SlideShare a Scribd company logo
Unit-IV
Germ plasm
Storage And
Cryopreservatio
n
Process of Cryopreservation:
⚫The cryopreservation of plant cell culture
followed by the regeneration of plants
involves the following steps:
⚫1. Development of sterile tissue cultures
⚫2. Addition of cryoprotectants and pre-
treatment
⚫3. Freezing
⚫4. Storage
⚫5. Thawing
⚫6. Re-culture
⚫7. Measurement of viability
⚫8. Plant regeneration
step I: Development of sterile
tissue culture:
⚫ One of the important steps is the selection of
plant species with reference to
morphological and physiological characters .
⚫ It directly influence the ability of explant to
survive cryopreservation.
⚫ Any tissue from a plant can be employed for
cryopreservation e.g. meristems,
endosperms, embryos, ovules, seeds,
cultured plant cells, calluses, protoplasts.
⚫ Out of these, meristematic cells and
suspension cell cultures which are in the late
lag phase or log phase are most appropriate.
step II: Addition of cryoprotectants
and pre-treatment:
⚫ The compounds that can prevent the damage
caused to cells by freezing or thawing are called as
cryoprotectants.
⚫ Cryoprotectants reduce the freezing point and
super-cooling point of water.
⚫ As a result, the ice crystal formation is delayed
during the process of cryopreservation.
⚫ Cryoprotectants used are dimethyl sulfoxide
(DMSO), glycerol, ethylene, propylene, sucrose,
mannose, glucose, proline and acetamide.
⚫ Among them, DMSO, sucrose and glycerol are
most commonly used.
⚫ Generally, a mixture of cryoprotectants instead of
a single one is preferred for more effective
cryopreservation without damage to cells/tissues.
step III: Freezing
⚫ The sensitivity of the cells to low temperature is
variable and largely relies on the plant species.
⚫ The different types of freezing methods used are as
follows:
⚫ 1. Slow-freezing method:
⚫ The tissue or the essential plant material is allowed to
slowly freeze at a slow cooling rates of 0.5-5°C/min
from 0°C to -100°C.
⚫ Then it is transferred to liquid nitrogen.
⚫ Slow-freezing method facilitates the flow of water
from the cells to the outside.
⚫ This avoids intracellular freezing and promotes
extracellular ice formation.
⚫ Because of this, the plant cells are partially
dehydrated and can survive better.
⚫ The slow-freezing technique is successfully
employed for the cryopreservation of suspension
cultures.
2. Rapid freezing method
⚫ This process is quite simple.
⚫ In this technique, the vial containing plant
material is plunged into liquid nitrogen.
⚫ During rapid freezing, reduction in
temperature from -300° to -1000°C/min
occurs.
⚫ The freezing process occurs so quickly that
small ice crystals are formed within the cells.
⚫ In addition to it, the growth of intracellular
ice crystals is also minimum.
⚫ Rapid freezing technique is applied for the
cryopreservation of shoot tips and somatic
embryos.
3. Stepwise freezing method
⚫This technique is a combination of slow
and rapid freezing procedures having the
advantages of both, and occurs in a
stepwise manner.
⚫Firstly, the plant material is cooled to an
intermediate temperature.
⚫Then it is kept there for about 30 minutes.
⚫Finally, it is rapidly cooled by plunging it
into liquid nitrogen.
⚫Stepwise freezing method has been
successfully applied for cryopreservation
of suspension cultures, shoot apices and
4. Dry freezing method
⚫It has been reported that the non-
germinated dry seeds can survive freezing
at very low temperature in comparison to
water-imbibing seeds which are sensitive
to cryogenic injuries.
⚫ In a similar way, dehydrated cells are
observed to have a better survival rate after
cryopreservation.
step IV: Storage
⚫ The frozen cultures should be maintained at the specific
temperature.
⚫ Generally, the frozen cells/tissues are maintained at
temperatures in the range of -70 to -196°C for storage.
⚫ Although, with temperatures above -130°C, ice crystal
growth may take place inside the cells which decreases
viability of cells.
⚫ The ideal storage is done in liquid N2 refrigerator at 150°C
in the vapour phase, or at -196°C in the liquid phase.
⚫ The final aim of storage is to halt all the cellular metabolic
activities and preserve their viability.
⚫ The temperature at -196°C in liquid nitrogen is regarded as
ideal for long term storage.
⚫ A regular and constant supply of liquid nitrogen to the
liquid nitrogen refrigerator is necessary.
⚫ It is essential to check the viability of the germplasm time
and again in some samples.
⚫ Proper documentation of the germplasm storage should be
done.
step V: Thawing
⚫ Thawing is usually performed by plunging the
frozen samples in ampoules into a warm water
(temperature 37-45°C) bath with robust
swirling.
⚫ By this process, rapid thawing (at the rate of
500- 750°C min-1) takes place, and this
preserves the cells from the damaging effects
from ice crystal formation.
⚫ As soon as the thawing occurs (ice completely
melts), the ampoules are transferred to a water
bath at temperature 20-25°C at the same
instant.
⚫ The cells get damaged if left in warm (37-45°C)
water bath for long time.
⚫ For the cryopreserved material (cells/tissues)
where the water content has been decreased to
an optimal level before freezing, the process
step VI: Re-culture
⚫To remove cryoprotectants, the thawed
germplasm is washed various times.
⚫Following standard procedures, this
material is then re-cultured in a fresh
medium.
⚫In some cases, the direct culture of the
thawed material is preferred without
washing.
⚫ It is so because certain vital substances,
released from the cells during freezing, are
assumed to enhance in vitro cultures.
step VII: Measurement of
viability:
⚫ The measurement of survival or viability of
the frozen materials can be performed at
any stage of cryopreservation or after
thawing or re-culture.
⚫ The techniques used to determine viability
of cryopreserved cells are the same as
applied for cell cultures.
⚫ The commonly used techniques are staining
techniques using triphenyl tetrazolium
chloride (TTC), Evan’s blue and fluorescein
diacetate (FDA).
⚫ The entry of cryopreserved cells into cell
division and regrowth in culture is the best
indicator to measure the viability of them.
⚫ This can be evaluated by the using following
expression.
step VIII: Plant regeneration
⚫The regeneration of the desired plant is
the ultimate purpose of cryopreservation
of germplasm.
⚫The cryopreserved cells/tissues have to be
carefully nursed, and grown for
appropriate plant growth and regeneration
.
⚫Along with maintenance of proper
environmental conditions, addition of
certain growth promoting substances is
often essential for successful plant
regeneratio
Limitations for
Cryopreservation:
⚫ An individual with good technical and
theoretical knowledge of living plant cells as
well as cryopreservation method is required.
⚫Precautions for cryopreservation:
⚫ The formation of ice crystals inside the cells
should be prevented as they are responsible
for causing injury to the organelles and the
cell.
⚫ Cells might be damaged if the intracellular
concentration of solutes is high.
⚫ Leakage of certain solutes from the cell
during freezing should be checked.
⚫ The physiological status of the plant
material is also essential.
Unit-IV Germplasm Storage and Cryopreservation.pptx
Unit-IV Germplasm Storage and Cryopreservation.pptx
Unit-IV Germplasm Storage and Cryopreservation.pptx

More Related Content

Similar to Unit-IV Germplasm Storage and Cryopreservation.pptx

Cryopreservation and reconstitution of preserved cell lines
Cryopreservation  and reconstitution  of preserved  cell linesCryopreservation  and reconstitution  of preserved  cell lines
Cryopreservation and reconstitution of preserved cell lines
Abdulrahman Muhammad
 
Topic 3 germplasm
Topic 3 germplasmTopic 3 germplasm
Topic 3 germplasm
Jan Mamun
 
Cryopreservation and reconstitution of preserved cell lines
Cryopreservation  and reconstitution of preserved  cell linesCryopreservation  and reconstitution of preserved  cell lines
Cryopreservation and reconstitution of preserved cell lines
Abdulrahman Muhammad
 
Cryopreservation and reconstitution of preserved cell lines
Cryopreservation  and reconstitution  of preserved  cell linesCryopreservation  and reconstitution  of preserved  cell lines
Cryopreservation and reconstitution of preserved cell lines
Abdulrahman Muhammad
 
Cryopreservation, germplasm storage
Cryopreservation, germplasm storageCryopreservation, germplasm storage
Cryopreservation, germplasm storage
KAUSHAL SAHU
 
Cryo
CryoCryo
Spermcryoperservation by Dr.Chandan
Spermcryoperservation by Dr.Chandan Spermcryoperservation by Dr.Chandan
Spermcryoperservation by Dr.Chandan
Morris Jawahar
 
Cryopreservation src
Cryopreservation srcCryopreservation src
Cryopreservation src
karishma purkayastha
 
Cryopreservation and its application to aquaculture.pptx
Cryopreservation and its application to aquaculture.pptxCryopreservation and its application to aquaculture.pptx
Cryopreservation and its application to aquaculture.pptx
Narsingh Kashyap
 
Introduction to Cryo and Organogenesis differentiation
Introduction to Cryo and Organogenesis differentiation  Introduction to Cryo and Organogenesis differentiation
Introduction to Cryo and Organogenesis differentiation
St. Bedes College , Shimla
 
pub-thebigfreeze-IPT-sept15
pub-thebigfreeze-IPT-sept15pub-thebigfreeze-IPT-sept15
pub-thebigfreeze-IPT-sept15
biocision
 
preservation.pptx
preservation.pptxpreservation.pptx
preservation.pptx
DhanaLakshmi787194
 
Deepshikha cryo final
Deepshikha cryo finalDeepshikha cryo final
Deepshikha cryo final
Deepshikha Keot
 
Cryopreservation
CryopreservationCryopreservation
Cryopreservation
megon94
 
Cryop ppt
Cryop pptCryop ppt
Cryop ppt
megon94
 
Cryopreservation of fruit crops
Cryopreservation of fruit cropsCryopreservation of fruit crops
Cryopreservation of fruit crops
annamalai university
 
Cryopreservation techniques in fruit crops
Cryopreservation techniques in fruit cropsCryopreservation techniques in fruit crops
Cryopreservation techniques in fruit crops
EkvVenkatraj
 
Sperm cryoperservation
Sperm cryoperservationSperm cryoperservation
Sperm cryoperservation
Yasminmagdi
 
Cryopreservation
CryopreservationCryopreservation
Cryopreservation
PARADHI
 
cryopreservation-170110143054.pdf
cryopreservation-170110143054.pdfcryopreservation-170110143054.pdf
cryopreservation-170110143054.pdf
SouravSwarnakar4
 

Similar to Unit-IV Germplasm Storage and Cryopreservation.pptx (20)

Cryopreservation and reconstitution of preserved cell lines
Cryopreservation  and reconstitution  of preserved  cell linesCryopreservation  and reconstitution  of preserved  cell lines
Cryopreservation and reconstitution of preserved cell lines
 
Topic 3 germplasm
Topic 3 germplasmTopic 3 germplasm
Topic 3 germplasm
 
Cryopreservation and reconstitution of preserved cell lines
Cryopreservation  and reconstitution of preserved  cell linesCryopreservation  and reconstitution of preserved  cell lines
Cryopreservation and reconstitution of preserved cell lines
 
Cryopreservation and reconstitution of preserved cell lines
Cryopreservation  and reconstitution  of preserved  cell linesCryopreservation  and reconstitution  of preserved  cell lines
Cryopreservation and reconstitution of preserved cell lines
 
Cryopreservation, germplasm storage
Cryopreservation, germplasm storageCryopreservation, germplasm storage
Cryopreservation, germplasm storage
 
Cryo
CryoCryo
Cryo
 
Spermcryoperservation by Dr.Chandan
Spermcryoperservation by Dr.Chandan Spermcryoperservation by Dr.Chandan
Spermcryoperservation by Dr.Chandan
 
Cryopreservation src
Cryopreservation srcCryopreservation src
Cryopreservation src
 
Cryopreservation and its application to aquaculture.pptx
Cryopreservation and its application to aquaculture.pptxCryopreservation and its application to aquaculture.pptx
Cryopreservation and its application to aquaculture.pptx
 
Introduction to Cryo and Organogenesis differentiation
Introduction to Cryo and Organogenesis differentiation  Introduction to Cryo and Organogenesis differentiation
Introduction to Cryo and Organogenesis differentiation
 
pub-thebigfreeze-IPT-sept15
pub-thebigfreeze-IPT-sept15pub-thebigfreeze-IPT-sept15
pub-thebigfreeze-IPT-sept15
 
preservation.pptx
preservation.pptxpreservation.pptx
preservation.pptx
 
Deepshikha cryo final
Deepshikha cryo finalDeepshikha cryo final
Deepshikha cryo final
 
Cryopreservation
CryopreservationCryopreservation
Cryopreservation
 
Cryop ppt
Cryop pptCryop ppt
Cryop ppt
 
Cryopreservation of fruit crops
Cryopreservation of fruit cropsCryopreservation of fruit crops
Cryopreservation of fruit crops
 
Cryopreservation techniques in fruit crops
Cryopreservation techniques in fruit cropsCryopreservation techniques in fruit crops
Cryopreservation techniques in fruit crops
 
Sperm cryoperservation
Sperm cryoperservationSperm cryoperservation
Sperm cryoperservation
 
Cryopreservation
CryopreservationCryopreservation
Cryopreservation
 
cryopreservation-170110143054.pdf
cryopreservation-170110143054.pdfcryopreservation-170110143054.pdf
cryopreservation-170110143054.pdf
 

Recently uploaded

Artificial Intelligence for XMLDevelopment
Artificial Intelligence for XMLDevelopmentArtificial Intelligence for XMLDevelopment
Artificial Intelligence for XMLDevelopment
Octavian Nadolu
 
5th LF Energy Power Grid Model Meet-up Slides
5th LF Energy Power Grid Model Meet-up Slides5th LF Energy Power Grid Model Meet-up Slides
5th LF Energy Power Grid Model Meet-up Slides
DanBrown980551
 
dbms calicut university B. sc Cs 4th sem.pdf
dbms  calicut university B. sc Cs 4th sem.pdfdbms  calicut university B. sc Cs 4th sem.pdf
dbms calicut university B. sc Cs 4th sem.pdf
Shinana2
 
Let's Integrate MuleSoft RPA, COMPOSER, APM with AWS IDP along with Slack
Let's Integrate MuleSoft RPA, COMPOSER, APM with AWS IDP along with SlackLet's Integrate MuleSoft RPA, COMPOSER, APM with AWS IDP along with Slack
Let's Integrate MuleSoft RPA, COMPOSER, APM with AWS IDP along with Slack
shyamraj55
 
Finale of the Year: Apply for Next One!
Finale of the Year: Apply for Next One!Finale of the Year: Apply for Next One!
Finale of the Year: Apply for Next One!
GDSC PJATK
 
How to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdf
How to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdfHow to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdf
How to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdf
Chart Kalyan
 
Best 20 SEO Techniques To Improve Website Visibility In SERP
Best 20 SEO Techniques To Improve Website Visibility In SERPBest 20 SEO Techniques To Improve Website Visibility In SERP
Best 20 SEO Techniques To Improve Website Visibility In SERP
Pixlogix Infotech
 
Deep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStr
Deep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStrDeep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStr
Deep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStr
saastr
 
AWS Cloud Cost Optimization Presentation.pptx
AWS Cloud Cost Optimization Presentation.pptxAWS Cloud Cost Optimization Presentation.pptx
AWS Cloud Cost Optimization Presentation.pptx
HarisZaheer8
 
WeTestAthens: Postman's AI & Automation Techniques
WeTestAthens: Postman's AI & Automation TechniquesWeTestAthens: Postman's AI & Automation Techniques
WeTestAthens: Postman's AI & Automation Techniques
Postman
 
June Patch Tuesday
June Patch TuesdayJune Patch Tuesday
June Patch Tuesday
Ivanti
 
Fueling AI with Great Data with Airbyte Webinar
Fueling AI with Great Data with Airbyte WebinarFueling AI with Great Data with Airbyte Webinar
Fueling AI with Great Data with Airbyte Webinar
Zilliz
 
Taking AI to the Next Level in Manufacturing.pdf
Taking AI to the Next Level in Manufacturing.pdfTaking AI to the Next Level in Manufacturing.pdf
Taking AI to the Next Level in Manufacturing.pdf
ssuserfac0301
 
Trusted Execution Environment for Decentralized Process Mining
Trusted Execution Environment for Decentralized Process MiningTrusted Execution Environment for Decentralized Process Mining
Trusted Execution Environment for Decentralized Process Mining
LucaBarbaro3
 
Ocean lotus Threat actors project by John Sitima 2024 (1).pptx
Ocean lotus Threat actors project by John Sitima 2024 (1).pptxOcean lotus Threat actors project by John Sitima 2024 (1).pptx
Ocean lotus Threat actors project by John Sitima 2024 (1).pptx
SitimaJohn
 
Letter and Document Automation for Bonterra Impact Management (fka Social Sol...
Letter and Document Automation for Bonterra Impact Management (fka Social Sol...Letter and Document Automation for Bonterra Impact Management (fka Social Sol...
Letter and Document Automation for Bonterra Impact Management (fka Social Sol...
Jeffrey Haguewood
 
Generating privacy-protected synthetic data using Secludy and Milvus
Generating privacy-protected synthetic data using Secludy and MilvusGenerating privacy-protected synthetic data using Secludy and Milvus
Generating privacy-protected synthetic data using Secludy and Milvus
Zilliz
 
HCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAU
HCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAUHCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAU
HCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAU
panagenda
 
leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...
leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...
leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...
alexjohnson7307
 
Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...
Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...
Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...
Jeffrey Haguewood
 

Recently uploaded (20)

Artificial Intelligence for XMLDevelopment
Artificial Intelligence for XMLDevelopmentArtificial Intelligence for XMLDevelopment
Artificial Intelligence for XMLDevelopment
 
5th LF Energy Power Grid Model Meet-up Slides
5th LF Energy Power Grid Model Meet-up Slides5th LF Energy Power Grid Model Meet-up Slides
5th LF Energy Power Grid Model Meet-up Slides
 
dbms calicut university B. sc Cs 4th sem.pdf
dbms  calicut university B. sc Cs 4th sem.pdfdbms  calicut university B. sc Cs 4th sem.pdf
dbms calicut university B. sc Cs 4th sem.pdf
 
Let's Integrate MuleSoft RPA, COMPOSER, APM with AWS IDP along with Slack
Let's Integrate MuleSoft RPA, COMPOSER, APM with AWS IDP along with SlackLet's Integrate MuleSoft RPA, COMPOSER, APM with AWS IDP along with Slack
Let's Integrate MuleSoft RPA, COMPOSER, APM with AWS IDP along with Slack
 
Finale of the Year: Apply for Next One!
Finale of the Year: Apply for Next One!Finale of the Year: Apply for Next One!
Finale of the Year: Apply for Next One!
 
How to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdf
How to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdfHow to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdf
How to Interpret Trends in the Kalyan Rajdhani Mix Chart.pdf
 
Best 20 SEO Techniques To Improve Website Visibility In SERP
Best 20 SEO Techniques To Improve Website Visibility In SERPBest 20 SEO Techniques To Improve Website Visibility In SERP
Best 20 SEO Techniques To Improve Website Visibility In SERP
 
Deep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStr
Deep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStrDeep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStr
Deep Dive: Getting Funded with Jason Jason Lemkin Founder & CEO @ SaaStr
 
AWS Cloud Cost Optimization Presentation.pptx
AWS Cloud Cost Optimization Presentation.pptxAWS Cloud Cost Optimization Presentation.pptx
AWS Cloud Cost Optimization Presentation.pptx
 
WeTestAthens: Postman's AI & Automation Techniques
WeTestAthens: Postman's AI & Automation TechniquesWeTestAthens: Postman's AI & Automation Techniques
WeTestAthens: Postman's AI & Automation Techniques
 
June Patch Tuesday
June Patch TuesdayJune Patch Tuesday
June Patch Tuesday
 
Fueling AI with Great Data with Airbyte Webinar
Fueling AI with Great Data with Airbyte WebinarFueling AI with Great Data with Airbyte Webinar
Fueling AI with Great Data with Airbyte Webinar
 
Taking AI to the Next Level in Manufacturing.pdf
Taking AI to the Next Level in Manufacturing.pdfTaking AI to the Next Level in Manufacturing.pdf
Taking AI to the Next Level in Manufacturing.pdf
 
Trusted Execution Environment for Decentralized Process Mining
Trusted Execution Environment for Decentralized Process MiningTrusted Execution Environment for Decentralized Process Mining
Trusted Execution Environment for Decentralized Process Mining
 
Ocean lotus Threat actors project by John Sitima 2024 (1).pptx
Ocean lotus Threat actors project by John Sitima 2024 (1).pptxOcean lotus Threat actors project by John Sitima 2024 (1).pptx
Ocean lotus Threat actors project by John Sitima 2024 (1).pptx
 
Letter and Document Automation for Bonterra Impact Management (fka Social Sol...
Letter and Document Automation for Bonterra Impact Management (fka Social Sol...Letter and Document Automation for Bonterra Impact Management (fka Social Sol...
Letter and Document Automation for Bonterra Impact Management (fka Social Sol...
 
Generating privacy-protected synthetic data using Secludy and Milvus
Generating privacy-protected synthetic data using Secludy and MilvusGenerating privacy-protected synthetic data using Secludy and Milvus
Generating privacy-protected synthetic data using Secludy and Milvus
 
HCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAU
HCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAUHCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAU
HCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAU
 
leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...
leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...
leewayhertz.com-AI in predictive maintenance Use cases technologies benefits ...
 
Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...
Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...
Salesforce Integration for Bonterra Impact Management (fka Social Solutions A...
 

Unit-IV Germplasm Storage and Cryopreservation.pptx

  • 2.
  • 3.
  • 4.
  • 5.
  • 6.
  • 7.
  • 8.
  • 9.
  • 10.
  • 11.
  • 12.
  • 13. Process of Cryopreservation: ⚫The cryopreservation of plant cell culture followed by the regeneration of plants involves the following steps: ⚫1. Development of sterile tissue cultures ⚫2. Addition of cryoprotectants and pre- treatment ⚫3. Freezing ⚫4. Storage ⚫5. Thawing ⚫6. Re-culture ⚫7. Measurement of viability ⚫8. Plant regeneration
  • 14. step I: Development of sterile tissue culture: ⚫ One of the important steps is the selection of plant species with reference to morphological and physiological characters . ⚫ It directly influence the ability of explant to survive cryopreservation. ⚫ Any tissue from a plant can be employed for cryopreservation e.g. meristems, endosperms, embryos, ovules, seeds, cultured plant cells, calluses, protoplasts. ⚫ Out of these, meristematic cells and suspension cell cultures which are in the late lag phase or log phase are most appropriate.
  • 15. step II: Addition of cryoprotectants and pre-treatment: ⚫ The compounds that can prevent the damage caused to cells by freezing or thawing are called as cryoprotectants. ⚫ Cryoprotectants reduce the freezing point and super-cooling point of water. ⚫ As a result, the ice crystal formation is delayed during the process of cryopreservation. ⚫ Cryoprotectants used are dimethyl sulfoxide (DMSO), glycerol, ethylene, propylene, sucrose, mannose, glucose, proline and acetamide. ⚫ Among them, DMSO, sucrose and glycerol are most commonly used. ⚫ Generally, a mixture of cryoprotectants instead of a single one is preferred for more effective cryopreservation without damage to cells/tissues.
  • 16. step III: Freezing ⚫ The sensitivity of the cells to low temperature is variable and largely relies on the plant species. ⚫ The different types of freezing methods used are as follows: ⚫ 1. Slow-freezing method: ⚫ The tissue or the essential plant material is allowed to slowly freeze at a slow cooling rates of 0.5-5°C/min from 0°C to -100°C. ⚫ Then it is transferred to liquid nitrogen. ⚫ Slow-freezing method facilitates the flow of water from the cells to the outside. ⚫ This avoids intracellular freezing and promotes extracellular ice formation. ⚫ Because of this, the plant cells are partially dehydrated and can survive better. ⚫ The slow-freezing technique is successfully employed for the cryopreservation of suspension cultures.
  • 17. 2. Rapid freezing method ⚫ This process is quite simple. ⚫ In this technique, the vial containing plant material is plunged into liquid nitrogen. ⚫ During rapid freezing, reduction in temperature from -300° to -1000°C/min occurs. ⚫ The freezing process occurs so quickly that small ice crystals are formed within the cells. ⚫ In addition to it, the growth of intracellular ice crystals is also minimum. ⚫ Rapid freezing technique is applied for the cryopreservation of shoot tips and somatic embryos.
  • 18. 3. Stepwise freezing method ⚫This technique is a combination of slow and rapid freezing procedures having the advantages of both, and occurs in a stepwise manner. ⚫Firstly, the plant material is cooled to an intermediate temperature. ⚫Then it is kept there for about 30 minutes. ⚫Finally, it is rapidly cooled by plunging it into liquid nitrogen. ⚫Stepwise freezing method has been successfully applied for cryopreservation of suspension cultures, shoot apices and
  • 19. 4. Dry freezing method ⚫It has been reported that the non- germinated dry seeds can survive freezing at very low temperature in comparison to water-imbibing seeds which are sensitive to cryogenic injuries. ⚫ In a similar way, dehydrated cells are observed to have a better survival rate after cryopreservation.
  • 20. step IV: Storage ⚫ The frozen cultures should be maintained at the specific temperature. ⚫ Generally, the frozen cells/tissues are maintained at temperatures in the range of -70 to -196°C for storage. ⚫ Although, with temperatures above -130°C, ice crystal growth may take place inside the cells which decreases viability of cells. ⚫ The ideal storage is done in liquid N2 refrigerator at 150°C in the vapour phase, or at -196°C in the liquid phase. ⚫ The final aim of storage is to halt all the cellular metabolic activities and preserve their viability. ⚫ The temperature at -196°C in liquid nitrogen is regarded as ideal for long term storage. ⚫ A regular and constant supply of liquid nitrogen to the liquid nitrogen refrigerator is necessary. ⚫ It is essential to check the viability of the germplasm time and again in some samples. ⚫ Proper documentation of the germplasm storage should be done.
  • 21. step V: Thawing ⚫ Thawing is usually performed by plunging the frozen samples in ampoules into a warm water (temperature 37-45°C) bath with robust swirling. ⚫ By this process, rapid thawing (at the rate of 500- 750°C min-1) takes place, and this preserves the cells from the damaging effects from ice crystal formation. ⚫ As soon as the thawing occurs (ice completely melts), the ampoules are transferred to a water bath at temperature 20-25°C at the same instant. ⚫ The cells get damaged if left in warm (37-45°C) water bath for long time. ⚫ For the cryopreserved material (cells/tissues) where the water content has been decreased to an optimal level before freezing, the process
  • 22. step VI: Re-culture ⚫To remove cryoprotectants, the thawed germplasm is washed various times. ⚫Following standard procedures, this material is then re-cultured in a fresh medium. ⚫In some cases, the direct culture of the thawed material is preferred without washing. ⚫ It is so because certain vital substances, released from the cells during freezing, are assumed to enhance in vitro cultures.
  • 23. step VII: Measurement of viability: ⚫ The measurement of survival or viability of the frozen materials can be performed at any stage of cryopreservation or after thawing or re-culture. ⚫ The techniques used to determine viability of cryopreserved cells are the same as applied for cell cultures. ⚫ The commonly used techniques are staining techniques using triphenyl tetrazolium chloride (TTC), Evan’s blue and fluorescein diacetate (FDA). ⚫ The entry of cryopreserved cells into cell division and regrowth in culture is the best indicator to measure the viability of them. ⚫ This can be evaluated by the using following expression.
  • 24. step VIII: Plant regeneration ⚫The regeneration of the desired plant is the ultimate purpose of cryopreservation of germplasm. ⚫The cryopreserved cells/tissues have to be carefully nursed, and grown for appropriate plant growth and regeneration . ⚫Along with maintenance of proper environmental conditions, addition of certain growth promoting substances is often essential for successful plant regeneratio
  • 25. Limitations for Cryopreservation: ⚫ An individual with good technical and theoretical knowledge of living plant cells as well as cryopreservation method is required. ⚫Precautions for cryopreservation: ⚫ The formation of ice crystals inside the cells should be prevented as they are responsible for causing injury to the organelles and the cell. ⚫ Cells might be damaged if the intracellular concentration of solutes is high. ⚫ Leakage of certain solutes from the cell during freezing should be checked. ⚫ The physiological status of the plant material is also essential.