cryopreservation

 Cryopreservation

Why Preservation is important?

• Until tow decades ago the genetic resources were getting depleted owing to the continuous depredation by man.
• It was imperative therefore that many of the elite Economically important and endangered species are preserved to make them available when needed.
• The conventional methods of storage of storage failed to prevent losses caused to various reason.
• A new methodology had to be devised for long term preservation of material.

There are various methods of storage

1. Cryopreservation:- Generally involves storage in liquid Nitrogen.
2. Cold storage:- It involve storage in low and non freezing temperature.
3. Low pressure:- It involves partially reducing the atmospheric pressure of surrounding.
4. Low oxygen storage:- It involves reducing the oxygen level but maintaining the pressure.

Cryopreservation;-

• Cryo is greek word, (Kroyes -frost)
• It literally means preservation in "frozen state"

The principle:- to bring plant cells or tissue to a zero metabolism and non dividing state by reducing the temperature in the presence of cryoprotectant.

It can be done:-

• Over solid carbon dioxide (at-79°C)
• Low temperature deep freezer (at-80°C)
• In vapor phase nitrogen (at-150°C)
• In liquid nitrogen (at-196°C)

Major advantages are:-

• Once the material is successfully conserved. Particular temperature it can be preserved indefinitely.
• Once in storage no chance of new contamination of fungus or Bacteria.
•  Minimal space required.
• Minimal labor required.

Mechanism of Cryopreservation:-

 

                             There cryopreservation technique followed by the regeneration of plants involves following steps:-

1. Selection of of material.
2. Addition of Cryoprotectant.
3. Freezing
4. Storage in liquid nitrogen.
5. Thawing.
6. Washing and reculturing.
7. Measurement of viability
8. Regeneration of plants.

1. Selection of plant material:-

             Two important factors depends on it such as 

                          a. Nature and

                          b. Density

      Any tissue can be selected for this purpose e.g., meristem, embryo, ovules, seeds, etc.

The density should be high.

2.Addition of Cryoprotectant:-

• They are chemical which prevent cryodestruction.
• These are sucrose, alcohol's, glycols, Some amino acid (proline), DMSO (dimethyl sulfoxide)
• Generally tow cryoprotectant should be used together instead of single one as they are more effective.

3. Freezing:-

      

               The sensitivity of cells to low temperature depends on the plant species.

  There are four different types of Freezing:-

• Slow freezing method:- The tissue or plant material is slowly frozen at slow cooling rate. The advantage is the plant cells are partially dehydrated and survive better.
• Rapid freezing method:- It involves pluming the vials in liquid nitrogen. The temperature decreases from -300°C to -1000°C  rapidly.
• Rapid freezing method:- This is combination of both slow and rapid freezing method. The process is carried out in step wise like manner.
• Dry freezing method:- In this method dehydrated cells and seeds are stored.

4.Storage:-

• The maintenance of the frozen cells or material at specific temperature is kept -70°C to -196°C
• Prolong storage is done at temperature of -196°C in liquid nitrogen.
• To prevent damage, continuous supply of Nitrogen is done.

5.Thawing:-

• Usually carried out by plunging the vials. into warm water bath with vigorous swirling.
• As thawing occurs the vials are transferred to another bath at 0°C degree.

6.Washing and reculturing:-

• The preserved material is washed few times to remove the cryoprotectant.
• This material is then recultured in a fresh medium.

7.Measurement of viability

• There is possibility of death of cells due to storage stress.
• Thus viability can be found at any stage.
• It is calculated by formula:-

  No. of cells growing/no of cells thawed ✖100

8.Plant regeneration:-

• The viable seeds are cultured on non specific growth medium.
• Suitable Environment Conditions are maintained. 

   Application

• It is ideal method for long term conservation of material.
• DISEASE FREE PLANT CAN BE CONSERVED AND PROPAGATE.
• Peculcitrant seeds can be maintained for long time.
• Endangered species can be maintained.
• Pollens can be maintained to increase longitivity.
• Rare germplasm and other genetic manipulations can be stored.

❄️ Cryopreservation MCQs (CSIR-NET Level)

1. Cryopreservation mainly involves storage at:

0°C −20°C −80°C −196°C

2. The word cryo is derived from Greek word meaning:

Ice Cold Frost Snow

3. The basic principle of cryopreservation is to:

Increase respiration Stop metabolism Increase enzyme activity Induce mutations

4. Which of the following is a commonly used cryoprotectant?

Agar DMSO Antibiotic Cytokinin

5. Use of two cryoprotectants together is preferred because:

They reduce temperature They increase toxicity They are more effective They increase contamination

6. Slow freezing is advantageous because:

Cells remain hydrated Cells are partially dehydrated Ice crystals increase Temperature rises slowly

7. Long-term storage of plant material is usually done at:

−40°C −70°C −150°C −196°C

8. Thawing of cryopreserved material is generally done by:

Keeping at room temperature Slow air drying Warm water bath Ice bath

9. Viability of cryopreserved cells is calculated as:

Cells thawed / cells growing ×100 Cells growing / cells thawed ×100 Total cells ×100 Dead cells / live cells ×100

10. One major application of cryopreservation is:

Short-term storage Mutation breeding Conservation of endangered species Increasing transpiration

🌱 Micropropagation — CSIR NET Quiz 🌱

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Essential (Indispensable) Amino Acids

Essential amino acids, also known as indispensable amino acids, are those amino acids that cannot be synthesized by the human body in sufficient amounts. Therefore, they must be obtained from dietary sources such as milk, eggs, meat, legumes, nuts, and cereals.

These amino acids are crucial for normal growth, development, tissue repair, muscle formation, and overall health. A deficiency of any essential amino acid can lead to poor growth, weakness, impaired immunity, and metabolic disorders.

                AMINO ACIDS

                     |

        --------------------------------

        |                                          |

   Essential                         Nonessential

 (From diet)                   (Body makes itself)

        |

  -------------------------------------------

  Arg  Val  His  Ile  Leu  Lys  Met  Phe  Thr  Trp

List of Essential Amino Acids in Humans (10):

·         Arginine

·         Valine

·         Histidine

·         Isoleucine

·         Leucine

·         Lysine

·         Methionine

·         Phenylalanine

·         Threonine

·         Tryptophan

Why are they important?

Essential amino acids play key roles in:
✔ Protein synthesis and muscle growth
✔ Tissue repair and wound healing
✔ Hormone and enzyme production
✔ Brain function and neurotransmission
✔ Immune system support

Easy Memory Trick (for exams)

“AVHILL MPT”
(Arginine–Valine–Histidine–Isoleucine–Leucine–Lysine–Methionine–Phenylalanine–Threonine–Tryptophan)

Exam Notes — Nonessential (Dispensable) Amino Acids

Definition (Very Important)

Nonessential amino acids are those amino acids that can be synthesized by the human body, so they do not need to be taken regularly from the diet.

The body can synthesize 10 nonessential amino acids through normal metabolic pathways.

Key Points for Exams

✔ Also called Dispensable amino acids
✔ Required for protein synthesis, metabolism, and tissue repair
✔ Although “nonessential,” they are biologically very important
✔ Their synthesis depends on availability of precursors and vitamins

List of 10 Nonessential Amino Acids (Must Remember)

Sl. No.	Amino Acid
1	Glycine
2	Alanine
3	Serine
4	Cysteine
5	Aspartate
6	Asparagine
7	Glutamate
8	Glutamine
9	Tyrosine
10	Proline

Memory Trick:
“G A S C A G G T P”
(Glycine–Alanine–Serine–Cysteine–Aspartate–Asparagine–Glutamate–Glutamine–Tyrosine–Proline)

Functions (Exam-Oriented)

Nonessential amino acids help in:

• ✔ Building body proteins
• ✔ Repair of tissues
• ✔ Enzyme and hormone production
• ✔ Energy metabolism
• ✔ Neurotransmission (especially Glutamate, Glycine)

Simple Diagram

AMINO ACIDS

|

--------------------------------

|                                            |

Essential                            Nonessential

          (From diet)                       (Body makes itself)

                                                     |

                                                        ---------------------------------

                                                     | | | | | | | | | |

                                                    Gly Ala Ser Cys Asp Asn

                                                        Glu Gln Tyr Pro

Caption you can write under the diagram:
“Figure: Classification of amino acids into essential and nonessential types.”

One-line Exam Answer (Very Useful)

Nonessential amino acids are those which the body can synthesize on its own and therefore are not required to be supplied regularly through diet (e.g., Glycine, Alanine, Serine, Cysteine, Aspartate, Asparagine, Glutamate, Glutamine, Tyrosine, Proline).

 

 

 

Recombinant DNA Technology: Revolution in Modern Biotechnology

Recombinant DNA Technology: Revolution in Modern Biotechnology

Introduction

Recombinant DNA (rDNA) technology is one of the most powerful and transformative tools in modern biotechnology. It allows scientists to combine DNA from different organisms to create new genetic combinations that do not exist in nature. This technology has revolutionized medicine, agriculture, industry, and research by enabling the production of valuable proteins, improved crops, and disease-resistant organisms.

In simple words, recombinant DNA technology is like “genetic engineering,” where useful genes are inserted into another organism to give it new and beneficial properties.

Discovery of Recombinant DNA Technology

The foundation of recombinant DNA technology was laid in the early 1970s by scientists Stanley Cohen and Herbert Boyer. They successfully transferred a gene from one bacterium into another using plasmids (small circular DNA molecules).

This breakthrough experiment marked the birth of genetic engineering and opened the doors to modern biotechnology. Since then, rDNA technology has grown rapidly and become a core tool in biological research.

Goals and Objectives of rDNA Technology

The main objectives of recombinant DNA technology include:

• Producing useful proteins like insulin and vaccines
• Improving crop quality and resistance
• Understanding gene function
• Treating genetic diseases
• Creating industrial enzymes

Basic Steps of Recombinant DNA Technology

The process of rDNA technology involves the following key steps:

1. Isolation of Desired Gene

A useful gene is identified and cut from the donor organism using restriction enzymes (molecular scissors).

2. Selection of Vector

A plasmid or viral DNA is chosen as a carrier (vector) to transport the gene into a host cell.

3. Cutting and Joining

Both the gene of interest and the vector are cut using the same restriction enzyme and then joined using DNA ligase.

4. Transformation

The recombinant plasmid is inserted into a host organism, usually bacteria like E. coli.

5. Multiplication and Expression

The host cell multiplies and starts producing the desired protein.

Key Enzymes Used in rDNA Technology

Some important enzymes include:

• Restriction enzymes – cut DNA at specific sites
• DNA ligase – joins DNA fragments
• DNA polymerase – copies DNA
• Reverse transcriptase – makes DNA from RNA

Applications of Recombinant DNA Technology

1. Medical Applications

• Production of human insulin for diabetes
• Growth hormones
• Blood clotting factors
• Vaccines
• Gene therapy for genetic disorders

2. Agricultural Applications

• Development of pest-resistant crops
• Herbicide-resistant plants
• Drought-tolerant varieties
• Improved nutritional value of food

Example: Golden rice enriched with Vitamin A.

3. Industrial Biotechnology

• Production of enzymes for detergents
• Biofuels
• Bioplastics
• Waste treatment

4. Environmental Uses

• Bioremediation of polluted soil and water
• Degradation of toxic chemicals

5. Fun and Novel Applications

• Fluorescent fishes like GloFish
• Glow-in-the-dark organisms used in research

Advantages of rDNA Technology

• Increases food production
• Helps cure diseases
• Reduces chemical pesticide use
• Produces life-saving medicines
• Improves quality of life

Limitations and Ethical Concerns

Despite its benefits, rDNA technology raises concerns such as:

• Environmental risks
• Ethical issues
• Genetic contamination
• Biosafety problems

Therefore, strict regulations are required.

Conclusion

Recombinant DNA technology is a cornerstone of modern biotechnology. It has transformed healthcare, agriculture, and industry. With responsible use and proper regulation, this technology can greatly benefit humanity in the future.

 

CSIR NET Interactive Quiz — Recombinant DNA Technology

Time Left: 10:00

Instruction: Attempt all questions before time runs out.

1) DNA ligase joins DNA fragments.
DNA polymerase
RNA polymerase
DNA ligase
Restriction enzyme

2) Restriction enzymes recognize:
Random sequence
Palindromic sequence
mRNA
tRNA

3) Most common vector is:
Chromosome
Plasmid
Ribosome
Histone

4) Best host in rDNA is:
Bacillus
E. coli
Yeast
Fungus

5) DNA cutting enzyme:
Ligase
Restriction enzyme
Helicase
Polymerase

6) Sticky ends produced by:
Blunt cut
EcoRI
Polymerase
Ligase

7) DNA from RNA made by:
Reverse transcriptase
Ligase
Helicase
Endonuclease

8) Selectable marker helps to:
Kill bacteria
Select transformants
Break DNA
Identify host

9) Common selectable marker:
Antibiotic resistance gene
Insulin gene
Histone
rRNA

10) Golden rice enriched with:
Vitamin A
Vitamin C
Iron
Protein