IN-VITRO GERMPLASM CONSERVATION [Autosaved].pptx

IN-VITRO GERMPLASM CONSERVATION [Autosaved].pptx

1. IN-VITRO GERMPLASM CONSERVATION 1

2. CONTENTS INTRODUCTION GERMPLASM CONSERVATION TYPES OF GERMPLASM CONSERVATION IN-VITRO GERMPLASM CONSERVATION TYPES OF IN-VITRO GERMPLASM CONSERVATION 2

3. INTRODUCTION Germplasm is defined as all the genotypes of a species that could be used for breeding a new genotype. It is represented by a collection of various strains and species. It contains the information for a species genetic makeup, a valuable natural resources of plant diversity. Plant germplasm is the genetic source material used by plant breeders to develop new cultivars. It includes: Seeds Other plant propagules such as i ) Leaf ii) Stem iii) Pollen iv) Cultured cells 3

4. INTRODUCTION CONT… 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 practices. It is important to conserve the endangered plants or some valuable genetic traits present in the primitive plants may be lost. 4

5. GERMPLASM CONSERVATION Germplasm conservation is a most effective way to maintain the genetic traits of endangered and commercially important plants . It provides raw material (genes) which the breeder used to develop commercial crop varieties. It has to be protected to ensure variability for future species improvement. For plants, germplasm are stored as pollen, seed, stem, callus and even a whole plant where as for animals, genes and body parts are stored in a gene bank or cryobank . Conventional germplasm is considered to store seeds at ambient temperature, low temperature and ultra-low temperature. But many seeds produce short leaves through this conventional germplasm method. Hence, in-vitro germplasm conservation is used as an alternative for seed banks and field banks. 5

6. NEED FOR CONSERVATION OF PLANT GERMPLASM Loss of genetic diversity among crop plant species. Human dependence on plant species for food and many different uses. Eg : Basic food crops, building materials, oils, lubricants, rubber and other latexes, resins, waxes, perfumes, dyes fibres and medicines. Species extinction and many others are threatened and endangered- deforestation. Great diversity of plants is needed to keep various natural ecosystems functioning stably- interactions between species. Aesthetic value of natural ecosystems and diversity of plant species. 6

7. TYPES OF GERMPLASM CONSERVATION Mainly there are two types of germplasm conservation based on locality of their storage: a) IN-SITU CONSERVATION b) EX-SITU CONSERVATION a) IN-SITU CONSERVATION: It is the on-site conservation and maintenance of germplasm in the natural population of plants like national parks, wildlife sanctuaries, biosphere reserves etc. It is the process of protecting an endangered plant in its natural habitat either by protecting or cleaning up the habitat itself or by defending species from predators. It is applied for the conservation of agricultural biodiversity in agro ecosystem by farmers who use unconventional farming practice. Disadvantage: Risk of germplasm being lost due to environment hazards. Cost of maintaining a large proportion of available genotypes in nurseries or fields may be extremely high. 7

8. IN-SITU CONSERVATION CONT… 8

9. b) EX-SITU CONSERVATION It is the off-site conservation and maintenance of germplasm as gene bank for long-term storage under suitable conditions. It is the process of protecting an endangered species of plant or animal outside of its natural habitat, eg : by removing part of population from a threatened habitat and placing it in a new location, which may be a wild area or within the care of humans. It involves transfer and easy accessibility of genetic material away from location where it is found. DISADVANTAGE Required costly and well developed lab. Loss of seed viability and seed destruction by pests. Some plants do not produce fertile seeds. Poor germination rate. Only useful for seed propagating plants. 9

10. EX-SITU CONSERVATION CONT… Ex-situ conservation can be carried out by several methods: Seed gene bank In-vitro storage DNA storage Pollen storage Field gene bank Botanical gardens Meristem gene bank 10

11. TYPES OF EX-SITU CONSERVATION CONT.. A . Gene Bank : It refers to a place or organization where germplasm can be conserved in living state. 1.Seed gene bank : a place where germplasm is conserved in the form of seeds. 2. Field gene bank : Also called plant gene bank. It is an area of land in which germplasm collections of land in which germplasm collections of growing plants are assembled. 3. Meristem gene bank : Germplasm of asexually propagating species can be conserved in the form of meristems. It is used in the conservation of horticultural crops. B. DNA Bank : It is where DNA can be stored as extracted uncut genomics. Such efforts have lead to storage of total genomic information of germplasm in the form of DNA libraries. 11

12. TYPES OF EX-SITU CONSERVATION CONT… C. Pollen Bank : Pollen can be preserved in limited space. Pollen preservation may be useful for base collections of species that do not produce orthodox seeds. D. Botanical Garden : More than 1,700 botanical gardens and institutions holding plant collections that serve both conservation and educational purposes globally. 12

13. EX-SITU CONSERVATION CONT… Usually , seeds are the most common and convenient materials to conserve plant germplasm. This is because many plants are propagated through seeds and seeds occupy relatively small space. Further, seeds can be easily transported to various places. LIMITATION IN THE CONSERVATION OF SEEDS Viability of seeds is reduced or lost with passage of time. Seeds are susceptible to insect or pathogen attack, often leading to their destruction. It is confined to seed propagating plants and thus it is of no use for vegetatively propagated plants eg : potato, ipomea, dioscorea It is difficult to maintain clones through seed conservation. 13

14. SEED CONSERVATION CONT… In seed banks, there are three types of conservation: 1. Short term storage (Working collections) 2. Medium term storage (Active collections) 3. Long term storage Short term storage Working collections are stored for short term (>3-5 years) at 10-15ºC at 10% moisture. The accessions being actively used in crop improvement programmes . These collections are maintained by breeders using them. 14

15. SEED CONSERVATION 2. Medium term storage The accessions in an active collection are stored at temperatures below 15ºC and the seed moisture is kept at 5%. Storage : 10-15 years. Collections are used for evaluation, multiplication and distribution of accessions. 15

16. IN-VITRO GERMPLASM CONSERVATION 16

17. IN-VITRO GERMPLASM CONSERVATION In-vitro germplasm conservation is an advanced technology for the preservation of genetic materials. Here, gene banks are made to preserve genetic material by non-conventional methods using tissue and cell culture procedures. Materials stored in-vitro may be isolated protoplasts, cells from suspension or callus cultures, meristem tips, somatic embryos, shoot tips and propagules at various stages of development or organised plantlets. It is important for vegetatively propagated and for non-orthodox seed plant species. Hence this preservation depends on the principle that plant cells which are mostly totipotent are kept alive for longer durations using in-vitro cultures. In-vitro storage of germplasm helps to assure access and safe transportation of plant material. Germplasm preserve can be maintained in an environment free from pathogens. 17

18. MATERIALS USED FOR IN-VITRO GERMPLASM Materials stored in-vitro may be isolated protoplasts, cells from suspension or callus cultures, meristem tips, propagules at various stages of development or organised plantlets. Cultured cells or shoots can be maintained by serial subcultures at 4-8 weekly intervals for virtually unlimited periods. Storage of germplasm by repeated cultures has some disadvantages: Risk of material loss due to human error or failure in maintenance of in-vitro. Genetic instability is also affected during serial subculturing of plant material. These genetic changes are either due to additions or deletions of gene sequences. 18

19. ADVANTAGES Requirement of little space for preservation of a large number of clonally multiplied plants. Maintenance of material in an environment free of pests or pathogens. Protection against dangers of natural environmental hazards. Sterile plants which cannot be reproduced in general can be maintained in-vitro. Availability of nucleus stock to propagate a large number of plants rapidly whenever necessary. Minimising obstacles imposed by quarantine systems on the movement of live plants. Since germplasm is kept under aseptic conditions, it can be easily transported. It requires constant electricity, skilled manpower and high technology. Formation of ice crystals is seen where it causes damage to germplasm. DISADVANTAGES: It requires constant electricity, skilled manpower and high technology and highly expensive. Formation of ice crystals is seen where it causes damage to germplasm. : 19

20. TYPES OF IN-VITRO GERMPLASM CONSERVATION Mainly there are three types of in-vitro germplasm conservation: Cold storage Low-pressure and low-oxygen storage Cryopreservation Other approaches: Slow Growth Cultures Desiccated Somatic Embryos (SE) and Artificial Seeds DNA clones 20

21. COLD STORAGE Cold storage involves germplasm conservation at a low and non-freezing temperatures. Low temperatures used is in a range of 1-9ºC but not the freezing temperature. This method slows down the growth process of cultured cells or tissues rather than fully stopping. Hence it is considered as a slow growth germplasm conservation method. Through this, genetic material can last for about 15 years and thus helps in preventing cryogenic injuries which lead to higher survival rates of germplasm and long term cold storage is simple and cost effective. It can store many in-vitro developed shoots or plants of fruiting trees like strawberries and grapes. With the addition of few drops of medium every 2-3 months, virus-free plants could be conserved at 10ºC for about 6 years. Several grape plants have been stored for over 15 years by cold storage (around 9ºC) by transferring them yearly to a fresh medium. 21

22. 22

23. LOW-PRESSURE STORAGE In low-pressure storage, the atmospheric pressure surrounding plant material is reduced. This results in partial decrease of pressure exerted by gases around germplasm. Lowered partial pressure reduces in-vitro growth of plants. Low- pressure storage systems are useful for short and long term storage of plant material. It increases shelf life of many plant materials. Germplasm grown in cultures can be stored for long term under low pressure. It also reduces activity of pathogenic organisms and inhibits spore germination in plant culture systems. 23

24. LOW-OXYGEN STORAGE In low-oxygen storage, oxygen concentration is reduced, but atmospheric pressure is maintained by addition of inert gases (nitrogen). Partial pressure of oxygen below 50mm Hg reduces plant tissue growth. This is because with reduced availability of O2, the production of CO2 is low. Thus photosynthetic activity is reduced thereby inhibiting plant tissue growth and dimension. LIMITATION OF LOS Long term conservation of plant material by low-oxygen storage is likely to inhibit plant growth after certain dimensions. 24

25. CRYOPRESERVATION Cryopreservation means preservation in the frozen state. Principle: To bring plant cell and tissue cultures to a zero metabolism or non-dividing state by reducing temperature in the presence of cryoprotectants. 25

26. SLOW GROWTH CULTURES Slow-growth of plantlets in-vitro provides an attractive alternative to freeze preservation of germplasm as it is simpler, cheaper and very effective. Slow growth may be achieved by maintaining plantlets either at a low temperature (4-9ºC) or on a medium having high osmotic concentration ( eg : 20% sorbitol or sucrose) or both. Nutritional status of the medium may be lowered to restrict the growth of plantlets. Under the conditions of slow growth, cultures may be attended to only once in several months. Its subculture may, be necessary only after long periods, once every 236 months. Slow growth approach is being utilized for germplasm conservation of specified root, tuber and tree species, in NBPGR, New Delhi. A national facility for plant tissue culture repository has been created for this purpose. Eg : garlic, banana, sweet potato etc. 26

27. DESICCATED SOMATIC EMBRYOS (SE) AND ARTIFICIAL SEEDS The techniques for desiccation of SE and for production of desiccated, artificial seeds are now becoming available. The desiccated SE and artificial seeds can be stored at low (4ºC) or ultralow (-20ºC) temperatures for prolonged periods in a manner similar to zygotic seeds. DNA CLONES Germplasm can also be conserved in the form of DNA segments cloned in a suitable vector, eg : cosmids , phasmids or YACs. The technique is highly sophisticated, technically demanding expensive. 27

28. APPLICATIONS OR SIGNIFICANCE OF IN-VITRO GERMPLASM CONSERVATION Preservation and maintenance of genetic diversity of a particular plant or genetic stock. Disease free plant materials can be frozen and propagated whenever required. Cryopreservation produces secondary metabolites. Recalcitrant seeds can be maintained for long. Conservation of somaclonal and gametoclonal variations in cultures. Plant materials from endangered species can be conserved. Establishment of germplasm banks for exchange of information at international level. IBPGR (International Bureau of Plant Genetic Resources) has been established for germplasm conservation and provides necessary support for collection, conservation and utilization of plant genetic resources through out the world. In NBPGR (National Bureau of Plant Genetic Resources), India has a cold storage facilities known as National Germplasm Repository. 28

29. REFERENCE 1. Bhojwani, S. S., & Razdan, M. K. (1986). Plant tissue culture: theory and practice. Elsevier. 2. Razdan, M.K. (2000). An introduction to plant tissue culture. Oxford &IBH publishing Co. Pvt. Ltd. New Delhi, Calcutta. Kumar, D. (2010). Plant breeding Biometry Biotechnology. New Central Book Agency Pvt. Ltd. Calcutta. 4. Misra, S. P. (2009). Plant tissue culture. Ane Books Pvt. Ltd. New Delhi. 29

30. THANK YOU 30