Ultra-low temperature freezers (typically operating between -80°C and -150°C, with some deep-freezing equipment reaching as low as -196°C using liquid nitrogen) are one of the core technological tools for germplasm preservation in gene banks. By creating an extremely low-temperature environment, these freezers significantly reduce the metabolic activity of biological samples, inhibit enzymatic reactions and microbial contamination, thereby ensuring long-term genetic stability of germplasm resources. Below is a detailed explanation of their specific applications in gene banks, covering aspects such as application scenarios, technical advantages, key operational points, and challenges.

 I. Core Requirements for Germplasm Preservation in Gene Banks

Germplasm resources (such as plant seeds, animal sperm/embryos, microbial strains, DNA/RNA samples, etc.) serve as carriers of biological genetic diversity. Their preservation must meet three major requirements: long-term stability (to avoid degradation or variation of genetic material), traceability (complete sample information), and high survival rate (normal functionality after revival). Traditional preservation methods (like room-temperature drying or 4°C refrigeration) are only suitable for short-term storage or certain hardy samples. In contrast, ultra-low temperature environments can effectively halt all biochemical reactions, enabling "indefinite" preservation.

 II. Specific Applications of Ultra-Low Temperature Freezers in Germplasm Preservation

 1. Plant Germplasm Resource Preservation

Plant germplasm includes seeds, pollen, embryos, callus tissues, shoot tips, etc. Among these, recalcitrant seeds (such as those from cocoa and mango) are not resistant to dehydration and low temperatures (they suffer damage even at 0-10°C), making traditional cold storage (-20°C) unsuitable. However, ultra-low temperature freezers (ranging from -80°C to -196°C), combined with vitrification freezing technology (which uses high concentrations of cryoprotectants to vitrify intracellular solutions and prevent ice crystal damage), enable long-term storage. For example:

- Seeds of model plants like *Arabidopsis thaliana* can be stored at -80°C for over 20 years while maintaining a germination rate above 90%.

- Excised tissues of rare medicinal plants (such as Dendrobium and Saussurea involucrata) can regenerate into whole plants through tissue culture after ultra-low temperature preservation, mitigating the risk of wild extinction.

 2. Animal Germplasm Resource Preservation

Animal germplasm encompasses semen, embryos, somatic cells (like fibroblasts), and germline stem cells (sperm/eggs). Ultra-low temperature preservation is a critical means for protecting livestock genetic resources and rescuing endangered species:

- Livestock Breeding: Semen from cattle and pigs can be stored in liquid nitrogen at -196°C for decades and used for artificial insemination and breed improvement.

- Endangered Species Conservation: Sperm or somatic cells from giant pandas and Siberian tigers, preserved under ultra-low temperatures, can help restore populations through cloning or assisted reproductive technologies.

- Human Genetic Resources: Umbilical cord blood stem cells and tumor tissue samples stored at -80°C or in liquid nitrogen are vital for disease research and precision medicine.

 3. Microbial and Viral Germplasm Preservation

Bacteria, fungi, viruses, and other microorganisms require long-term maintenance of their activity and genetic traits. Ultra-low temperature freezers (especially -80°C units and liquid nitrogen tanks) are the mainstream preservation methods:

- Standard Strains (such as *Escherichia coli* K-12 and *Saccharomyces cerevisiae*): When mixed with glycerol (15-30%) as a cryoprotectant and frozen at -80°C, they remain viable for over 10 years with stable phenotypes upon revival.

- Pathogenic Microorganisms (like novel coronavirus strains and *Mycobacterium tuberculosis*): Must be stored below -80°C to prevent mutations, ensuring accuracy in vaccine development and diagnostic reagents.

- Industrial Microorganisms (such as enzyme-producing strains and fermentation cultures): Ultra-low temperature storage prevents degradation during subculturing, guaranteeing stability in industrial production.

 4. Nucleic Acid and Biobank Preservation

Molecular samples like DNA, RNA, and proteins in gene banks are highly sensitive to temperature. Ultra-low temperature environments suppress nuclease activity (e.g., DNase, RNase), preventing degradation:

- Genomic DNA: Purified DNA samples stored at -80°C can remain intact for over 20 years, with minimal fragmentation and base damage.

- RNA Samples: Require storage in liquid nitrogen at -196°C (as -80°C may still allow slow degradation), especially crucial for transcriptomics research.

- Single-Cell Samples: Single-cell suspensions, protected by cryoprotectants and stored under ultra-low temperatures, support cutting-edge techniques like single-cell sequencing.

 III. Technical Advantages of Ultra-Low Temperature Preservation

Compared to traditional methods (such as agar slant subculturing or freeze-drying), ultra-low temperature freezers offer irreplaceable advantages:

- Long-Term Stability: Theoretically enables "permanent" storage (samples in liquid nitrogen can last for decades or even centuries).

- Minimal Metabolic Loss: At temperatures below -80°C, cellular metabolism completely stops, avoiding genetic drift or phenotypic loss during subculturing.

- Broad Applicability: Suitable for almost all types of germplasm resources, including plants, animals, microorganisms, and nucleic acids.

- Operational Flexibility: Supports handling from small batches (e.g., centrifuge tubes, cryovials) to large-scale operations (using programmable coolers and liquid nitrogen tanks).

 IV. Key Operations and Precautions

To ensure effective ultra-low temperature preservation, strict control over the following aspects is necessary:

1. Pretreatment: Samples must undergo quality checks (e.g., viability, purity) and be treated with appropriate cryoprotectants (like DMSO or glycerol) to prevent ice crystal damage.

2. Cooling Rate: Most cells require slow cooling (at -1°C/min down to -30°C, followed by rapid cooling to -80°C) to balance intracellular and extracellular osmotic pressure.

3. Storage Management: Use barcode/RFID labels for sample identification, coupled with database records of location, source, and preservation time to avoid mix-ups.

4. Equipment Maintenance: Regularly calibrate temperatures (tolerance ±5°C), inspect cooling systems (compressors, liquid nitrogen replenishment), and equip with backup power and alarm systems to guard against power outages or temperature fluctuations.

5. Revival Verification: Periodically test revived samples for viability (germination rate, colony formation ability) and genetic integrity (via SSR markers or whole-genome sequencing) to confirm preservation effectiveness.

 V. Future Developments

- Energy-Saving Technologies: Innovations like variable-frequency refrigeration and heat recovery systems will reduce energy consumption.

- Automated Equipment: Integration of robotic arms and Automated Storage/Retrieval Systems (AS/RS) will enhance sample management efficiency.

- Hybrid Preservation Strategies: Combining ultra-low temperature with freeze-drying (for some microbes) or liquid phase storage (e.g., silicone oil) will expand applicability.

 Conclusion

Ultra-low temperature freezers are the "core infrastructure" of gene bank germplasm preservation. Through extreme low-temperature environments, they achieve long-term stabilization of germplasm resources, supporting advancements in biodiversity conservation, breeding innovation, and disease research. As technology progresses, their applications will become more efficient and intelligent, providing stronger guarantees for the global protection and utilization of genetic resources.