in vitrogermplasm conservation

germplasm and its conservation

1. IN-VITRO Germplasm conservation PREPARED BY: KALPATARU NANDA I.D NO. : 03PBG/16 Department of Plant Biotechnology MBB -508 1 st yr M.Sc.(Ag)

2. What is germplasm ? The sum total of all the genes present in a crop and its related species constitutes its germplasm . It is ordinarily represented by a collection of various strains and species.  Plant germplasm is the genetic source material used by the plant breeders to develop new cultivars. They may include: Seeds Other plant propagules (Which can be grown into mature plant) are: Leaf Stem Pollen Cultured cells Germplasm provides the raw material (genes) which the breeder used to develop commercial crop varieties . Therefore, germplasm is the basic indispensable ingredient of all breeding programmes . Thus a great emphasis is placed on collection, evaluation and conservation of germplasm . 

3. The conservation of germplasm involves the preservation of the genetic diversity of a particular plant or genetic stock. It can be used at any time in future. It is important to conserve the endangered plants or else some of the valuable genetic traits present in the existing and primitive plants will be lost. Some crops produce recalcitrant or short lived seeds . Similarly, in case of clonal crops seeds are not the best material to conserve due to their genetic heterogeneity and unknown worth. Their genes need to be conserved. The roots and tubers loose viability rapidly. Their storage requires large space, low temperature and is expensive. In addition, materials modified by genetic engineering may some, times be   unstable. Such materials are needed to be conserved intact for future .A global organization- International Board of Plant Genetic Resources  (IBPGR)  has been established for germplasm conservation and provides necessary support for collection, conservation and utilization of plant genetic resources through out the world. APPLICATIONS OR SIGNIFICANCE OF GERMPLASM CONSERVATION

4. in vitro method for germplasm conservation In vitro method employing shoots, meristems and embryos are ideally suited for the conservation of germplasm.The plant with recalcitrant seeds and genetically engineered can also be preserved by this in vitro approach. There are several advantages associated with in vitro germplasm conservation :- Large quantities of material can be preserved in small space. The germplasm preserved can be maintained in an environment free from pathogens. It can be protected against the nature’s hazards. From the germplasm stock large number of plants can be obtained whenever needed.

5. There are three main approaches for the in vitro conservation of germplasm CRYOPRESERVATION COLD STORAGE LOW –PRESSURE AND LOW OXYGEN –STORAGE Other approaches:- Slow Growth Cultures   Desiccated Somatic Embryos (SE) and Artificial Seeds DNA Clones

6. In vitro conservation of germplasm ..

7. CRYOPRESERVATION Cryopreservation ( Greek,krayos -frost)literally means in the frozen state. The principle involved in cryopreservation to bring the plant cells and tissue cultures to a zero metabolism or non-dividing state by reducing the temperature in the presences of cryoprotectants (DMSO ( dimethyl sulfoxide ), glycerol, ethylene, propylene, sucrose, mannose, glucose, praline, acetamide etc ). CRYOPRESERVATION broadly means the storage of germplasm at very low temperature using :- Over solid carbon dioxide(at 79°C) Low temperature deep freezer(at -80°C) Using vapour nitrogen (at- 150 °C ) In liquid nitrogen( at -196°C)

8. Among these, the most commonly used cryopreservation is by employing liquid nitrogen . At the temperature of liquid nitrogen(at -196°C), the cell stay in a completely inactive state and thus can be conserved for longer period. Infact cryopreservation has been successfully applied for germplasm conservation of some plant species e.g rice,wheat,peanut,sugarcane,coconut . 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. Mechanism of cryopreservation

9. TECHNIQUE OF CRYOPRESERVATION The cryopreservation of plant cell culture followed the regeneration of plants broadly involves the following stages. Development of sterile tissue culture. Addition of cryoprotectant and pretreatment. Freezing Storage Thawing Reculture Measurement of survival/viability Plant regeneration

10. 1.Development of sterile tissue culture The selection of plant species and the tissue with particular references to the morphological and physiological characters largely influences the ability of the explants to survive in cryopreservation.Any tissue from a plant can be used for cryopreservation e.g.meristems,embryos,endosperm,ovules,seeds,culture plants. 2.ADDITION OF CRYOPROTECTANT Cryoprotectant are the compound that can prevent the damage caused to cells by freezing or thawing .There are several cryoprotectant which include: (DMSO, GLYCEROL, ETHYLENE, PROPYLENE, SUCROSE, MANNOSE, GLUCOSE…..)

11. 3.freezing The sensitivity of the cells to low temperature is visible and largely depends on the plant species.Four different types of freezing are used. Slow freezing method Rapid freezing method Stepwise freezing method Dry freezing method 4.STORAGE Maintenance of the frozen cultures at the specific temperature is as important as freezing.In general,the frozen cells/tissues are kept for storage at temperature in the range of -72 to-196°C. Storage is ideally done in liquid nitrogen refrigerator at -150°C in the vapour phase,or at -196°C in the liquid phase. The ultimate objective of storage is to stop all the cellular metabolic activities and maintain their viability .For long term storage temperatue at -196°C in liquid nitrogen is ideal.

12. Look of storage technique!

13. 5.THAWING Thawing is usually carried out by plunging the frozen sample in ampoules into the warm water (temp 35-45°C) bath with vigorous swirling. By this approach,rapid thawing(at the rate of 500-750°Cmin-1)occurs, and this protects the cell from the damaging effects ice crystal formation. As the thawing occurs (ice completely melts) the ampoules are quickly transferred to a water bath at temperature 20-25°C. This transfer is necessary since the cells get damaged if left for long in warm(35-45°C) water bath. 6.RECULTURE In general thawed germplasm is washed several times to remove cryoprotectant . The material is then cultured in a fresh media.

14. 7.Plant regeneration The ultimate purpose of cryopreservation of germplasm is to regenerate the desired plant. For appropriate plant growth and regeneration,the cryopreserved cell/tissue have to be carefully nursed and grown. Addition of certain growth promoting substances ,besides maintenance of appropriate environmental conditionis often necessary for successful plant regeneration.

15.   Cold Storage Cold storage is a slow growth germplasm conservation method. It conserves the germplasm at a low and non-freezing temperature (1- 9 ° C). The growth of the plant material is slowed down in cold storage in contrast to complete stoppage in cryopreservation . Thus it prevents cryogenic injuries. Long term cold storage is simple, cost effective. It yields germplasm with good survival rate. Virus free strawberry plants could be preserved at 10 ° C for about 6 years. Several grape plants have been stored for over 15 years by using a cold storage at temperature around 9°C and transferring them in the fresh medium every year.

16. Low pressure and low oxygen storage In low- pressure storage, the atmospheric pressure surrounding the plant material is reduced. In the low oxygen storage, the oxygen concentration is reduced. The lowered partial pressure reduces the in vitro growth of plants . In the low-oxygen storage, the oxygen concentration is reduced and the partial pressure of oxygen below 50 mmHg reduces plant tissue growth . Due to the reduced availability of 0 2 , and reduced production of CO 2 , the photosynthetic activity is reduced. It inhibits the plant tissue growth and dimension. This method has also helped in increasing the shelf life of many fruits, vegetables and flowers.

17. 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 the plantlets either at a low temperature (4-9°C or Ca. I5°C) or on a medium having high osmotic concentration (e.g., 20% sorbitol or sucrose) or both. In addition, the 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.

18. The slow-growth approach is being utilized for germplasm conservation of specified root, tuber and tree species by the NBPGR, New Delhi. A National Facility for Plant Tissue Culture Repository has been created for this purpose. It has so far developed the slow-growth protocols for ginger, garlic, banana, sweet potato, etc The techniques for desiccation of SE s and for production of desiccated, artificial seeds are now becoming available. The desiccated SEs and artificial seeds can be stored at low (4°C) or ultralow (-20°C) temperatures for prolonged periods in a manner similar to zygotic seeds..  Desiccated Somatic Embryos (SE) and Artificial Seeds

19. DNA Clones Germplasm can also be conserved in form of DNA segments cloned in a suitable vector, e.g., cosmids , phasmids or YACs . The technique is highly sophisticated, technically demanding and expensive. It is likely to be used for conservation of valuable  . genes or DNA segments from threatened species. It can also be used for the conservation of the entire genomes of various germplasm lines of different species.

20. Limitation of in-vitro germplasm conservation 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 .

21. Storage stability using cryopreservation: a case study in papaya  [2011] Ashmore , S.E.Drew , R.A.Kaity , A. Abstract Ex situ conservation of Carica papaya and its crop wild relatives has been the subject of ten years of research in our laboratory. This paper summarises the achievements and key findings of this work. Both clonal and seed materials have been investigated to allow storage of elite clonal material as well as maximum genetic diversity. Clonal material from several genotypes has been successfully stored using micropropagation followed by shoot tip cryopreservation, with >70% regeneration after 12 months storage in LN. The regeneration and acclimatisation of cryopreserved plants has been achieved for field testing and observations of morphological characteristics, including flowering and fruiting, have not detected any changes associated with cryopreservation. Genetic stability following shoot tip cryopreservation has also been monitored using both RAF (Random Amplified DNA Fingerprinting) and AMP (Amplified DNA Methylation Polymorphism) techniques. Whilst both RAF and AMP changes were observed following cryopreservation, these were not associated with cryopreservation per se and did not correlate with any modification in plant morphology. Investigations of papaya seed storage indicate that seed is essentially orthodox, but dormancy breaking treatments were required for germination. However, germination post storage for 12 months was >68% at ultra-low temperatures (LN), but <5% at conventional seed storage temperatures (-20°C). This is in line with recent evidence that cryopreservation enhances storage stability and longevity, even in orthodox seeds, when compared with standard seed bank approaches. This highlights the importance of cryopreservation for the long-term management of genetic resources of both clonal and seed genetic resources of papaya.  

22. Cryopreservation of Orchid Genetic Resources by Desiccation: A Case Study of Bletilla formosana Rung‐Yi Wu, Shao ‐Yu Chang, Ting‐Fang Hsieh, Keng ‐Chang Chuang,IeTing , Yen‐Hsu Lai and Yu‐ Sen Chang Abstract Many native orchid populations declined yearly due to climate change. This resulted in some wild orchids being threatened. In order to maintain the orchid genetic resources, development of proper methods for the long-term preservation is urgent. Low temperature or dry storage methods for the preservation of orchid genetic resources have been implemented but are not effective in maintaining high viability of certain orchids for long periods. Cryopreservation is one of the most acceptable methods for long-term conservation of plant germplasm . Orchid seeds and pollens are ideal materials for long-term preservation (seed banking) in liquid nitrogen (LN) as the seeds and pollens are minute, enabling the storage of many hundreds & thousands of seeds or pollens in a small vial, and as most species germinate readily, making the technique very economical . This article describes cryopreservation of orchid genetic resources by desiccation and a case study of Bletilla formosana .

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