Germplasm and cryopreservation

Sakeena Asmi
MSc Biotechnology

1. GERMPLASM CONSERVATION AND CRYOPRESERVATION SAKEENA ASMI T MAHATMA GANDHI UNIVERSITY

2. GERMPLASM? Germplasm broadly refers to the hereditary material (total content of genes) transmitted to the offspring through germ cells. In other words, germplasm refers to a whole library of different alleles of a species . Germplasm contains the information for a species‘ genetic makeup, a valuable natural resource of plant diversity. Germplasm provides the raw material for the breeder to develop various crops. For plants, the germplasm may be stored as a seed collection(even a large seed bank ). For trees, in a nursery.

3. WHY CONSERVATION? Until two decades ago the genetic resources were getting depleted owing to the continuous depredation by man. Great diversity of plants are needed to keep the various natural ecosystems functioning stably. Humans are dependent on plant species for food and many different uses . E.g : Basic food crops, building materials, oils, lubricants, rubber and other latexes, resins, waxes, perfumes, dyes fibres and medicines . Aesthetic value of natural ecosystems and the diversity of plant species are also important.

4. Conti… 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 . LIKE………

5. 1. ATTACK BY PEST AND PATHOGENS 2. CLIMATE DISORDER 3. NATURAL DISASTERS 4. POLITICAL AND ECONOMIC CAUSES The conventional methods of germplasm preservation are prone to possible catastrophic losses because of: So new methodologies have been devised for long term preservation of materials.

6. WHAT IS GERMPLASM CONSERVATION? Germplasm conservation refers to maintain the collected germplasm in such a state that there is minimum risk for its loss and that either it can be planted directly in the field or it can be prepared for planting with relative ease when ever necessary . The very objective of germplasm conservation (or storage) is to preserve the genetic diversity of a particular plant or genetic stock for its use at any time in future. In recent years, many new plant species with desired and improved characteristics have started replacing the primitive and conventionally used agricultural plants. It is important to conserve the endangered plants or else some of the valuable genetic traits present in the primitive plants may be lost.

7. GERMPLASM CONSERVATION

8. There are basically two approaches for germplam conservation of plant genetic materials: In-situ conservation 2. Ex-situ conservation

9. In-situ conservation Conservation of germplasm under natural habitat is referred to as in situ conservation . In-situ means in the natural origin, place or position. It involves conservation of plants and animals in their native ecosystems . In situ conservation is the preservation of species and populations of living organisms in a natural state in the habitat where they naturally occur . It includes : 1. BIOSPHERE RESERVES 2. NATIONAL PARKS 3. GENE SANCTUARIES 4. ON FARM CONSERVATION

10. BIOSPHERE RESERVES A biosphere reserve is an area proposed by its habitats, ratified by a national committee and designated by UNESCO’s Man and Biosphere(MAB) program in 1971, which demonstrates innovative approaches to living and working in harmony with nature. The term “Biosphere” refers to All the land, water and atmosphere that supply life on earth . The word “reserve” means that it is a special area recognised for balancing conservation with sustainable use. Each biosphere reserve demonstrates practical approarches to balancing conservation and human use of an area.

11. BIOSPHERES IN INDIA

12. NATIONAL PARKS National park may be defined as an area , declared by state , for the purpose of protecting, propagating or developing wildlife there in , or its natural environment for their scientific ,educational and recreational value . India's first national park was established in 1936 as Hailey National Park , now known as Jim Corbett National Park, Uttarakhand . By 1970, India only had five national parks. As of July 2015, there were 103 national parks encompassing an area of 40,500 km2 (15,600 sq ), comprising 1.23% of India's total surface area.

13. JIM CORBETT NATIONAL PARK SUNDARBAN NATIONAL PARK KAZIRANGA NATIONAL PARK

14. GENE SANCTAURY A gene sanctuary is an area where plants are conserved. India has set up its first gene sanctuary in the Garo Hills of Meghalaya for wild relatives of citrus. Efforts are also being made to set up gene sanctuaries for banana, sugarcane, rice and mango. The genetic diversity is sometimes conserved under natural habitat

15. GARO HILLS, MEGHALAYA

16. ON-FARM CONSERVATION On-farm conservation is defined as “A form of in situ conservation in the place where the domesticated or cultivated species have developed their distinctive properties.” The maintenance of domesticates such as landraces or local crop varieties in farmers fields often referred to as on-farm conservation ( Maxted et al. 2002), in agro or ‘inter situ’ ( Blixt 1994). There is an urgent need to also pay attention to the many economically important wild species that are neither on farm nor in protected areas.

17. ADVANTAGES OF IN-SITU CONSERVATION Plants are conserved in their natural environment. Biodiversity permanently protected. Representative examples of ecosystems also permanently protected. Natural and cultural heritage protected permanently. Ecological integrity is maintained and managed. Opportunities may arise for ecologically sustainable land uses . ( which come with associated economic benefits ) Facilitates scientific research of the site. It may be possible to improve the ecological integrity of the area and restore it if it has been damaged by poaching etc.

18. DISADVANTAGES OF IN-SITU CONSERVATION Endangered habitats may be fragmented so the area may not be large enough to ensure the survival of these species. Genetic diversity may have already been dramatically decreased. Conditions that threatened the organisms in the area may still be present, e.g. disease or interspecific competition. Poachers and ecotourists may see the thriving area as an opportunity and may cause damage.

19. EX SITU CONSERVATION Ex situ means away from the natural , original place or position. E x situ conservation which include conservation of plants and animal away from their native ecosystem. Ex situ conservation is the conservation of genes or plant genotypes outside their environment of natural occurrence , for current or future use.

20. TYPES OF EX SITU CONSERVATION GENE BANK Gene bank refers to a place or organization where germplasm can be conserved in living state. Gene banks are mainly three types:- 1. SEED GENE BANK : A place where germplasm is conserved in the form of seeds is called seed bank . 2 . FIELD GENE BANK : Field gene banks also called plant gene bank are area of land in which germplasm collections of growing plants are assembled.

21. 3. MERISTEM GENE BANK : Germplasm of asexually propagating species can be conserved in the form of meristems. It is used in conservation of horticultural crops. Conti… DNA BANK - Where DNA can be stored as extracted uncut genomic DNA. Such efforts have lead to storage of total genomic information of germplasm in the form of DNA libraries. POLLEN BANK - pollen can be preserved limited space. Pollen preservation may be useful for base collections of species that do not produce orthodox seeds .

22. Botanical garden – There are more than 1,700 botanical gardens and institutions worldwide holding plant collections that serve both conservation and educational purposes . Botanical garden and zoos are the most conventional methods of ex situ conservation like NBRI, Lucknow, Acharya Jagadish Chandra Bose Indian Botanical Garden Kolkata, IARI Dehli , FRI , Dehradun, Royal botanical Garden, Kew, England

23. NBRI, Lucknow Acharya Jagadish Chandra Bose Indian Botanical Garden, Kolkata

24. Royal Botanical Garden, Kew, England

25. WHAT IS ARTIFICIAL SEED ..? • Artificial seed can be defined as artificial encapsulation of somatic embryos, shoot bud or aggregates of cell of any tissues which has the ability to form a plant in in-vitro or ex-vivo condition. • Artificial seed have also been often referred to as synthetic seed .

26. • Artificial seeds were first introduced in 1970’s as a novel analogue to the plant seeds . • The production of artificial seeds is useful for plants which do not produce viable seeds. It represents a method to propagate these plants. • Artificial seeds are small sized and these provides further advantages in storage, handling and shipping. • The term, “EMBLING” is used for the plants originated from synthetic seed . • The use of synthetic varieties for commercial cultivation was first suggested in Maize (Hays & Garber , 1919). HISTORY

27. 1. DESICCATED SYNTHETIC SEEDS Desiccated synthetic seeds are produced nacked or polyoxyethylene glycol encapsulated somatic embryos. This type of synthetic seeds is produced in desiccation tolerant species plant . 2. HYDRATED SYNTHETIC SEEDS Hydrated synthetic seeds are produced by encapsulating the somatic embryos in hydrogels like sodium alginate , potassium alginate , carrageenan, sodium pectate or sodium alginate with gelatine . BASED ON THE TECHNIQUES TWO TYPES OF ARTIFICIAL SEEDS ARE PRODUCED

28. Development of artificial seed production technology is currently considered as an effective and efficient method of propagation in several commercially important agronomic and horticultural crops. These artificial seed would also be a channel for new plant lines produced through biotechnological advances to be delivered directly to greenhouse and field . High volume propagation potential of somatic embryos combined with formation of synthetic seeds for low-cost delivery would open new ways for clonal propagation in several commercially important crop species. NEED FOR ARTIFICIAL PRODUCTION TECHNOLOGY

29. BASIC REQUIREMENT FOR THE PRODUCTION OF ARTIFICIAL SEEDS. • One pre-requisite for the application of synthetic seed technology in micropropagation is the production of high quality , Vigorous Somatic Embryos that can produce plants with frequencies comparable to natural seeds . 2. Inexpensive production of large numbers of high quality somatic embryos with synchronous maturation . 3. Encapsulation and coating systems, though important for delivery of somatic embryos, are not the limiting factors for the development of synthetic seeds . 4. Commercialization of synthetic seeds.

30. Establish somatic embryogenesis Mature somatic embryogenesis Synchronize and singulate somatic embryos Mass production of somatic embryos Standardization of encapsulation Standardization of artificial endosperm Mass production of artificial seeds Greenhouse and field planting PROCEDURE FOR PRODUCTION OF ARTIFICIAL SEEDS

31. Methods for artificial seed encapsulation Dropping method Somatic embryos are dipped in hydrogel, this step encapsulate SEs . Hydrogel used may be any of the following. Alginate – sodium alginate, agar from see weeds, seed gums like guar gum, locust bean gum . - Sodium alginate solution (1 – 5%), prepared in MS basal medium solution. - SEs are dipped in this solution . - These coated beads are added one by one into a complexation solution flask kept on magnetic stirrer and kept such for around 20-30 minutes.

32. Embryos get covered by calcium alginate which is a stable complex due to ionic bond formation, become harder, s eeds become harder . Then gelled embryos are washed with water or MS basal medium. The synthetic seeds are ready . Molding method • This method follows simple procedure of mixing of embryos with temperature dependent gel (e.g. gel rite, agar ). • Cells get coated with the gel at lowering of the temperature.

33. POTENTIAL USES OF ARTIFICIAL SEEDS DELIVERY SYSTEMS : Reduced costs of transplants. Direct greenhouse and field delivery of : - elite, select genotypes. - hand-pollinated hybrids. - genetically engineered plants. - sterile and unstable genotypes. Large-scale mono cultures. Mixed-genotype plantations. Carriers for adjuvants such as microorganisms, plant growth regulators, pesticides, fungicides, nutrients and antibiotics. Protection of meiotically -unstable, elite genotypes. Can be conceivably handled as seed using conventional planting equipment.

34. POTENTIAL USES OF ARTIFICIAL SEEDS ANALYTICAL TOOLS : Comparative aid for zygotic embryogeny . Production of large numbers of identical embryos. Determination of role of endosperm in embryo development and germination. Study of seed coat formation. Study of somoclonal variation.

35. ADVANTAGES OF EX-SITU CONSERVATION I t is possible to preserve entire genetic diversity of crop species at one place. Less interaction with environment , so less chance to loss of genetic resources. Ex situ conservation removes targets from their natural habitat and conserves them in a seed bank, botanical garden etc. Handling germplasm is easy. Small propagules stored in less space, no need to generate plants every season. The germplasm preserved can be maintained in an environment free from pathogens . It can be protected against the natural hazard.

36. DISADVANTAGES OF EX-SITU CONSERVATION Monitoring of viability frequently is essential in seed gene banks. High cost of maintenance. 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 . Conditions that threatened the organisms in the area may still be present, e.g. disease or interspecific competition.

37. IN VITRO STORAGE

38. V arious methods of in-vitro conservation Cryopreservation - generally involves storage in liquid nitrogen. 2. Cold storage - it involves storage in low and non freezing temperature . 3. Low pressure – it involves partially reducing the atmospheric pressure of surrounding. 4. Low oxygen storage - it involves reducing the oxygen level but maintaining the pressure .

39. Cryopreservation Cryo is a Greek word ( krayos – frost ). It literally means preservation in “frozen state.” Cryopreservation is the technique of freezing cells and tissues at very low temperatures (sub-zero temperature, typically - 196 o C) at which the biological material remains genetically stable and metabolically inert, while minimizing ice crystal formation . Any biological activity including the biochemical reactions that would cause cell death are effectively stopped.

40. HISTORY Ernest John Christopher Polge , an English biologist, was the first person to solve the mystery of how to preserve living cells at very low temperatures in 1949. He accidently discovered the cryoprotective properties of glycerol on fowl sperm. During early 1950s, James E. Lovelock suggested that increasing salt concentrations in a cell as it dehydrates to lose water to the external ice might cause damage to the cell. He also proposed that the mechanism of action of cryoprotectants . In 1953, the research work of Jerome K. Sherman led him to successfully freeze and thaw human sperm . In 1983, Alan Trounson , was credited for successfully achieving a pregnancy after freezing early human embryos one to three days after fertilization.

41. Principle : T o bring plant cells or tissue to a zero metabolism and non dividing state by reducing the temperature in the presence of cryoprotectant . 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.

42. It can be done : Over solid carbon dioxide (at -79 degree) 2. Low temperature deep freezer (at -80 degree ) 3. In vapor phase nitrogen (at -150 degree) 4. In liquid nitrogen (at -196 degree)

43. Why Liquid nitrogen ? Chemically inert Relatively low cost Non toxic Non flammable Readily available Liquid nitrogen is most widely used material for cryopreservation. Dry ice can also be used.

44. Mechanism of cryopreservation The technique of freeze preservation is based on the transfer of water present in the cells from a liquid to solid state . Due to the presence of salts and organic molecules in the cells, the cell water requires much more lower temperature to freeze (even up to -68°C) compared to the freezing point of pure water (around 0°C ). When stored at low temperature , the metabolic processes and biological deteriorations in the cells/ tissues almost come to standstill.

45. STEPS INVOLVED IN CRYOPRESERVATION

46. Conti…

47. 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.

48. FACTORS: o Tissue must be selected from healthy plants. o Small & young. o Rich in cytoplasm. o Meristematic cells can survive better than the larger. o Highly vacuolated cells.

49. 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 . o Water content of cell or tissue used for cryopreservation should be low freezable water , tissues can withstand extremely low temperatures.

50. 2. 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 : A bscisic acid Proline T rehalose

51. 3. ADDITION OF CRYOPROTECTANTS A cryoprotectant is a substance that is used to protect biological tissue from freezing & thawing damage ( damage due to ice formation ). They acts like antifreeze. They lower freezing temperature. Increase viscosity and Prevents damage to the cells.

52. Penetrating cryoprotectants Penetrating cryoprotectants are so called because they penetrate the cell membrane and enter the cytosol . They are exclusively small molecules . They form hydrogen bonds with water to prevent ice crystallisation . They act by replacing water and therefore controlling cell size changes as well as preventing intracellular ice formation and prevent excessive dehydration during cell cryopreservation. Common penetrating cryoprotectants are DMSO- (Dimethyl sulfoxide ), glycerol, ethylene glycol .

53. Non-Penetrating cryoprotectants This type of cryoprotectants do not penetrate the cell membrane . They are larger molecules, usually polymers such as polyethylene glycol or saccharides such as sucrose. Non-penetrating cryoprotectants are thought to act by dehydrating the cell before freezing, thereby reducing the amount of water that the cell needs to lose to remain close to osmotic equilibrium during freezing . They inhibit ice growth by the same mechanism as penetrating cryoprotectants , but they do not enter cells . They help to dehydrate the cell prior to cryopreservation by altering the osmotic balance and also help to prevent damage to cells during recovery from cryopreservation by preventing solutes, particularly larger protoplasmic elements , from escaping the cell too rapidly.

54. There are several cryoprotectant which include (DMSO), glycerol, ethylene, propylene , sucrose, mannose, glucose , proline and acetamide . Among these DMSO, sucrose and glycerol are most widely used.

55. 4. 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.

56. 5. 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.

57. 6. ENCAPSULATION AND DEHYDRATION This involves the encapsulation of tissues in calcium alginate beads . W hich 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 .

58. 7. FREEZING After addition of cryoprotectants , freezing is done in such a way that it does not cause intracellular freezing & crystal formation . The following types of freezing is done in the process of cryopreservation : 1. Rapid Freezing 2. Slow Freezing 3. Stepwise Freezing

59. Ampoules used during freezing FREEZERS FREEZING OF CELLS

60. 8. 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.

61. 9. 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 . 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.

62. 10. 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.

63. Advantages 1. Once the material is successfully conserved to particular temperature it can be preserved indefinitely. 2. Once in storage no chance of new contamination of fungus or bacteria . 3 . Minimal space required . 4. Minimal labour required.

64. Applications It is ideal method for long term conservation of genetic 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 . Biodiversity conservation. Conservation of plant germplasm .