This document provides an overview of germplasm collection, conservation, and cryopreservation. It defines germplasm as living genetic resources such as seeds or tissues maintained for plant breeding and research. Germplasm collection involves collecting diverse crop varieties with different gene alleles. Methods of germplasm conservation include in-situ conservation of genetic resources in natural habitats and ex-situ conservation of germplasm outside natural habitats, such as in seed banks or through cryopreservation. Cryopreservation involves freezing plant material at liquid nitrogen temperatures to preserve viability, with the goal of long-term storage and regeneration of plants from the stored genetic material.

1. GERMPLASM COLLECTION
AND CRYOPRESERVATION
PRESENTED BY :
HARSHITHA A M
REG NO : P03MS21S0011

2. CONTENT
• GERMPLASM : DEFINITION
• GERMPLASM COLLECTION
• GERMPLASM CONSERVATION
• CRYOPRESERVATIONN
• METHODS
• APPLICATIONS

3. GERMPLASM
• GERMPLASM ARE LIVING GENETIC RESOURCES SUCH AS SEEDS OR TISSUES THAT ARE
MAINTAINED FOR THE PURPOSE OF PLANT BREEDING, PRESERVATION, AND OTHER RESEARCH
USES.

4. GERMPLASM COLLECTION
• GERMPLASM COLLECTION IS A COLLECTION OF LIVE CROP
VARIETIES WITH DIVERS ALLELES OF GENES .
• A VERY GOOD COLLECTION IS HELPFUL IN PICKING UP THE DESIRED
TRAIT FOR INCORPORATION IN NEW IMPROVED VARIETIES

6. INDIAN INSTITUTE OF SPICES RESEARCH
POSSESSES WORLD’S LARGEST GERMPLASM
COLLECTION
• ALL SPICES -180
• CARDAMOM – 416
• CLOVE – 223
• CINNAMON – 408
• GARCINIA – 61
• GINGER – 700
• NUTMEG - 482
• PAPRIKA – 130( 96 INDIGENOUS & 34 EXOTIC)
• TURMERIC – 899
• BLACK PEPPER - 468

7. • THINGS TO BE CONSIDERED FOR GERMPLASM
COLLECTION
1 SEED & VEGETATIVE SAMPLES SHOULD BE CHECKED ON A
REGULAR BASIS FOR INSECT AND FUNGAL ATTACK.
2 IF SEED SAMPLES BEING ACTIVELY DRIED,SILICA GEL MAY HAVE
TO BE CHANGED AND DRY SAMPLES REMOVED.
3 SAMPLES OF FLESHY FRUITS IN PLASTIC BAGS WILL NEED TO BE
AERATED REGULARLY.
4 DRYING PAPERS IN HARBARIUM PRESSES MUST BE CHANGED
EVERY COUPLE OF DAYS.
5 SAMPLES MAY NEED TO BE SENT BACK TO THE GENE BANK AT
VARIOUS STAGES.

8. GERMPLASM CONSERVATION
• GERMPLASM CONSERVATION IS THE MOST
SUCCESSFUL METHOD TO CONSERVE THE GENETIC
TRAITS OF ENDANGERED AND COMMERCIALLY
VALUABLE SPECIES.

10. TYPES OF GERMPLASM CONSERVATION:
THERE ARE MAINLY TWO TYPES OF GERMPLASM CONSERVATION:
• IN-SITU CONSERVATION
• EX-SITU CONSERVATION

11. IN-SITU CONSERVATION
• THE CONSERVATION OF GERMPLASM IN THEIR NATURAL HABITAT BY
CONSTRUCTING NATIONAL PARKS/GENE SANCTUARIES IS TERMED AS IN-
SITU CONSERVATION.
• IT IS REGARDED AS A HIGH PRIORITY GERMPLASM PRESERVATION
PROGRAMME.
• IT HELPS IN THE CONTINUATION OF PLANT LIFE IN THE ECOLOGICAL
COMMUNITY.
• ONE OF THE ADVANTAGES OF IN-SITU CONSERVATION IS THAT IT
CONTINUES THE EVOLUTION OF THE SPECIES ALONG WITH THE
ALLOWANCE OF APPEARANCE OF NEW RECOMBINANT FORM.

12. EX-SITU CONSERVATION:
• IT IS THE MAJOR METHOD FOR THE PRESERVATION OF GERMPLASM
OBTAINED FROM BOTH WILD AND CULTIVATED PLANT MATERIALS.
• THE GENETIC MATERIALS CAN BE CONSERVED EITHER BY
COLLECTING PLANTS AND KEPT IN NORMAL GROWING
CONDITIONS OR IN THE FORM OF SEEDS IN SEED BANKS, THROUGH
TISSUE CULTURE AND LOW TEMP MAINTAINED BY LIQUID N2. THIS
TYPE OF CONSERVATION IS TERMED AS EX-SITU CONSERVATION.
• SUGARCANE, COCOA AND RUBBER ARE STORED IN THIS WAY.

13. CRYOPRESERVATION
• IN CRYOPRESERVATION, PLANT MATERIAL IS FROZEN AND
MAINTAINED AT THE TEMPERATURE OF LIQUID NITROGEN (-196
DEGREE CELSIUS.
• IN THIS TEMPERATURE,CELLS ARE INACTIVE STATE
• THE PART USED FOR CRYOPRESERVATION ARE SHOOT, EMBRYOS
AND YOUNG PLANTLETS.

14. PROCESS OF CRYOPRESERVATION
1.
•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

15. 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.

16. 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.
• 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

17. 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.

18. RAPID FREEZING METHOD:THIS PROCESS IS QUITE
SIMPLE.
1 IN THIS TECHNIQUE, THE VIAL CONTAINING
MATERIAL IS PLUNGED INTO LIQUID NITROGEN.
2 DURING RAPID FREEZING, REDUCTION IN
FROM -300° TO -1000°C/MIN OCCURS.
3 RAPID FREEZING TECHNIQUE IS APPLIED FOR THE
CRYOPRESERVATION OF SHOOT TIPS AND SOMATIC
EMBRYOS.

19. . STEPWISE FREEZING METHOD:THIS TECHNIQUE IS A COMBINATION OF
SLOW AND RAPID FREEZING PROCEDURES HAVING THE ADVANTAGES
OCCURS IN A STEPWISE MANNER.
1 FIRSTLY, THE PLANT MATERIAL IS COOLED TO AN INTERMEDIATE
2 THEN IT IS KEPT THERE FOR ABOUT 30 MINUTES.
3 FINALLY, IT IS RAPIDLY COOLED BY PLUNGING IT INTO LIQUID
4 STEPWISE FREEZING METHOD HAS BEEN SUCCESSFULLY APPLIED FOR
CRYOPRESERVATION OF SUSPENSION CULTURES, SHOOT APICES AND

20. . DRY FREEZING METHOD:IT HAS BEEN REPORTED THAT THE
NON-GERMINATED DRY SEEDS CAN SURVIVE FREEZING AT
TEMPERATURE IN COMPARISON TO WATER-IMBIBING SEEDS
SENSITIVE TO CRYOGENIC INJURIES.
* IN A SIMILAR WAY, DEHYDRATED CELLS ARE OBSERVED TO
BETTER SURVIVAL RATE AFTER CRYOPRESERVATION.

21. 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.
• 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.

22. 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.

23. 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

24. 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).

25. 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 REGENERATION.

26. 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.

27. APPLICATION
1 MAINITIN BIODIVERSITY AND ENDANGERED SPECIES-ITBIS EASY METHOD TO
MAINTAIN ENDANGERED AND GENET BIODIVERSITY OF PLANT.
2 MAINTAIN PATHOGEN FREE PLANT- THIS IS EASY METHOD TO MAINTAIN
PATHOGEN FREE PLANT.
3 EASY TRANSPORT TO OTHER COUNTRIES- BECAUSE IT IS PATHOGEN FREE AND
NEED LITTLE SPACE FOR STORAGE AND EASY TO TRANSPORT.
4 CRYOPRESERVATION SERVE AS LARGE STORAGE AND WHEN WE NEEDED GROW
IT.

28. REFERENCE
• PLANT TISSUE CULTURE- S S BHOJWANI AND M K RAZDAN
• BIOTECHNOLOGY BY U SATYANARAYANA
• HTTPS://WWW.NCBI.NLM.NIH.GOV › PMCCRYOPRESERVATION: A REVIEW ARTICLE
– PMC
• SAGE JOURNALSHTTPS://JOURNALS.SAGEPUB.COM › FULLCRYOPRESERVATION:
AN OVERVIEW OF PRINCIPLES AND CELL-SPECIFIC ...