Skip to main content

❥ preservation for germplasm conservation. Cryopreservation of vegetative propagated and recalcitrant seed species.

preservation for germplasm conservation. Cryopreservation of vegetative propagated and recalcitrant seed species. 


❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥

1. Introduction


Germplasm conservation is the systematic preservation of genetic resources for present and future use. Many economically important plants are vegetatively propagated (banana, potato, sugarcane, cassava) or produce recalcitrant seeds (cocoa, rubber, coconut), which cannot be conserved by conventional seed storage.
Cryopreservation offers a safe, long-term and genetically stable method for conserving such germplasm by storing living tissues at –196°C in liquid nitrogen (LN).


2. Need for Preservation of Germplasm

Prevention of genetic erosion
Conservation of elite, endangered and rare species
Backup of field and in-vitro collections
Support to crop improvement and breeding
Preservation of pathogen-free planting material
Conservation of plants with non-orthodox seeds


3. Limitations of Conventional Storage Methods
Seed Banks
Not suitable for vegetatively propagated crops
Ineffective for recalcitrant seeds (desiccation-sensitive)
Field Gene Banks
High cost and space requirement
Vulnerable to pests, diseases, climate change
Risk of genetic drift
In-vitro Slow Growth Storage
Limited duration
Genotype-specific responses
Risk of somaclonal variation


4. Cryopreservation – Concept and Principle

Definition

Cryopreservation is the storage of viable plant material at ultra-low temperature (–196°C) in liquid nitrogen, where all metabolic and biochemical activities are completely arrested.
Principle
Cellular dehydration
Prevention of ice crystal formation
Vitrification (glass-like state)
Use of cryoprotectants


5. Cryopreservation of Vegetatively Propagated Crops
Examples
Banana
Potato
Cassava
Sugarcane
Yam
Garlic
Suitable Explants
Shoot tips (apical meristems)
Axillary buds
Somatic embryos
In-vitro nodal segments
Reasons for Cryopreservation
No true seeds or sterile plants
High heterozygosity
Risk of disease accumulation
Need for clonal fidelity

Cryopreservation Methods Used


1. Vitrification

Treatment with plant vitrification solutions (PVS2, PVS3)
Rapid cooling in LN
No ice crystal formation

2. Droplet Vitrification

Explants placed in droplets on aluminum foil
Ultra-rapid cooling
High survival and regeneration rates

3. Encapsulation–Dehydration

Explants encapsulated in calcium alginate beads
Partial dehydration
Direct immersion in LN


Advantages


Maintains genetic stability
Long-term conservation
Minimal space requirement
Protection from contamination

6. Cryopreservation of Recalcitrant Seed Species


Recalcitrant Seeds – Definition
Seeds that lose viability when dried below a critical moisture content and cannot tolerate low temperature storage.


Examples
Cocoa (Theobroma cacao)
Rubber (Hevea brasiliensis)
Coconut
Mango
Jackfruit


Problems in Conservation
High moisture content
Desiccation sensitivity
Rapid loss of viability
Short storage life
Materials Used for Cryopreservation
Excised embryos
Embryonic axes
Plumules
Shoot apices from seedlings


Cryopreservation Techniques


1. Excised Embryo Cryopreservation
Removal of seed coat and endosperm
Partial dehydration
Freezing in LN


2. Vitrification of Embryonic Axes


Treatment with cryoprotectants
Rapid freezing and thawing


3. Encapsulation–Vitrification


Combines protection of alginate beads and vitrification
Advantages for Recalcitrant Seeds
Overcomes desiccation sensitivity
Enables long-term storage
Preserves rare and endangered species


7. Cryoprotectants in Germplasm

Cryopreservation
Penetrating Cryoprotectants
DMSO
Glycerol
Ethylene glycol
Non-penetrating Cryoprotectants
Sucrose
Mannitol
Sorbitol
PEG

Function:


Protect membranes
Reduce ice crystal formation
Stabilize cellular structures


8. Thawing and Recovery


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


9. Advantages of Cryopreservation for Germplasm Conservation


Long-term and secure storage
High genetic fidelity
Minimal maintenance
Suitable for vegetatively propagated and recalcitrant seed species
Effective backup system


10. Limitations


High initial cost
Need for skilled personnel
Species-specific protocols
Post-thaw regeneration challenges

11. Applications


Conservation of endangered plant species
Preservation of elite clones
Germplasm exchange and quarantine safety
Conservation of transgenic plants
Support to biodiversity conservation programs


12. Conclusion


Cryopreservation plays a crucial role in germplasm conservation, especially for vegetatively propagated crops and recalcitrant seed species that cannot be stored by conventional methods. By arresting metabolic activity at ultra-low temperatures, cryopreservation ensures long-term genetic stability, safety and sustainability of plant genetic resources, making it an indispensable tool in modern plant biotechnology and conservation biology.



50 MCQs – Germplasm Conservation & Cryopreservation


1. Germplasm conservation mainly aims to
A. Increase fertilizer use
B. Preserve genetic diversity
C. Increase mutation rate
D. Eliminate wild species
✔ Answer: B
2. Vegetatively propagated crops are difficult to conserve because
A. They produce many seeds
B. Seeds show dormancy
C. Seeds do not breed true
D. They lack genetic variation
✔ Answer: C
3. Which crop is vegetatively propagated?
A. Wheat
B. Rice
C. Banana
D. Maize
✔ Answer: C
4. Recalcitrant seeds are characterized by
A. Low moisture content
B. High desiccation tolerance
C. Sensitivity to drying
D. Long storage life
✔ Answer: C
5. Which is a recalcitrant seed species?
A. Wheat
B. Rice
C. Cocoa
D. Barley
✔ Answer: C
6. The ideal method for long-term conservation of vegetative crops is
A. Field gene bank
B. Seed bank
C. Cryopreservation
D. Cold storage
✔ Answer: C
7. Cryopreservation temperature is
A. 0°C
B. –20°C
C. –80°C
D. –196°C
✔ Answer: D
8. Liquid nitrogen is used because it
A. Enhances growth
B. Prevents contamination
C. Arrests metabolic activity
D. Improves regeneration
✔ Answer: C
9. Suitable explant for cryopreservation of vegetative crops is
A. Mature leaf
B. Root hair
C. Shoot tip
D. Senescent tissue
✔ Answer: C
10. Major injury during freezing is due to
A. Cell elongation
B. Ice crystal formation
C. Chlorophyll loss
D. Protein synthesis
✔ Answer: B
11. Cryoprotectants are used to
A. Kill cells
B. Promote cell division
C. Prevent freezing damage
D. Increase temperature
✔ Answer: C
12. Which is a penetrating cryoprotectant?
A. Sucrose
B. Mannitol
C. PEG
D. DMSO
✔ Answer: D
13. A non-penetrating cryoprotectant is
A. Glycerol
B. Ethylene glycol
C. Sucrose
D. DMSO
✔ Answer: C
14. Vitrification results in
A. Ice crystal formation
B. Glassy state of cytoplasm
C. Cell rupture
D. Rapid cell division
✔ Answer: B
15. PVS2 solution is used in
A. Slow freezing
B. Seed storage
C. Vitrification
D. Cold storage
✔ Answer: C
16. Droplet vitrification involves
A. Slow cooling
B. Use of aluminum foil strips
C. High temperature storage
D. No cryoprotectant
✔ Answer: B
17. Encapsulation–dehydration uses
A. Agar
B. Alginate beads
C. Charcoal
D. Gelatin
✔ Answer: B
18. Best explant for recalcitrant seed cryopreservation is
A. Whole seed
B. Mature endosperm
C. Embryonic axis
D. Seed coat
✔ Answer: C
19. Recalcitrant seeds cannot be stored in seed banks because
A. They are dormant
B. They require light
C. They are desiccation-sensitive
D. They are too small
✔ Answer: C
20. Example of recalcitrant seed crop is
A. Sorghum
B. Maize
C. Rubber
D. Mustard
✔ Answer: C
21. Metabolic activity during cryostorage is
A. Increased
B. Reduced
C. Completely arrested
D. Irregular
✔ Answer: C
22. Thawing of cryopreserved samples should be
A. Slow
B. Very slow
C. Rapid
D. At room temperature
✔ Answer: C
23. Preferred thawing temperature is
A. 5–10°C
B. 20–25°C
C. 35–40°C
D. 60°C
✔ Answer: C
24. Cryopreservation ensures genetic stability because
A. Cells divide actively
B. DNA replication stops
C. Growth hormones are added
D. High temperature is used
✔ Answer: B
25. Major advantage of cryopreservation is
A. Frequent subculturing
B. Unlimited storage duration
C. High mutation rate
D. Large space requirement
✔ Answer: B
26. Field gene banks are risky due to
A. Genetic stability
B. Climate and pest hazards
C. Easy maintenance
D. Low cost
✔ Answer: B
27. Cryopreservation is most suitable for
A. Annual seed crops
B. Vegetatively propagated crops
C. Weed species
D. Fast-growing plants
✔ Answer: B
28. Coconut seeds are classified as
A. Orthodox
B. Recalcitrant
C. Intermediate
D. Dormant
✔ Answer: B
29. Cryopreservation overcomes problems of
A. Dormancy
B. Seed viability loss
C. Desiccation sensitivity
D. Germination failure
✔ Answer: C
30. Encapsulation–vitrification is
A. Seed drying
B. Field storage method
C. Combination of alginate beads and vitrification
D. Cold storage
✔ Answer: C
31. Example of vegetatively propagated crop conserved by cryopreservation
A. Wheat
B. Rice
C. Potato
D. Sorghum
✔ Answer: C
32. Which structure is ideal for clonal fidelity?
A. Callus
B. Leaf disc
C. Shoot meristem
D. Root tissue
✔ Answer: C
33. Cryopreservation minimizes somaclonal variation because
A. Cells are actively growing
B. Cells remain dormant
C. DNA mutates rapidly
D. Hormones are removed
✔ Answer: B
34. Which cryoprotectant stabilizes membranes?
A. Sucrose
B. Auxin
C. Cytokinin
D. Gibberellin
✔ Answer: A
35. Rubber (Hevea brasiliensis) seeds are
A. Orthodox
B. Recalcitrant
C. Dormant
D. Hard-coated
✔ Answer: B
36. Major limitation of cryopreservation is
A. Genetic instability
B. Need for skilled expertise
C. High contamination
D. Frequent transfer
✔ Answer: B
37. Cryopreserved materials are stored in
A. Deep freezers
B. Refrigerators
C. Liquid nitrogen tanks
D. Incubators
✔ Answer: C
38. Excised embryo technique is mainly used for
A. Orthodox seeds
B. Recalcitrant seeds
C. Vegetative propagation
D. Callus culture
✔ Answer: B
39. PEG acts as
A. Growth regulator
B. Nutrient
C. Cryoprotectant
D. Antibiotic
✔ Answer: C
40. Cryopreservation provides backup to
A. Seed banks only
B. Field gene banks
C. In-vitro cultures
D. All conservation systems
✔ Answer: D
41. Banana germplasm is conserved mainly through
A. Seed storage
B. Field banks only
C. Cryopreservation of shoot tips
D. Pollen storage
✔ Answer: C
42. High moisture content in recalcitrant seeds causes
A. Dormancy
B. Desiccation tolerance
C. Storage problems
D. Rapid germination
✔ Answer: C
43. Cryopreservation is a form of
A. Medium-term storage
B. Short-term storage
C. Long-term storage
D. Temporary storage
✔ Answer: C
44. Ideal cooling rate in vitrification is
A. Very slow
B. Slow
C. Ultra-rapid
D. Moderate
✔ Answer: C
45. Genetic erosion refers to
A. Increase in diversity
B. Loss of genetic diversity
C. Mutation induction
D. Hybridization
✔ Answer: B
46. Somatic embryos are useful in cryopreservation because
A. They are highly differentiated
B. They regenerate efficiently
C. They lack genetic material
D. They are non-viable
✔ Answer: B
47. Cryopreservation is essential for
A. Hybrid seed production
B. Conservation of endangered species
C. Fertilizer production
D. Weed control
✔ Answer: B
48. Which factor is most critical for success of cryopreservation?
A. High light intensity
B. Proper dehydration
C. High temperature
D. Rapid growth
✔ Answer: B
49. Mango seeds are classified as
A. Orthodox
B. Recalcitrant
C. Dormant
D. Intermediate
✔ Answer: B
50. Germplasm conservation ultimately supports
A. Genetic erosion
B. Sustainable agriculture
C. Monoculture
D. Habitat loss
✔ Answer: B


❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥

Comments

Post a Comment

Popular Posts

AFLP--Amplified Fragment Length Polymorphism

AFLP is a PCR-based DNA fingerprinting technique combining restriction digestion and selective PCR amplification of genomic DNA fragments. Developed by Vos et al., 1995. AFLP detects DNA polymorphisms at the genomic level and is highly reproducible and sensitive. Used in genetic mapping, diversity studies, phylogenetics, and marker-assisted selection. Principle AFLP relies on restriction digestion of genomic DNA, followed by ligation of adaptors and PCR amplification of a subset of fragments. Polymorphism arises due to variations in restriction sites, fragment length, insertions, or deletions. Key idea: Restriction digestion → Adaptor ligation → Selective amplification → Gel separation → Detection of polymorphic bands Materials Required Genomic DNA Restriction enzymes (usually EcoRI and MseI) Adaptors complementary to restriction sites PCR reagents: Taq polymerase, dNTPs, buffer, Mg²⁺ Primers complementary to adaptors with selective nucleotides Thermal cycler Polyacrylamide or agarose ...
Post a Comment
Read more

❥ Southern Blotting Notes

Southern Blotting  ❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥ 𓆞❥  Introduction Southern blotting is a molecular biology technique used for the detection of specific DNA sequences in a complex mixture of DNA. It was developed by Edwin M. Southern in 1975. The method involves restriction digestion of DNA, separation by gel electrophoresis, transfer (blotting) onto a membrane, and hybridization with a labeled DNA probe. Principle of Southern Blotting The technique is based on the principle of complementary base pairing. A single-stranded labeled DNA probe hybridizes specifically with its complementary DNA sequence immobilized on a membrane. Detection of the label confirms the presence and size of the target DNA fragment. Steps Involved in Southern Blotting. 1. Isolation of DNA Genomic DNA is extracted from cells or tissues. DNA must be pure and intact to ensure accurate results. 2. Restriction Enzyme  Digestion DNA is digested using specific restriction endonucleases. Produces DNA f...
1 comment
Read more

Secondary Databases (PROSITE, PRINTS, BLOCKS)

Secondary Databases (PROSITE, PRINTS, BLOCKS  Secondary Databases Introduction Biological databases are broadly classified into primary and secondary databases. Primary databases store raw experimental data (e.g., nucleotide or protein sequences), whereas secondary databases contain derived information obtained by analyzing primary sequence data. Secondary databases are mainly used to: Identify protein families Detect conserved motifs, patterns, and domains Predict protein function Study structure–function relationships Examples of secondary databases include PROSITE, PRINTS, BLOCKS, Pfam, etc. 1. PROSITE Database Definition PROSITE is a secondary database that documents protein domains, families, and functional sites in the form of patterns and profiles. Developed by Swiss Institute of Bioinformatics (SIB) Maintained along with UniProt Principle PROSITE is based on the idea that functionally important regions of proteins are conserved during evolution. These conserved regions can ...
Post a Comment
Read more

DNA-Mediated Gene Transfer – Detailed Notes

DNA-Mediated Gene Transfer – Detailed Notes 1. Definition DNA-mediated gene transfer refers to the direct introduction of exogenous DNA into a host cell’s genome or cytoplasm without using viral or bacterial vectors. It is a physical or chemical approach to achieve gene delivery. Also called direct gene transfer. 2 . Principle Foreign DNA is delivered into host cells through physical or chemical methods. DNA may integrate into the host genome (stable transformation) or remain episomal (transient expression). Expression depends on: DNA sequence and promoter Type of host cell Delivery efficiency 3. Types of DNA-Mediated Gene Transfer A. Physical Methods These methods use physical forces to introduce DNA into cells. Microinjection DNA is injected directly into the nucleus or cytoplasm using a glass micropipette. Used in: animal embryos, oocytes, plant protoplasts Advantages: Precise, can deliver large DNA fragments Limitations: Labor-intensive, requires specialized equipment, low throughp...
Post a Comment
Read more

Single Nucleotide Polymorphisms (SNPs) – Detailed Notes

Single Nucleotide Polymorphisms (SNPs) – Detailed Notes 1. Definition SNPs are single base-pair variations in the DNA sequence that occur at a specific position in the genome among individuals of a species. Example: At a specific locus, one individual may have A while another has G: Copy code Individual 1: …A T C G A T…   Individual 2: …A T C G G T… SNPs are the most common type of genetic variation in most organisms. 2. Characteristics of SNPs Single base change: Involves substitution of one nucleotide for another (A↔G, C↔T). Biallelic nature: Most SNPs have only two alleles in a population. Widespread in the genome: Found in coding regions (exons), non-coding regions (introns, promoters, intergenic regions). Stable inheritance: Passed from generation to generation like other genetic markers. Frequency: Occur approximately every 100–300 bp in the human genome. 3 . Types of SNPs SNPs are categorized based on location or effect on gene function: A. Based on genomic location Cod...
Post a Comment
Read more

SSR (Simple Sequence Repeat) Marker

SSR (Simple Sequence Repeat) Markers – Detailed Notes Introduction SSR markers, also called microsatellites, are short tandem repeats (1–6 bp) of DNA sequences found throughout the genome. Examples: (A)n, (CA)n, (GATA)n, where n is the number of repeat units. SSRs are highly polymorphic, co-dominant, and locus-specific, widely used in genetic mapping, variety identification, population genetics, and marker-assisted selection (MAS). SSRs are similar to STRs; in plants and animals, the term SSR is more commonly used in molecular breeding, while STR is used more in forensics and human genetics. Structure of SSR Repeat motif: 1–6 bp Number of repeats: Variable among individuals → basis of polymorphism Flanking regions: Conserved sequences used to design specific PCR primers SSR loci are generally abundant in non-coding regions, though some occur in genes. Principle SSR markers exploit variation in the number of repeat units at a specific locus. PCR amplification using primers flanking the...
Post a Comment
Read more

Protein Structure Database (PDB)

Protein Structure Database (PDB) Introduction The Protein Structure Database (PDB) is the primary global repository for the three-dimensional (3D) structures of biological macromolecules such as proteins, nucleic acids, and protein–ligand complexes. These structures are determined experimentally using techniques like X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and Cryo-Electron Microscopy (Cryo-EM). PDB plays a vital role in understanding: Protein structure and function Molecular interactions Drug discovery and design Structural biology and bioinformatics History and Development Established in 1971 Founded by Brookhaven National Laboratory (USA) Initially contained only 7 protein structures Now maintained by the Worldwide Protein Data Bank (wwPDB) Members of wwPDB RCSB PDB (USA) PDBe (Europe) PDBj (Japan) BMRB (Biological Magnetic Resonance Data Bank) Objectives of PDB To collect, store, and distribute 3D structural data of biomolecules To provide free and ope...
Post a Comment
Read more

GEL RETARDATION ANALYSIS

GEL RETARDATION ANALYSIS (EMSA – Electrophoretic Mobility Shift Assay) Introduction Gel retardation analysis, also known as Electrophoretic Mobility Shift Assay (EMSA), is a widely used in vitro technique for studying DNA–protein and RNA–protein interactions. The method is based on the observation that a DNA–protein complex migrates more slowly than free DNA during non-denaturing gel electrophoresis, resulting in a mobility shift or “retardation”. EMSA is extensively used to study transcription factor binding, regulatory DNA elements, and binding specificity. Definition Gel retardation analysis (EMSA) is a technique used to detect and analyze binding interactions between nucleic acids and proteins by observing the reduced electrophoretic mobility of nucleic acid–protein complexes compared to free nucleic acids. Principle A labeled DNA or RNA probe is incubated with a specific binding protein. When binding occurs, a nucleic acid–protein complex is formed. This complex has a larger size ...
Post a Comment
Read more

Agrobacterium & CaMV-Mediated Gene Transfer –

Agrobacterium and CaMV-Mediated Gene Transfer – Detailed Notes 1. Introduction Gene transfer in plants is often achieved by exploiting natural genetic mechanisms of Agrobacterium tumefaciens and Cauliflower Mosaic Virus (CaMV). These systems allow stable introduction of foreign genes into plant genomes for transgenic plant development. 2. Agrobacterium-Mediated Gene Transfer 2.1 Definition Agrobacterium-mediated gene transfer uses the natural ability of Agrobacterium tumefaciens, a soil bacterium, to transfer a part of its DNA (T-DNA) into plant cells. T-DNA integrates into the plant nuclear genome, enabling stable transformation. 2.2 Mechanism Recognition and attachment Agrobacterium detects phenolic compounds secreted by wounded plant cells. These compounds activate virulence (vir) genes on the Ti (tumor-inducing) plasmid. Activation of vir genes VirA (sensor kinase) and VirG (response regulator) induce expression of other vir genes (VirB, VirC, VirD, VirE). T-DNA processing and tran...
Post a Comment
Read more

SCAR (Sequence Characterized Amplified Region) Markers

SCAR (Sequence Characterized Amplified Region) Markers   Introduction SCAR markers are PCR-based DNA markers derived from RAPD, AFLP, or other random markers. Developed by Paran and Michelmore in 1993 to convert dominant, less reproducible markers into specific, reproducible, co-dominant markers. SCAR markers are locus-specific, reproducible, and sequence-characterized, making them ideal for marker-assisted selection (MAS). Principle SCAR markers are designed based on known DNA sequences obtained from cloned RAPD/AFLP fragments. Specific primers (18–24 bp) are synthesized to amplify a single, defined locus. The PCR amplification of this region generates a distinct band, which is highly reproducible and can distinguish homozygotes from heterozygotes if designed as co-dominant. Key idea: Random marker (e.g., RAPD) → Cloning & sequencing → Design specific primers → PCR → SCAR marker Materials Required Genomic DNA from the organism Specific primers (18–24 bp) designed from sequence...
Post a Comment
Read more