Cryopreservation is a process where biological materials like cells, tissues, and organs are preserved at very low temperatures, typically in liquid nitrogen at -196°C. This process stops all metabolic activities and allows long-term preservation. The key steps involve selection of suitable plant material, addition of cryoprotectants to prevent ice crystal formation, slow freezing or vitrification to solidify water in an amorphous glassy state without crystallization, storage in liquid nitrogen, and thawing for regeneration of plants. Cryopreservation has many applications in conservation of genetic resources, maintenance of disease-free stock, and long-term storage of cell cultures and germplasm in seed banks and gene banks.
Until two decades ago the genetic resources were getting depleted owing to the
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 failed to prevent losses caused due to various reasons.
A new methodology had to be devised for long term preservation of material.
Cryopreservation in plant culture and techniques - introduction, definition , steps of cells cryopreservation, methods of cryopreservation, Techniques of cryopreservation , application, advantage and disadvantage, diagram of cryopreservation.
Gametoclonal variation in Plant tissue culture - Variation in gametes clones # Origin # Production # Application of Gametoclonal Variation in plants with their examples.
Please watch the slides and don't forget to follow our channel to getting more updates.
HYBRIDIZATION & HAPLOID PRODUCTION
Introduction
WIDE HYBRIDIZATION
INTER-SPECIFIC HYBRIDIZATION
Barriers to distant hybridization
Techniques to overcome barriers
Haploids and Doubled Haploids in Plant
Production of haploids and doubled haploids
a) Induction of maternal haploids
Wide hybridization
3. In vitro induction of maternal haploids – gynogenesis
Induction of paternal haploids – Androgenesis
Production of Homozygous Diploid Plants
Application of Haploids in Plant Breeding
Importance and Implications of Anther and Pollen Culture
Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
Multiplication of Apical or Axillary bud, Shoot tip or meristem culture
Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
Until two decades ago the genetic resources were getting depleted owing to the
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 failed to prevent losses caused due to various reasons.
A new methodology had to be devised for long term preservation of material.
Cryopreservation in plant culture and techniques - introduction, definition , steps of cells cryopreservation, methods of cryopreservation, Techniques of cryopreservation , application, advantage and disadvantage, diagram of cryopreservation.
Gametoclonal variation in Plant tissue culture - Variation in gametes clones # Origin # Production # Application of Gametoclonal Variation in plants with their examples.
Please watch the slides and don't forget to follow our channel to getting more updates.
HYBRIDIZATION & HAPLOID PRODUCTION
Introduction
WIDE HYBRIDIZATION
INTER-SPECIFIC HYBRIDIZATION
Barriers to distant hybridization
Techniques to overcome barriers
Haploids and Doubled Haploids in Plant
Production of haploids and doubled haploids
a) Induction of maternal haploids
Wide hybridization
3. In vitro induction of maternal haploids – gynogenesis
Induction of paternal haploids – Androgenesis
Production of Homozygous Diploid Plants
Application of Haploids in Plant Breeding
Importance and Implications of Anther and Pollen Culture
Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
Multiplication of Apical or Axillary bud, Shoot tip or meristem culture
Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
Cryopreservation Prepared by Md. Ali HaidarAli Haidar
I am Md. Ali Haidar student at faculty of Agriculture, EXIM Bank Agricultural University Bangladesh. I am a future Agriculturist. I published my Presentation for helping other student.
Preservation of industrially important microorganisms, methods of preservation, periodic transfer, storage in saline suspension, storage in sterile soil, cryopreservation
According to Lokesh, the goal of pure culture preservation is to preserve genetic stability and microbiological viability by use of several methods. Important techniques include the straightforward and inexpensive short-term storage of refrigeration at 4°C on agar slants; the long-term preservation of deep freezing at -70°C to -80°C with cryoprotectants like glycerol or DMSO, which guarantees little genetic changes over years; and the freeze-drying process, or lyophilization, in which cultures are frozen and dehydrated under a vacuum to preserve viability and stability over long periods of time. Research, clinical diagnostics, and industrial applications all depend on these techniques to guarantee the availability of clean microbial cultures when required.
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
2. Cryo is Greek word. (krayos – frost)
It literally means preservation in “frozen state.”
It is a process where tissues, organelles, cells, extracellular
matrix, organs or any other biological constructs susceptible to
damage caused by unregulated chemical kinetics are preserved by
cooling to very low temp -196 degree Celsius using liquid nitrogen.
Cryopreservation-
2
3. What is germplasm conservation?
Plant germplasm is the genetic sources material used by the
plant breeders to develop new cultivars.
They may include:
Seeds
Other plant propagules are:
Leaf
Stem
Pollen
Cultured cells
Which can be grown into mature plant?
Germplasm provide the raw material( genes) which the
breeder used to develop commercial crop varieties.
3
4. Why preservation is
important ?
Until two decades ago the genetic resources were getting depleted owing to the
continous 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 failed to prevent losses caused due to
various reasons.
A new methodology had to be devised for long term preservation of material.
4
5. Germplasm
A germplasm is a collection of genetic resources for
an organism.
For plants, the germplasm may be stored as a seed
collection (even a large seed bank).
For trees, in a nursery.
Animal as well as plant genetics may be stored in a
gene bank or cryobank.
5
6. Cryopreservation
Cryopreservation is a non-lethal storage of biological
material at ultra-low temperature. At the temperature
of liquid nitrogen (-196 degree) almost all metabolic
activities of cells are ceased and the sample can then
be preserved in such state for extended peroids.
However, only few biological materials can be frozen to
(-196 degree) without affecting the cell viability.
6
7. HISTORY
Theoreticians of cryopreservation was James Lovelock
(born 1919).
Osmotic stress
Salt concentration
Christopher Polge, carried out cryopreservation of
first fowl sperm.
Later, in 1950s they tried it on humans where
pregnancy was obtained after insemination of frozen
sperm.
7
8. However, the rapid immersion of the samples in liquid
nitrogen did not produce the necessary viability to
make them usable after thawing.
Importance of controlled or slow cooling to obtain
maximum survival on thawing of the living cells.
Cryoprotectants were introduced.
8
9. INTRODUCTION
In recent years with tremendous increase in
population:
Forest
Land resources
Population of medicinal plants
Aromatic plants species
9
10. The conventional methods of germplasm
preservation are prone to possible catastrophic
losses because of:
1. Attack by pest and pathogens
2. Climate disorder
3. Natural disasters
4. Political and economic causes
10
11. The conservation of germplasm can be done
by two methods.
1. In-situ preservation: preservation of the
germplasm in their natural environment by
establishing biospheres, national parks etc.
2. Ex-situ preservation: in the form of seed or in vitro
cultures.
11
12. Ex-situ has following disadvantages
Some plants do not produce fertile seeds.
Loss of seed viability
Seed destruction by pests, etc.
Poor germination rate.
This is only useful for seed propagating plants.
It’s a costly process.
12
13. Advantages
Small areas can store large amount of material.
Protection from environmental methods.
13
14. Liquid nitrogen is most widely used material for
cryopresevation.
Dry ice can also be used.
Why Liquid nitrogen ?
Chemically inert
Relatively low cost
Non toxic
Non flammable
Readily available
14
15. STEPS INVOLVED IN CRYOPRESERVATION
SELECTION OF PLANT MATERIAL
PREGROWTH
ADDITION OF
CRYOPROTECTANTS
VITRIFICATION
CRYOPROTTECTIVE
DEHYDRATION
15
18. SELECTION OF PLANT MATERIAL
Morphological and physiological conditions of plant
material influence the ability of explants to survive
during cryopreservation.
Different types of tissues can be used for
cryopreservation such as:
Ovules
Anther/pollen
Embryos
Endosperm
Protoplast, etc.
18
19. FACTORS:
o Tissue must be selected from healthy plants.
o Small
o Young
o Rich in cytoplasm
o Meristematic cells can survive better than the larger
o Highly vacuolated cells.
19
20. o Callus derived from tropical plant is more resistant to
freezing damage.
o A rapidly growing stage of callus shortly after 1 or 2
weeks of subculture is best for cryopreservation.
o Old cells at the top of callus and blackened area should
be avoided.
o cultured cells are not ideal for freezing. Instead,
organized structures such as shoots apices, embryos or
young plantlets are preferred.
20
21. o Water content of cell or tissue used for
cryopreservation should be low freezable water,
tissues can withstand extremely low temperatures.
21
22. PREGROWTH
Pregrowth treatment protect the plant tissues against
exposure to liquid nitrogen.
Pregrowth involves the application of additives known
to enhance plant stress tolerance.
E.g.
abscisic acid
praline
trehalose
22
23. Partial tissue dehydration can be achieved by the
application of osmotically active compounds.
The addition of low concentration of DMSO (1-5%)
during pre-growth often improves shoot tip recovery,
E.g.
C. roseus cells are precultured in medium containing
1M sorbitol before freezing. (Chen et al., 1984)
Digitalis cells were precultured on 6% Mannitol
medium for 3 days before freezing. (Seitz et al., 1983)
Nicotiana sylvestris with 6% sorbitol for 2-5 days
before freezing. (Maddox et al., 1983)
23
24. ADDITION OF A CRYOPROTECTANT
A cryoprotectant is a substance that is used to
protect biological tissue from freezing damage
(damage due to ice formation).
They acts like antifreeze
They lower freezing temperature
Increase viscosity and
Prevents damage to the cells.
24
25. There are two potential sources of cell damage
during cryopreservation.
1. Formation of large ice crystals inside the cell.
2. Intracellular concentration of solutes increase to
toxic levels before or during freezing as a result of
dehydration.
25
27. VITRIFICATION
The term “vitrification” refers to any process
resulting in “glass formation”, the transformation from
a liquid to a solid in the absence of crystallization.
According to this definition, cells that are properly
slow frozen become “vitrified”.
A process where ice formation cannot take place
because the aqueous solution is too concentrated to
permit ice crystal nucleation. Instead, water solidifies
into an amorphous ‘glassy’ state.
27
28. CRYOPROTECTIVE DEHYDRATION
Dehydration can be achieved by growth in presence
of high concentration of osmotically active compounds
like
sugars
polyols and/or
In a sterile flow cabinet
over silica gel.
28
29. Dehydration reduces the amount of water
Depresses
its freezing
temperature and
Promotes
vitrification
If cells are sufficiently dehydrated they may be able to
withstand immersion in liquid hydrogen.
Ice formationIncreases the
osmotic pressure
29
30. ENCAPSULATION AND DEHYDRATION
This involves the encapsulation of tissues in calcium
alginate beads.
Which are pre-grown in liquid culture media
containing high concentration of sucrose.
After these treatments the tissues are able to withstand
exposure to liquid nitrogen without application of
chemical cryoprotectants.
30
31. FREEZING: RAPID FREEZING
The plant material is placed in vials and plunged into
liquid nitrogen and decrease of -300 to -10000c or
more occurs.
The quicker the freezing is done , the smaller the
intracellular ice crystals are.
Dry ice can also be used in a similar manner.
31
32. This method is technically simple and easy to handle.
Rapid freezing has been employed for
cryopreservation of shoot tips of potato , strawberry ,
brassica species
32
33. SLOW FREEZING
Tissue is slowly frozen with decrease in temperature
from -0.1 to 10°c/min.
Slow cooling permits the flow of water from the cells
to the outside , thereby promoting extracellular ice
formation instead of lethal intracellular freezing.
This method has been successfully employed for
cryopreservation of meristems of peas , potato ,
cassava , strawberry etc.
33
34. In a normal ice making process, the surface of the cube
freezes up much faster than the interior.
Which “cramps” the interior, clouding it.
By using very hot (and pure) water inside an insulated
environment, you are assuring yourself a very slow
freezing that allows the interior to cool down at a rate
far closer to that of the exterior, and that lack of
“cramping” is what produces such clear ice.
34
35. STEPWISE FREEZING
In this method slow freezing down to -20 to 40c.
A stop for a period of approximately 30 min and then
additional rapid freezing to -196c is done by plunging
in liquid nitrogen.
Slow freezing permits protective dehydration of the
cells and rapid freezing prevents the growing of big ice
crystals.
The Stepwise freezing gives excellent results in
strawberry and with suspension cultures.
35
36. STORAGE
Storage of frozen material at correct temperature is as
important as freezing.
The frozen cells/tissues are kept for storage at
temperature ranging from -70 to -196°c.
Temperature should be sufficiently low for long term
storage of cells to stop all the metabolic activities and
prevent biochemical injury.
Long term storage is best done at -196°c. …
36
37. THAWING
It is done by putting ampoule containing the sample in
a warm water bath (35 to 40°c).
Frozen tips of the sample in tubes or ampoules are
plunged into the warm water with a vigorous swirling
action just to the point of ice disappearance.
It is important for the survival of the tissue that the
tubes should not be left in the warm water bath after
ice melts .
37
38. just a point of thawing quickly transfer the tubes to a
water bath maintained at room temperature and
continue the swirling action for 15 sec to cool the warm
walls of the tube.
Tissue which has been frozen by
encapsulation/dehydration is frequently thawed at
ambient temperature.
38
39. DETERMINATION OF SURVIVAL/VIABILITY
Regrowth of the plants from stored tissues or cells is
the only test of survival of plant materials.
Various viability tests include Fluorescien diacetate
(FDA) staining , growth measurement by cell number ,
dry and fresh weight.
Important staining methods are:
Triphenyl Tetrazolium Chloride (TTC)
Evan’s blue staining.
39
40. TRIPHENYL TETRAZOLIUM CHLORIDE (TTC)
ASSAY
Cell survival is measured by amount of red formazan
product formed due to reduction of TTC assay which is
measured spectrometrically.
Only the viable cells which contain the enzyme
mitochondrial dehydrogenase which reduces TTC to
red formazan will be stained and dead cells will not
take up the dye.
40
41. EVAN’S BLUE STAINING
One drop of 0.1% solution of Evan’s blue is added to
cell suspension on a microscope slide and observed
under light microscope.
Only non viable cells (dead cells) stain with Evan’s
blue. % of viable cells = Number of fluorescent cells ×
1oo total no of cells(viable + non-viable).
Individual cell viability assayed with Evan's blue dye
and fluorescein diacetate.
41
42. MEASUREMENT OF GROWTH OF CELL
CULTURES
Fresh and dry weight measurements
Increase in cell number
Packed cell volume (PCV)
Molecular protein and DNA
Mitotic index
Medium component calibration
Conductivity of medium
Cellular protien
42
43. APPLICATIONS OF CRYOPRESERVATION:
CONSERVATION OF GENETIC MATERIAL
Cryopreservation provides an opportunity for
conservation of endangered medicinal plants.
Cryopreservation has been used successfully to store a
range of tissue types , including meristems ,
anthers/pollens and embryos.
43
44. FREEZE STORAGE OF CELL CULTURES
A cell line to be maintained has to be subcultured and
transferred periodically and repeatedly over an
extended period of time.
cryopreservation is an ideal approach to suppress cell
division to avoid the need for periodical subculturing
44
45. MAINTAINENCE OF DISEASE FREE
STOCK
Pathogen free stocks of rare plant material could be
frozen and propagated when needed.
Cold acclimatization and frost resistance
A cryopreserved tissue culture would provide a
suitable material for selection of cold resistant mutant
cell lines , which could later differentiate into frost
resistance plants.
45
46. SEED BANK
A seed bank stores seeds as a source for planting in
case seed reserves elsewhere are destroyed.
It is a type of gene bank.
The seeds stored may be food crops, or those of rare
species to protect biodiversity.
The reasons for storing seeds may be varied.
46
47. Seeds are dried to a moisture content of less than 5%.
The seeds are then stored in freezers at -18°C or below.
Because seed (DNA) degrades with time, the seeds
need to be periodically replanted and fresh seeds
collected for another round of long-term storage.
47
48. GENE BANK
Gene banks are a type of bio repository which
preserve genetic material.
In plants, this could be by freezing cuts from the plant,
or stocking the seeds.
In animals, this is the freezing of sperm and eggs
in zoological freezers until further need..
48
49. In an effort to conserve agricultural biodiversity, gene
banks are used to store and conserve the plant genetic
resources of major crop plants and their crop wild
relatives.
There are many gene banks all over the world, with the
Svalbard Global Seed Vault being probably the most
famous one.
49
50. Major advantages are :
1. Once the material is sucessfully conserved to particular temperature it can
be preserved indefinately.
2. Once in storage no chance of new contamination of fungus or bacteria.
3. Minimal space required.
4. Minimal labour required.
51. Application
It is ideal method for long term conservation of material.
Disease free plants can be conserved and propagated.
Recalcitrant 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.
52. National Seed Storage Laboratory (NSSL) (Fort
Collins, Colorado, USA): 2,100 accessions of apple
(dormant buds) .
National Clonal Germplasm Repository (NCGR) of
Corvallis (USA): 104 accessions of pear (shoot tips).
International Potato Centre (CIP) (Lima, Peru) : 345
potato accessions.
Tissue Culture BC Research Inc .(Vancouver, BC,
Canada) : 5000 accessions representing 14 conifer
species.
52
53. Limitation of germplasm
The expensive equipment needed to provide
controlled and varible rates of cooling/warming
temperatures can however be a limitation in the
application of in vitro technology for large scale
germplasm conservation.
Formation of ice crystal inside the cell should be
prevented as they cause injury to the cell.
Sometimes certain solutes from the cell leak out
during freezing.
Cryoprotectant also effect the viability of cells.