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

Genetically modified microbes - biodegradation, biopesticides, bioremediation, mineral leaching and biofertilizers.

 Genetically Modified Microbes (GMMs) covering biodegradation, biopesticides, bioremediation, mineral leaching and biofertilizers.  Genetically Modified Microbes (GMMs) Introduction Genetically Modified Microbes (GMMs) are microorganisms such as bacteria, fungi, yeast or algae whose genetic material has been altered using recombinant DNA technology to enhance or introduce desirable traits. These microbes are engineered to improve efficiency, specificity and speed of biological processes useful in agriculture, industry and environmental management. GMMs play a vital role in sustainable development by reducing dependence on chemical fertilizers, pesticides and polluting industrial processes. 1. Genetically Modified Microbes in Biodegradation Definition Biodegradation is the microbial breakdown of complex organic pollutants into simpler, non-toxic substances. Role of GMMs Natural microbes often degrade pollutants slowly. Genetic modification enhances: Enzyme activity Substrate sp...
Post a Comment
Read more

Mapping of DNA

DNA MAPPING   1. Introduction DNA mapping refers to the process of determining the relative positions of genes or DNA sequences on a chromosome. It provides information about the organization, structure, and distance between genetic markers in a genome. DNA mapping is an essential step toward genome sequencing, gene identification, disease diagnosis, and genetic engineering. DNA maps serve as roadmaps that guide researchers to locate specific genes associated with traits or diseases. 2. Objectives of DNA Mapping To locate genes on chromosomes To determine the order of genes To estimate distances between genes or markers To study genome organization To assist in genome sequencing projects. 3. Principles of DNA Mapping DNA mapping is based on: Recombination frequency Physical distance between DNA fragments Hybridization of complementary DNA Restriction enzyme digestion Use of genetic markers The closer two genes are, the less frequently they recombine during meiosis. 4 . Types of DNA...
2 comments
Read more

protoplast fusion

Protoplast Fusion – Detailed Notes 1. Definition Protoplast fusion is a technique in which two or more protoplasts (cells without cell walls) are fused to form a single hybrid cell. It is widely used in plant biotechnology for hybridization, gene transfer, and somatic hybrid production. Also called somatic hybridization or somatic cell fusion. 2. Principle Cell wall removal: Plant cells are treated with cell wall-degrading enzymes (cellulase, pectinase) to generate protoplasts. Fusion of protoplasts: The naked cells are induced to fuse physically or chemically. Hybrid cell formation: Nuclei from different protoplasts combine to form a heterokaryon. Regeneration: The hybrid cell regenerates a new cell wall and divides, eventually forming a somatic hybrid plant. Key Concept: Protoplast fusion bypasses sexual incompatibility barriers, allowing hybridization between distant species or genera. 3. Steps in Protoplast Fusion Step 1: Isolation of Protoplasts Plant tissues (leaves, callus, cell...
Post a Comment
Read more

✩‧₊ Plaque Blotting Technique

Plaque Blotting Technique *ੈ✩‧₊˚༺☆༻*ੈ✩‧₊˚*ੈ✩‧₊˚༺☆༻*ੈ✩‧₊˚ Introduction Plaque blotting is a molecular biology screening technique used to identify specific DNA or RNA sequences present in bacteriophage plaques formed on a bacterial lawn. It is especially useful in the screening of recombinant phage libraries such as λ (lambda) phage genomic or cDNA libraries. This technique combines: Plaque assay (to isolate individual phage clones) Blotting technique (to transfer nucleic acids onto a membrane) Hybridization (to detect specific sequences using labeled probes) Principle of Plaque Blotting The principle of plaque blotting is based on nucleic acid hybridization. Each plaque represents a clone of phage particles containing identical DNA. DNA from phage particles in plaques is: Released Denatured into single strands Transferred onto a nitrocellulose or nylon membrane The membrane is incubated with a labeled DNA/RNA probe complementary to the target sequence. Hybridization between probe and t...
Post a Comment
Read more

Protein Sequence DatabasesPIR, SWISS-PROT and TREMBEL

Protein Sequence Databases PIR, SWISS-PROT and TREMBEL 1. Introduction Protein sequence databases are biological databases that store information about amino acid sequences of proteins, along with their functional, structural, and biochemical characteristics. Since proteins are the functional molecules of the cell, protein databases are essential for understanding gene expression, metabolism, enzymatic activity, signaling pathways, and evolution. Protein sequence databases mainly contain data derived from translated nucleotide sequences and experimental protein studies. 2. Types of Protein Sequence Databases Protein sequence databases are broadly classified into: A. Primary Protein Databases Contain original protein sequence data Minimal or no manual annotation B. Secondary Protein Databases Derived from primary databases Provide curated functional and structural information C. Composite Protein Databases Combine protein data from multiple sources Reduce redundancy 3. Protein Informati...
Post a 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

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

♡Introduction to Bioinformatics: Definition and History of Bioinformatics Internet. Computational Biology and Bioinformatics

Introduction to Bioinformatics: Definition and History of Bioinformatics Internet.  Computational Biology and Bioinformatics ﮩ٨ـﮩﮩ٨ـ♡ﮩ٨ـﮩﮩ٨ـﮩ٨ـﮩﮩ٨ـ♡ﮩ٨ـﮩﮩ٨ﮩ٨ـﮩﮩ٨ـ♡ﮩ٨ـﮩ Definition of Bioinformatics Bioinformatics is an interdisciplinary field that combines biology, computer science, mathematics, and statistics to collect, store, analyze, and interpret large volumes of biological data. It mainly deals with molecular biology data such as DNA, RNA, protein sequences, gene expression data, and biological networks. Bioinformatics helps in understanding biological processes at the molecular level using computational tools. It plays a crucial role in modern biological research, especially after the availability of whole genome sequences. According to NIH, “Bioinformatics is the application of computational tools to capture and interpret biological data.” History and Evolution of Bioinformatics Early Beginnings (Pre-1970) The roots of bioinformatics date back to the 1950s–1960s. In 1953, Wat...
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

Fourth Semester M.Sc. Degree Examination, June 2022BotanySpecial Paper II - Elective BO 242 a BIOTECHNOLOGY

N-6278 Fourth Semester M.Sc. Degree Examination, June 2022 Botany Special Paper II - Elective BO 242 a BIOTECHNOLOGY - (2013-2018 Admission) Time: 3 Hours Max. Marks 75 Give Illustrations wherever necessary. 1. Answer the following questions. 1. Define a calliclone. 2. Which fungi helps in fermentation? 3. How is hairy root culture induced in plants? 4. Name a microbe that produces organic acid. 5. What is the advantage of chromosome elimination technique? 6. Describe the significance of PIPS. 7. What is elicitation in plant tissue culture? 8. Describe the term bioconversion. 9. Which is the most successful chemical used for protoplast fusion? 10. Define cybridization. (10x1 =10 Marks) II. Answer the following questions in not more than 50 words . 11. (a) Give an account on signature tagged mutagenesis. OR (b) Write notes on cytoplasts and their importance? 12. (a) Give an account of gene theft. OR (b) Describe induced androgenesis and gynogenesis for haploid plant production. 13. (a) ...
Post a Comment
Read more