Rapid Freezing In Liquid Nitrogen Research Articles

Evaluating stability and bioactivity of Rehmannia-derived nanovesicles during storage

Plant-derived nanovesicles (PDNVs) have garnered growing attention in the biomedical field owing to their abundance in plant-derived ribonucleic acids (RNA), proteins, lipids and metabolites. The question about the preservation of PDNVs is a crucial and unavoidable concern in both experiments’ settings and their potential clinical application. The objective of this research was to examine the impact of varying storage temperatures on the stability and bioactivity of Rehmannia-derived nanovesicles (RDNVs). The results showed that RDNVs aggregated after 2 weeks of storage period at 4 °C, and the particle size of some RDNVs gradually increased with time, along with the increase of solution potential. After 2 months of storage, all RDNVs exhibited varying levels of aggregation irrespective of storage temperature. The bioactivities of nanovesicles under different temperature storage conditions revealed a gradual decline in cell proliferation inhibition bioactivity over time, significantly lower than that of freshly prepared RDNVs. In contrast, the preservation of anti-migratory activity in RDNVs was found to be more effective when subjected to rapid freezing in liquid nitrogen followed by storage at − 80 °C, as opposed to direct storage at − 80 °C. These findings suggest that temperature alone may not be sufficient in safeguarding the activity and stability of RDNVs, highlighting the necessity for the development of novel protective agents for PDNVs.

Alginate Cryogels as a Template for the Preparation of Edible Oleogels.

A high consumption of solid fats is linked to increased inflammation and a risk of cardiovascular diseases. Hence, in recent years, there has been increasing interest in the development of oleogels as a fat substitute in food products. Oleogels are edible gels that contain a large amount of liquid oils entrapped in a 3D network and that can potentially be applied to spreads, bakery goods, meat, and dairy products in order to lower their saturated fat content while maintaining a desirable food texture and mouthfeel. In this work, alginate cryogels were studied as templates for three different edible oils in the process of oleogel formation. Two different freezing regimes to obtain cryogels were employed in order to evaluate better the textural and morphological capabilities of cryogels to adsorb and retain edible oils. It was shown that rapid freezing in liquid nitrogen produces alginate cryogels with a lower density, higher porosity, and a greater ability to adsorb the tested oils. The highest uptake and holding oil capacity was achieved for olive oil, which reached a value of 792% and 82%, respectively. The best chewiness was found for an oleogel containing olive oil, whereas oleogels with the other two tested oils showed better springiness. Hence, the results presented in this work demonstrated that alginate-based cryogels can be effectively used as templates for oleogels and potentially find applications in the food industry.

Comparison of different freezing methods for micro-volume semen

BACKGROUND: Mico-volume semen freezing is essential and used popularly for fertility preservation of patients suffering cancer or undergoing male reproductive system related surgeries, and for other reasons that may risk fertility potential in ART cycles. However, clinicians and embryologists still face some unresolved technical and theoretical issues about the frozen-thawed efficiency. OBJECTIVE: To choose the appropriate freezing method for different volumes of normal semen samples. MATERIALS AND METHODS: We investigated the frozen-thawed outcomes of semen with different volumes (20 μL, 50 μL, 100 μL, 200 ??L, 500 μL and 1 mL) using two freezing methods (FLNV, static liquid nitrogen vapour cooling followed by liquid nitrogen preservation; RFLN, direct rapid freezing in liquid nitrogen) and analyzed the vitality, progressive motility and DNA fragmentation index of thawed sperm. RESULTS: We found that semen freezing with volumes more than 100 μL had better outcomes than volumes less than or equal to 50 μL after thawing. FLNV presented a higher efficiency for cryopreservation of semen with volumes less than 50 μL. CONCLUSION: For smaller (micro) volumes, the FLNV technique is better than the RFLN method.

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures.

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beamrapidly induces radiation damage of photosensitivecofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (> 0.1 mm3) has been grown. We demonstrate mounting of hydrogenated proteincrystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length. Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-of-flight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron data-only or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.

Comparison of methods for preservation of activated sludge samples for high-throughput nucleic acid sequencing and bacterial diversity analysis

Preservation of environmental samples is an important step in maintaining the original microbial community until the nucleic acid extraction in the laboratory. Here, we collected activated sludge samples to both immediately extract nucleic acids (control) and submit to different storage/preservation methods for 48 h before nucleic acids extraction: room temperature storage, storage on ice, direct storage at −20 °C, rapid freezing in liquid nitrogen followed by storage at −20 °C, and preservation with TRIzol®, RNAlater®, and Nucleic Acid Preservation (NAP) buffers. Bacterial community was compared by high-throughput 16S rRNA gene sequencing from total DNA and RNA. Among the evaluated methods, TRIzol® and rapid freezing with liquid nitrogen were the least indicated, since they led to significant changes in the microbial community structure and abundance of functional groups involved in ammonia metabolism. Compared to control, storage on ice and NAP preserved more than 80% and 75% of genera at the DNA and RNA level respectively, indicating that these methods are the most suitable for storage/preservation of activated sludge samples intended for nucleic acids sequencing.

DNA recovery from Droplet Digital™ PCR emulsions using liquid nitrogen.

Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a simple and environmentally friendly protocol for recovering DNA from ddPCR.

Decay behavior and stability of free radicals of silk fibroin with alkali/urea pretreatment induced by electron beam irradiation

Silk fibroin determines the perseverance of silk-based materials. The samples of silk fibroin were pretreated combined alkali/urea system with rapid freezing in liquid nitrogen. The structural changes of unpretreated and pretreated silk fibroins were investigated by XRD, SAXS and FTIR. The results indicate that part of the amorphous region was transformed into silk-II crystalline structure, and the radius of gyration decreased due to the folding of molecular chains by hydrogen bonds. The free radicals of unpretreated and pretreated silk fibroin induced by electron beam irradiation were studied by electron spin resonance (ESR). The results indicate that the radical concentration increased by 10 times at 25 kGy and attained 8.04*1019 spins·g−1 at 50 kGy after pretreatment, which is comparable to the saturated radical concentration of unpretreated silk fibroin above 200 kGy. And the half-life time of free radicals is 10 times longer than that of the unpretreated samples. The higher stability of the free radicals is considered to be related to the protein structure changes induced by pretreatment and this can provide convenience for silk irradiation grafting. The new pretreatment method may provide inspiration for designing protein structures to regulate free radical formation.

Phosphorylated Connexin 43 is Associated with Increased Susceptibility for Atrial Fibrillation in Mice Treated with a Single High Dose of Ibrutinib but not Acalabrutinib

Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase is a highly effective treatment of B‐Cell lymphoproliferative disorders. However, therapy has been shown to increase the incidence of Atrial Fibrillation (AF) in patients. Second generation drugs such as Acalabrutinib have been suggested to have less AF propensity, although there may be a signal for enhanced ventricular arrhythmias. Previously, we demonstrated an increased susceptibility to electrically induced AF with acute Ibrutinib treatment in mice. As conduction slowing due to alterations in connexin (Cx) content or phosphorylation state have been associated with AF, we hypothesised that such Cx changes would be one mechanism underlying AF susceptibility. Experiments followed the Canadian Council on Animal Care Guidelines for the Care and Use of Laboratory Animals, and the protocol was approved by the Animal Care Committee of Western University. Using previously published techniques, western blot was used to determine if a single dose by oral gavage (10 mg/kg), of Ibrutinib or Acalabrutinib reduced the content of Cx43 and/or Cx40 and/or the phosphorylated forms of Cx43 (S368 and Y265), increase of which have been shown to decrease Cx43 permeability and pore closure. Two‐month old C57BL/6, male (n=18), and female mice (n=18), were given Trappsol vehicle control, Ibrutinib or Acalabrutinib by oral gavage. After 90 min, they were anaesthetized with an intraperitoneal (IP) mixture of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). The heart was removed under anaesthesia, blotted to remove excess blood, the atria separated and weighed before rapid freezing in liquid nitrogen for later homogenization and protein analysis. Proteins were separated on polyacrylamide SDS gels, transferred to Polyvinylidene fluoride (PVDF) membranes and reacted with specific Cx antibodies followed by specific secondary antibodies linked to horseradish peroxidase (HRP). Content was detected by enhanced chemiluminescence (ECL). The content was expressed as the ratio to α‐actinin, as an internal loading control. Statistical evaluation was done with 2‐way ANOVA followed by post‐hoc Tukey test. Single dose Ibrutinib or Acalabrutinib did not alter the protein content of Cx43 or the content of Cx43 (Y265) or its ratio to total Cx43 in either male or female mice. However, the content of Cx43 (S368) and the ratio of the phosphorylated to total Cx43 content was higher in Ibrutinib but not the Acalabrutinib treated mice. Further the content of Cx40 was higher in Ibrutinib treated female than male mice. There was a treatment by sex interaction. These preliminary results suggests that off‐target phosphorylation at the S368 site of Cx43 may be one contributor to the acute pro‐arrhythmic effect of Ibrutinib, the magnitude of which may be sex‐dependent. The process by which this occurs and the lack of Acalabrutinib effect as well as potential ventricular targets that may contribute to ventricular arrhythmia requires further investigation.

Degree of moisture in seeds for the cryopreservation of orchids native to Brazil

ABSTRACT: The objective of this study was to determine the ideal moisture degree for the cryopreservation of Cattleya labiata Lindl. and Miltonia regnellii Rchb.f. orchid seeds. Orchid seeds were standardized in relation to the desired moisture degrees (4, 6, 8, 10, 12, and 15%) and were subjected to rapid freezing in liquid nitrogen (-196 °C) for 24 h; then, the seeds were defrosted and analyzed. For both orchid species, the seeds that were immersed in liquid nitrogen with 4% moisture content had the highest viability percentage, averaging 95% and 68% of viable seeds for C. labiata and M. regnellii, respectively. The seeds with 12% and 15% moisture content had no viable seeds. For the fresh mass of seedlings, there was no significant difference between moisture content treatments for either species.

Short Communication. An efficient axillary shoots propagate system for the living fossil plant - Wollemi pine (Wollemia nobilis)

Aim of study: The aim of the work was to determine the optimum axillary shoot induction system by optimizing different factors.Area of study: The different factors which were pretreatment methods, media types, kinds of cytokinins and dark treatmentMaterial and methods: After pretreating and sterilizing, the stems of Wollemi pine which about 3cm long were cultured on three kinds of media (GD, DCR AND ms) with different concentration and type of cytokines (BA, ZT and TDZ), as well as dark treatment (0, and 4 weeks) and then exposure to tissue culture room. The experiments were randomized block designed and the results were analyzed with ANOVA.Main results: Rapid freezing in liquid nitrogen was the most effective pretreatment method, dark treatment for the first 4-weeks of stem explants cultured in induction media could increase the frequency of axillary shoot induction significantly. The highest frequency of axillary shoot induction (63%) was obtained when explants were cultured in 1/2GD medium with 6.8 μM TDZ combining 4-weeks dark treatment. Research highlights: The optimized conditions for shoot production in vitro can be useful for conservation in vitro of Wollemia nobilis.Keywords: Wollemia nobilis; in vitro conservation; tissue culture; an endangered relict conifer.

Shoot tip cryopreservation by vitrification in Kaempferia galanga L. An endangered, overexploited medicinal plant in Tropical Asia

Shoot tips from the established in vitro shoot cultures of Kaempferia galanga L., an endangered medicinal plant were successfully cryopreserved using the vitrification procedure. Effect of concentration of sucrose for preculture, vitrification treatment with PVS2(Plant vitrification solution 2) and recovery medium on cryopreservation of shoot tip meristems has been analyzed. Overnight preculturing of the dissected shoot tips in MS medium containing 0.4 M sucrose, osmoprotection with loading solution for 20 minutes, dehydration with PVS2 for 20 minutes at 0 oC were found to be optimum among the various treatments tested. The cryoprotected shoot tips upon rapid freezing in LN (liquid nitrogen) followed by rapid thawing produced 50-60% survival and 30-40% regeneration rates. Incorporation of GA3 (giberellic acid) in the post-thaw culture medium was essential to induce shoot emergence in the initial phase. Subculturing of the recovered shoot tips in MS medium supplemented with 1.0 mgl -1 BA (6-benzyl adenine) and 0.5 mgl -1 NAA (α-naphthaleneacetic acid) resulted plantlet production. The regenerated shoots grew to mature plants were free of any morphogenetic variation upon field transfer.

Survival and genetic stability of Picea abies embryogenic cultures after cryopreservation using a pregrowth-dehydration method

A vitrification method enabled efficient cryopreservation of embryogenic tissue (ETs) of Norway spruce (Picea abies L.) at −196 °C in liquid nitrogen (LN). Correctly formed, normal somatic embryos were generated from ETs that had been thawed after removal from LN. The pregrowth-dehydration method involved preculture of ETs with sucrose (0.25–1.00 M) in the presence or absence of 10 μM abscisic acid (ABA), followed by air-drying for 2 h and rapid freezing in LN. Pretreatment of ETs with both sucrose and ABA promoted ET growth after preculture and thawing more effectively than treatment with sucrose alone. Survival of ETs after thawing from LN using both sucrose and ABA was 54.4 % compared to pretreatment with sucrose alone which was 20 %. Addition of ABA in the preculture medium also improved the ability of ETs to form cotyledonary stage somatic embryos. The somatic embryos, which had normal shoot and root apices and the correct number of cotyledons, were indistinguishable from regenerants obtained from control cultures. Genetic analysis of control and cryopreserved ETs, as well as somatic embryos derived from cryopreserved ETs, indicated that the cryopreservation method had no effect on any of the five microsatellite loci (SpAGC1, SpAGC2, SpAGG3, SpAC1H8, and SpAC1F7) tested. The cryopreservation protocol outlined should enable the long-term storage of valuable clones of Norway spruce in LN, potentially for hundreds of years.

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.

The evaluation of periodontal ligament cells of rat teeth after low-temperature preservation under high pressure

The purpose of this study was to evaluate the viability of periodontal ligament cells of rat teeth after low- temperature preservation under high pressure by means of MTT assay, WST-1 assay. 12 teeth of Sprague- Dawley white female rats of 4 week-old were used for each group. Both side of the first and second maxillary molars were extracted as atraumatically as possible under tiletamine anesthesia. The experimental groups were group 1 (Immediate extraction), group 2 (Slow freez- ing under pressure of 3 MPa), group 3 (Slow freezing under pressure of 2 MPa), group 4 (Slow freezing under no additional pressure), group 5 (Rapid freezing in liquid nitrogen under pressure of 2 MPa), group 6 (Rapid freezing in liquid nitrogen under no additional pressure), group 7 (low-temperature preservation at 0℃ under pressure of 2 MPa), group 8 (low-temperature preservation at 0℃ under no additional pres- sure), group 9 (low-temperature preservation at -5℃ under pressure of 90 MPa). F-medium and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in 37℃ water bath, then MTT assay, WST-1 assay were processed. One way ANOVA and Tukey HSD method were performed at the 95% level of confidence. The values of optical density obtained by MTT assay and WST-1 were divided by the values of eosin staining for tissue volume standardization. In both MTT and WST-1 assay, group 7 (0℃/2 MPa) showed higher viability of periodontal ligament cells than other group (2-6, 8) and this was statistically significant (p < 0.05), but showed lower viability than group 1, immediate extraction group (no statistical significance). By the results of this study, low-temperature preservation at 0℃ under pressure of 2 MPa suggest the possibility for long term preservation of teeth. ABSTRACT

Formation of specialized propagules resistant to desiccation and cryopreservation in the threatened moss Ditrichum plumbicola (Ditrichales, Bryopsida).

Successful cryopreservation of bryophytes is linked to intrinsic desiccation tolerance and survival can be enhanced by pre-treatment with abscisic acid (ABA) and sucrose. The pioneer moss Ditrichum plumbicola is naturally subjected to desiccation in the field but showed unexpectedly low survival of cryopreservation, as well as a poor response to pre-treatment. The effects of the cryopreservation protocol on protonemata of D. plumbicola were investigated in order to explore possible relationships between the production in vitro of cryopreservation-tolerant asexual propagules and the reproductive biology of D. plumbicola in nature. Protonemata were prepared for cryopreservation using a four-step protocol involving encapsulation in sodium alginate, pre-treatment for 2 weeks with ABA and sucrose, desiccation for 6 h and rapid freezing in liquid nitrogen. After each stage, protonemata were prepared for light and electron microscopy and growth on standard medium was monitored. Further samples were prepared for light and electron microscopy at intervals over a 24-h period following removal from liquid nitrogen and re-hydration. Pre-treatment with ABA and sucrose caused dramatic changes to the protonemata. Growth was arrested and propagules induced with pronounced morphological and cytological changes. Most cells died, but those that survived were characterized by thick, deeply pigmented walls, numerous small vacuoles and lipid droplets in their cytoplasm. Desiccation and cryopreservation elicited no dramatic cytological changes. Cells returned to their pre-dehydration and cryopreservation state within 2 h of re-hydration and/or removal from liquid nitrogen. Regeneration was normal once the ABA/sucrose stimulus was removed. The ABA/sucrose pre-treatment induced the formation of highly desiccation- and cryopreservation-tolerant propagules from the protonemata of D. plumbicola. This parallels behaviour in the wild, where highly desiccation-tolerant rhizoids function as perennating organs allowing the moss to endure extreme environmental conditions. An involvement of endogenous ABA in the desiccation tolerance of D. plumbicola is suggested.

The Metallo-β-lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes able to hydrolyze and inactivate most beta-lactam antibiotics. The large diversity of active site structures and metal content among MbetaLs from different sources has limited the design of a pan-MbetaL inhibitor. Here we report the biochemical and biophysical characterization of a novel MbetaL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MbetaLs. Contrasting all other related MbetaLs, GOB-18 is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MbetaLs.

Resistance to Nitric Oxide-induced Necrosis in Heme Oxygenase-1 Overexpressing Pulmonary Epithelial Cells Associated with Decreased Lipid Peroxidation

Increased expression of heme oxygenase-1 (HO-1) increases NO resistance in several cell types, although the biochemical mechanism for this protection is unknown. To address this issue, we have measured different molecular markers of nitrosative stress in three stably transfected cell lines derived from the human lung epithelial line A549: two lines that overexpress rat HO-1 (L1 and A4), and a control line with the empty vector (Neo). Compared with the control Neo cells, L1 and A4 cells had, respectively, 5.8- and 3.8-fold greater HO activity accompanied by increased resistance to NO-induced necrosis. Compared with the Neo control, the HO-1-overexpressing cells also showed significantly less lipid peroxide formation and decreased perturbation of transition metal oxidation and coordination states following a cytotoxic NO exposure. These effects were blocked by the HO-1 inhibitors Zn- and Sn-protoporphyrin IX. In contrast, HO-1 overexpression did not significantly affect total reactive oxygen or nitrogen species, the levels of the nucleobase deamination products in DNA (xanthine, inosine, and uracil) following NO exposure, or NO-induced protein nitration. While increased HO-1 activity prevented NO-induced fluctuations in transition metal homeostasis, addition of an iron chelator decreased NO toxicity only slightly. Our results indicate that lipid peroxidation is a significant cause of NO-induced necrosis in human lung epithelial cells, and that the increased NO survival of L1 cells is due at least in part to decreased lipid peroxidation mediated by HO-1-generated biliverdin or bilirubin.

Molecular, Biochemical, and Functional Characterization of a Nudix Hydrolase Protein That Stimulates the Activity of a Nicotinoprotein Alcohol Dehydrogenase

The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.

CONSERVATION OF BANANA GERMPLASM THROUGH CRYOPRESERVATION

Since most banana landraces (Musa spp.) do not produce seed and are vegetatively propagated, germplasm must be maintained clonally. Therefore field and in vitro collections were established. Currently, the world's largest banana collection (1080 accessions) is kept as in vitro proliferating meristems under limited growth conditions at the laboratory of Tropical Crop Improvement of K.U.Leuven (Katholieke Universiteit Leuven, Belgium). Cryopreservation is a welcome alternative since during storage at ultra-low temperatures (-196°C), all metabolical, chemical and physical processes are arrested. As such, the material under storage does not need to be subcultured, thus avoiding risks of contamination and human error, and no somaclonal variation will take place. We developed an extremely simple cryopreservation system relying on a two-week-preculture phase of highly proliferating meristems on media with elevated sugar concentrations followed by rapid freezing in liquid nitrogen. This cryopreservation protocol was successfully tested on 20 accessions belonging to different genomic groups. Normal plants were regenerated from each of them. Regeneration frequencies vary from 7.4 to 68.9 %, depending on the cultivar. The mode of action of the sugar preculture phase with respect to the cryopreservation ability of different cultivars is under investigation. It is demonstrated that lipid, sugar and protein contents of non-precultured and sugar precultured plants differ.

Cryopreservation of embryogenic tissue of a range of genotypes of sweet potato (Ipomoea batatas [L] Lam.) using an encapsulation protocol.

Embryogenic tissue of nine sweet potato [Ipomoea batatas (L.) Lam] genotypes from Asia, Africa and the Americas was established from in vitro axillary buds on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. Embryogenic aggregates, 1.0-2.0 mm in diameter, were encapsulated in alginate gel, precultured on medium containing elevated levels of sucrose and dehydrated prior to rapid freezing in liquid nitrogen. The maximum survival of embryogenic tissue ranged from 4% to 38%, depending on the genotype. With the incorporation of a slow-cooling step, survival was generally much higher than that obtained after rapid freezing alone. Five of eight genotypes tested with this protocol gave survival percentages in excess of 55%, and a further two in excess of 33%, all after evaporative dehydration. The most effective sucrose treatment(s), however, varied with the genotype.