Collection, evaluation and documentation of germplasm

FSC 503: Biodiversity and conservation of fruit crops
Collection: Tapping of genetic diversity from various sources and assembling at one place is called germplasm collection.
Evaluation: It deals with the assessing the agronomic potential of an accession including quality parameters and response to various abiotic and biotic stresses.
Documentation:Germplasm conservation, in its various stages, includes a range of activities for which information is required or from which information is derived. This may refer to species, their sites of origin, or activities or stages of conservation. The action of recording, organizing, and analyzing conservation data is known as documentation.

1. WELCOME FSC 503: Biodiversity and conservation of fruit crops Collection, Evaluation and Documentation of Germplasm Submitted by: Patel Yash N. Reg. no.: 2020219035 M. Sc. Horticulture (Fruit Sci.) Submitted to: Dr. A. K. Pandey Assistant Professor Department of Fruit Science

2. India has a rich and varied heritage of biodiversity, encompassing a wide spectrum of habitats from tropical rainforests to alpine vegetation and from temperate forests to coastal wetlands. Out of 18 biodiversity hot spots identified in the world, four hotspots , i.e. Western Ghats, Western Himalaya, Indo-Burma region, and Nicobar islands (Sunderland) are in India. It is estimated that there are 8.7 million species of the world’s biota. Out of them only 1.7 million have been described to date, and their distribution is highly uneven. India contributes significantly to the biodiversity of the world by accounting 7.31 % of the global plant diversity from 2.4% of the world’s area.

3. Primary centre Mango, Citrus, Jack Fruit, Bael , Aonla , Ber , Khejri , Jamun , Tamarind, Phalsa , Lasoda , Karonda , Wood apple, Pilu , Bilimbi , Garcinia , Under Utilized Fruits Secondary centre Banana, Pomegranate, Mulberry, Malus , Pyrus , Prunus , Rubus India-Centre of Diversity of fruit Crops: (Source: Singh et al. 2009)

4. COLLECTION OF GERMPLASM

5. REASONS FOR GERMPLASM COLLECTING The demand of germplasm is unpredictable and dynamic but in practice, some prioritization is necessary both at species level and geographic regions. It is in danger of extinction or even erosion; A clear need exists for it, as it was expressed by farmers; The diversity it represents is missing from or insufficiently represented in, existing ex-situ germplasm collections; Definition: Tapping of genetic diversity from various sources and assembling at one place is called germplasm collection.

6. EXPLORATION PROGRAMME : STEPS Planning Making contacts with local research organization Gathering equipment and preparation Meeting with local researchers in area to be surveyed Sorting out of collected samples Reporting to the Headquarters Preparation & publication of reports Distribution/conservation of collected samples

7. AREAS TO BE SURVEYED Diversity/Variability rich area Under explored area Tribal dominated area Hot spots Threatened habitats Farmers’ field

8. PLANNING OF EXPLORATION MISSION Prioritization of Species and Areas after Gap Analysis: The areas to be explored and crops/species to be collected should be prioritized after thorough gap analysis based on information from different sources including database/National Genebank status at NBPGR. The explorer should be well-versed with the nature and extent of diversity and breeding behaviour of the crop/species to be collected.

9. Plan well in advance to facilitate the preparations of the proposed missions except those to be carried out under special situations like rescue collecting. Visit to herbaria should be made to know the range of distribution, localities, diversity pattern and period of collection particularly for wild species. Phytosanitary regulations should be followed in case of transportation of material from foreign country.

10. FINALIZATION OF MISSION Gathering eco-geographic information: Information on topography, climatic conditions, vegetation, crops in cultivation and their maturity, etc. needs to be gathered to finalize the tour path of collecting mission. Besides, explorers should establish local contacts especially at grass root level to seek the social, cultural, ethnic and other information of interest. Types of survey: Coarse grid survey should be conducted in unexplored areas to capture the overall variability . Fine grid survey should be carried out to build-up more collections for specific trait(s) known to exist in identified pockets in previously explored areas.

11. PERIOD OF COLLECTION: For seed producing crops and species, exploration should be undertaken when these are physiologically mature and ready for harvest. In case of species with shattering nature, missions are executed rather earlier (7-10 days depending on crop/species) before their maturity. Further, longer duration (2-3 weeks) mission and repeat visits are suggested for collection of wild species. For vegetatively propagated crops/species, the targeted areas should be surveyed first for identification and marking of elite types at the time of flowering/fruiting and subsequently the collections are made at appropriate time. Within the country should be of at least 10-15 working days (excluding journey period) and more than a month when organised in foreign countries.

12. Team composition: Team consisting of 2-3 members including a collaborator and need-based local-aid be formed preferably a botanist/ breeder as leader. Area and route of exploration: This should be fine-tuned in consultation with the subject experts of local bodies as soon as the team reaches to the starting point keeping in view the targeted species and areas of the proposed mission. Items and equipments required: As per the nature of the germplasm to be collected (fruit/ seed/ vegetative propagule / in vitro/ live plants) and the area(s) to be explored, the required items and equipments have been listed. Domestic quarantine: All precautions including need-based domestic quarantine should be followed for pest-free collection and its transportation.

13. TYPES OF COLLECTIONS Currently three types of collections are held at most genetic resources centres . 1. Base collection The base collection is defined as a set of accessions, which in terms of genetic integrity, is as close as possible to the sample provided originally, which is preserved for long term future. Seeds can be conserved under long term ( 50 to 100 years ), at about - 20˚C with 5% moisture content .  They are disturbed only for regeneration.

14. 2. Active collection An active collection comprises accessions which are immediately available for multiplication and distribution for use. Accessions are stored for short to medium periods of time ( generally upto 20 years ). Active collections should be kept at such a conditions at least 65% germination can be obtained after for 10 to 20 years. Active collections are generally held at temperatures between 0-10˚C depending upon the species stored, the prevailing ambient environment and cost factors.

15. 3. Working collection Seeds are stored for 3-5 years at 5-10˚C and the usually contain about 10% moisture .  Such materials are regularly used in crop improvement programmes . These collections are maintained by breeders.

16. SAMPLING METHOD 1. Random Sampling Random sampling is usually carried out by randomly selecting a starting point at a collection site and taking a single spike or few pods at every second and third place along a number (50-60) transacts evenly dispersed in a target sets and bulking them to form a random sample. 2. Biased sampling This method is generally followed for sampling rare phenotype variants or observable spontaneous mutants occurring in the target population. The attention is given to identify specific variants for direct utilization.

17. 3. Clustered Sampling This method is applicable for collection of wild relatives or weedy species. The collection site is divided into several evenly spread clusters. Samples are taken independently from each cluster and then bulked together to form a multiple sample. 4. Coarse Grid Sampling This technique of sampling is used to collect random bulk samples from a site by harvesting parts/plants from several spots of the site. In coarse grid sampling is made at wide interval over the entire region. 5. Fine grid sampling This sampling technique is used in interested or intensive areas of variation identified after the coarse grid survey. Collector may walk across the site or field twice in cross or zigzag manner avoiding sampling from borders.

18. COLLECTING WILD RELATIVES OF CROP SPECIES B y wild relatives and related taxa can be classified into primary, secondary and tertiary gene pools: 1. Primary genepool (GP-1) Wild species in the primary genepool can produce fertile hybrids with cultivated types and hence are easy to exploit. These are wild progenitors closely related to crops. 2. Secondary genepool (GP-2) The wild species are relatively distantly related and cross compatible and hence contribute germplasm less easily. 3. Tertiary genepool (GP-3) Distantly related and unrelated taxa of different genera/species which can only be used with difficulty for some crops for a limited number of genetic traits are considered as tertiary gene pool. Hybrids are sterile . The wild species and the weed races represent the highest level of genetic heterozygosity and heterogeneity among the different classes of germplasm .

19. COLLECTION TECHNIQUES Seed propagated, cultivated and wild species Randomly selected sample of 50-100 plants of self pollinated crop and 100-200 plants of cross pollinated crops. Collect fully and physiologically mature seeds. Collect about 50 seeds (as per the availability) from each plant and bulk to make a population sample of 2000-4000 seeds in self pollinated and 4000-8000 seeds in cross pollinated crops. Record passport data and important plant traits for each sample. Sample as many sites as possible as per the availability of time. If considerable morphological variation is present make separate samples of each type.

20. Add biased sample if some morphotypes are not included in random Make herbarium specimens wherever possible. Take photographs of the important variants materials. Write desired field notes b) Vegetatively propagated crops: Sample each distinct morphotype in the area. Supplement with seed collections wherever possible give separate number for same plants or seeds from several plants (bulk samples) The collected materials for grafting with 3-4 days send by speed post or special messengers

21. No. Crop NAG site* (NAGS) No. of accessions currently available 1. Vegetables NBPGR, Pusa Campus, New Delhi 16,139 2. Potato CPRI, Shimla ( Himachal Pradesh ) 2,375 3. Spices NRC for Spices, Marikunnu , Calicut ( Kerala ) 2,847 4. Plantation Crops CPCRI, Kasaragod ( Kerala ) 307 5. Medicinal & Aromatic Plants AICRP on Medicinal & Aromatic Plants, NBPGR, New Delhi 375 6. Fruits (semi-arid ) All India Coordinated Project (Semi Arid Fruits ), HAU, Hisar ( Haryana ) 541 7. Fruits (Subtropical & Temperate ) NBPGR Regional Station, Phagli , Shimla ( HP ) 454 8. Fruits (all) IIHR, Bangalore ( Karnataka ) 13,118 9. Citrus NRC for Citrus, Nagpur ( Maharashtra ) 51 10. Mango CIHNP, Lucknow ( UP ) 587 11. Tuber Crops CTCRI, Sreekariyam , Trivandrum ( Kerala ) 3,586 Indian Plant Genetic Resources System - Directory of National Active Germplasm Sites (NAGS)

22. EVALUATION OF GERMPLASM

23. Definition: It deals with the assessing the agronomic potential of an accession including quality parameters and response to various abiotic and biotic stresses. Evaluation of germplasm resources is necessary to identify the appropriate germplasm with a target trait for their further utilization. Evaluation of germplasm is a multi-disciplinary approach and it should be done in collaborative mode involving germplasm curator, plant breeder, physiologist, pathologist, entomologist, biochemist etc. Proper plant spacing, fertilizer application, weeding, irrigation, plant protection measures need to be followed.

24. The number and row length should be more for cross pollinated species than those for self-pollinated ones. The observations should be recorded on the plants from the middle row to avoid the border effects. After evaluation, the promising accessions should be further validated through multi- locational , multi-season and multi-year evaluations. Evaluation trails should be carried out in at least three diverse environments to minimize Genotype x Environment (G x E) interaction .

25. Identification of resistance source against a particular race/strain/biotype within a particular location does not guarantee its resistance response in other locations may vary depending upon the agro-meteorological conditions. Evaluation for biotic stresses: Therefore, screening of germplasm for biotic stresses should be accompanied with identification race/strain/biotype of the pest and pathogens existing at that particular location. These four factors i.e. susceptible host, virulent pathogen, environment and time, which cause disease called is called “ Disease Quadrangle ”.

26. Under these circumstances, repeated field screening with proper experimental design followed by stringent screening under artificial challenged inoculation condition under field and glasshouse are essential to establish a real resistance or tolerance response of germplasm . For recording of the observations on fungal, bacterial and viral diseases, standard evaluation system (SES) scale should be followed. For recording of the data on defoliators, percentage infestation in each accession should be recorded. In case of sucking insect pests, the number of insects per unit plant/leaves/inflorescence should be counted.

27. Preliminary screening/ phenotyping should be done for one year with large number of accessions under field conditions. To narrow down the large number of germplasm accessions, preliminary screening for abiotic stress should be carried out for one year with large number of accessions under field conditions peculiar to the stress under study. Evaluation of germplasm for tolerance to different abiotic stresses is to be carried out under well-defined controlled conditions so that the optimum stress can be imposed at the desired stage. Evaluation for abiotic stresses:

28. Like, evaluation for light and temperature stresses should be carried out under phytotron facility . Evaluation for drought tolerance should be undertaken in drought plots with rain out shelters and with well defined moisture conditions. Microplots with well defined electrical conductivity including specific salt/ions should be used for evaluating germplasm under saline/alkaline conditions. Standard checks identified for specific abiotic stress should be used for proper comparison of the germplasm . Phytotron facility

29. Evaluation for quality parameters: Quality evaluation plays an important role for in identification of value rich germplasm keeping in view of the nutritional and health security. Quality traits like oil, fatty acids, protein, phenols, sugar, amino acids, vitamins, minerals and anti-nutritional factors requires specialized equipments. Properly labeled and packed material containing complete experimental details along with passport information should be made available for quality analysis.

30. DOCUMANTATION OF GERMPLASM

31. Germplasm conservation, in its various stages, includes a range of activities for which information is required or from which information is derived. This may refer to species, their sites of origin, or activities or stages of conservation. The action of recording, organizing, and analyzing conservation data is known as documentation. WHAT IS DOCUMENTAION?

32. OBJECTIVES Understand what documentation of plant genetic resources (PGRs) signifies, and its importance in the routine management and scientific use of a germplasm bank Define the stages of constructing a documentation system Document the most common operational procedures of a germplasm bank

33. Stages for the establishment of a documentation system: Obtaining information on the bank’s needs. Defining documentation objectives. Analyzing procedures. Identifying significant descriptors. Developing data formats and recording forms. Developing documentation procedures and implementing the system.

34. Documenting common procedures The most common procedures in germplasm banks include activities are: Registering samples (data of accessions) collection cleaning drying viability testing storage Characterization and evaluation regeneration distribution

35. Accession: An accession is a group of related plant material from a single species which is collected at one time from a specific location. Each accession is an attempt to capture the diversity present in a given population of plants. Accession Number: A unique number assigned to each accession.

36. MULTI CROP PASSPORT DISCRIPTORS: Institute code (INSTCODE) Accession number (ACCENUMB) Collecting number (COLLNUMB) Collecting institute code (COLLCODE) Genus (GENUS) Species (SPECIES) Species authority (SPAUTHOR) Subtaxa (SUBTAXA) Subtaxa authority (SUBTAUTHOR) Common crop name (CROPNAME) Accession name (ACCENAME) Acquisition date [YYYYMMDD] (ACQDATE) Country of origin (ORIGCTY) Location of collecting site (COLLSITE) Latitude of collecting site (LATITUDE)

37. Longitude of collecting site (LONGITUDE) Elevation of collecting site ( masl ) (ELEVATION) Collecting date of sample [YYYYMMDD] (COLLDATE) Breeding institute code (BREDCODE)Biological status of accession (SAMPSTAT) Ancestral data (ANCEST) Collecting/acquisition source (COLLSRC) Donor institute code (DONORCODE) Donor accession number (DONORNUMB) Other identification (numbers) associated with the accession (OTHERNUMB) Location of safety duplicates (DUPLSITE) Type of germplasm storage (STORAGE) Remarks (REMARKS) Continue…

38. Registering samples The registration of samples consists of assigning each sample (or accession) a unique identification number and recording data received with the samples, including those known to be descriptors of the accession. The data recorded would include: Accession number Other numbers associated with the accession (e.g., code numbers for collectors and donors) Scientific name (genus, species, subtaxa , and authorities) Common name(s) of the cultivated species Cultivar name(s) Date of acquisition of sample (incorporation into the germplasm bank) Date of last regeneration

39. Collection data Collection data are also known as passport data and refer to the data reported when the sample was first collected. These collection data or descriptors can be numerous, depending on the degree of detail in which information is needed. FAO and IPGRI have jointly prepared a list of passport descriptors of many crops to provide uniform coding systems for common passport descriptors of various crops (FAO and IPGRI 2001). The passport data most commonly documented on registering samples include: Collection date Collector’s name, number, and institute Country and province or state of collection Locality, latitude, longitude, and altitude of collection site Origin of sample (e.g., household garden, market, or farm) State of sample (e.g., wild, landrace, or advanced cultivar) Number of sampled plants

40. SEED CLEANING The seeds to be conserved in a germplasm bank should be, as far as possible, clean and free of broken seeds, residues, or infested or infected seeds. To save time, some banks do not document this procedure; others consider that such data have little practical or scientific value. Some descriptors suggested for this procedure are: Accession number Date of procedure Method used Total number of seeds Empty seeds (%) Operator (name of person who carried out the test)

41. SEED DRYING In a germplasm bank, orthodox or intermediate seeds are dried to reduce their moisture content to acceptable levels without affecting their viability. The initial moisture content is first determined. If this is very high, then the seeds are dried, using a suitable method, to reduce moisture content to the desired level. Once seeds are dried, some banks determine the total weight of the dried seeds and the 100- (Seed index) or 1000-seed weight (Test Weight), depending on their size. The most commonly used descriptors for seed drying are: Accession number Initial moisture content Drying method Date of measurement Final moisture content Total dry weight of seeds 1000-seed weight 100-seed weight (for large seeds)

42. SEED VIABILITY Germination under laboratory conditions is defined as the emergence and development of those essential structures that indicate, for the class of seed being analyzed, the seed’s ability to become a normal plant under favourable conditions. The results of this test indicate the percentage of live seeds of an accession that can produce plants under appropriate conditions. Accession number Lot reference (any date, code, or number that uniquely identifies the accession’s regeneration or multiplication cycle) Collection type (e.g., whether base or active collection) Reference for method used (e.g., absorbent tissue or tetrazolium test) Viability (%) Operator (name of person who carried out the test)

43. STORAGE Once the seeds have been dried and cleaned, and their percentage of viability recorded, they are stored in cold rooms (or under normal conditions, according to case). The following data or descriptors are recorded: Accession number Lot reference Collection type Location in cold room Total quantity of seeds stored per accession 1000-seed weight 100-seed weight (for large seeds) Minimum quantity permitted for seeds (this parameter helps determine when more seeds should be produced or multiplied)

44. GERMPLASM CHARACTERIZATION AND EVALUATION Germplasm characterization refers to the recording of highly inheritable descriptors (or data) that are readily seen and are expressed in all environments. They mostly include: Accession number Plant descriptors (morphological characterization) Susceptibility to abiotic stress (evaluation) Susceptibility to biotic stress (evaluation) Biochemical markers (molecular characterization) Molecular markers (molecular characterization) Cytological characteristics

45. REGENERATION OR MULTIPLICATION Regeneration or multiplication is carried out in response to data obtained through the seed monitoring control or during the growth cycle of the vegetative propagated species, which is conducted at given intervals of time to test the viability of each accession in storage and ascertain the quantity of seeds it has. The principal data or descriptors used to record regeneration or multiplication are: Accession number Lot reference Collection type Regeneration site Plot reference (of field, furrow, and plot number) Planting date Planting density Germination in the field (%) Established plants (no.) Days from planting to flowering (no.) Harvest date Cultural practices

46. GERMPLASM DISTRIBUTION: Linked to the information mentioned above on storage, information on the distribution of germplasm that the bank carries out should also be recorded. For example, some banks continually distribute germplasm for improvement programmes or for exchange with other banks. In these cases, to maintain efficient control over the bank’s holdings of materials, a record must be kept of the materials being distributed. Typical descriptors that should be considered are: Accession number Lot reference Date of exit of material Quantity of seed sent Data on receiver Plant health certificate number (if applicable)

47. REFERENCES FAO; IPGRI. 1994. Genebank standards. Rome. 15 p. Also available at http://www.ipgri.cgiar.org/ Konopka J; Hanson J. (1985) Information handling systems for genebank management. In Konopka J; Hanson J, eds. Proc. Workshop held at the Nordic Genebank , Alnarp , Sweden, 21-23 Nov 1984. IBPGR, Rome. pp 21-28 Stalker HT; Chapman C. (1989) Scientific management of germplasm : characterization, evaluation and enhancement. IBPGR, Rome. 194 p