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π“†ž Germplasm Storage and Cryopreservation - In vitro strategies, short, medium and long term (cryopreservation)

Germplasm Storage and Cryopreservation - In vitro strategies, short, medium and long term (cryopreservation)

❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ 


1. Introduction

Germplasm refers to the total genetic resources of plants, including seeds, pollen, embryos, tissues, organs, and cells capable of regeneration.

Conservation of germplasm is essential for:

Preservation of genetic diversity

Crop improvement and breeding programs

Conservation of endangered and elite genotypes

Protection against biotic and abiotic stresses

In-vitro germplasm storage offers a reliable
 alternative to field gene banks, especially for vegetatively propagated, sterile, or recalcitrant seed–producing plants.


2. Need for In-vitro Germplasm Storage

Loss of genetic resources due to climate change, pests, diseases

High maintenance cost and risk in field gene banks

Somatic mutations and contamination risks

Requirement for pathogen-free elite planting material

Conservation of species with recalcitrant or no seeds

3. In-vitro Germplasm Conservation Strategies


In-vitro conservation involves maintaining plant material under aseptic conditions with reduced growth or metabolic activity.

Classification based on duration : 

Short-term storage
Medium-term storage
Long-term storage (Cryopreservation)


4. Short-Term In-vitro Germplasm Storage

Duration:  Few weeks to 3–6 months

Stored material  


Shoot cultures
Callus
Cell suspensions
Protoplasts

Strategies  


Regular subculturing
Transfer to fresh medium at fixed intervals
Standard growth conditions
Normal MS medium
25 ± 2°C
16 h photoperiod


Advantages  

Simple and rapid
Suitable for active research and multiplication

Limitations

Labor-intensive
Risk of contamination
Increased chance of somaclonal variation
High maintenance cost

5. Medium-Term In-vitro Germplasm Storage (Slow-Growth Storage)

Duration : 6 months to 2–5 years

Principle
Reduction of plant growth and metabolic activity without affecting viability or genetic stability.

Methods of Slow-Growth Storage

1. Low Temperature Storage

Temperature: 4–15°C
Reduces respiration and cell division
Widely used for potato, banana, cassava

2. Osmotic Growth Retardation

Addition of osmotic agents:
Mannitol
Sorbitol
High sucrose concentration
Reduces water availability

3. Growth Retardants

Abscisic acid (ABA)
Paclobutrazol
CCC (Chlormequat chloride)

4. Reduced Nutrient Concentration

Diluted MS medium (½ or ¼ strength)
Reduced nitrogen and phosphate levels

5. Reduced Light Intensity
Low light or dark conditions

Advantages

Less frequent subculturing
Reduced cost and labor
Maintains clonal fidelity

Limitations

Not suitable for very long-term storage
Genotype-specific response
Risk of physiological disorders


6. Long-Term Germplasm Storage – Cryopreservation

Definition
Cryopreservation is the storage of viable plant material at ultra-low temperatures (–196°C) in liquid nitrogen (LN), where all metabolic activities are completely arrested.


Plant Materials Used
Shoot tips / apical meristems
Somatic embryos
Zygotic embryos
Pollen
Seeds
Cell suspensions
Protoplasts

7. Principles of Cryopreservation

Avoidance of ice crystal formation
Cellular dehydration
Vitrification (glass formation)
Use of cryoprotectants


8. Cryoprotectants


Protect cells from freezing injury.
Penetrating cryoprotectants
DMSO
Glycerol
Ethylene glycol
Non-penetrating cryoprotectants
Sucrose
Mannitol
Sorbitol
PEG
9. Methods of Cryopreservation

1. Slow Freezing Method

Gradual cooling (0.5–1°C/min)
Storage in LN
Suitable for cell suspensions

2. Rapid Freezing

Direct immersion into LN
Used for pollen and seeds

3. Vitrification

Use of highly concentrated cryoprotectant solutions (e.g., PVS2)
Avoids ice formation
Widely used for shoot tips

4. Encapsulation–Dehydration

Explants encapsulated in alginate beads
Partial dehydration
Direct freezing in LN

5. Encapsulation–Vitrification

Combination of encapsulation and vitrification
High survival rate

6. Droplet Vitrification

Explants placed in droplets on aluminum foil
Ultra-rapid cooling
High recovery efficiency

10. Thawing and Recovery

Rapid thawing at 35–40°C
Removal of cryoprotectants
Culture on recovery medium with growth regulators
Gradual acclimatization


11. Advantages of Cryopreservation

Long-term (theoretical indefinite) storage
Genetic stability maintained
Minimal space requirement
Protection from contamination and mutations
Ideal for endangered species

12. Limitations of Cryopreservation

High initial cost
Requires technical expertise
Species- and tissue-specific protocols
Post-thaw regeneration problems

13. Applications
Conservation of endangered plant species
Maintenance of elite germplasm
Backup for field and in-vitro collections
Preservation of transgenic lines
Support to plant breeding programs

14. Conclusion
In-vitro germplasm storage and cryopreservation are powerful tools for plant genetic resource conservation. Short- and medium-term storage ensures safe maintenance of actively used germplasm, while cryopreservation provides a secure, cost-effective, and genetically stable solution for long-term conservation, making it indispensable in modern plant biotechnology.




Germplasm Storage & Cryopreservation – 50 MCQ


1. Germplasm refers to
A. Plant nutrients
B. Genetic material of plants
C. Plant hormones
D. Culture medium
✔ Answer: B

2. The main objective of germplasm conservation is
A. Increase yield
B. Preserve genetic diversity
C. Produce hybrids
D. Eliminate mutations
✔ Answer: B

3. In-vitro germplasm storage is especially useful for
A. Seed propagated crops
B. Recalcitrant seed species
C. Annual crops only
D. Weeds
✔ Answer: B

4. Short-term in-vitro storage usually lasts for
A. 1–2 weeks
B. 3–6 months
C. 5–10 years
D. Indefinitely
✔ Answer: B

5. Frequent subculturing is required in
A. Cryopreservation
B. Medium-term storage
C. Short-term storage
D. Seed bank
✔ Answer: C

6. Major drawback of short-term in-vitro storage is
A. High genetic stability
B. Risk of contamination
C. No regeneration
D. Low cost
✔ Answer: B

7. Medium-term storage is also known as
A. Fast growth storage
B. Active storage
C. Slow growth storage
D. Cryogenic storage
✔ Answer: C

8. Growth retardation in medium-term storage is achieved by
A. Increasing temperature
B. Increasing nutrients
C. Reducing metabolic activity
D. Adding fertilizers
✔ Answer: C

9. Temperature commonly used for slow growth storage is
A. 25–30°C
B. 37°C
C. 4–15°C
D. –196°C
✔ Answer: C

10. Osmotic agents used in slow growth storage include
A. Auxins
B. Cytokinins
C. Mannitol and sorbitol
D. Vitamins
✔ Answer: C

11. Abscisic acid (ABA) is used in
A. Rapid growth
B. Slow growth storage
C. Cryopreservation only
D. Seed germination
✔ Answer: B

12. Reduced nutrient concentration in medium-term storage helps in
A. Enhancing photosynthesis
B. Delaying senescence
C. Reducing growth rate
D. Increasing mutations
✔ Answer: C

13. Long-term storage of germplasm is achieved by
A. Subculturing
B. Cold storage
C. Cryopreservation
D. Field gene banks
✔ Answer: C

14. Cryopreservation temperature is
A. 0°C
B. –20°C
C. –80°C
D. –196°C
✔ Answer: D

15. Liquid nitrogen is used in cryopreservation because
A. It is cheap
B. It stops metabolic activity
C. It promotes growth
D. It is sterile
✔ Answer: B

16. Major problem during freezing of cells is
A. Desiccation
B. Ice crystal formation
C. Cell elongation
D. Photosynthesis
✔ Answer: B

17. Cryoprotectants are used to
A. Kill cells
B. Increase temperature
C. Prevent freezing injury
D. Promote mutations

✔ Answer: C

18. A penetrating cryoprotectant is
A. Sucrose
B. Mannitol
C. DMSO
D. PEG
✔ Answer: C

19. Non-penetrating cryoprotectant is
A. Ethylene glycol
B. Glycerol
C. DMSO
D. Sucrose
✔ Answer: D

20. Vitrification refers to
A. Ice formation
B. Glassy state without ice
C. Cell division
D. Cell death
✔ Answer: B

21. PVS2 is used in
A. Slow freezing
B. Rapid thawing
C. Vitrification
D. Seed germination
✔ Answer: C

22. Encapsulation–dehydration involves
A. Free cells
B. Alginate bead formation
C. Pollen storage
D. Field storage
✔ Answer: B

23. Droplet vitrification is characterized by
A. Slow cooling
B. Use of aluminum foil
C. High temperature storage
D. No cryoprotectant
✔ Answer: B

24. Rapid thawing is preferred because
A. It promotes ice formation
B. It reduces ice recrystallization
C. It kills cells
D. It delays recovery
✔ Answer: B

25. Suitable explant for cryopreservation is
A. Mature leaves
B. Root hairs
C. Shoot tips
D. Senescent tissues
✔ Answer: C


26. Metabolic activities during cryostorage are
A. Increased
B. Reduced
C. Completely arrested
D. Variable
✔ Answer: C

27. Major advantage of cryopreservation is
A. High mutation rate
B. Unlimited storage period
C. Frequent subculturing
D. High contamination risk
✔ Answer: B

28. Cryopreservation helps in conserving
A. Only seeds
B. Only pollen
C. Vegetatively propagated crops
D. Only annual crops
✔ Answer: C

29. Genetic stability during cryopreservation is
A. Poor
B. Moderate
C. High
D. Unpredictable
✔ Answer: C

30. Field gene banks are NOT suitable for
A. Seed crops
B. Vegetative crops
C. Long-term storage
D. Short-term trials
✔ Answer: C

31. Somaclonal variation is more common in
A. Cryopreservation
B. Short-term storage
C. Seed banks
D. Pollen storage
✔ Answer: B

32. Cryopreservation is most useful for
A. Rapid multiplication
B. Germplasm backup
C. Acclimatization
D. Callus induction
✔ Answer: B

33. One limitation of cryopreservation is
A. Low survival rate always
B. Need for skilled personnel
C. Genetic instability
D. Large space requirement
✔ Answer: B


34. Cryopreserved materials are usually stored in
A. Freezers
B. Refrigerators
C. Liquid nitrogen tanks
D. Incubators
✔ Answer: C

35. Which plant part is commonly cryopreserved?
A. Old leaves
B. Flowers
C. Apical meristems
D. Bark
✔ Answer: C

36. Medium-term storage reduces subculturing frequency by
A. Increasing nutrients
B. Slowing growth
C. Increasing light
D. Raising temperature
✔ Answer: B 

37. PEG used in 
cryopreservation acts as
A. Hormone
B. Nutrient
C. Cryoprotectant
D. Antibiotic
✔ Answer: C

38. Which method avoids ice crystal formation completely?
A. Slow freezing
B. Rapid freezing
C. Vitrification
D. Cold storage
✔ Answer: C

39. Thawing temperature is usually
A. 0–5°C
B. 10–15°C
C. 35–40°C
D. 60°C
✔ Answer: C

40. Recovery of cryopreserved explants requires
A. No culture medium
B. Special recovery medium
C. Field planting directly
D. Dark storage only
✔ Answer: B

41. Cryopreservation is considered safe because
A. No chemicals are used
B. Cells do not divide
C. DNA replication continues
D. Growth is promoted
✔ Answer: B

42. Encapsulation–vitrification is a combination of
A. Seed storage and freezing
B. Alginate beads and vitrification
C. Cold storage and drying
D. Field storage and LN
✔ Answer: B

43. Long-term germplasm storage ensures
A. Rapid multiplication
B. Continuous growth
C. Conservation for future use
D. Immediate planting
✔ Answer: C

44. Cryopreservation is NOT useful for
A. Endangered species
B. Elite clones
C. Genetic erosion prevention
D. Rapid field multiplication
✔ Answer: D

45. In slow growth storage, light intensity is
A. Increased
B. Normal
C. Reduced
D. Eliminated completely always
✔ Answer: C

46. Which crop is commonly conserved by in-vitro slow growth?
A. Wheat
B. Rice
C. Potato
D. Maize
✔ Answer: C
47. The key requirement for cryopreservation success is
A. High temperature
B. Ice formation
C. Proper dehydration
D. Continuous growth
✔ Answer: C

48. Cryopreservation is also called
A. Cold storage
B. Ultra-low temperature storage
C. Dry storage
D. Field storage
✔ Answer: B

49. Pollen can be preserved effectively by
A. Field storage
B. Subculturing
C. Cryopreservation
D. Callus culture
✔ Answer: C

50. Germplasm conservation ultimately supports
A. Genetic erosion
B. Sustainable agriculture
C. Monoculture
D. Habitat destruction
✔ Answer: B


❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥ π“†ž❥

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