Genomic imprinting

Genomic imprinting is an epigenetic (study of changes in organisms caused by modification of gene expression rather than alternation of the genetic code itself )phenomenon which in most cases is belived to occur in gametogenesis.

 It occurs when both maternal and paternal alleles are present ,but one allele will be expressed while the other remains inactive.This process is necessary for development and may somehow regulate growth in the embryo .

Evidence - It came from experiments with androgenotes ( embryos with two paternal genomes ) and gynogenotes ( embryos with two maternal genomes ) , which were produced by nucler transplantation .These zygotes are formed , but neither type was able to undergoes further development.

Example -
1. Gene Igf2 is an imprinted gene being expressed only from the paternal chromosome .
2. H19 gene is an imprinted gene expressed only from the maternal chromosome .  

                                                       Image source credit-http://curekatelyn.com/how-close-are-we-to-a-cure/

It is also called as uniparental disomy as only one allele is expressed either from father or from mother .

Genomic imprinting is rare in mammals because most of the genes are not imprinted. From a nuclear transplantation experiment in mouse during  1980 s it was confirmed that foe normal mammalian development both the maternal and paternal genomes are required.

In case of flowering plants some genes are found to be expressed from maternal genomes while some others are expressed exclusively from the alone paternal copy .( In case of Sweetbay -Magnolia virginiana )

In case of insects like honeybees and  ants can reproduce either through fertilized eggs or unfertilized eggs. This sex determining system develops males from unfertilized eggs and females develop from fertilized eggs. The queen honey bee decides whether to fertilize an egg or to leave it unfertilized .

Improper imprinting in humans leads to some developmental abnormalities-
1.Prader-Willi-syndrome (when fathers copy is missing or there are 2 maternal parents.)
2.Angelman syndrome( when mothers copy is missing and there are 2 paternal parents .)


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Hypervolume niche

Hypervolume Niche- The limits, for all important environmental conditions, within which individuals of a species can survive, grow and reproduce is termed as niche . If each condition, resource or other species is seen as a dimension, the niche is a n-dimensional hypervolume. 

Types of hypervolume niches-

 a. Fundamental niche 
 b. Realized. niche

 Fundamental niche is the the actual entire niche that an oraganism/ species occupy ,when species is not competing with others for its resources. But individual or a species normally remains in competition (either interspecific or intraspecific or both) and under such conditions, only a part of the niche is realized by the species. This smaller hypervolume occupied by the species is called realized niche.

Thus,each species has both fundamental niche within a community to which it is adapted in the evolutionary process, but because of competition it occupies a smaller niche, namely realized niche.
 In  competing zone due to competition, the reproductive success of each species and hence chances of survival reduced.

Individuals, who remain outside the overlapping competitive zone, are likely to have a greater survival rate and reproductive success.Natural selection favors individuals lying in the non-competing zone.

The fundamental niche is larger then the realized niche. The realized niche can be called a subset of the fundamental niche. 

                                 Imge source credit -https://quizlet.com/75558972/bio-lecture-17-flash-cards/

When all of the boundaries of tolerance of diverse environmental influences are assembled into a single, multivariate factor. This is known as  the multidimensional zone (also known as a hypervolume) of environmental tolerance, in which an individual can potentially survive or in which a species can maintain viable populations. We can quantify in this the niche breadth and niche overlap .



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Transgenic plants

Transgenic plants are genetically modified plants by introduction or expression of a foreign gene. It is also called as genetically engineered plants. It use the recombinant Dna technology  so we can say it is a result of modern biotechnology .

Production of transgenic plants -

1. Isolation - isolate and clone the gene of intrest. The gene is called transgene.
2.Addition of segments- Add the Dna segments for the initiation of the gene expresiion.
3.Selectable markers - Selectable markers are those which allow the selection of transformed cells, or tissue explants, by their ability to grow in the presence of an antibiotic or a herbicide. The most frequently used selectable markers are kanamycin and hygromycin. In addition to selecting for transformants, such markers can be used to follow the inheritance of a foreign gene in a segregating population of plants.
4.Transformation -Transfer the gene construct into plant cell .
5.Selection of transfered cells or tissues .
6.Regenerate a new whole plant .

Advantage -
1. It improves the size of nutrients.
2.Reduce the percentage of waste gives a longer shelf life .
3.Plant cells are totipotent in nature that means a whole plant can generate  from an explant .
3. Increase the yield of crops
4.Improves the resistant power against disease,pests and herbicides.
5.Decrease pollution -Some genetically modified plants are made to prevent heavy metal pollution from contaminated soil.
6.Tolerance to drought and salinity .

Examples-

1.Bt corn varieties (for pest resistance ) 1995 for example, contain a gene from a bacterium (Bacillus thuringiensis) found in the soil that causes the transgenic corn to produce an insecticidal protein. The gene confers resistance to ball worms.

2.Slow ripening tomato (Flavr savr )1994 It is the first commersialized GM crop .

• It improves the texture.
• It delays the ripening process.
• It helps in facilitation of transportation .

3.Golden rice ( Improves nutritional quality ). 

4.GM maize (drought tolerance)- Transgenic maize expresses a Rna chaperone gene in tolerant to salt and water stress . 
1.

Q. The transgenic animals are those which have
(a) foreign DNA in some of its cells
(b) foreign DNA in all its cells
(c) foreign RNA in all its cells
(d) DNA and RNA both in the cells
Ans.b
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Gastrulation

It is the process of cell and tissue movements whereby the cells of the blastula are rearranged in different manner.

• The blastula consists of numerous cells , the positions are established during cleavage.
• The three germ layers such as ectoderm(outside), endoderm and mesoderm are first produced in the process of gastrulation .
• Skin and nervous system system are spread over its outside surface.
• The movements of gastrulation involve the entire embryo and cell migrations in one part of the gastrulating embryo which  coordinate with other movements occuring simultaneously.

Gastrulation usually involves some combination of the following types of movements -

1. Invagination - (sea urchin endoderm)The infolding of a cells ,much like the indenting of a soft rubber bait when it is pocked .(inward buckling of an epithelium.)

                                Image source credit-https://www.studyblue.com/notes/note/n/developmental-bio-exam-1/deck/5569140

2.Involution - (Amphibian mesoderm) The inward movement of an expanding outer layer so that it spreads over the internal surface of the remaining external cells. It requires migration .(movement of individual cells over other cells ).

                                     Image source credit-http://biology.kenyon.edu/courses/biol114/Chap14/Chapter_14.html

3.Ingression - (Sea urchin mesoderm and Drosophila neuroblasts) Migration of individual cells from the surface layer into the cativy or interior of the embryo .

                             Image source credit -http://www.biology.arizona.edu/developmental_bio/problem_sets/developmental_mechanisms/03t.html

4.Delamination -( Mammalian and bird hypoblast formation ) Spilliting of 1 cell sheet into 2 or more parallel sheets. 

5.Epiboly- (Ectoderm formation in amphibians, sea urchins and tunicates )Spread of an outside cell layer(epidermal sheets) to envelop a yolk mass or deeper layer.or expansion of one cell sheet over other cells.

Types of cell movements during gastrulation 

                                  Image source credit -http://courses.biology.utah.edu/bastiani/3230/DB%20Lecture/Lectures/a7Gast.html


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TWO PIGMENT SYSTEMS

Two Pigment Systems: 

In result of red drop and Emerson effect it was concluded that at least two pigment systems are involved in photosynthesis. These two pigment systems are -

1. PS1 (piment system 1)

2.PS2(pigment system 2)

Presence of two such systems has been supported by studies based on chloroplast fractionation process which showed two types of particles within the chloroplast membrane. Acc . to Salisbury and Ross in 1986 smaller and lighter particles of PS I(located in non-appressed regions (stroma lamella) of  thylakoids) and larger and heavier particles of PS II(in appressed regions (grana ) of thylakoids ).

PS I complex -

• Consists of 200 chlorophylls, 50 carotenoids and  a molecule of P 700.
• Cytochrome f, one plastocyanin, two cyt-b, FRS (ferredoxin reducing substance), one or two membrane bound ferredoxin molecules etc.are also present.
• Primary electron acceptor is Fd .
• The carotenoids collect light energy for both the systems. Reduced carotenoids such as carotenes are found in PS-I. 
• It is rich in chl-a, iron and copper.  PS I controls the process of producing a strong reductant to reduce NADP into NADPH + H+.
• It involves in cyclic as well as non cyclic phosphorylation .
• It is not associated with photolysis of water .
• Main function is ATP sunthesis and production of NADPH.

Z-scheme of elctron transport chain 

Image source credit -http://www.bio.miami.edu/dana/226/226F08_10print.html

PSII complex -

• Consists of 200 chlorophylls, 50 carotenoids, a molecule of P 680, and a primary electronacceptor Q as plastoquinone, four plastoquinone equivalents four Mn++molecules bound to one or more proteins, two cyt-b6 559, one cyt-b6 and chloride. 
•  PS II is concerned with generation of strong oxidant and weak reductant coupled with the release of oxygen.
• More oxidised forms as xanthophylls, violaxanthin and neoxanthin are found in PS-II. 
• It involves in both non-cyclic phosphorylation .
• It is associated with photolysis of water.

(water molecules lyse using light energy, produces H+ ions (protons) e- (electrons) and oxygen. )

                                                 Image source credit -http://www.memrise.com/mem/2084591/photolysis-of-water/

• Main function is ATP synthesis and hydrolysis of water.

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Aquaporins

  

Image source credit- https://www.studyblue.com/notes/note/n/bio-111-study-guide-2012-13-wan/deck/9724270

Aquaporins are membrane proteins of a cell which helps in regulating the flow of water. It is a 28Kda protein . This is a kind of water channels which facilitate the movement of water by incresing the permiability of water towards membrane . It is found in all kingdom of life like plants, animals and micro-organisms.

The structure of Aquaporin is made up of 6 membrane helices.

Functions -
1. In cell migration .
2.Skin hydration . (AQP3 &AQP7)
3.Cell proliferation particularly AQP3.
4.AQP7 found to be involved in fat metabolism .
5.Conduct water molecules in and out of the cell and preventing the flow of ions and other molecules.
6. AQPs have also been proposed to transport other small molecules and gases, including carbon dioxide, ammonia, nitric oxide and hydrogen peroxide .
7.The AQP4 water channel is expressed in astrocytes throughout the central
nervous system.

The strucuture of aquaporins have been defined by mutagenesis, spectroscopy and freeze fracture electron microscopy method.
                                          CSIR QUESTIONS                Q.Which of the following statements describe the characteristics of  an aquaporin-

A)they are membrane proteins that are soluble in water. 
B)they are protein molecules with channels through which water molecules can diffuse.
C)their numbers in a membrane can change due to differing conditions or hormonal stimulation.
D)B& C

Ans. (D) They are actually hydrophobic proteins and therefore not soluble in water. Aquaporins are especially abundant in kidney and intestinal epithelial cells where water transport occurs extensively across tubular and epithelial cells. Dehydration condition can stimulate the kidney tubules to increase the number of water channels under stimulation by the posterior pituitary hormone ADH.

Q2.Aquaporins are a class of proteins that are relatively abundant in plant membranes .Following are certain statements -
a.Aquaporins form water channels in membrane.
b.some aquaporins also transport uncharge molecules such as ammonia.
c.Activity of aquaporins is not regulated by phosphorylation.
d.Activity is regulated bu calcium concentration and reactive oxygen species .
Which of the combinations are correct -
a.A,B& D                    C.A,C&D
b.B,C &D                          D.A,B& C

Ans. a (A,B &D ) the aquaglyceroporins transport uncharged molecules such as ammonia,urea,glycerol and CO2.

NOTE- Aquaporin proteins are composed of a bundle of six transmembrane α-helices. They are embedded in the cell membrane. The amino and carboxyl ends face the inside of the cell. The amino and carboxyl halves resemble each other, apparently repeating a pattern of nucleotides. Some researchers believe that this was created by the doubling of a formerly half-sized gene. Between the helices are five regions (A – E) that loop into or out of the cell membrane, two of them hydrophobic (B, E), with an asparagine–proline–alanine ("NPA motif") pattern. They create a distinctive hourglass shape, making the water channel narrow in the middle and wider at each end.

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Fats and Oils

Fats, oils and waxes are the general terms describing a structurally diverse bio-macromolecule called ‘lipids’. Lipids are esters of glycerol and fatty acids. Lipids with saturated fatty acids (saturated fat) tend to be solid at room temperature (25oC) and hence we generally call it as ‘fat’. On the other hand, lipids with unsaturated fatty acids (unsaturated fat) tend to be liquid at room temperature and thus they are called ‘oils’. Lipids are present in all living organisms including Archaebacteria. 

                    Imagesourcecredit-http://images.flatworldknowledge.com/ballgob/ballgob-fig17_x009.jpg

Similarities -

1. Both animal and plant fats are triglycerides (one glycerol esterified with three fatty acid residues)

2. Both are food reserve of the cell

3.  Unsaturated and saturated fats occurs in both plants and animals .
Difference -

Animal fats (Fats) Butter fat, Beef fat

•  Animals fats are relatively rich in saturated fatty acids.
• Animals fats due to their saturation, tend to stay solid at room temperature, hence commonly known as ‘fats’.
• Iodine number of animal fats will be relatively less (iodine number denote the degree of un-saturation in fatty acids).
•  Oxidative rancidity is observed more frequently in animal fats.
•  Animal fats are stored in liver, beneath the skin etc.
•  Animals have specialized cells for storing fat reserve called adipocytes.
• Vanderwall forces between the molecules are stronger .

Plant fats or Vegetable fats (Oil ) Coconut oil, Olive oil, Sunflower oil

• Plant fats are comparatively rich in unsaturated fatty acids.
•  Plant fats due to their high un-saturation, tend to stay liquid at room temperature, hence commonly known as ‘oils’.
•  Iodine number of plant fats will be more when compared to animal fats.
•  Oxidative rancidity is relatively less in plant fats.
•  Plant fats are stored in fruits and seeds.
•  Plants do not have adipocytes for storing fats, fats are stored as granules (oil droplets) in the cell cells of endosperm of seeds or other cells.
• Vanderwall forces between the molecules are weaker.

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Archaebacteria

A kind of bacteria which can live in very harsh environment . They do not require oxygen and they can sustain in very hot condition ,extreme salty enviornment and deep sea hydrothermal vents  . They are unicellular prokaryotes .




                     Image source credit -http://lady-rosales.blogspot.in/2010/08/three-domains-system-three-domain.html


Methanophiles (in sewage plant to help in breakdown of sewage ), halophiles (high conc. of salt ) and thermophiles (extreme hot areas ) are the examples of archaebacteria. These are looks like bacteria in microscope .

It differs from eubacteria in the respect of  rna which is present in ribosomes and arranged differently. They are thought to be oldest living organisms on earth .

Characters -

1. It stain  Gram + ve or -ve .
2. shape - sperical, lobed, spiral, rod shape
3. multiplication is asexual by binary fission or budding or fragmentation .
4. Circular DNA like bacteria and lacks histone protein .
5.GΨC content varies from 21-68%.
6. in T C arm of t- rna - pseudouridine/ 1- methyl pseudo uridine is present .
7. I t is sensitive to anisomycin and insensitive to kanamycin and chloramphenicol.
8. EF-2 reacts with diptheria toxin .
9. It obtain energy from sulphur and ammonia.
10. Methanogens live in deep mud without oxygen .
11.Halophiles can utilize light energy for the direct synthesis of ATP.
12.It is responsible for producing methane in bio gas ferementer.
13.Cell walls made up principally of pseudomurein.( N-acethyltalosaminuronic acid)Lacks D amino acids.
14.Ether linkage is present instead of ester linkage.
15.RNA pol enzyme contains 5-11 different subunits .


MCQ. 
Q.Archaebacteria differ from eubacteria in
1.cell membrance structure
2.mode of nutrition
3.cell shape
4.mode of reproduction

ans. 1

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CHECKPOINTS , CYCLINS AND CDK( CYCLIN DEPENDENT KINASE)

Cell cycle checkpoints-Cell cycle is a sequential step that taking place in a cell leading to the accurate duplication of genetic materials (DNA).  Checkpoints halt the progress of cell cycle if

 (1) any of the chromosomal DNA is damaged

 (2)  DNA replication during S phase or chromosome alignment during M phase, have not been properly completed.  

 Cell cycle checkpoints functions:

•  The nuclear genome is intact (without any mutation)
•  The conditions are appropriate for a cell to divide (enough nutrients is there for the daughter cells)
•   Genetic material is replicated only once in a cell cycle
•  No mutations occurred in the replicated chromosomes.If mutations are occurred, these mutations will be rectified by DNA repair system
•  Chromosomes are correctly oriented in the metaphase plate
•  All chromosomes are correctly attached to the spindle fibres.

The cyclins are so named because their amount varies throughout the cell cycle. To be active, the cyclin dependent kinases (cdks) controlling the cell cycle must bind to a specific cyclin. Cyclins fluctuate during cell cycle.

Different types of checkpoints in cell cycle:

                            Image source credit -http://gleesonbiology.pbworks.com/w/page/7537859/C9

There are three checkpoints in a cell cycle.

(1). G1 checkpoint (restriction checkpoint)

(2). G2 checkpoint (G2-M DNA Damage Checkpoint)

(3). Metaphase (M)-checkpoint (Spindle assembly checkpoint)

(1). G1 checkpoint:

G1 checkpoint(restriction point). operates at the end of G1 phase of cell cycle. It checks whether the conditions are favorable for the cell to divide. It also checks the DNA for any damage before it is going for a cycle of DNA replication in the next phase (S phase). If DNA damage is detected, checkpoint proteins will prevent the formation of active cyclin/cdk complexes. Inhibition of cyclin/cdk complex formation stops the progression of the cell cycle. The cells are then direct the DNA repair mechanism to rectify the DNA damage. If the environmental conditions are not good, the cell may enter into G0 phase. In yeast cells, G1 checkpoint is also called as start point.

(2). G2 checkpoint

G2 operates at the end of G2 phase. ( G2-M DNA damage checkpoint.) G2 checkpoint checks the DNA for any damage that might be occurred during the DNA replication in the previous cell cycle phase (S phase). G2 checkpoint also ensures that the entire DNA has been replicated completely. Apart from this, G2 checkpoint monitors the levels of proteins and growth factors that are needed in the next phase (M phase) of cell cycle. If any of the above factors are not satisfactory, the G2 check point hold the cells at G2 phase and initiate machineries to rectify the problems.  

(3). Metaphase (M) checkpoint (spindle assembly checkpoint)

Metaphase checkpoint is also called as spindle assembly checkpoint. It operates at the end of M phase. Metaphase checkpoint senses the integrity of the spindle apparatus in the cell. Spindle apparatus is involved in sorting of chromosomes during cell division. Correct orientation of chromosomes in the metaphase plate of cell is very essential for the proper segregation of chromosomes. If chromosomes are not correctly attached to the spindle apparatus, the metaphase checkpoint will stop the cell cycle. Thus, M checkpoint prevents cells from incorrectly sorting their chromosomes during division.So, M-Cdk inhibits mitosis .

 Importance of cell cycle checkpoints-

•  They can induce apoptosis (programmed cell death) .
•  Cell cycle checkpoint ensure only one round replication of DNA per cell cycle.
• Almost all cancers are due to the improper functioning of either one or many proteins involved in cell cycle regulation. (Eg. P53 – guardian of genome, a tumor suppressor gene).

Q.1 Tyr-15 is dephosphorylatedby ? 

a. cdc25

 b. cdc2 

c. Both

 d. Cdk1

And. A
Q2.When cell has stalled DNA replication fork, which checkpoint should be predominantly activated?

A)M
B)Both G¹M and M
C)G¹/S
D)G²/M
Ans.D

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Haemoglobin

Haemoglobin-(Hb )( Respiratory pigment )

It is a globular protein present in RBC which helps in transport oxygen by the help of histidine  . It is a kind of quaternary protein with 4 polypeptide chains (2 alpha and 2 beta ). The heme unit is a ring of atoms which carry an iron atom .(Fe 2+).
Heme binds o2 reversibly .
Co binds to heme more strongly than o2.
Haemoglobin has 2 states-
1.Oxyhaemoglobin (o2 bound 4 nos. )
2.Deoxy- haemoglobin (No O2 bound )

It is found in 2  major conformation forms -
1. R- form (Relaxed form )
-Fewer  interactions ,more flexible
-Higher affinity for O2.

2. T-form (Tense form/ taut stage )
-More interactions ,more stable
-Lower affinity for O2.

Sickle cell disease is a genetic alteration that causes some hemoglobin molecules to be defective (the defective hemoglobin is termed hemoglobin S). 

The iron atom in heme binds to the 4 nitrogen atoms in the centre of the porphyrin ring. Cooperative binding property( uptake of one ligand influences the affinities of the remaining unfilled binding sites. ) Haemoglobin is allosterically inhibited by CO2, protons( which is released from carbonic acid dissociation ) and the body needs oxygen .

 Image source credit -http://cikgurozaini.blogspot.in/2011/09/carbon-dioxide-transport.html


 When oxygen is bound to the first subunit of hemoglobin it change the quaternary structure of the protein this  helps subsequent molecule of oxygen to bind to the next subunit.  This phenomenon is called an allosteric (through space interaction).Iron atom is attached to the proximal histidine, this causes the local helix to move also.

Deoxy Hb   -------  Oxy Hb

Image source credit -http://www.chemistry.wustl.edu/~edudev/LabTutorials/Hemoglobin/MetalComplexinBlood.html

Haemoglobin dissociation curve is S-shaped or sigmoidal.
when Hb combines to one Oxygen, its shape distorts slightly, and that makes it easier for the second Oxygen and so on upto 4th o2 molecule .Fourth can't combine with any more Oxygen molecules since 4 is the maximum number of O2 molecules that can combine with one molecule of Haemoglobin. This diagram reflects the idea:

                              http://www.s-cool.co.uk/a-level/biology/transport/revise-it/blog

Q1. When oxygen haemoglobin curve shift to left it represents -(2008 DEC)
a.Decrease in pH
b.Decrese in Co2 level 
c.Rise in concentration of 2,3 BPG
d.more affinity for oxygen 
Ans.  c

Q2.The major role of 2,3 BPG formed during glycolysis in RBC is for haemoglobin is -
a.Increasing affinity for oxygen
b.Decreasing affinity for oxygen
c.Increasing affinity for Co2
d.Decreasing affinity for Co2
Ans. b

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CRYOPRESERVATION

It is a process of preservation of  cells or whole tissues ,  which are preserved by cooling the temperature to low- subzero .(-196c  ) the boiling point of liquid nitrogen . in this all biological process are suspended and material does not decompose .Cryopreserved cells are fragile it should be handle carefully . 

Advantage of freezing cells-
1. Reduced risk of Genetic change .
2.Reduced risk of Senescence leading to extinction of cell line. 
3.Reduced risk of Transformation to tumor related properties. 
4.Reduced risk of Contamination 
5.Saving reagents time 
6.Prevent Cross contamination by other cell line.

Process of cryopreservation -
1.The selection of plant species or tissues with particular  morphological and physiological characters . ( meristems, embryos, endosperms, ovules, seeds, cultured plant cells, protoplasts, calluses.) Among these, meristematic cells and suspension cell cultures, in the late lag phase or log phase are most suitable.

2.Cryoprotectants are the compounds that can prevent the damage caused to cells by freezing .The freezing point of water are reduced by the presence of cryoprotectant. As a result, the ice crystal formation is retarded during the process of cryopreservation.
Cryoprotectants  are dimethyl sulfoxide (DMSO), glycerol, ethylene, propylene, sucrose, mannose, glucose, proline and acetamide. Among these, DMSO, sucrose and glycerol are most widely used. 

3.The sensitivity of the cells to low temperature is variable and largely depends on the plant species.

4.The frozen cells/tissues are kept for storage at temperatures in the range of -70 to -196°C. Storage is ideally done in liquid nitrogen refrigerator — at 1 50°C in the vapour phase, or at -196°C in the liquid phase to maintain viability .For long term storage, temperature at -196°C in liquid nitrogen is ideal. A regular and constant supply of liquid nitrogen to the liquid nitrogen refrigerator is essential. 

5.Thawing is usually carried out by plunging the frozen samples in ampoules into a warm water (temperature 37-45°C) bath with vigorous swirling. By this approach, rapid thawing (at the rate of 500- 750°C min-1) occurs, and this protects the cells from the damaging effects ice crystal formation.

6.Thawed germplasm is washed several times to remove cryoprotectants and  then re-cultured in a fresh medium following standard procedures. 

7.The viability/survival of the frozen cells can be measured at any stage of cryopreservation or after thawing or re-culture.The techniques employed to determine viability of cryopreserved cells are the same as used for cell cultures .Staining techniques using triphenyl tetrazolium chloride (TTC), Evan’s blue and fluorescein diacetate (FDA) are commonly used.

8.Regeneration - For appropriate plant growth and regeneration thr cryopreserved cells should  be carefully nursed, and grown. Addition of certain growth regulators also help in regeneration.

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