Cryo letters最新文献

Background: Cicer microphyllum Benth. is a crop wild relative (CWR) of chickpea (C. arietinum L.), that possess useful genes for cold and drought tolerance. The species is being conserved in the In Vitro Active Genebank for short- to medium-term conservation. Cryopreservation would be a useful complementary approach for its long-term conservation.

Objective: The current work aimed to develop an efficient cryoconservation protocol for cryobanking of C. microphyllum shoot tips.

Materials and methods: In vitro shoot tips excised from 4-month old shoot cultures grown on B5 + 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO₃ medium were cryoconserved using a droplet-vitrification technique. Post-thaw regrowth was evaluated after: (i) preculture medium (B5 basal, B5 + 3, 4, 6 and 10% sucrose), (ii) preculture incubation temperature (25 ± 2, 10, 8 and 22/5 degree C), (iii) PVS2 duration (10, 20, 30. 40, 50 and 60 min) and (iv) regrowth medium (B5) supplemented with 0.5 mg/L KIN + 0.1 NAA mg/L; 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO₃; 0.2 mg/L BAP + 10 mg/L AgNO₃; 0.2 mg/L BAP + 20 mg/L AgNO₃ and 0.2 mg/L BAP + 30 mg/L AgNO₃.

Results: In vitro shoot tips grown on B5 + 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO₃, precultured on B5 + 6% sucrose at 10 degree C for 3 days, followed by PVS2 treatment for 20 min, unloading solution for 60 min and regrowth on B5 + 0.2 mg/L BAP + 20 mg/L AgNO₃ resulted in highest survival (57%) and regrowth (40%) after cryoconservation.

Conclusion: The standardized protocol was successfully used for cryobanking of in vitro shoot tips of C. microphyllum in the In Vitro Base Genebank of ICAR-NBPGR, New Delhi. Doi.org/10.54680/fr23610110412.

Background: The search for compounds that can prevent cold stress-attributed apoptosis is of immediate interest. In this regard, the use of neuropeptides, in particular synthetic leu-enkephalin, as protectors is promising, due to their ability to prevent the development of apoptosis under some stresses.

Objective: To study apoptotic phenomena after cold stress and to evaluate the protective effect of dalargin on these processes.

Materials and methods: The study was performed on a L929 fibroblast line. The impact of cold stress and the protective effect of dalargin on apoptosis against cold stress were evaluated using morphological parameters, distortion of cell membrane asymmetry and release of cytochrome C into the cell cytoplasm. To assess the proliferative potential of fibroblasts, mechanical damage to the monolayer was modeled as a scratch wound.

Results: The study showed that cold stress induced apoptosis in L929 fibroblasts and reduced proliferation in the fibroblast monolayers. Conspicuous apoptotic changes were found to develop only after a certain time after cold exposure, when the cells were returned to normothermia. Dalargin was demonstrated to exert a protective effect on proliferation and against apoptosis during cold stress. Using the opioid receptor antagonist naloxone, we revealed that the protective mechanism of dalargin appeared to be due to activation of delta-opioid receptors of L929 fibroblasts, which affected the development of apoptosis.

Conclusion: In addition to their fundamental value, these findings are of practical importance since neuropeptides, in particular dalargin, added to perfusion solutions and media for hypothermic preservation of organs and cells, can improve their efficiency. Doi.org/10.54680/fr23610110212.

Background: Cryopreservation currently represents the most suitable strategy for the long-term conservation of plant germplasm. While much effort has focused on the development of protocols to enable successful cryostorage, there are few, if any reports, that consider the effect of cryogenic temperatures on the phytohormone status of the seed and developing seedlings.

Objective: To investigate the effect of cryopreservation on external seed coat features as well as levels of indole-3-acetic acid (IAA), abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in maize.

Materials and methods: Two groups of seeds at 6% moisture content were compared: one was maintained at 4 degree C (control) while the other was exposed to LN within cryo-vials.

Results: Seeds exposed to cryogenic temperatures were characterized by the presence of large cracks in the seed coat compared with control seeds. Cryogenic exposure also resulted in a reduction in biomass and plant height. Results from the phytohormone analysis showed an initial reduction in the levels of IAA, ABA and ACC after 7 days of growth followed by sharp increase in levels relative to the control by 14 days. Whilst the roles of ABA and ethylene (and by extension, its precursor ACC) are well studied as stress response molecules, much less is known about the potentially vital role of auxins in regulating plant growth under conditions of low temperature stress.

Conclusion: It is postulated that the interaction of all three hormones modulate crosstalk between various stress responses and recovery pathways to ameliorate the damage caused by freezing stress and enable plant survival. Given the dearth of information on phytohormones in cryobiology, more studies are needed to fully elucidate these relationships in the context of freezing stress caused by liquid nitrogen. Doi.org/10.54680/fr23610110612.

Background: Amides are low molecular weight cryoprotectants. N-methylacetamide (MA) is one of the cryoprotectant agents in this group.

Objective: To investigate the cryoprotective effect of MA in rabbit semen.

Materials and methods: For this purpose, six ejaculates from six New Zealand rabbits were collected and pooled using an artificial vagina. Pooled semen was divided into four equal parts and diluted with TCG+ egg yolk. CPA was added to form the following groups: Control with 6% DMSO; Group 1 with 1% MA; Group 2 with 2% MA; and Group 3 with 3% MA. After the addition of CPA, the semen eqilibration procedure was started. Sperm were then drawn into 0.25 mL straws, frozen by automatic semen freezing and stored in a liquid nitrogen container. Pipettes were thawed after 24 h and analyses were performed.

Results: Total, progressive and rapid motility values of the Control group were higher than those of the MA groups (p<0.05). However, there was no statistical difference between the Control and Group 2 in terms of these parameters. While there was no statistical difference between the groups in terms of acrosome damage and mitochondrial membrane potential, the best results were observed in Control, Group 2, Group 1 and Group 3, respectively. When we compared all groups, no difference was found in terms of MDA, CAT and GSH-Px. There was a statistical difference between Group 3 and the Control in terms of GSH level (p<0.05).

Conclusion: DMSO appeared to be more useful for the cryopreservation of rabbit semen compared to MA. Doi.org/10.54680/fr23610110812.

Background: Tarenaya spinosa is a medicinal species used for treating respiratory and inflammatory diseases. Various biotechnological studies have been developed for in vitro establishment of plants and long-term conservation of this species.

Objective: This study aimed to establish a new cryopreservation protocol using the D cryo-plate technique for in vitro-grown shoot tips of T. spinosa.

Materials and methods: Different steps of the cryopreservation procedure were evaluated in this work: preculture; sucrose concentration of calcium alginate gel; concentration and time of exposure to osmoprotective loading solution; time of exposure to silica gel; and regrowth on recovery medium.

Results: The optimal procedure included preculture with increasing sucrose concentration (from 0.25 to 0.50 M), encapsulation and dehydration over silica-gel for 60 min. Increasing sucrose concentration in the loading solution or exposure duration had a negative effect on recovery of cryopreserved shoot tips. However, the association of calcium alginate gel enriched with 0.6 M sucrose with post-rewarming culture with BAP for 2 weeks resulted in the most efficient cryopreservation protocol (76% survival and 70% shoot recovery).

Conclusion: The plants developed after cryopreservation maintained their in vitro multiplication capacity and demonstrated the efficiency of long-term conservation by D cryo-plate technique for T. spinosa. Doi.org/10.54680/fr23610110512.

The process of freezing biological material at extremely low temperatures is known as cryopreservation. To ensure the preservation of cells and tissues over an extended period of time, low temperatures are applied since biological processes, including the biochemical ones, come to a halt under cryogenic conditions and thus it is possible to maintain their structural and functional integrity. The field of cryopreservation gained more prominence in the 20th century and emerged as an unavoidable technology for different applications such as cell therapy, tissue engineering, or assisted fertilization. In this work we provide an overview of various technologies in the field of cryotechnology with regard to the freezing, storage and thawing of living cells. The first part covers the freezing process, starting with cryoprotective agents regarding their protection mechanisms and compositions, passing by cryo-imaging, micro-fluidic systems, and the currently available freezing and biobanking equipment. The second part focusses on the thawing process as well as the hypothermic preservation for the short-term storage of biological materials and constructs. Doi.org/10.54680/fr23610110112.

Background: Chitin is the second largest carbon source on the earth, and chitosan oligosaccharides produced by its degradation have good application prospects in medicine, cosmetics, and agricultural production.

Objective: The discovery of a chitinase with high efficiency, high stability and clear degradation mechanism is of great help to promote the research of chitin derivatives and the development of the industrial chain.

Materials and methods: In this experiment, a low-temperature chitinase-producing strain Photobacterium sp. LG-29 was isolated from deep-sea mud in the Bohai Sea, and studied by means of molecular biology, biochemistry and bioinformatics.

Results: Purification of chitinase yielded an enzyme solution with a concentration of 0.918 mg per mL and a specific activity of 21.036 U per mg. The optimum action temperature is 35 degree C, and it is still active at 4 degree C, showing low-temperature enzymatic activity, and also has certain thermal stability. The optimum pH is 8.0, and it maintains more than 70% of the enzyme activity at pH 11, which is very stable in an alkaline environment. Mn²⁺, Ca²⁺, and Mg²⁺ are the main activators of enzymes, while Fe²⁺, Zn²⁺, etc. have extremely significant inhibitory effects on enzymes. The Km and Kcat of chitinase were determined to be 269.05 μmol/L and 0.49 min^-1, respectively. Chitinase PbCHI5 has both endonuclease and exonuclease activity. The theoretical pI of the enzyme is 4.16, which is a stable hydrophilic protein.

Conclusion: This experiment laid a theoretical foundation for the development and utilization of new low-temperature chitinases. Doi.org/10.54680/fr23510110212.

Background: Lilium candidum L. is a perennial ornamental plant that has various medicinal properties and is used in the cosmetic industry. The species is facing threats from urbanization and climate change and requires urgent protection. The most secure and efficient technology for the long-term storage of plant genetic resources is cryopreservation, which involves preserving genetic material at extremely low temperatures.

Objective: Today, plant biodiversity is endangered because of the narrowing of its natural distribution areas and/or destruction for different purposes. This study concentrated on creating a cryopreservation process using shoot tips and calluses as explant sources for the long-term conservation of L. candidum species.

Materials and methods: Populations of L. candidum naturally distributed from three different regions of Turkey (Kepsut, Balikesir; the area surrounding Bafa Lake, Aydin; and Fethiye-Mugla) were grown in vitro to supply shoot tip and callus explants. Prior to freezing by droplet-vitrification and vitrification techniques, shoot tips and calluses were treated with MS nutritional medium supplemented with 0.4 M sucrose 7 g per L agar and plant vitrification solution 2 (PVS2).

Results: Cryopreserved shoot tips showed the highest levels of regeneration (71.8%) after a PVS2 treatment of 90 min, while calluses showed the highest levels of regrowth (63.9%) after a PVS2 exposure of 60 min.

Conclusion: High levels of regrowth are produced when the various cryopreservation procedures described here are used to preserve both shoot tip and callus explants. This potentially makes the method promising for the long-term preservation of endangered L. candidum varieties. Doi.org/10.54680/fr23510110512.

Background: N. wightii (Leguminosae) is valued as a cover crop and as a potential source of protein in food insecure countries. However, plantlet establishment is limited by physical dormancy. Our previous work has shown that exposure of N. wightii seeds to cryogenic temperatures is able to overcome physical dormancy.

Objective: The current study is an extension of that work where the field performance and nutritional composition of plants regenerated from N. wightii seeds was investigated.

Results: It was evident that plants regenerated from cryopreserved seeds displayed faster growth rates than those from control seeds. In addition, cryopreservation did not alter the nutritional profile of plants produced from cryo-stored seeds.

Conclusion: Collectively, the results indicate that cryopreservation serves as a suitable strategy for the preservation of seeds of N. wightii with the added benefit of also serving as a dormancy breaking mechanism upon retrieval from cryogenic temperatures. Doi.org/10.54680/fr23510110712.

Background: Semen cryopreservation is a complex process during which there is alteration in the expression of sperm and seminal plasma proteins, molecular weight of protein or loss of membrane proteins during the process. In order to compensate for these changes, different membrane stabilizers are used in freezing semen extenders. However, there is scarcity of such studies during cryopreservation of goat semen.

Objective: To investigate the effect of membrane stabilizers on sperm membrane protein expression during cryopreservation of goat semen.

Materials and methods: A total of 36 semen ejaculates from nine Assam Hill Goat bucks aged 2 to 2.5 years was collected by artificial vagina method. Three membrane stabilizers, each at two different concentrations viz. 50 and 80 mM sucrose, 50 and 100 mM trehalose, and 100 and 150 ng per mL IGF-1 (insulin-like growth factor 1 protein) were added to Tris-citric acid fructose egg yolk glycerol (TCFEYG) extender and semen samples were cryopreserved. The sperm membrane protein profile was studied in fresh and cryopreserved semen by SDS-PAGE.

Results: SDS- PAGE of sperm membrane extract of fresh semen revealed the presence of 24 protein bands with molecular weights ranging from 10 kDa to 240 kDa. Samples supplemented with 50 mM sucrose and 80 mM sucrose revealed 21 protein bands with molecular weights ranging from 10 kDa to 240 kDa. All the 21 protein bands were same as those observed in the sperm membrane of fresh spermatozoa, except that the 23 kDa, 29 kDa and 42 kDa bands were absent in frozen semen. Similarly, frozen semen extended with 50 mM trehalose and 100 mM trehalose revealed 22 protein bands with molecular weights ranging from 10 kDa to 240 kDa, but lacking the 29 kDa and 42 kDa bands. Proteins with molecular weights of 29 kDa, 130 kDa and 240 kDa were absent in frozen semen supplemented with 100 ng per mL IGF-1 and 150 ng per mL IGF-1.

Conclusion: The present study revealed that supplementation of tris basic extender with trehalose at 100 mM and or IGF-1 at 100 ng/mL or 150 ng per mL improves the post-thaw semen characteristics and protects certain fertility related sperm membrane proteins. Doi.org/10.54680/fr23510110612.