Ex situ conservation

presenation only for exsitu conservation includes topic (Components of ex-situ conservation
Plant genetic resources conservation in gene banks, national gene banks and gene repositories
Preservation of genetic materials under natural conditions, Perma-frost conservation
Guidelines for sending seeds to network of active/ working collections
Orthodox and recalcitrant seeds- differences in handling
Clonal repositories
genetic stability under long term storage condition)

1. Ex-situ Conservation Submitted by: Kiran (2019A46D) Submitted to: Dr. Somveer Nimbal

2. Contents Components of ex-situ conservation Plant genetic resources conservation in gene banks, national gene banks and gene repositories Preservation of genetic materials under natural conditions, Perma -frost conservation Guidelines for sending seeds to network of active/ working collections Orthodox and recalcitrant seeds- differences in handling Clonal repositories genetic stability under long term storage condition.

3. Introduction Ex-situ conservation is the preservation of components of biological diversity outside their natural habitats This involves conservation of genetic resources (wild and cultivated species Such strategies include establishment of botanical gardens, zoos, conservation strands and gene, pollen seed, seedling, tissue culture and DNA banks.

4. Components of Ex-situ conservation

5. GENE BANKS

6. Gene banks It refers to a place or organisation where germplasm to be conserved in living state The most fundamental activity of gene bank is to treat a new sample in a way that will prolong its viability as long as possible while ensuring its quality Plant samples must periodically be grown out, regenerated and new seeds harvested to maintain the sample

7. Field gene banks Field gene banks also called plant gene banks Conservation of germplasm by growing in feild Those plant species that have recalcitrant seeds, vegetatively propagated and are long lived perennials conserved in field gene banks. In field gene banks, germplasm is maintained in the form of plants as a permanent living collection. Field gene banks are often established to maintain working collections of living plants for experimental purposes. They are used as source of germplasm for species such as coconut, rubber, mango, cassava, yam and cocoa. Field gene banks have been established in many countries for different crops.

8. Horticulture Crop Field gene bank Field gene bank in Phillippines

9. Advantages Disadvantages Suitable for species with recalcitrant and intermediate seeds Vulnerable to changes in management practices Convinient fr characterisation and evaluation Susceptible to pests, diseases and other natural or human driven calamities such as drought, neglect and war Easily accesible for use Limited amount of genetic diversity conserved Possible to combine conservation and research High maintainance costs, not suitable for long term conservation

10. Seed Gene Banks Germplasm is conserved in form of seeds The largest seed bank in world is the Millennium Seed Bank housed at the  Wellcome Trust Millennium Building (WTMB), located in the grounds of  Wakehurst Place in West Sussex, near London Advantage: allows large population to be preserved and minimises the genetic erosion by providing optimum conditions and reduces the need for regeneration

11. In-vitro Gene Bank Conservation of PGR in the form of small parts economizes the space and allows conserving vegetatively propagated an medium and long term storage conditions. Two major techniques in In vitro storage: a) In vitro Bank: In this technique, buds, protocorm and meristematic cells are preserved through particular light and temperature arrangements in a nutrient medium. This technique is used to preserve seedless plants and plants that reproduce sexually. b) Cryobank : In this technique, a seed or embryo is preserved at very low temperatures. It is usually preserved in liquid nitrogen at -196 °C. This is helpful for the conservation of species facing extinction.

12. Cryo presevation

13. DNA Bank DNA can be stored as extracted uncut genomic DNA or may be maintained in genomic library on bacteria or virus. Storage is performed by immobilisation of the DNA onto nylon membrane. DNA bank is a particular type of genetic resource bank that preserves and distribute the DNA molecule and provides associated information Advantages Disadvantages Minimum space required for storage Not a practical germplasm conservation method Applicable to all plant species Well established facilities and skills is required Effective and rapid method Large number of sample can be handled

14. Pollen Bank 'Pollen Banks' are used to store pollen grains for a short as well as very long period of time in a viable conditions.  Advantages Disadvantages More effective in case of recalcitrant species It may lead to loss of organelle genome, thus require effective plant generation protocol Minimum space required for storage Tricellular pollen storage extremely difficult Intra nad inter population variation can be conserved Propagules not readily available Only half of the genome conserved

15. Purpose of Gene Banks Physical facilities for maintaining collections of live plant materials – domesticated cultivated plants – wild plant species (crop wild relatives and other wild plant species useful for food and agriculture and other end uses) – entire plants, seeds, pollen, embryos, meristems , cells, or DNA, depending on the biology of the species It is a facility for maintaining crop diversity . Usually this diversity is in the form of seeds, stored and conserved in a frozen state Some gene banks use normal household freezers for this purpose The ideal temperature is between -10and -20 Each different type is stored in its own container Such as a bottle, a can or a sealed aluminum foil package

16. Maintaining material in genebanks is often termed ‘ex situ conservation’ – defined as ‘the conservation of components of biodiversity outside their natural habitats’ (CBD, 1992) Whereas in situ conservation is maintenance of viable population in their natural surroundings – a dynamic system which allows the biological resources to evolve and change over time through natural selection processes Both concepts are therefore fundamentally different but are complementary.

17. Activities in Gene Bank Collecting and Acquisition – assembling the collection Processing – assessing the quantity, viability, health of samples and preparation for storage Storage – in a cold store, laboratory or in the field Regeneration and Multiplication – periodically rejuvenating and increasing the material Characterization and Evaluation Documentation, Inventory – maintaining and making available detailed records on each sample Distribution – of clean, disease-free seeds, or other planting material, to requestors

18. National Gene banks for major crops CROP CENTRE Wheat IIWBR, Karnal Rice NRRI, Cuttack Potato CPRI, Shimla Cotton CICR, Nagpur Sugarcane SBI, Coimbatore Tobacco CTRI, Rajahmundry Pulses IIPR, Kanpur Tuber crops( except potato) CTCRI, Trivandrum Plantation crop CPCRI, Kasorgod Oilseed crop IIOr , Hyderabad Horticulture crop IIHR, Bangalore Soybean DOSR, Indore Groundnut DOGR,Junagarh Maize IARI, New Delhi

19. BOTANICAL GARDENS

20. Botanical gardens A  botanical garden  is a place where plants, especially ferns, conifers and flowering plants, are grown and displayed for the purposes of research and education The botanical garden in  Nashik  is the first of its kind in the country and only one in South East Asia with a unique laser show. Botanical garden Guru Nanak Dev University, Amritsar Punjab Botanical garden Punjabi University, Patiala Punjab and National Cactus and Succulent Botanical Garden and Research Centre in Punchkula (Haryana) There are other botanical garden like NBRI, Lukhnow , Indian Botanical Garden, Kolkata, IARI,Delhi , FRI, Dehradun and Royal Botanical garden, Kew England There are over 2500 botanical gardens in 148 countries

21. Permafrost

22. Permafrost Permafrost is any ground that remains completely frozen—32°F (0°C) or colder—for at least two years straight. These permanently frozen grounds are most common in regions with high mountains and in Earth’s higher latitudes—near the North and South Poles Permafrost is made of a combination of soil, rocks and sand that are held together by ice. The soil and ice in permafrost stay frozen all year long These are the places for storage of germplasm without involving high cost in refrigeration Eg . Genebank located in Svalbard Seed Vault in the island of Spitsbergen. Samples are stored in glass ampules and moisture content is around 2-4%. Thetemperature in the mine is constant from -2 to 3 ⁰C.

23. Permafrost in india Doomsday vault present at 17,500 m high on a cliff top in Himalayas, Chang-La has sub zero temeprature and low humidity necessary to suspend seed life for future generation.

24. Classification of seed banks

25. Base collections:  Seeds can be conserved under long term (50 to 100 years), at about -20 ⁰ C with 5% moisture content.  They are disturbed only for regeneration. Active collection:  Seeds are stored at 0 ⁰ C temperature and the seed moisture is between 5 and 8%.  The storage is for medium duration, i.e., 10-15 years.  These collections are used for evaluation, multiplication, and distribution of the accessions. Working collections:   Seeds are stored for 3-5 years at 5-10 ⁰ C and the usually contain about 10% moisture.  Such materials are regularly used in crop improvement programmes . Orthodox seeds:   Seeds which can be dried to low moisture content and stored at low temperature without losing their viability for long periods of time is known as orthodox seeds. ( eg .) Seeds of corn, wheat, rice, carrot, papaya, pepper, chickpea, cotton, sunflower. Recalcitrant:  Seeds which show very drastic loss in viability with a decrease in moisture content below 12 to 13% are known as recalcitrant seeds. ( e.g ) citrus, cocoa, coffee, rubber, oilpalm , mango, jack fruit etc.

26. Guidelines for sending seeds to network of active/working collections Plant germplasm proposed to be registered should be submitted to NBPGR Material is to be accompanied with properly filled Form –A (Annexure I) duly signed by the applicant and Head of the institution with official rubber seal (15 copies, each attached documentary evidences submitted). Form A accompanied complete description of the germplasm material using standard descriptors (as per concerned crop AICRP or NBPGR descriptors). Include photograph(s) of plant/plant parts/crop and /or fingerprints (DNA or biochemical profile), if available. A declaration to the effect that working-stock for supply to users would be maintained by the instiution associated with the development of the material. It may be ensured by the Director/PD/PC or Competent Authority of the concerned organization. Another declaration is that such germplasm does contain any gene or gene sequence involving terminator technology would also be mandatory.

27. Guidelines for submitting the orthodox seed material Seed material dried to low moisture level without loss of seed viability. Minimum number of 4000 seeds in case of cross-pollinated crop species, 2000 in self –pollinated and 100-1000 in difficult species, such as some vegetables, wild relatives etc. submitted. Seed supplied from a fresh harvest and should not be more than 60 days old. Seeds supplied sound, healthy, physiologically mature and collected from healthy plants For providing good quality healthy seeds. It is advised to dry the seed material in shade immediately after the harvest. The potential viability of seeds should be more than 85% in most crop species except in special cases, such as cotton, forage grasses, sugarcane some vegetable crops etc. Seeds should not be treated with chemicals. Seeds packed in good quality paper, muslin cloth or plastic packet(s) with proper identity. If required, the packets should be packed in card-board boxes to minimize damage and moisture absorption .

28. Guidelines for Submission of Recalcitrant / Intermediate Seed Material The guidelines to be follows are given below: Preferably, more than 1000 seeds supplied. However, recognizing the importance of material, even small quantity may be acceptable. Supply of additional seeds may help develop DNA profiles. To avoid any injury to the fruit surface they should be sent in aerated polythene bags/cardboard boxes in the form of complete fruit. If fruits are bulky and difficult to transport, the seeds should be extracted without causing any injury and should be transported within 48 hrs, packed in saw dust/charcoal /peat moss etc. Avoid transporting at high temperature (above 30 ⁰C ). Store and transport should be preferably in moist conditions between 15-20 ⁰C temperature conditions. Extracted seeds should be treated with suitable fungicide (0.1 % Captan or Thiram powder). Avoid air drying and washing of seeds. In remaining cases the genetic material should be supplied to relevant NAGS in the form of propagules establishment in the field gene bank following the guidelines given below.

29. Guidelines for Submission of Propagules 10-25 propagules (depending on crop) supplied to the concerned NAGS( Nationa Active Germplasm Site) for their maintenance in field repository or in-vitro repository (if available) with a request for an acknowledgement. Concerned NAGS should be informed in advance about the supply of material to facilitate processing and establishment of germplasm . Genetic material, stocks, propagules of non-orthodox seed producing crops are generally being maintained in the form of grafts, slips, propagules , seedlings and plants. While supplying genetic material following steps and precautions are to be followed depending on the crop: The slips, grafts, propagules or plants supplied to the NAGS should be free from insects, weeds and diseases as far as possible. The material should be welllabelled and packed properly in aerated polythene bags. During the dry summer the grafts of crafts should be wrapped in moist moss grass to retain the moisture.

30. In case of crops like coconut, the material should be sent either as embryos or seedlings. If the embryos need to be transferred from the field, the embryos embedded in the endosperm should be packed in the sterile plastic bag with sterile moist cotton. These should be kept in the refrigerator overnight and transferred in the same box with proper labels on it. In case of seedlings the embryos should be grown using the river sand in plastic bags/boxes. Once the seedlings are established these should be transferred to bigger pots. The healthy, vigorous seedlings should be supplied. The material should be packed in small wooden/card-board boxes with proper aeration. Also, these boxes should be well marked with labels at 3 or 4 places “To be handled carefully: seedlings’’ in order to avoid any damage during transit. The material should be sent to the NAGS immediately after harvest. To avoid any delay in transfer, use speed post or reliable courier services or airlies .

31. Handling of orthodox seeds

32. Drying and storage : Drying to equilibrium in a controlled environment of 5-20°C and 10-25 % of relative humidity, depending upon species Storage - in air-tight container for long term storage Long-term conditions (base collections); temperature of -18 ± 3°C , relative humidity of 15% ± 3 For medium-term conditions (active collection) samples are stored under refrigeration at 5-10 °C and relative humidity of 15 % ± 3% – medium-term storage conditions are adequate for 30 years. Seed viability Monitoring: Timing After cleaning and drying or at the latest within 12 months after receipt of the sample 200 seeds for initial germination tests (ISTA, 2008) followed by sequential testing, if the initial germination is less than 90 % (Ellis et al. 1985) during storage 100 or smaller seed samples If there are not sufficient seeds; conducted with replications. Viability: Variability- initial germination threshold - above 85% for most cultivated crop species; lower value acceptable for specific accessions, wild and forest species Regeration : The viability drops below 85 % of the initial viability The remaining seed quantity is less than what is required for three sowings of representative population of the accession. The most-original-sample to be used In practice when accession does not have sufficient seeds for long-term storage (e.g. 1500 seeds for a self-pollinating species and 3000 for an out-crossing species) Deposit in long-term storage for reference purposes - at least 50 seeds of the original and the subsequent most original samples.

33. Handling of Recalcitrant seeds

34. Assessment of water content, vigour and viability: The storage category of the seed should be determined immediately by assessing its response to dehydration. The water content should be determined individually, on separate components of the propagule , and in a sufficient number of plants. The vigour and viability should be assessed by means of germination tests and in a sufficient number of individuals. During experimentation, cleaned seed samples should be stored under conditions that do not allow any dehydration or hydration. As a first step to preservation, it is important to ascertain the seed storage category by assessing the response of the propagule to dehydration. The response to drying will in turn determine the treatment needed for cryostorage . The rate and uniformity of germination of a seed sample, or of seed-derived explants, is a reliable indicator of vigour , while the totality of germination (i.e. what proportion/percentage of seeds or explants tested finally germinated) reveals the overall viability of the sample. Viability should not be not less than 80% in a sample. The recommended temperatures for tropical/sub-tropical species and those of temperate origin are 25 °C and 15 °C respectively (Pritchard et al. 2004).

35. Hydrated storage of recalcitrant seeds Hydrated storage, and is achieved by holding the seeds in a closed under saturated relative humidity (RH) conditions. Hydrated storage should be carried out under saturated RH conditions, and seeds should be maintained in air-tight containers, at the lowest temperature that they will tolerate without damage All seeds should be decontaminated prior to hydrated storage and infected material should be eliminated. Stored seeds must be inspected and sampled periodically to check if any fungal or bacterial contamination has occurred, and whether there has been any decline in water content and/or vigour and viability. The basic principle for maximising the storage life span of recalcitrant seeds is that water contents should be retained at essentially the same levels as those characterising the newly-harvested state. Thus the seeds must not lose water either before or after being placed in storage. Ideally sealing polythene bags with an inner paper bag inside (‘bag within a bag’) or sealing plastic buckets of appropriate size for the seed numbers, are favoured for storage ( Pasquini et al. 2011).

36. Storage temperatures should be the lowest that seeds of individual species will tolerate, without any deleterious effect on vigour and viability For recalcitrant seeds of temperate origin, temperatures of 6 ± 2 °C are generally suitable for storage, while for the majority of seeds of tropical/subtropical origin, 16 ± 2 °C is normal range Under hydrated storage conditions, fungi (or less frequently bacteria) are likely to proliferate, so vigilance and appropriate action to curtail seed-to-seed infection is required. If infected seeds are not removed they will contaminate the entire batch in a storage container. This renders the stored seeds useless and eliminates their potential for supplying explants for cryopreservation Depending on the duration of hydrated storage, containers should be briefly and periodically ventilated to avoid development of anoxic conditions at which time the contents of containers must be inspected and any contaminated seeds discarded. Storing seeds in a monolayer is ideal, but if seeds are stored in several layers, the seeds should be mixed about during aeration.

37. National clonal germplasm repositories

38. Clonal repositories contain active collections that hold and propagate agriculturally important germplasm , such as strawberries, raspberries, fruit trees, coffee and nuts that for a variety of reasons are not usually held in active collections as seed. The primary responsibilities of the repositories are to collect, identify, propagate, preserve, evaluate, document and distribute clonal germplasm as part of the NPGS. This includes maintenance of an information file on each accession in the clonal collection. The repositories are charged with developing active global collections of appropriate wild species and domestic cultivars and to assemble a maximum level of genetic diversity possible for each genus and species for which they are responsible. They also conduct research to improve evaluation, propagation, characterization and preservation of clonal germplasm . The national clonal germplasm repositories are intended to carryout by the regional stations. Unlike seeds held at the laboratory, however, backups of clonal collections are limited to the same sites were the active collections are maintained.

39. Many clonal crops can be conserved as seed, but they are impossible to maintain true to type by raising plants from seed. Many clonally propagated species take a long time to mature, and they are best preserved as mature live plants for plant breeding and research. Clonal collections are expensive to establish and they have many of the same problems that confront seed collections. Accessions must be maintained as plants in the field , which can require large tract of land or in screen houses or green houses. Accessions may also be maintained as lives sticks of budwood held under refrigeration or as tissue cultures. There may be many losses during maintenance from insects and disease, freezing temperatures, electric power failures and grazing animals. Clonal preservation is more expensive and labour intensive than seed storage. Clonal collections have been threatened as facility or land use priorities have been changed. By establishing the national clonal repositories, a mechanism for stable, longterm maintenance for many important clonally propagated species has been provided

40. Genetic stability under long term storage condition

41. “ Clonal stability depends on the method of preservation, the length of time, and the inherent genetic stability of the clone Mutations can occur as a result of background gamma- and X-ray irradiation, exposure to mutagenic chemicals, and other environmental influences. Each clone has its own natural rate of mutation, so generalizations cannot be made.” Westwood (1989)

42. Monitor genetic stability: Genetic shifts in the collection can arise from genetic instability of accessions due to chromosomal changes and gene mutations causing morphological or biochemical variation. These genetic instabilities are manifested as somaclonal variation in tissue culture. Avoid propagating off-types: Individuals of a clone should be inspected regularly for off-types. If the accession appears to be a mixture of genotypes, characterization data should be used to determine which is the correct propagule . Herbarium specimens or photographs of accessions may also be useful for verifying the identity of questionable specimens.

43. Accessions from which seeds are collected for propagation (grasses and legumes) can lose genetic information over time if too few individuals of an accession are maintained and self pollination occurs in a percentage of cases. This problem is lessened in accessions that primarily produce seed apomictically . Species that inter cross easily lose superior genotypes when cross-pollination is not controlled and seeds are used. Studies on seed physiology and reproductive biology would identify grasses that could be stored as bulked seed and would reduce the size of field collections. Crops of this type need to establish protocols for maintaining accessions according to breeding behaviour (Sackville Hamilton and Chorlton 1997).

44. Evaluate genetic stability: Monitoring collections for genetic stability is not an easy task. Each crop has specific descriptors that can be used to characterize the accessions. A well-identified collection may be used to develop RNA or DNA fingerprinting, which may be used for future evaluations. The relevance of the existing techniques is discussed in a publication entitled ‘Using Molecular Marker Technology in Studies on Plant Genetic Diversity’ (de Vicente and Fulton 2003). Avoid mixing propagules : Mixing can easily occur in fields where roots, stolons , rhizomes and runners can invade adjacent plots. Repotting or planting errors and growth of propagules such as runners into adjacent pots may occur in screenhouses and greenhouses. Wide spacing of pots can be used to limit growth of stolons or runners into adjacent pots. Careful attention by staff members will minimize planting errors.

45. Minimize labelling and handling mistakes: More than one identification number (i.e. plot number and accession number) should be used when planting or harvesting fields. A field map should be available showing the sequence of planting, and, if possible, labels should be printed from computer files, or carefully checked to minimize the problem. Labels should be indelible and as indestructible as possible. Advance planning before any planting or repotting effort will minimize errors.

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