)crystallization during warming but increases the probability of over-swelling of the cell due to fast inflow of water. The survival rates of mouse zygotes after SLF or VIT were compared using either (i) various warming rates or (ii) various concentrations of SUC in the warming dilution medium. The ICCP in mouse zygotes during the VIT procedure prior to plunging them in liquid nitrogen was ∼2.14 M, i.e. one-third of the concentration in the VIT solution. After SLF, the warming rate did not affect the zygote survival rate. In contrast, only 3/30 vitrified zygotes survived when warmed slowly but as many as 30/30 zygotes survived when warming was fast (>20 000°C/min). Vitrified zygotes showed significantly higher survival rates than slow-frozen zygotes when they were placed directly in the culture medium or in solutions containing low concentrations of SUC (P < 0.01). These two experiments demonstrate a lower ICCP after VIT than after SLF. The results should not be directly extrapolated to other stages of development or to other species due to possible differences in membrane permeability to water and CPs. The low ICCP we observed after VIT removes the concern about high ICCP after VIT, at least in murine zygotes and helps to explain the observed efficiency and lack of toxicity of VIT. The study was funded by the FNRS (National Funds for Scientific Research). The authors declare that they have no competing interests.

Acclimation-induced changes in cell membrane composition and influence on cryotolerance of in vitro shoots of native plant species

Cell membranes are the primary sites of cryopreservation injury and measuring changes to membrane composition arising from cold acclimation may assist with providing a rationale for optimising cryopreservation methods. Shoot tips from two south-west Western Australian species, Grevillea scapigera and Loxocarya cinerea, and Arabidopsis thaliana (reference species) were subjected to cryopreservation using the droplet vitrification protocol. Two pre-conditioning regimes involving a constant temperature (23 °C, CT with a 12 h light/dark cycle) or an alternating temperature (AT) regime (20/10 °C with a 12 h light/dark cycle) were compared. Soluble sugars, sterols and phospholipids present in the shoot tips were analysed. Use of AT pre-conditioning (acclimation) resulted in a modest decrease in cryotolerance in A. thaliana, increased cryotolerance in G. scapigera, and increased survival in the non-frozen control explants of L. cinerea in comparison to CT pre-conditioning. Increased cryotolerance was accompanied by a higher total sugar sterol and phospholipid content, as well as an increase in strong hydrating phospholipid classes such as phosphatidylcholine. The double bond index of bound fatty acyl chains of phospholipids was greater after AT pre-conditioning, mostly due to a higher amount of monoenes in A. thaliana and trienes in G. scapigera and L. cinerea. These findings suggest that AT pre-conditioning treatments for in vitro plants can have a positive influence on cryotolerance for some plant species and this may be related to observed changes in the overall composition of cell membranes. However, alternative factors (e.g. oxidative stress) may be equally important with other species (e.g. L. cinerea).

The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification

This report highlights the first successful cryopreservation protocol for shoot tips of Garcinia mangostana L. achieved by using vitrification technique. We investigated the effects of different temperatures and exposure periods to a plant vitrification solution 2 (PVS2), sucrose concentrations and preculture periods, and unloading treatments in steps of the vitrification protocol on the survival of G. mangostana shoot tips after cryopreservation. Exposure to PVS2 for 25 min gave beneficial effects with 10.4 ± 1.8 % survival at 0 °C with average water content of 1.1 ± 0.3 g g−1 dry mass. Survival was 13.7 ± 5.5 % when using preculture medium with full-strength Murashige and Skoog (MS) medium supplemented with 0.6 M sucrose for 2 days. A significant difference was observed in survival of shoot tips when treated with various sucrose concentrations in preculture which strengthens their importance towards enhancing survival of shoot tips after cryopreservation. MS with 0.4 M sucrose and 2 M glycerol applied as an unloading solution increased the survival of shoot tips to 44.1 ± 6.5 %. Experiments on the effect of ascorbic acid were also conducted for each step of vitrification. Our results showed higher survival of 45.8 ± 3.8 % but there were no significant effects compared with the control (without ascorbic acid). Further study on the recovery dark/light period was conducted. Survival of shoot tips significantly increased to 50.0 ± 16.7 % when subjected to 7 days in the dark before transferring to 16 h/8 h light/dark photoperiod. These studies strengthen suggestions that cryopreservation through vitrification is possible for ex situ conservation of germplasm of this tropical recalcitrant species.

Clinical efflux of cryoprotective agents from vitrified human articular cartilage

In previous research, we successfully cryopreserved intact human articular cartilage on its bone base with high chondrocyte viability using a vitrification protocol that entailed sequential exposure to several cryopreserving agents (CPAs) at lowering temperatures resulting in a high final concentration of CPA. The CPA must be removed from the cartilage at warming due to its toxicity to cells in the cryopreserved tissue and the post-transplant adjacent tissues. The current experiment explores the relationship between removal solution volume and time required for complete removal of CPA from bone–cartilage samples.Osteochondral dowels of 10mm diameter from five patients undergoing total knee arthroplasty were vitrified using our protocol resulting in 6.5M CPA within the matrix. In the primary experiment, the warmed dowels were immersed in 10mL of X-VIVO for 30min and this was repeated 5times (the last wash being 5min only). Removal solution osmolality was recorded at various times and compared to controls of pure X-VIVO. Changes in removal solution osmolality over time were normalized to tissue volume. In a secondary experiment, the procedure was repeated using double the volume of removal solution (20mL X-VIVO).Results showed a rapid change in the osmolality of the removal solution indicating a rapid efflux of CPA from cartilage. The efflux rate decreased with time and during subsequent immersions until equilibrium was reached during the 4th immersion indicating effectively complete removal of CPA. Doubling the amount of removal solution demonstrated the effective removal of CPAs by the third immersion.The results of this study yield a practical relationship between the amount of removal solution and the time and number of immersions required to remove CPA from the transplantable tissue.

Evaluation of a New Vitrification Protocol for Oocyte Cryopreservation

During the last years, vitrification was introduced into clinical practice. Although vitrification counts several years of experimental studies, this method is still under development. In this study, a new vitrification protocol was evaluated. Materials and methods: An experimental study conducted in a University laboratory evaluated a new, short vitrification protocol including two vitrification and two thawing solutions, made in-house. The vitrification solutions contained dimethyl sulfoxide, ethylenoglycol and human serum albumin. The thawing solutions contained sucrose and human serum albumin. Rat oocytes, allocated in two groups, were used. In the control group, the oocytes were vitrified using a standard protocol. In the experimental group, the oocytes were vitrified using the new protocol. The endpoint was the survival rate of the oocytes just after thawing and 48 hours later. Results: The survival rate just after thawing was similar in the two groups (62.5% for the control group and 60% for the experimental group, p=0.18). However after 48 hour incubation, there was a significant difference between the two groups: 55% survival for the control group and 45% for the experimental group (p=0.008). Conclusion: The two vitrification protocols achieved similar survival rates just after thawing. However, after 48 hours incubation of thawed oocytes, the standard method had higher survival rates, than the in-house one.

Closed vitrification of mouse oocytes using the CryoLogic vitrification method: A modification that improves developmental competence

ObjectiveTo compare the mouse oocyte vitrification outcomes of the CryoLogic vitrification method (CVM) and the conventional open method using a Cryotop. Two CVM methods (original CVM and modified CVM) were tested.MethodsMature oocytes obtained from female BDF-1 mice were vitrified by two-step exposure to equilibrium and vitrification solutions. Three vitrification protocols were tested on three groups: the CVM-kit, modified CVM, and Cryotop groups. After exposure to the two solutions, the oocytes were vitrified. After warming, the oocytes were fertilized in vitro, and the embryo development was assessed. Blastomeres positive for caspase were counted using an in situ assay kit. The spindle morphology and chromosome configurations of warmed vitrified oocytes were also assessed.ResultsThe modified CVM and Cryotop groups showed similar developmental capacities, and similar proportions of cells with intact spindles and chromosome configurations. The modified CVM protocol was superior to the original CVM protocol for developmental competence and intact spindle preservation. However, the CVM group showed a relatively higher number of apoptotic cells in blastocysts.ConclusionClosed vitrification using the modified CVM protocol may be used as an alternative to the conventional open method, but strategies to decrease apoptosis in the blastomere need to be investigated.

The new Rapid-i carrier is an effective system for human embryo vitrification at both the blastocyst and cleavage stage

BackgroundThe Rapid-i is a new FDA cleared closed carrier for embryo vitrification. The cooling rate of - 1220°C/min is far lower than that reported with open vitrification systems such as the cryoloop (−15,000°C/min). Little published data is currently available on this device. This study presents our initial clinical data, as well as live birth outcomes, with the Rapid-i. The efficacy of this device for the cryopreservation of cleavage, as well as blastocyst stage human embryos is also analyzed. We further compare outcomes to those achieved with the cryoloop, an “open” vitrification system routinely used in our laboratory.MethodsHuman embryos were vitrified at either the 8–10 cell stage or else the blastocyst stage. The vitrification protocol was: 7.5% DMSO/7.5% ethylene glycol (EG) (2–3 min) followed by incubation in 15% DMSO /15% EG (45 sec) before loading on the vitrification carrier. Cryoprotectant was removed during warming by sequential washes in 0.25 M and 0.125 M sucrose in culture medium. Clinical outcome data for frozen cycles between January 2011 and August 2012 were stratified according to carrier and cell stage. The student t-test and chi square test were used to compare results. P value of < 0.05 was considered significant.ResultsA total of 486 vitrified-warmed embryos were assessed and 92% of them were transferred. The clinical pregnancy rate (CPR) and implantation rate (IR) with Rapid-i vitrified blastocysts were 59% and 49%, versus 47% and 37%, respectively for cleavage stage embryos. This was not statistically different from results with the cryoloop vitrified blastocysts (CPR 46%, IR 38%) nor the cleavage stage vitrified embryos (CPR 49%, IR 35%). To date, there have been 31 deliveries of 34 healthy infants from Rapid-i vitrified embryos, with another 12 pregnancies still on-going.ConclusionsThe Rapid-i offers an excellent alternative to existing open vitrification devices for embryo cryopreservation at the 8–10 cell stage as well as the blastocyst stage. Use of this type of “closed” sealed system that prevents direct contact between the embryos and liquid nitrogen reduces the potential risk of sample cross-contamination or infection. These preliminary data and live birth outcomes have paved the way toward transitioning to a closed vitrification system in our own IVF program.

Histology and scanning electron microscopy observations of cryopreserved protocorm-like bodies of Dendrobium sonia-28

The genus Dendrobium possesses horticultural importance. Dendrobium sonia-28 is an important ornamental orchid in the flower industry. Cryopreservation is a favoured long-term storage method for orchids with propagation problems. Protocorm-like bodies (PLBs) of Dendrobium sonia-28 were cryopreserved using the vitrification technique. Histology and scanning electron microscopy (SEM) observations were conducted on stock, non-cryopreserved (control), and cryopreserved PLBs of Dendrobium sonia-28 to detect cryoinjuries resulting from the vitrification protocol. Histological observations of control PLBs indicated that the preculture, osmoprotection, and dehydration steps were not physically damaging to the PLBs. Histological and SEM analyses of cryopreserved PLBs indicated that the freezing and thawing cycles inflicted damages on the parenchymatic regions of the PLBs. Only embryogenic cells survived the treatment. Scanning electron microscopy studies of the control and cryopreserved PLBs indicated that both osmotic and freezing injuries occurred only in the interior regions of the PLBs.

Comparison of clinical outcomes following vitrified warmed day 5/6 blastocyst transfers using solid surface methodology with fresh blastocyst transfers

OBJECTIVES:The literature regarding clinical outcomes following day 5/6 vitrified warmed blastocysts transfer has been conflicting. We decided to evaluate and compare the clinical outcomes following vitrified warmed day 5/6 blastocyst transfer using a solid surface vitrification protocol with fresh blastocyst transfers.SETTINGS:University teaching hospital.STUDY DESIGN:A total of 249 women were retrospectively analyzed: 146 fresh day 5 blastocyst (group 1), 57 day 5 vitrified warmed blastocyst (group 2), and 46 vitrified warmed day 6 blastocyst (group 3) transfer cycles. Vitrification was done using solid surface methodology (non immersion protocol). The main outcomes were implantation rates, clinical pregnancy, and live birth rate per embryo transfer.RESULTS:The baseline clinical characteristics were similar among all three groups. The implantation and clinical pregnancy rates following vitrified warmed day 6 blastocyst transfers (20.9% and 32.6%) were significantly lower as compared to day 5 fresh and vitrified warmed day 5 blastocyst transfers (40.3% and 56.1%, 36.3%, and 52.6%). However, there was no significant difference in the live birth rates across the three groups (group 1: 37.6%, group 2: 40.3%, and group 3: 28.2%).CONCLUSION:No statistically significant difference was observed in live birth rates between fresh day 5 blastocyst transfers and vitrified warmed day 5/6 blastocyst transfers. Vitrification of blastocysts using solid surface methodology is an efficient method of cryopreservation.

Refinement of a vitrification protocol for protocorm-like bodies of Dendrobium sonia-28

The hybrid Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, it faces the risk of producing heterozygous progenies through conventional seed propagation or somaclonal variation. Cryopreservation is a favoured long-term storage method in the preservation and maintenance of orchids with specific desired traits. This paper presents a successful cryopreservation protocol for protocorm-like bodies (PLBs) of Dendrobium sonia-28 through the vitrification technique and the use of ascorbic acid and charcoal. Survival assessments were conducted through growth evaluations and visual and spectrophotometric 2,3,5-triphenyltetrazolium chloride assays. Results of this study, an improvement over the previous protocol applied to this orchid hybrid, showed that the addition of ascorbic acid to the preculture, loading, dehydration, unloading, and regeneration media, coupled with the addition of charcoal to the regeneration medium, helped boost recovery rates of cryopreserved PLBs of Dendrobium sonia-28.

Review Structure, function, and localization of aquaporins: their possible implications on gamete cryopreservation

The discovery of water channels (aquaporins, AQPs) was a landmark event for the clarification of water transport through the plasma membrane. AQPs belong to a family of intrinsic membrane proteins that act as selective channels for water and for solutes such as glycerol and urea. AQPs were found in different tissues and organs, including male and female reproductive systems. In the swine female reproductive system, the AQPs were localized in the uterus, oviduct, and ovary, as well as in the granulosa cells from primordial follicles. Knowing the involvement of AQPs with the male and female germ cells, as well as their acknowledged role in transporting water through the plasma membrane, the research of these proteins in cryopreservation processes becomes essential. Thus, this review aims to describe the structure and function of AQPs in membranes, highlighting their role in the reproductive system (male and female). We also discuss the involvement of AQPs in cryopreservation, focusing on the effect and importance of these proteins on the rates of vitrification protocols for preantral follicles present in the ovarian tissue of domestic mammals.

Optimized protocol for cryopreservation of human eggs improves developmental competence and implantation of resulting embryos

BackgroundSuccessful egg cryopreservation has many potential benefits to a variety of patients. However, a superior standard protocol describing all aspects of oocyte cryopreservation has not yet been identified. Oocyte cryopreservation is still a technical challenge for many infertility clinics. To maintain satisfactory clinical outcomes, there is a need to develop an easy to use, yet efficient laboratory protocol. The present study was designed to examine if human embryos resulting from eggs frozen with an optimized vitrification protocol have similar developmental competence as those from fresh eggs.MethodsTwenty recipients received donated eggs vitrified with a protocol in which short exposure time to the vitrification solution was used and 23 recipients received donated eggs and 6 patients had their own eggs vitrified with a modified protocol in which long exposure time to the vitrification solution was used. After warming, egg survival, fertilization, cleavage, blastocyst formation, clinical pregnancy and implantation rates were compared. The developmental competence of eggs vitrified with the optimized protocol was further compared with fresh eggs donated from the same donors.ResultsThere was no difference in the oocyte survival, fertilization, cleavage, clinical pregnancy or implantation rates between the short and long protocol groups. However, blastocyst formation rate was significantly (P < 0.001) higher in the long protocol group (50.8%) than that in short protocol group (26.5%), resulting in more blastocysts being transferred and frozen. When frozen eggs vitrified with long protocol and fresh eggs from the same donors (12) were compared in 39 recipients, no differences were observed in terms of fertilization (86.4 vs 80.1%), blastocyst formation (50.0 vs 59.2%), clinical pregnancy (63.2 vs 60.0%) and implantation (41.7 vs 44.7%) rates. Four out of 6 patients had ongoing pregnancy after transfer of embryos from their own frozen eggs with a 46.2% implantation rate.ConclusionsThese results indicate that blastocyst development is an appropriate measure for egg survival after cryopreservation and frozen eggs have similar developmental potential as fresh eggs if they are frozen with an optimized method.

Achieving oocyte survival and stable spindles after vitrification using closed pulled straws regardless of zona status

ObjectivesEmbryo cryopreservation is a well-established technique at in vitro fertilization centers but the best protocol for vitrification of oocytes and zona manipulation remains inconclusive. This study aimed to determine if the closed pulled straws (CPS) method and zona drilling for vitrification provide a higher survival rate for thawed oocytes. Materials and MethodsFemale MF1 mice were superovulated by injection of gonadotropins. Cumulus-oocyte complexes were derived from excised fallopian tubes and the oocytes were divided into the following three groups: (1) one-hole zona drilling by laser, (n = 40); (2) intact zona (n = 48); and (3) zona digestion by pronase (n = 43). The control group consisted of 40 nonvitrified oocytes. After thawing, surviving oocytes were stained for spindles and chromosomes after 1-hour and 3-hour incubations, and compared to controls. ResultsThere were no significant differences in the survival rates among Groups 1 (34/40, 85%), 2 (34/48, 71%), and 3 (30/43, 70%), but there were significant differences compared with the control oocytes (100%). After 1-hour and 3-hour incubations, vitrified oocytes in the three groups did not have significantly fewer normal spindles than the controls (1 hour: Group 1, 70.5%; Group 2, 64.7%; and Group 3, 66.6% vs. control 90%). There was also no significant difference in the percentage of oocytes with a normal spindle shape between the 1-hour and 3-hour recovery times (3 hours: Group 1, 88.2%; Group 2, 88.2%; and Group 3, 80%; control, 95%). ConclusionsThe zona pellucidum with CPS method has no effect on spindle injury and oocyte survival. Sufficient culture time for the recovery of the meiotic spindle is imperative for fertilization of vitrified oocytes. A CPS vitrified oocyte has the advantages of high survival and preserved good spindles.

Vitrification of Domestic Cat Oocytes – Effect on Viability and Integrity of Subcellular Structures

Cryopreservation of female gametes is a crucial part of assisted reproduction techniques. The domestic cat is a model for both wild felids and human research. The aim of this study was to evaluate the effect of different vitrification protocols applied to feline oocytes at different maturational stages on the preservation of oocyte viability and integrity of subcellular structures. The vitrification solutions consisted of 20% ethylene glycol, 20% DMSO, 20% FCS, and 1.5 m Trehalose with and without 10% Ficoll PM-70. Markers for cell viability (PI/FDA), cytoskeleton organization (Anti-α-Tubulin-FITC antibody, Phalloidin-TRITC), as well as nuclear configuration (DAPI) were used for evaluation of vitrified-warmed oocytes. Our results show that 52% and 41% of live mature and immature oocytes, respectively and until 32% of microtubules, 28% of nuclear configuration and 36% of microfilaments in the normal pattern can be obtained with protocol described in this paper. According to our data, Ficoll PM-70 essentially improves the oocytes survival upon vitrification.

73. Evaluation of the survival of somatic embryos of Petiveria alliacea L. cryopreserved after different pretreatments with sucrose and PVS2

Petiveria alliaceae L. (Phytolacaceae) is a herbaceous plant species, native to humid tropical regions, traditionally used in folk medicine due to its broad spectrum of pharmacological actions, mainly immunomodulatory, anti-inflammatory, cytotoxic and neurotoxic. In Brazil, this species is produced only in home gardens. For this reason, the harvest predatory for use as a drug is causing genetic erosion in natural populations. In this context, biotechnology techniques for crop production are suitable for the production and conservation of germplasm. In vitro culture via somatic embryogenesis, was previously established for this species and the purpose of this study was to evaluate the survival and recovery of embryos cryopreserved after treatment with cryoprotectants. Somatic embryos from leaf explants cultured on MS medium supplemented with PIC (5 mg/l) were subcultured and maintained for one week in 1/2 MS medium with 2% Phytagel in a growth chamber at 30 °C ± 2 °C, with luminous intensity of 46 μM m −2 s −1 and photoperiod of 16 h. Then the embryos were distributed in vials containing MS medium supplemented with different concentrations of sucrose (0.3–0.5–0.7 M). Then the material was submitted to PVS2 cryoprotectant solution at different time periods (0, 15, 30, 45 or 60 min) and then frozen in liquid nitrogen (LN 2 ) for 24 h. In each sample 5–10 embryos were used to evaluate the water content, by the ratio between fresh and dry weight. Thawing was rapid at 40 °C for 2 min in a water bath. The survival of embryos was assessed, after the pre-treatment and after freezing in LN 2 , by testing the TTC (2,3,5 triphenyltetrazolium chloride hydrochloride), by change of color visually detected in embryonic tissues. The recovery was determined by cell proliferation, after 30 days in culture conditions described. Pretreatment with sucrose in embryos caused dehydration proportional to the concentrations tested, and does not affect their survival. Treatment with PVS2 provided a 100% survival, when the embryos were exposed for 15 min, however, exposure for a longer period was lethal to these tissues. The recovery of these embryos was recorded after 30 days of culture by number of embryos observed. Given that there are no previous reports of cryopreservation of Petiveria alliacea germplasm, the optimization and tolerance to dehydration of explants to cryoprotectant solutions are key steps for establishing a vitrification protocol, aiming at long-term conservation of the botanical material, and can benefit the herbal industry.

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.