Herbarium Management- Role in Plant Genetic Resources Study

Herbarium Management:

Methods and Current Trends

Herbariumof Cultivated Plants National

Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources Pusa Campus, New Delhi 110012, India

Training Programme on Herbarium Management: Methods and Current Trends

ICAR-NBPGR, New Delhi, July 15-20, 2019

For the official from: The Directorate of Seed Testing and Certification Ministry of Agriculture, Bagdad Government of Iraq

Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources Pusa Campus, New Delhi 110012, India

Citation Anjula Pandey (2019) Herbarium Management: Methods and Current Trends. The Training Manual, ICAR-National Bureau of Plant Genetic Resources, New Delhi, India

Technical Assistance: Shashi K Sharma and Rita Gupta Layout and Design: Shashi K Sharma

Disclaimer: This publication contains information provided by various authors “as it is” with editorial inputs. Contents of some chapters have been sourced by the authors from their prior publications, and ICAR- NBPGR is not liable for any copyright infringement whatsoever.

Contents

S.No. Title Page No.

About the Training Manual ---- i 1 Herbarium Management- Role in Plant Genetic Resources Study 1 Anjula Pandey 2 Methods for Management of Herbarium 28 Anjula Pandey, K Pradheep and Rita Gupta 3 Taxonomic Literature: Role in Plant Systematics Study 48 Anjula Pandey and K Pradheep 4 The relevance of Field and Ecological Studies in Herbarium 58 E. Roshini Nayar 5 Modern Tools in Plant Taxonomy 65 S Rajkumar 6 Chemotaxonomy - A Tool for Taxonomic Delineation and 80 Identification of Plant Genetic Resources Poonam Suneja, Rakesh Bhardwaj* and Anjula Pandey 7 Applications of Geographic Information Systems (GIS) Tools in Harbarium 87 Studies DP Semwal, Anjula Pandey, Rita Gupta and NS Panwar 8 Illustrative Field Aids for Identification of Plants 93 Anjula Pandey, S Nivedhitha and Pavan Malav

9 Floras, Monographs, Taxonomic Revisions and Identification Keys 102 Anjula Pandey 10 Biosystematic Studies on Crop Plant Taxa 113 Anjula Pandey and K Pradheep 11 Digitizing Specimens in A Herbarium: Procedures and Approaches 126 Anjula Pandey, Sunil Archak, Rita Gupta and ER Nayar 12 Exploration and Germplasm Collection of Plant Genetic Resources: 137 Prospects and Procedures SP Ahlawat and Anjula Pandey 13 Field Visits: Guidelines for Execution 150 KC Bhatt, RS Rathi and Anjula Pandey Training Programme ii List of the Participants-Herbarium Management iv Faculty members and other contact persons v Committees to Organize the Training Programme vi

About the Training Manual ----

National and international databases serve as biodiversity information resources and collectively provide data for visualization analysis of patterns of biodiversity. Amongii these data records associated with herbarium specimens’ label have now become available for large scale innovative research at the global level. Despite realizing the value of herbarium decades ago only in the past few years the knowledge hidden with the herbarium repositories the potential resources for new findings have been explored now using e-resources and vast floristic literature. Various global database facilities such as the Global Biodiversity Information Facility (GBIF), the Atlas of Living Australia (ALA), the US Geological Survey’s portal (Biodiversity Information Serving Our Nation; BISON), and iDigBio (Integrated Digitized Biocollections) provide digital biodiversity data, including information from herbarium specimens. Digitized herbarium specimen data serve as resources for synthetic analyses, link the phylogenetic, climatological and genomic resources to address several unresolved queries in plant sciences. Researchers in plant systematics are now benefitted with the enhanced understanding of diversity distribution, plant collecting strategies, seed storage behaviour, and taxonomic identification. Value of herbarium as a powerful tool in plant genetic resources programme was realised as early as 1948 when a build-up of herbarium voucher specimens generated through research activity was maintained as referral collection in the Botany Department of IARI (which later named as the Plant Introduction Division, IARI, Pusa). Later, the institute was rechristianization as ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR) in 1976 and since then it has been instrumental in disseminating knowledge on use of herbarium through teaching and training programmes, formal and informal technical discussions, brainstorming sessions and know-how on herbarium methodology. In continuation to these efforts, the ICAR-NBPGR is now organizing a short training course on “Herbarium Management” for participants from Directorate of Seed Testing and Certification, Ministry of Agriculture, Baghdad, Iraq with financial support from Government of Iraq from July15-20, 2019. For the benefit of participants a training manual entitled “Herbarium Management: Methods and Current Trends” is published with an overall perspective on the management of the herbaria. A total of eleven chapters including practical exercises and demonstrations will contribute towards a better understanding of herbarium management. Use of taxonomic keys in plant identification, herbarium and field methods, and biosystematics tools in species delimitation will help in build- up of the herbarium, besides enhancing their expertise. Topics like advances in modern tools, illustrative guides and herbarium digitization will help to boost the confidence of participants in setting up of state-of-the-art facility with their organisation(s). This training would sensitize the participants to create effective interest in the management of herbarium. We are highly thankful to the authors for contributing the contents of various chapters and bringing their views in this form. We hope that this publication will serve as a valuable reference to the course trainees as well as other users associated with PGR sciences.

(Anjula Pandey)

i Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

Herbarium Management- Role in Plant Genetic Resources Study Anjula Pandey Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources New Delhi 110 012, India Introduction There are approximately 4,000 recognized active global herbaria collectively holding 35,00,00,000 herbarium specimens. India represents over 3.5 million herbarium specimens including~23,000 type specimens (source:http://sciweb.nybg.org/science2/IndexHerbariorum.asp). These major global herbaria are committed to providing herbarium of economic species accessible to users (K, P, MO, S, B, UC/JEPS), and yet some others focus on regional flora (F,BM, PE, E, CAL). Among the cultivated plant herbaria, only a few of them are rich in the representation of cultivated plants (H- cultivated ornamentals and The Gatersleben Herbarium (GAT- Crop Plant Genetics mainly). GAT is located in the Department of Genebank of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) and is one of the largest specialized herbaria which serve as a source of reference and working material for the reproduction of accessions maintained in the genebank and other institutional research programmes. The holdings include over 0.430 million specimens of cultivated plants and its wild relatives, seed and fruit collection (about 0.1 million samples) and the spike collection (55,000 samples) (http://www.ipk-gatersleben.de/en/gbisipk- gaterslebendegbis-i/herbarium/).List of selected herbaria with significant information is given in Appendix I. Management of a herbarium includes all the activities pertaining to build-up, maintenance and research undertaken to establish a facility. While developing a herbarium state-of-the-art facility, the activity begins with the build-up of the collection followed by all other activities. A herbarium is a repository of plant specimens to serve as valuable resources for plant and data used for scientific research. For a quality specimen, an ideally dried herbarium should have character representation (vegetative characters: roots, tubers, bulbs and rhizome, leaf, stipule, spine, bark, etc. and floral: inflorescence, flower- spathe, scape, stamen, sepal, petal/tepals; and fruit characters: pericarp,

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey) placentation, seed) needed for taxonomic studies (Lawrence 1951; Davis and Heywood 1963; Holmgren and Holmgren 1998). Information on plant species with respect to the area of availability, variability pattern, flowering/fruiting time, status on rare/endangered/endemism, other ecological features, economic uses, indigenous traditional knowledge (ITKs), etc. gathered from herbarium label data serves as resource for basic and applied research, referral use and for educational programme. The information available on the herbarium label could be helpful in the planning and management of genetic resources and may serve the users in various ways. ‘Voucher specimens’ used in experimental studies as well as those collected from diverse areas and deposited in herbaria can be used for further study by others. Herbaria located in teaching/ educational institutions will have specimens generated through basic study and applied sciences. On the other hand, institutions dealing with the survey, exploration and collection of germplasm will have specimens collected from diverse phytogeographical location and depict distributional records. Herbarium specimens collected a century ago from a location that is currently fully urbanized can reflect on the data on species occurrences of the then location of plant species preserved as voucher herbarium specimens. With the digitization of herbarium holdings and the possibility of using DNA extracted from tissues of the preserved plant material makes them invaluable resources to study the ecological and evolutionary species in responses to the global environmental change (Savolainen et al. 1995). The herbarium of cultivated plants at ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi, also known as National Herbarium of Cultivated Plants (code ‘NHCP’) occupies an important place among the 25 major Indian herbaria (Singh 2010). The NHCP is listed in the ‘Index Herbariorum’ which is a global directory of public herbaria in different regions (Holmgren and Holmgren, 1998;http://sciweb.nybg.org/science2/IndexHerbariorum.asp). It holds significant collections mainly taxa of cultivated and wild relatives/ weedy relatives of both native and exotic origin, and potential species identified through PGR programme. Besides, seed and carpological samples/ economic products of plant genetic resources (PGR) relevance are available as a complementary collection. Additionally, the virtual or digitization herbarium images of selected specimens can be used for research activities at the institute. The National Herbarium for Cultivated Plants (NHCP), New Delhi at ICAR-NBPGR differs in its mandate from the other herbaria across the

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey) country in representing wide range of variability in crop plants depicted as modern cultivars, primitive types/ landraces, wild/semi-domesticated forms and crop wild relatives (CWR)/ weedy types taxa which are being poorly represented in traditional herbaria. Besides underutilized or species of minor economic value (potential domesticates, wild economic plant species) collected from different agro-ecological regions of India, local flora of Delhi, weed flora, and experimental voucher deposited including the exotic germplasm introduced under various research programme are well represented. Herbaria in genetic resource study Depending upon the interest of the organization or institution housing herbarium they may be named as: (i) herbaria of organizations, (ii) regional herbaria, (iii) local herbaria, and (iv) herbaria of institutions, university, colleges and educational institution. The contents of a herbarium may represent: (a) plant representing local flora, (b) drugs and medicinal plants; and (c) crop plants, weeds in cultivated fields; ethnobotanically important plants. Likewise, depending on location and contents of herbaria the users may be students (in basic/ applied research), breeders (crops and wild relatives), taxonomists, agronomists, weed scientists, pathologists foresters, medicine men, pollination biologists, genetic resource personnel, conservators, and molecular biologists. The Herbarium of plant genetic resources facilitates in: 1. locating areas of distribution and diversity; collecting species with a narrow range of distribution, endemic cultigens, wild taxa, etc. 2. gathering data on flowering and fruiting, variation for plant vs. area/collection time (phenological data) 3. study on ecogeography vs. morphological variation 4. gap identification for germplasm collection and herbarium 5. bioprospecting based on ethnobotanical records in the herbarium labels 6. education and research: a source of material for analyses of anatomy, disease and disease control, biochemistry, evolutionary relationships, conservation, plant pollinators, host specificity 7. modelling plant distribution over time, act as evidence to insight on crop evolution to demonstrate wild forms to native domesticates 8. novel scientific activities in plant genetic resources vs. crop modulation; 9. developing taxonomic treaties and field identification aids for teaching programmes in taxonomy

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

NHCP- journey from inception Traditional herbaria rarely focus on herbarium collections depicting variability within cultivars, primitive forms, landraces, and as also obsolete cultivars. Keeping this in view the herbarium of Plant Introduction Division of Indian Agricultural Research Institute was set up in 1948. During 1948-1974 nearly 5,000 specimens were added through collections made under various plant genetic resource programmes viz. plant introduction, explorations, germplasm evaluation and post-entry quarantine activities. The herbarium was rechristened in 1985 as the National Herbarium of Cultivated Plants with its location at the ICAR-National Bureau of Plant Genetic Resources, New Delhi. During 1985 major thrust on building-up of infrastructure facilities were laid under the institutional project entitled “Establishment, build-up and maintenance of herbarium and seed museum of cultivated plants” under the leadership of Dr E Roshini Nayar as the curator of NHCP. NHCP started its journey from a room measuring 3x3m in ICAR- NBPGR, in the old building with selected herbarium specimens and seed/ economic products assembled and housed in three pigeon hole almirah were to the present state. With the major thrust on collections of landraces, CWR and rare cultivars from unrepresented areas (as neglected regions, across eco-geographical regions, tribal/ north-eastern region) presently NHCP has grown as a well established herbarium of cultivated plant repository (Fig. 1), enlisted in the global list ‘Index Herbariorum’ (code-NHCP). Specialized training attained by the scientific staff of the herbarium at various national and international herbaria such as the Royal Botanic Gardens at Kew, and New York Botanical Garden and Arnold Arboretum in USA and Wealth of India Herbarium, New Delhi and various herbaria - National Botanical Survey of India and Forest Research Institute, Dehradun and National Botanical Research Institute, Lucknow, Uttar Pradesh facilitated in up-gradation of infrastructure. The insect-pest- and dust-free storage cabinets gradually replaced the traditional pigeonhole cabinets. With the addition of five new-space saver compactors during 2004-2008, the capacity of herbarium has increased to up to 40,000 specimens. Build-up of herbarium specimens The National Herbarium of Cultivated Plants (NHCP) presently has herbarium approximately 24,000 specimens representative of 267 families, 1552 genera and 4,277 species (as on June 2019). NHCP is linked to the Index Herbariorum (Thiers 2016). Selected 7,000 herbarium sheets of different categories plant specimens were imaged

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey) with database and available on the herbarium website. Important taxa of plant genetic resource (PGR) relevance include over 500 crop taxa and ~700 species of crop wild relatives (CWR)/weedy relatives (Pandey et al. 2018). Additionally, the seed and the economic botany museum provide a reference collection of crop, wild and weedy plants. The build-up of material is through specimens/ seeds collected during explorations in different agro-ecological zones of India, material introduced from abroad under various research/ breeding programmes and also vouchers of the systematic studies on crop-groups. Some important publications which have contributed as baseline for build-up of material in the NHCP include: cultivated taxa (and variability within them) of crop/economic species (Ambasta et al. 1986; Nayar et al. 2003), wild relatives of major crop taxa (Arora and Nayar 1984; Pandey et al. 2005) and wild edible and economic taxa (Arora and Pandey 1996). System of the arrangement of herbarium specimens differs from that of the other herbaria; specimens are arranged by families, then by genera and then by species; all in alphabetical order. This was found more convenient for wide use by PGR workers, para-botanists, non- taxonomists and the beginners. For efficient access to the resources, documentation of the herbarium holdings as soft data, images in the virtual herbarium and Index Cards and inventory of digitised taxa (Nayar et al. 2011) can be referred. Facilities are available as net-house to grow- out for identification, raising material received as vegetative propagules/seed and for PGR research and teaching programme. In addition standardization of methodology for economic/ eco-friendly storage, specialized groups such as landraces (variation), difficult groups (succulents, large-fruited types, aquatic plants, plants tending to leaf fall on drying), etc. (Pandey et al. 2013) are in progress. Guidelines for effective use of the herbarium for consultation/visit to the NHCP and identification/authentication of species are available (Pandey 2015b,c; 2016a; [email protected]; [email protected]); annexure I- VII.

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

Figure1. (top) Layout of National Herbarium of Cultivated Plants (NHCP) facility at National Bureau of Plant Genetic Resources, New Delhi; (bottom) compactors used for storage of herbarium specimens Current holdings in NHCP

Year-wise holdings of herbarium specimens maintained in the NHCP (as on December 31, 2018) are given in Fig. 2.

10000 9000 8000 7000 6000 5000 4000 3000 2000 1000

0 Herbarium Herbarium (no.) specimens

Years

Herbarium specimens (no.)

Figure 2.Year-wise holdings of herbarium specimens maintained in the NHCP (December 2018) Some significant collections Besides herbarium specimens of cultivated plants, some neglected groups: less-known domesticated species viz. Flemingia procumbens/Moghania vestita (Soh-phlong), Digitaria (Raishan), Coixlacryma-jobi in north-eastern hills, and others such as Malva

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey) verticillata, Inula racemosa, Hodgsonia heteroclita, Brachiaria mutica, Aisandra butyracea (Cheura), Adansonia digitata (Gorakh imli), Setaria glauca, Momordica dioica, Allium spp., rice bean, winged bean, Vigna vexillata, and taxa of potential/commercial value are represented. Important taxa of crop wild relatives (CWR) in the NHCP include: Oryza, Sorghum, Vigna, Cajanus/Atylosia, Solanum, Abelmoschus, Cucumis, Luffa, Allium, Trichosanthes, Sesamum, Curcuma, Piper, Amaranthus, Melilotus, Medicago and Trifolium. Some speciality collections include wild Vigna from north-western Himalaya, fodder grasses and forage legumes from north-western areas and peninsular India, and wild Allium from high altitude areas of western and eastern Himalaya; and wild Triticeae from western Himalaya. Type collections of newly described taxa by ICAR-NBPGR under genera viz.Curcuma, Abelmoschus, Vigna, Cucumis, Herpetospermum, Momordica, etc. are some valuable collections maintained in the NHCP. Eragrostiella bifaria (Vahl) Bor (HS3007) collected from the Delhi Ridge area (10.9.1939 by Dr HB Singh), Indigofera viscosa (HS5390- the oldest herbarium specimen collected in 1933 from Baluchistan, now in Pakistan), Vicia hyaeniscyamus (HS8539- exotic material raised in Plant Quarantine Experimental Fields, ICAR-NBPGR, Delhi), rare/ endangered taxa- Cycas beddomei, Podophyllum hexandrum (anti-cancer plant), etc. form some significant holdings. Herbarium specimens of PGR of exotic origin include those prepared under the Plant Introduction (PI) Scheme operational in the Botany Division of the IARI commenced functioning in 1946. Some important genera/crop groups represented under this group included Oryza from Philippines; Avena from Australia; Brassica from Canada; Medicago from Portugal and Australia; Trifolium from Australia, Portugal and UK; Vicia from Australia; Solanum from USA and Sri Lanka; Lycopersicon from South America; others such as Amaranthus, chenopods from USA; dwarf peaches from Australia and Agathis, Calluna, Corynocarpus from New Zealand (Nayar et al., 2011). Virtual herbarium specimens of 298 taxa representing 482 exotic germplasm accessions bearing Exotic Collection (EC) numbers are uploaded in website for wider use (http://192.168.5.92/NHCP/Advancesearch.aspx). Herbarium specimens gathered during special programmes: Grassland Survey Scheme of PL-480 (1960-70s); Flora of Karnataka Project (1980s); Project on Collection of Fodder Grasses and Forage Legumes from North-western region (1948-86); National Agricultural Technology Project (NATP) on Plant Biodiversity (1999-2005); Subproject on Biosystematics of the Genera- Vigna, Cucumis, and Abelmoschus

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey) under National Agricultural Innovation Project (NAIP) (2011-14); Herbarium of Dr YS Rao’s Collection on Aquatic Plants (1948-1986) have significantly contributed to NHCP holdings. Evaluation and regeneration activity taken up under NATP programme (1999-2005) and ICAR-NBPGR evaluation programme has facilitated in the build-up of collections of some groups like wheat, paddy, sorghum, Hordeum, as seed and spike/head collection. Fruit and seed variability in many cereals, legumes and vegetables represented in the collection can be referred for identification. Variability depicted as dried fruits (okra, Luffa), seeds (pulses), boles of cotton is displayed as mounted material for demonstration. Seed collection (3,090) and economic products (696 samples) are maintained as a complementary collection in NHCP. Seed samples are maintained in plastic boxes in dry form depicting species under genus: Oryza, Vigna, Phaseolus, Pisum, Cicer, Solanum, Moringa, Cucurbita, Capsicum, Trichosanthes, Momordica, Allium, Luffa, Brassica, Sesamum, Ricinus, Gossypium, Crotalaria, etc. Economic botany collection is maintained as bulky herbarium (as wet preservation or dry form). Some significant ones include- Lagenaria siceraria (90cm long fruit; dry fruit pericarp as carpological sample); Luffa (species collection as dry fibrous fruit), Entada phaseoloides (pod 60 cm; seed; stem) and Parkia roxburghii (dried pods); Moringa oleifera (fruit and seed collection of cultivated and wild types); Musa bulbisiana (inflorescence and fruits); Juglans regia and Prunus amygdalus (fruits), coconut (fruit, coir); arecanut (fruit, saucer made from spathe); Ravenalamadagascariensis (inflorescence); Trapa bispinosa (dry fruit); Diospyros (fruits of four species); Aleurites moluccana (nuts); Glycyrrhiza glabra (roots), Flemingia procumbens and Pachyrrhizus erosus (tubers), sorghum and okra (landrace diversity as head/fruit); Gossypium arboreum (bolls of different races); Triticum and related taxa (species diversity as spike); Curcuma, Zingiber (rhizome); Pandanus odoratissimus (male inflorescence); Saussurea lappa (rootstock); Tecomella undulata (bark), Commiphora mukul (gum crystals), Saraca indica (fruits); Garcinia gummi-gutta/ kokam (fruits) and Terminalia (species diversity as fruit). Virtual herbarium Digital images are being maintained in the virtual herbarium. These images are classified in their respective folders (arranged in alphabetical order in family, genus and species) with unique identity numbers (herbarium specimen number) and linked to the database. For detail refer to chapter 10.

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

Development of protocol for safe preservation Difficult-to-store groups are a priority for developing a protocol for pest- free storage, ideal storage conditions for families which are sensitive to pest damage, standardization of the use of low temperature (-20°C) using methods - deep freezer, the dusting of naphthalene powder, etc. Difficult-to-represent taxa like bulbous group, tuberous/rhizomatous taxa, Musa, Agave, etc. are being worked out for good representation of diverse material in the NHCP. Traditional processing techniques with a combination of microwave drying techniques have been successfully standardized for difficult material (Jain and Rao 1977; Pandey et al. 2006a). The material used for biosystematics study, biochemical/ phytochemical and molecular study the efforts have been on the minimum use of hazardous chemicals (contact poisons/chemicals, and insect repellants/naphthalene bolls, deep freezing methods). Significant documents Publications in the form of books, manuals, chapters, and research papers on new records on geographical distribution and new taxa have been brought out from time to time. Some significant ones include: Wild Relatives of Crop Plants of India (Arora and Nayar 1984), Wild Edible Plants of India (Arora and Pandey 1996), Wild Relatives of Crop Plants- Collection and Conservation (Pandey et al. 2008), Genetic Resources of Rosaceae of India (Pandey et al. 2007); Importance of Voucher Specimens of Introduced Germplasm (Nayar et al. 2003; 2014). For the benefit of different users on services provided by the NHCP, guidelines are in place (Pandey et al. 2015a; krishikosh.egranth.ac.in/.../1/2035781; krishikosh.egranth.ac.in/.../1/.../1/8.pdf). The taxonomic works were undertaken in the project mode on ‘Genetic Resources Study of Economically Important Plant Families- Cucurbitaceae, Malvaceae, Rosaceae and Poaceae’ during 1984-1995 (Pandey et al. 2006b). Study of crop taxa of Indian region (Asiatic Vigna, Crotalaria, Allium, Prunus and wild Triticeae); and Check-lists of Indian Crop Plants and Crop Wild Relatives pin-pointed the gaps in collection and facilitated prioritization for the build-up of herbarium holdings (Arora and Nayar 1984; Nayar et al. 2003). Teaching, services and linkages Since 1999 the NHCP facility is available for conducting the teaching programme on taxonomy, ethnobotany and economic botany with Post- Graduate (PG) School, ICAR-Indian Agricultural Research Institute, New Delhi. Besides the expert consultation service in the field of taxonomy,

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

NHCP is actively involved in providing technical input on identification/ authentication, validation of taxa of PGR relevance; it also provides hands-on exercise on herbarium procedures to a large number of collage and school students and researchers especially those working in fields of pharmacy, pathology, entomology, breeding, etc. Beneficiaries from other fields of science especially for seeking identification/authentication of material as host-plant species relationship, introduced germplasm, weed science, agronomy, conservation, etc. The NHCP is a specialized herbarium and the digital resources provide identification aids to the collectors with images and data. This has been done in some groups where there have been a high degree of ambiguity in identity of material received from collection trips viz. oilseed Brassica, Sesamum, Allium, legumes in general and grain legumes in particular, and rare cereals and millets. Herbarium staff keeps an update on knowledge through visits, training and linkages with national and international experts. The NHCP maintains links with many ICAR institutes, State Agriculture Universities and traditional universities, Botanical Survey of India (BSI), Forest Research Institute (FRI), and Herbarium Cryptogamae Indiae Orientalis (HCIO- a national-fungal herbarium facility at IARI, New Delhi), and the Herbarium of Wealth of India, CSIR-NISCAIR, New Delhi. References/selected readings Ambasta SP, K Ramachandran, K Kashyapa, Ramesh Chand (eds) (1986) Useful Plants of India. Publication and Information Directorate, Central Scientific and Industrial Research, New Delhi, India. Arora RK and A Pandey (1996) Wild Edible Plants of India: Diversity, Conservation and Use. National Bureau of Plant Genetic Resources, New Delhi, 294 p. Arora RK and ER Nayar (1984) Wild Relatives of Crop Plants in India. Sci. Monogr. 7: 90pp. Bhatt KC, Anjula Pandey, OP Dhariwal, NS Panwar and DC Bhandari (2009a) ‘Tum-thang’ (Crotalaria tetragona Roxb. ex Andr.): A Little Known Wild Edible Species in the Northeastern Hill Region of India. Genet Resour Crop Evol 56: 729-733. Bhatt KC, N Sharma and Anjula Pandey (2009b) ‘Ladakhi tea’ Bidens pilosa L. (Asteraceae): A Cultivated Species in the Cold Desert of Ladakh Himalaya, India. Genet Resour Crop Evol 56: 879-882.

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

Davis PH and VH Heywood (1963) Principles in Angiosperm Taxonomy. University of Edinburgh Press, Great Britain, p 556. Holmgren, PK and NH Holmgren (1998) Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff. New York Botanical Garden, New York, USA. Jain SK and RR Rao (1977) A Handbook of Field and Herbarium Methods. Today and Tomorrow Printers and Publishers, New Delhi, p157. Lawrence GHS (1951) Taxonomy of Flowering Plants. Oxford & IBH Publishing Co., p 823. Malav PK, APandey, KCBhatt, SGopala Krishnan and ISBisht (2015) Morphological Variability in Holy Basil (Ocimum tenuiflorum L.) from India. Genet Resour Crop Evol 62: 1245-1256. Nayar ER (2015) Crop Wild Relatives in Indian Gene Centre: An Overview. In: Pradheep K, A Pandey, KC Bhatt, SP Ahlawat, DP Semwal and KC Bansal (eds) Crop Wild Relatives: Identification, Collecting and Utilization. ICAR-National Bureau of Plant Genetic Resources, New Delhi, pp 18-26. Nayar ER, A Pandey, K Pradheep and Rita Gupta (2011) Inventory of Digitized Taxa in the NHCP. National Bureau of Plant Genetic Resources, New Delhi. Nayar ER, A Pandey, Kamala Venkateswaran, Rita Gupta and BS Dhillon (2003) Crop Plants India: A Check-list of Scientific Names. Agro- biodiversity (PGR)-26. National Agricultural Technology Project on Sustainable Management of Plant Biodiversity, National Bureau of Plant Genetic Resources, New Delhi, 48p. Nayar ER, A Pandey, K Pradheep, R Gupta and SK Sharma (2014) National Herbarium of Cultivated Plants (NHCP): Importance of Voucher Specimens of Introduced Germplasm. Indian J Plant Genet Resour 27: 163-170. Pandey Anjula (2015a)Plant Systematics: Field Inventory, Herbarium Preparation and Management of Important Herbaria and Botanical Gardens of the World and India. Institute of Life Long Learning, Delhi University (http://vle.du.ac.in/mod/resource/view.php?id=13116) ISSN NO. 978-93-85611-90-2. Pandey Anjula (2015b)Plant Systematics: Documentation: Flora, Monographs, Journals, Online Journals and Keys. Institute of Life Long Learning, Delhi University

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

(http://vle.du.ac.in/mod/resource/view.php?id=13116) ISSN NO. 978-93-85611-90-2. Pandey Anjula and ER Nayar (1994) Some Observations on Systematics of Genus Crotalaria. Indian J Pl Genet Resour 7(2):133-144 (spl. issue.) Pandey Anjula and Ruchira Pandey (2005) Wild Useful Species of Allium in India- Key to Identification. Indian J Plant Genet Resour 18(2): 175-178. Pandey Anjula, ER Nayar and Rita Gupta (2006a) An Efficient Methodology for Processing of Herbarium Specimens of Cultivated Plants. Indian J Plant Genet Resour 19:47-49. Pandey Anjula, E Roshini Nayar and K Venkateswaran (2006b) Plant Genetic Resources of Rosaceae in India. National Bureau of Plant Genetic Resources, New Delhi, 53p. Pandey Anjula, E Roshini Nayar and K Venkateswaran (2007) Plant Genetic Resources of Rosaceae in India. National Bureau of Plant Genetic Resources, New Delhi, 53p. Pandey Anjula and KC Bhatt (2008) Diversity Distribution and Collection of Genetic Resources of Cultivated and Weedy Type in Perilla frutescens var. frutescens and their Utilization in Indian Himalaya. Genet Resour Crop Evol 55: 883-892. Pandey Anjula, Rakesh Singh, J Radhamani and DC Bhandari (2010) Exploring the Potential of Ziziphus nummularia (Burm. f.) Wight et Arn. from Drier Regions of India. Genet Resour Crop Evol 57(6): 929-936. Pandey Anjula, Ajay Tomer, DC Bhandari and SK Pareek (2008) Towards Collection of Wild Relatives of Crop Plants in India. Genet Resour Crop Evol 55: 187-202. Pandey A, DC Bhandari, KC Bhatt, SK Pareek, AK Tomar and BS Dhillon (2005) Wild Relatives of Crop Plants in India: Collection and Conservation. Agro-biodiversity (PGR) 41. National Agricultural Technology Project on Sustainable Management of Plant Biodiversity, National Bureau of Plant Genetic Resources, New Delhi, 73p. Pandey A, K Pradheep and DP Semwal (2014a) Notes on Luffa (Cucurbitaceae) Genetic Resources in India: Diversity Distribution, Germplasm Collection, Morphology and Use. Indian J Plant Genet Resour 27: 47-53.

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

Pandey A, K Pradheep and N Sharma (2014b) Potential Introduced Medicinal Plant African Bitter Leaf (Vernonia amygdalina Delile) in India: Botany, Propagation and Uses. Med Pl 6: 272-276. Pandey A, K Pradheep and R Gupta (2014c) Chinese chives (Allium tuberosum Rottler ex Sprengel): A Home Garden Species or A Commercial Crop in India. Genet Resour Crop Evol 61: 1433-1440. Pandey A, K Pradheep and R Gupta (2015a) Manual on National Herbarium of Cultivated Plants. National Bureau of Plant Genetic Resources, New Delhi, 50 p + i-vi. Pandey A, KS Negi, K Pradheep and MC Singh (2015b) Note on Occurrence of Fragrant False Garlic (Nothoscordum gracile (Aiton) Stearn) in India. Indian J Pl Genet Resour 28: 351-355. Pandey Anjula, DP Semwal, KC Bhatt, Rita Gupta and SP Ahlawat (2016) A New Report on Cultivation of “Sukhlai” [Abelmoschus manihot (L.) Medik. subsp. tetraphyllus (Roxb. ex Hornem.) Borss. Waalk.]: A Species Used as Organic Clearant in jaggery Industry in India. Genet Resour Crop Evol 63(8): 1447-1455. Pandey Anjula, K Pradheep, Rita Gupta, E Roshini Nayar and DC Bhandari (2011a) Drumstick Tree (Moringa oleifera Lam.): A Multipurpose Potential Species in India. Genet Resour Crop Evol 58 (3): 453-460. Pandey Anjula, V Joshi and U Lachungpa (2011b) Notes on Cultivation, Variability and Conservation of Scarlet Runner Bean (Phaseolus coccineus L.): A Fast Disappearing Minor Pulse of India. Annals of Forest 19(1): 34-38. Pandey Anjula, ER Nayar, K Pradheep and Rita Gupta (2013a) Preparation of Herbarium Specimens of Cultivated Plants. In: Jacob et al. (eds.) Training Manual on Management of Plant Genetic Resources National Bureau of Plant Genetic Resources, New Delhi, India, pp 14-20. Pandey Anjula, Rakesh Singh, Rekha Chaudhury and SK Malik (2013b) Systematic Studies on Crotalaria tetragona Roxb. ex Andr. (Fabaceae-Crotalarieae): A Wild Relative of Sunnhemp. Indian J Pl Genet Resour 26(1): 68-75. Pandey Anjula, K Pradheep and R Gupta (2016a) Herbarium Procedures (Guidelines for Beginners) National Herbarium of Cultivated Plants. Division of Plant Exploration and Germplasm Collection, National Bureau of Plant Genetic Resources, New Delhi, 6p.

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

Pandey Anjula, K Pradheep and Rita Gupta (2016b) Methodology for Collecting and Preparing Herbarium Specimen of Allium. Indian J Plant Genet Resour 29: 32-39. Pandey Anjula, K Pradheep, Rita Gupta and SP Ahlawat (2017) National Herbarium of Cultivated Plants: A Resource for Study of Crop Genepools. Indian J Plant Genet Resour 30(3): 246-252 Pandey Anjula, K Pradheep and Rita Gupta (2018) Two-leaf Nightshade (Solanum diphyllum L.) - An Addition to the Flora of Delhi, India and Weed Risk Assessment of the Species Indian J Plant Genet Resour 31(2): 164-168 Pradheep K and Soyimchiten (2016) Occurrence of Juglans sigillata and Caryota obtusa in Nagaland: New Distributional Records and Economic Notes. Ind Forest 142(7): 680-683. Pradheep K, Anjula Pandey and DC Bhandari (2011a) Notes on Naturalized Taxa of Plant Genetic Resource Value in Himachal Pradesh. Indian J Pl Genet Resour 24(3): 293-298. Pradheep K, PK Singh, Anjula Pandey and DC Bhandari (2011b) Collecting Genetic Resources of Wild Moringa oleifera Lam. from Western Himalayas. Indian J Pl Genet Resour 24(1): 75-81. Pradheep K, A Pandey, KC Bhatt and ER Nayar (2014) Herpetospermum operculatum (Schizopeponeae, Cucurbitaceae): A New Species from India, Myanmar and China. Blumea 59: 1-5. Pradheep K, DR Pani and DC Bhandari (2013) Addition of Gymnopetalum chinense (Lour.) Merr. to the Flora of Odisha. Ind Forest 139 (5): 465-466. Pradheep K, DR Pani and KC Bhatt (2015) Taxonomic Notes on the Trichosanthes cucumerina Group (Cucurbitaceae) from India. Novon 24: 39-45. Pradheep K, RS Rathi, Soyimchiten and ER Nayar (2015c) “Meetha patta”(Plukenetia corniculata Sm.): A New Report of Leafy Vegetable Crop from North-Eastern Region of India. Genet Resour Crop Evol 62: 1113-1120. Rathi RS, K Pradheep, S Roy, SK Singh and AK Misra (2016). Stahlianthus involucratus (King ex Baker) Craib ex Loes.: A New Record to the Flora of Mizoram, Indian J Threat. Taxa 8(3): 8629- 8631. Savolainen V, P Cue´noud, R Spichiger, MDP Martinez, M Cre´vecoeur and JF Manen (1995) The Use of Herbarium Specimens in DNA

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

Phylogenetics: Evaluation and Improvement. Pl Syst Evol 197:87- 89. Semwal DP, KC Bhatt, DC Bhandari and NS Panwar (2014) A Note on Distribution, Ethnobotany and Economic Potential of Hodgsonia heteroclita (Roxb.) Hook.f. & Thoms. (Cucurbitaceae) in North- eastern India. Ind J Natl Prod Resour 5: 88-91. Singh HB (2010) Handbook on Herbaria in India and Neighbouring Countries. National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. Soyimchiten, K Pradheep, K Joseph John and ER Nayar (2015) An Occurrence of Indo-Chinese Taxon Momordica subangulata Blume subsp. subangulata (Cucurbitaceae) in Nagaland: A New Distribution Record from India. J Threat Taxa 7: 8182-8184. Thiers B (2016) Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff. New York Botanical Garden’s Virtual Herbarium. http://sweetgum.nybg.org/science/ih/ Appendix I

List of Important Museum/ Herbaria, Code, Specialty and Date of Foundation

Name of museum (code) Specimen Specialty Date of (no.) foundation

Museum of Natural 8,000,000 Vascular plants 1635 History, Paris (P) worldwide, especially Africa (northern, western, and equatorial), southeastern Asia, Royal Botanic Gardens, 7,000, 000 Types and collections 1852 Kew (K) from Africa, tropical Asia, and Australasia Komarov Botanical 7,106,000 Institute, Leningrad

Conservatory and 6,000,000 Mediterranean, Middle 1824 Botanical Garden, East, South America, Geneva (G) Africa, Madagascar, and regional flora

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

Combined Herbaria, 5,005,000 Worldwide collection of Harvard University, vascular plants with Cambridge (GH) emphasis on New World plants New York Botanical 7,300,000 Tropical America and 1891 Garden, Bronx (NY) North Americas

U. S. National 1,25,000 Seed plant families 1908 Herbarium, Washington worldwide (BARC) British Museum of 5,200,000 Europe, Africa, North 1753 Natural History, London America, West Indies and (BM) the Himalaya Missouri Botanical 5, 870 000 Central America 1859 Garden, Saint Louis (MO)

Field Museum of Natural 2 ,700 000 Tropical and North 1893 History, Chicago (F) America

University of Uppsala, 3,100,000 All groups worldwide 1785 Uppsala (UPS)

National Botanic Garden 3,000,000 Vascular plants of Belgium, Belgium

Botanical Garden and 3,500,000 All groups worldwide, 1815 Botanical Museum, especially central Europe, Berlin (B) Mediterranean area, southwestern Asia, Africa Academy of Natural 245,000 Diatoms worldwide 1860 Science, Philadelphia (ANSP) Museum of Natural 8,000,000 History, Paris (P)

Royal Botanic Garden, 2,000,000 Southwestern and 1839 Edinburg (E) southeastern Asia, Arabia, Turkey, Bhutan, Brazil, Britain, China, Himalayas, Mediterranean, Chile, Argentina, and southern Africa

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

University of California, 96,000 Vascular plants of 1950 Berkeley (JEPS) California

University of Michigan, 1,700,000 Worldwide, especially 1837 Ann Arbor (MICH) temperate North America and the Great Lakes region Botanical Survey of 2,000,000 Economically important 1897 India, Calcutta (CAL) angiosperms; marine algae of coastal India

List of Important Herbaria in India* S. Indian herbarium (code) no.

1. The Central National Herbarium, Botanical Survey of India, Calcutta (CAL)

(i) Eastern Circle Herbarium, Shillong (ASSAM)

(ii) Southern Circle Herbarium, Coimbatore (MH)

(iii) Arid Zone Circle, Jodhpur (BSJO)

(iv)Western Circle Herbarium, Pune (BSI)

(v) Northern Circle Herbarium, Dehradun (BSD)

(vi) Andaman and Nicobar Circle, Port Blair, A&N (PBL)

(vii) Central Circle Herbarium, Allahabad (BSA)

2. Forest Research Institute, Dehradun (DD)

3. Blatter Herbarium, Botany Department, St. Xavier’s College, Mumbai, Maharashtra (BLAT)

4. National Botanical Research Institute, Lucknow, Uttar Pradesh (LWG)

5. Herbarium of Industrial Section, Indian Museum, Calcutta, West Bengal (BSIS)

6. Medicinal Plant Herbarium, Central Drug Research Institute, Lucknow, Uttar Pradesh (CDRI)

7. Herbarium of State Forest Research Institute, Jabalpur, Madhya Pradesh

8. Birbal Sahni Institute of Paleobotany, Lucknow, Uttar Pradesh (BSIP)

Herbarium Management- Role in Plant Genetic Resources Study (Anjula Pandey)

9. Herbarium of the Agharkar Research Institute, Department of Botany, Pune, Maharashtra (AHMA)

10. Herbarium of the Department of Ecology, French Institute of Pondicherry, Puducherry, Tamil Nadu (HIFP)

11. National Herbarium of Crop Plants, National Bureau Plant Genetic Resources, New Delhi (NHCP) *Source: Singh (2010) with modification

Guidelines for consulting the NHCP

Annexure I Guidelines for consulting the NHCP Contact herbarium curator through request letter for visit to NHCP and intended taxa of study. This communication can be made through letter or e-mail ([email protected]; [email protected]). A tentative date for consultation should be indicated and visit be made only after approval. 1. On getting the approval for a consultation visit the herbarium, on arrival make an entry in the visitors’ register; fill a mandatory form (visitor’s information) and deposit the same with the curator. 2. Check for the species available in herbarium using computerized catalogue and /or index cards. 3. Consult species catalogue/databases and also digital herbarium of taxa. Later herbarium folder may be requested as desired. 4. Locate the block where family is placed (map); family folders have species arranged alphabetic order; genus folders (in six colours) have been used to categorize the specimens (Blue for wild and weedy specimens: Green for Cultivated types; Yellow for local Delhi collections; Brown for Exotic materials; Red for Type specimens; and Mustard brown for illustrations, photographs and photocopies); ask for specific folder of your interest. 5. After the consultation, ensure that the material is handed over to curator. ‘Determinavet slips’ are available in the herbarium for notes or confirmation of the identity of specimens checked or studied by the users. Put determinavet slip or notes if you are sure of any discrepancy in identification. Any additional input on the taxa may also be recorded in the space available with the specimen. 6. If interested in any photograph or material of interest put a requisition slip in provided format (with signed letter of undertaking). Information on the species or photographs can be supplied on request on a case-by-case basis after due approval by the authorities. 7. Suggestions on any aspects are most welcomed and remarks can be written in visitors register or sent by mail.

Guidelines for consulting the NHCP

Annexure II Guidelines for identification/ authentication/ issue of authentication certificate This is regarding the plant specimen brought by a visitor for identification/authentication or/ and issue of authentication certificate. The indentor has to consider the following points: 1. Send/submit the letter of request from authority with proposed study (thrust area) and detailed information on collector name, date of collection, source locality/ habitat, part used for study etc. (through a letter or e-mail ([email protected]). 2. Deposit completely dry/ processed plant material in the form of herbarium specimen (refer Jain and Rao 1977; compulsorily with flowers/fruit and other identifiable parts (product etc.) 3. Do not deposit any raw material/ or part of the material like leaf, bark, stem sample for the issue of authentication certificate. 4. Provide a hard copy of field view of plant population (if wild), accompanied by a close up photo (hard or soft copy) with the specimen. 5. Sign a copy of the undertaking before leaving. After the issue of a certificate, the material is not maintained for the future. Processing time 15-30 days in general (may extend in special case). 6. Provide at least two email addresses, for dispatch of the Authentication Certificate and for future correspondence. Annexure III Guidelines for deposition of standard herbarium samples NHCP Collection of plant material for the preparation of herbarium specimens  Herbarium specimens should be representative of the diversity observed. Therefore, unique and distinctive material from farmer’s field or of wild species (representing features noted in the field) may be made into herbarium specimens.  Twenty-twenty five cm long specimens are recommended for drying, especially immediately after collection. Representation of all parts especially flowers, or fruits or both is recommended.  Collection of at least three specimens from a locality is recommended. In the situation where only one or two plants are available in the wild, only a twig need be collected but with flowers or fruits represented, (in highly variable material, e.g. introgressed material mass collection. i.e. collection of twigs from large no. of plants may be done for better representation of the population variability).

Guidelines for consulting the NHCP

 The plant must be labelled immediately after collection. Price tags are readily available and easy to use for this purpose.  Locality, date of collection and field notes should be clearly recorded for each herbarium specimen. Plant material can vary depending on the season, climate, and habitat conditions. The reference no. of the seed sample collection from the same area should also be clearly recorded for each. Economic products such as fruits, dried flowers fibres, gums etc. may also be collected and represented.  Characters of the plant which are lost on drying, or which may not be represented in the herbarium specimen (height of plant for trees or shrubs), flower colour, leaflets (which may be shed on drying) should be noted on the herbarium record sheet. Drying of plant material  When drying, the overlap of plant parts should be avoided (pieces of blotting paper may be placed between these parts). One of two nodes with branches, 2-3 leaves (showing upper or lower surface), inflorescence, buds and flowers, and developing fruits should be available on a specimen. Excess parts may be trimmed off.  Sheets should be changed every day in wet conditions or every alternate day in dry conditions (in the shade) for a week or 10 days till completely dry.  The drying process may be hastened by placing specimen bundles under 100 Watt bulb or near a room heater (not more than 45oC) for a few minutes (less than 5-10 minutes). Dispatch of dried specimens  Dried specimens may be made into bundles (of 30-35 specimens/bundle) within a herbarium press in between thick sheets of cardboard, tied tightly once they are ready for transportation. In the event of specimens not being completely dry (even after 3-4 changes), extra sheets may be placed between specimens till they are brought to the destination.  Fruits, seed samples and economic products, which may often be large and bulky, could be dried or preserved separately and sent.  Specimens should be thoroughly dry, free of infection and infestation, well-labeled with complete collection details, and notes on plant, locality and habitat features. The material should preferably be brought personally (and not dispatched through post/courier) to ensure proper handling.  List of specimens along with all details should be provided along with the material.

Guidelines for consulting the NHCP

 Only specimens fulfilling all these criteria would be included in the herbarium. These will be given a herbarium specimen number, which shall be communicated to the collector after processing, indexing and inclusion at the NHCP.  It may not always be possible to collect material suitable for the herbarium during a seed or germplasm collection trip, as by the time, the plants may already be dry. In such cases, it is recommended that herbarium specimens be prepared from plants raised in the experimental area/ net house, during preliminary evaluation. Annexure IV Guidelines for preparation and deposition of herbarium samples of difficult-to-process Diversity in cultivars: Since the cultivated plants represent the spectrum of diversity in the form of landraces, cultivars, etc. efforts should be made to represent the ideal specimens. Complementary material of cultivars, elite types, breeding lines should be represented in the form of diversity charts, folders etc. and cross-referred to the representative material (with a number). For example specimens of cultivars of cereals may be represented by few ideal specimens with variability in panicle depicted in the form of display material, mounted on herbarium boards. In maize since the plants are tall, cobs are bulky, representative specimens with portions of stilt roots with underground roots and other aerial parts could be included; material representing shanks, silk colour and kernel colour is to be displayed together, besides variability photographs to complement to main herbarium collection. Large specimens: pruning to be done to the required size; give turn to form V or N shape where folding is possible (e.g. grasses). Specimens with big leaves/ parts like Musa, papaya, leafy brassicas, Araceae and Arecaceae are accommodated in standard herbarium sheet after mounting. The selected specimen may be split into two or three parts and pressed separately but with the same number. Plants with large leaves can be represented by cutting the portion of leaf tip, base and striped from the middle. The spathe of banana and ligule of bamboos, the bark of trees are significant for identification and can be dried and put on the full sheet with hand-drawn outline on the label. Rhizomatous/tuberous/bulbous types: vegetatively propagated material/ bulky underground parts like Dioscorea, Amorphophallus, taros, etc. and those 3-dimensional parts that lose their identity on drying may be depicted by photographs/outlines. Small sections of the peels, transverse section of succulent tuber/root can accompany the

Guidelines for consulting the NHCP herbarium. In bulbous plants, Allium bulb with a coat, a portion of bulb cut open in longitudinal section, also the leaf if hollow/ flat can be noted, as on drying this character is difficult to note. In rhizomatous taxa, Curcuma, Zingiber, Canna and Xanthosoma, leaf with petiole, rhizome cut in section and inflorescence (if available) can be represented. Root material like carrot, radish, sugar beet, turnip, etc. can be represented as sections giving an outline of the root shape and peel mount of the surface. Aquatic plants: need to be collected along with water, transferred in muslin cloth and sheets (many folds) replace quickly to avoid any infection. Fleshy fruits- Trapa bispinosa, Euryale ferox, and others need special drying with heavy blotters put. If the plants are very large, specimens can be cut into parts and dried in the same way but extra care has to be taken to avoid any fungal infection. Fleshy material: mature fruit with a longitudinal slit, horizontal slit opening the cavity to expose seeds are helpful in identification as well as easy drying. A peel section with fruit surface showing the texture of fruit wall, any ornamentation, etc. can be included in the mature stage and dried separately with the same number and mounted with original material. Citrus fruits where the rind characters are very important for identification can be additionally photographed or drawn the outline of fruit is included. Flower as identification markers: Cucumis species can be identified at the flowering or early fruiting with characters of the ovary that can be represented with just open flower, and peel of fruit etc. can also be represented. Gamopetalous flowers to be opened wide by giving a slit midway. Different cultivars of okra/Abelmoschusesculentus are distinct in fruit morphology; representation of dried/ mature fruits viz. photographs could be the best option. Delicate, small flowers like those of Sesamumspp. can be pressed using tissue paper and handled carefully till they dry. Problem taxa: processing of certain taxa is difficult due to the problem of leaf fall during drying (e.g.) Citrus, many legumes, Moringa, etc. Specimens can be treated using microwave heat at the beginning of processing to avoid the formation of abscission layer and improve the quality of processed specimen (Fuller and Barber 1981).

Guidelines for consulting the NHCP

Annexure V Guidelines for visit to NHCP  Request for a visit to herbarium by individual/ or a group from an institution (s) is entertained. Based on the purpose and need of the visitor (s) the arrangements are made to put demonstrations/ display of material etc. The following points are discussed for this purpose:  Contact the herbarium curator through request letter for visit to NHCP and intended purpose of visit. This communication can be made through letter or e-mail with a tentative date and time with persons visiting (e-mail: [email protected]).  Date of arrival should be fixed only after the approval date of intended visit; can contact for the enquiry through mail/ fax/ phone.  To facilitate the contents of address during the visit, level of visitors (college, school, farmer, etc.) and a number of team members should be clearly spelt out.  On arrival first of all the group leader (associates) is (are) advised making an entry in the Visitors Register.  Fill the Feedback Proforma for any suggestion on the usefulness of the facility. Annexure VI Guidelines for participation in training/technical know-how on herbarium procedures  Training/technical know-how on herbarium procedures are imparted to different indentors only need-based. Therefore the NHCP does not provide any certificate for training attended or participation by the indentor. The details are as follows:  Contact the herbarium curator through request letter or personal request for getting training on herbarium procedures with intended interest in the subject (through a letter or e-mail- [email protected]).  Tentative date/time should be fixed before arrival so that approval is accorded for the purpose. On getting the approval for a consultation visit the herbarium.  On arrival, first of all, make an entry in the Visitors Register.  A formal training is imparted for trainees of the basic/ core course, school students, college students through visual media or demonstrations.

Guidelines for consulting the NHCP

 After the training fruitful suggestions or general improvement through filling the Feedback Performa (available with NHCP) are welcome. Annexure VII Proforma to be filled for taking photographs of herbarium specimens for study (NHCP) ICAR-NBPGR © Copyright Declaration Name and designation: ------Complete postal address: ------BRIEF DESCRIPTION OF HERBARIUM SPECIMENS TO BE PHOTOGRAPHED ------1. In consideration of the NHCP, ICAR-NBPGR granting me the right to take photographs of the herbarium specimens, I assign with full title guarantee to NHCP copyright © and publication rights in all photographic and electronic images that I make of the herbarium specimens. 2. Notwithstanding that I have assigned the copyright to ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi agrees that I may use the images but only for non-commercial, scientific and educational purposes. 3. I understand that should I wish to reproduce the images in any publication, I must obtain permission from Director, ICAR-NBPGR prior to the publication of any image. I will also acknowledge the source of the images with the words “by permission of the Director, ICAR-NBPGR. 4. I understand that I shall duly acknowledge the Director, ICAR- NBPGR in all published works resulting from images data. 5. I undertake to provide the NHCP, ICAR-NBPGR with copies of all photographs or electronic images in soft copy that I take off items belonging to the NHCP. 6. I agree to provide information of intended use of this/ these images and a copy of the eventually published work if required to the NHCP- NBPGR

Guidelines for consulting the NHCP

Signature ------(Indentor) Place ------Date ------TERMS AND CONDITIONS OF USE OF HERBARIUM IMAGES  The use of herbarium specimen images supplied by the NBPGR- NHCP is subject to the terms and conditions as given.  All images are the copyright of the Director, ICAR-NBPGR.  Images are supplied for non-commercial, scientific and educational use only.  Permission from the Director, ICAR-NBPGR is required for using these images in any publication brought out by the indenter.  Users must acknowledge the Director, ICAR-NBPGR in all published works resulting from images/data supplied by ICAR- NBPGR.

Methods for Management of Herbarium (Anjula Pandey et al.)

2 Methods for Management of Herbarium Anjula Pandey, K Pradheep and Rita Gupta Division of Plant Exploration and Germplasm Collection National Bureau of Plant Genetic Resources New Delhi-110 012, India Introduction

The management of herbarium involves activities linked to build-up, maintenance and use. The standard procedure for preparation of herbarium specimen are: 1) Collection and processing of specimen; 2) Taxonomic identification; 3) Indexing and documentation; 4) Housing/ preserving/inclusion in a herbarium, and 4) Dissemination of knowledge. Establishing a herbarium depends on aims and objectives as well as the target users. One has to ensure that the specimens included are quality material, correctly identified, safely preserved, indexed and accessible for efficient use. The herbarium specimens are prepared (using standard procedures) with unique characters (vegetative characters- roots, tubers, bulbs and rhizome, stipule, spine, spathe, bark, etc.; floral characters- flower/inflorescence and fruit characters) (refer Jain and Rao 1977; Rao and Sharma 1990). An ideal herbarium specimen should represent all plant parts and information on locality, habitat and other details on uses in the area of collection. Where plant parts are difficult to process using standard methods, for examples preparing herbarium specimens of cultivated plants with bulky parts, the procedures may need certain additional steps (Appendix I, II). There are two methods for processing of herbarium specimens: the “dry processing method” and “wet processing method”. The dry method involves pressing the plants in fields immediately using thick blotters/newspapers (this may be useful for plants that tend to wild quickly-cucurbits, climbers). Polythene bag or ‘Vasculum’ (metal collecting vessel) is also used to collect the material. The “wet processing method” is applied when the collector is unable to process the specimens immediately or the material is perishable. In this method, collected specimens are placed between sheets of newspaper and about 40-50 specimens are tied together. These bundles of specimens are then placed in large polythene bags with formaldehyde solution added. These bags are then sealed and stored until the collectors return to their base. The wet specimens are then carefully taken out and spread on fresh dry

Methods for Management of Herbarium (Anjula Pandey et al.) blotting sheets and dried (Saldanha and Nicolson 1976). If there is any probable delay in pressing of specimen immediately after collection a sealed container/ pot or airtight packs (Ziploc bags) containing 5-10% formaldehyde at the base are used. Plant specimens must be processed for drying right in the field (using field press) or at least on the same day (using heavy lab press). Method using standard drying procedure has advantages as plants made as herbarium specimens: 1) are easy to handle, refer and study; 2) sending/ exchange is simple; 3) drying and mounting is convenient and more economical; 4) space and money economical as compared to the bulky collection. But it is disadvantageous as: 1) characters are lost on drying; 2) representation of part population or few plants; and 3) needs regular maintenance for pest-free/dust-free storage. The representation herbarium specimens may vary depending on the type of material and purpose of collection. In contrast to the above standard herbarium, “Bulky herbarium” may be prepared as wet collection in 4 per cent formalin/ rectified spirit/formalin or special dried form/ cut section (ex. flower, fruit, bamboo culms, spathe, trunk of a tree) to represent characters difficult to preserve using standard drying procedures. It may be pickled, air-dried, naturally dried plants/ parts (woody flowers, cones, fruits, capsules); vegetatively propagated and tuberous forms need to be represented in the herbarium for teaching purpose. In some herbaria, they may be part of carpological samples/ economic botany collections. In case of bulky herbarium preservation method has advantages due to: 1) shape and other structural characters are retained for study; 2) represent the nearest possible features for study. But disadvantages are: 1) cumbersome and difficult to handle for identification or study; 2) soft material and fragile material lose colour after a long time; 3) routine maintenance such as refilling of a storage medium; and 4) more space consuming. Build-up of herbarium collections Major additions to the herbarium collections may depend on the activity of the affiliating institution. The flow chart (Fig.1) depicted here provides the steps followed from collection to inclusion of herbarium specimen at the NHCP. Ideally, specimen selected for making into herbarium should have both flowers and young fruits; therefore, the specimen should be taken during the late flowering stage.

Methods for Management of Herbarium (Anjula Pandey et al.)

Figure 1. Flow chart of herbarium processing: collection to inclusion Selection of material depends on the essential or desirable additional parts to be collected for identification. Additions to the herbarium are made through collections from fields, material raised in experimental gardens and voucher samples and plants raised from seed deposited by researchers (Fig. 2). If herbarium is associated with collage or research organization, contents may vary accordingly.

Methods for Management of Herbarium (Anjula Pandey et al.)

Figure 2. Voucher samples prepared and ready to mount

Collecting populations of plants representing a wider range from diverse ecologies is a routine method; however, biased sampling can be done to collect a selected type. Using a strong knife, a pair of pruning shears or secateurs, the plant is dugout to take the underground parts. A tag with collector number and date of collection is attached to individual plants while collecting. Latitude-longitude, soil, temperature, rainfall data should be recorded in the site of collection. Ideally about 20-25 cm length of plant/ parts may favourably fit in a herbarium sheet. Normally a minimum of two specimens of the same collection should be made; however, for the unique variant(s), 4-5 specimens from same plant/locality are desirable (describing as new taxa). For dry or wet method may be selected as per need. A field record book containing information on range of occurrence, plant features (tree canopy, leaf fall), soil and associated vegetation forms, data on herbarium label (altitude, latitude, longitude and slope/direction), soil and type of vegetation should be recorded. The specimen should consist of whole plants with roots, stem, leaves, flowers and if possible fruits (if immature, then split open). Where the plants are shrubs or trees, it is necessary to select a representative specimen to accommodate the area on the mounting sheet (bark sample for identification). In the case of dioecious plants try to collect both

Methods for Management of Herbarium (Anjula Pandey et al.)

Figure 3. Herbarium processing in field from collecting to drying: (top row) selecting the ideal material, putting price tags/labelling; (middle row) putting the collected material in polythene bag, pressing material between the blotters; (bottom row) pressing herbarium specimens in field press, using corrugated sheets in-between the blotters to provide support and aeration.

Methods for Management of Herbarium (Anjula Pandey et al.)

Figure 4. Mounting and including herbarium specimens: (top row) mounting specimen on board, disinfecting herbarium specimen using poisoning method; (middle row) deep-freezing under -20 degrees temperature, storing in herbarium cabinets; (bottom row) scanning for virtual herbarium; data documentation in the virtual herbarium. male and female plants/ branches. They should be accessioned with different herbarium number. When flowers and fruits are too large to be pressed with the leaves, they should be processed separately (leaf and inflorescence of banana) or it can be dried (especially sponge gourd,

Methods for Management of Herbarium (Anjula Pandey et al.) smooth gourd, okra, many fruits) stored as a bulky collection (in boxes). Extra specimens of the same collection can be made depending on the requirement; duplicate samples when collected may be used for exchange/studies. When plucked from the plant, the specimen should be arranged in such a way that all the characters are well represented and plant parts are well spread using blotters or newspaper sheets. The ‘plant press’ containing the bundle of specimens (20-30 cm height) is left tied using a strong belt/strap is again put in the press for 24-36 hours. A number of blotting sheets may depend on the type of material, for examples, the cereals generally dry easily while the vegetables especially the ones with fleshy material need repeated changes of blotters to avoid fungal growth during initial processing. Generally during wet season more sheets and use of artificial heat is desirable for good results. Care should be taken to avoid overlapping of plant parts; corrugated sheets are placed between the blotters and the specimen if the processing is done using artificial drying method or during the wet season. The whole bundle is tied using a strong belt/strap and placed in a plant press (Fig. 3-4).

Processing of cultivated plants, especially with fleshy parts/fruits (cucurbits, solanaceous fruits), those with large leaves/ flower buds (cauliflower, cabbage) and succulent leaves (spinach, portulaca, Malabar spinach), are difficult to press. There is a choice to represent them in the wet collection (4-10 per cent formalin) or in died form/ cut-sections of economic parts to represent characters. Drying methods Pressing and drying are the two simultaneous processes in the herbarium methodology and chiefly contribute to the quality of the herbarium specimen. During pressing (sweating period) maximum water loss takes place from plant due to transpiration. When first opened, all plant parts are neatly rearranged on fresh blotters. Care must be taken to remove the damaged or pest-infected/infested portion if any. Bulky parts, e.g., bulbs, corms, tubers and fleshy rhizomes may be reduced by slicing off in such a manner not to affect the exposed surface. Some leaves should be turned upside down in order to make visible both the surfaces in the final specimen. The process is repeated for change of sheets followed by rearranging the parts for about a week or until drying is complete (especially in succulent/ fleshy material). Used blotters may be recycled after drying (ensuring no infection). Extra blotters and corrugated sheets used between the specimens can enhance the drying process, especially

Methods for Management of Herbarium (Anjula Pandey et al.) during the humid or wet season. In case of any unintentional delay, wet preservation method is helpful. Pads of paper and absorbent cotton are placed around the bulky portions of thick specimens/parts to avoid shrinkage. The excess moisture is lost (naturally or under artificial heat or microwave) (Fuller and Barber 1981). Gradual increase of pressure aids in water expression from parts and prevents curling and crushing of plant tissues. Herbarium press for lab use or field use varies in their weight and purpose. During exploration trips, due to overweight or bulky material, carrying hard metal or wooden press is difficult to use. Instead of hardwood frames, lightwood frames may aid in the better pressing of specimens; one can also prepare his own press using thick cardboards or sun pack boards which can be can be tied together by straps/cord/bolts which. The specimen during the processing may change the colour, especially of leaf and flower. Rapid drying for 1-2 days not only helps to minimize this reversion but also aids in preventing blackening of leaves, control of pest and insect free. Artificial heat prevents mould infection due to high humidity. In an alternative method, specimens after 24 hours of placement in the press are rearranged and placed over a heat source (drying chambers, ovens, stoves, etc. (temperature adjusted to 46-50 degrees C). The corrugated sheets placed between the blotters and specimens facilitate passing of hot air. Time taken for drying varies with respect to the type of material, season and habitat of collection and this procedure may be modified based. The technique is standardized using micro-wave for succulents or fleshy material such as fleshy roots in vegetables, big-sized fruits as in cucurbits or large-leaved plants like aroids roots/ tuberous material for effective drying (Fuller and Barber 1981; Pandey et al. 2006).

Fully dried specimens are grouped into bundles (containing 25-30 specimens/bundle) within thick sheets of cardboard and tied tightly. Depending on the weather they are to be retained for build-up or dispatched elsewhere they can be labelled on the outside sheet to designate them for further action. For safe handling, the packed bundle for dispatch may be clearly labelled with the instruction “Handle With Care” during transport (Fig.5a).

Methods for Management of Herbarium (Anjula Pandey et al.)

Figure 5a. Fully dried specimens grouped into bundles for storage (left); Packaging of the packed bundle for transport (right) Besides the bulky parts like fruits, succulent roots, stem etc. should be sectioned in longitudinal and transverse sections. Sectioning flowers (longitudinally) and pressing them flat to exhibit the inner parts. They can be dried separately and while mounting of specimen linked on same mounting sheet. Preservation The collected specimens are poisoned immediately after collection or at the time of mounting using chemical treatment (alcohol is particularly done to avoid the microbial damage under high humidity conditions). Dipping/ spraying with a saturated solution of mercuric chloride in ethyl alcohol and processing the same for drying is generally practiced (Fig. 4). For poisoning/dipping specimens a brush may also be used. Pouring 10% formalin over specimens contained in the press and placed in an airtight polythene bag. Specimens housed in the cabinets over a period of time may need monitoring and fumigated when an infestation of pests is high. Volatile poisonous liquids like carbon disulphide, methyl bromide, carbon tetrachloride are also used. Specimens should remain in airtight condition for 3-4 days. These chemicals pose a health hazard and should be handled with great care. A practice of deep-freezing subjected to a period of 48-72 hours (at -20 degree C) has been found effective in the control of pests/pathogens during processing or under storage. This method is also used in new material received from outside. Regular dusting of the repellants like powdered naphthalene kept in small muslin bags and is found to be effective to control mild infection by the storage pests; storage spaces can be repeatedly swiped with rectified alcohol. A roster of a chart prepared for this routine poisoning and dusting helps in checking the damage. Some families like Solanaceae, Cucurbitaceae, Apiaceae, and Lamiaceae are more prone to infection than others and therefore need frequent

Methods for Management of Herbarium (Anjula Pandey et al.) infestation, whereas grass family, zingibers, gymnosperms etc. are good storers. Mounting of specimens For mounting completely dried, poisoned specimens on a standard-sized mounting sheets/boards (of 29x41 cm) glue, paste, narrow strips of glued linen, a needle and thread, etc. can be used (Fig. 5b). The glue or gum paste is usually applied using a brush. An alternative procedure is to spread the glue over a sheet of glass over which the lower side of the plant is placed before mounting on sheet. Stiff/bulky plant parts are usually tied on the mounting sheet using needle and thread and other parts as a leaf or softer stem can be glued. The herbarium label (11x6.5cm) containing information on plant name, family, local name, date of collection, place of collection, collector number, status (flowering/vegetative) should be pasted on the bottom right-hand corner with information typed or filled with permanent ink. A paper pouch with extra plant parts as buds, flowers, peels of tubers, etc. may be pasted on the bottom left-hand corner. Herbarium label gives basic information on the specimen when collected from its natural habitat, the location, date of collection, collector's name, identity etc. (III). Additional notes as collector's name and collection number, place and date of collection, and features of the plant not shown in the dried specimen as soil types, associated flora, distribution, etc. should be recorded in the field notebook. The mounting board with a pre-prepared label (typed/ handwritten) with the botanical name written with correct author citation is pasted with glue and fixed on the lower right-hand corner before the specimen. In the label, the latest accepted name should be filled. For mounting, three different procedures are quite popular. Gluing/pasting is best for flat leaves and evenly placed. It is not easy to remove but difficult to mount. An efficient and effective way is to coat the surface of a glass or copper plate with glue (using a paintbrush), place the specimen on the plate and tap gently with forceps. The specimen (bottom side) is then lifted using forceps, inspected to check even distribution of glue and placed on the sheet. Leaves in many plants may be pasted/ glued to resemble their original position. Bulky herbarium can be placed in separate cabinets with a number indicating its main herbarium sheet. The specimens after being strapped (with linen tape) and sewing with a heavy linen thread can be fixed on mounting sheet. Sewing may also be exclusively used for fixing heavy stems, overlapping leaves, rhizomes, and matted bases of grasses, large fruits, cones, heads, where the use of plastic or glue is impractical.

Methods for Management of Herbarium (Anjula Pandey et al.)

Figure 5b. Herbarium specimen (mounted and processed) Herbarium disinfectants may be used by dipping or painting with a brush with an alcoholic solution (2-4%) of mercuric chloride. Specimens so treated should always be clearly labeled. Part of the material used for the study if removed (microscopic study, sectional work) should be placed back in pouches for study by others. Any additional records like notes, observations can be drawn with pencil on the mounting sheet, or photograph etc can be put in an illustrative folder, filed at the end of family or genus section. Identification of the plant specimen Identification methods involve a study of the plant characters; careful examination and comparison of the characters vis-a-vis the description in the regional floras using family, genus and species keys, cross-matching with already available identified specimens. When no clues are available floras of the adjacent regions are consulted and reference is made to larger herbaria. Unidentified specimens can be sent to different institutions/ experts for identification. Specimens sent for identification must be: (a) completely dry, (b) free of infection and infestation, (c) well-labelled with complete collection details, and notes on plant, locality and habitat features. Else we may make a personal approach to take material for identification or sent by post (ensure proper postage handing) to the institution/ specialist. Herbarium specimens to be dispatched for study, donation, loan or for identification, needs proper packaging. The box used for this purpose should be nearly of the same size of the specimen size to avoid rattling while movement or

Methods for Management of Herbarium (Anjula Pandey et al.) transit. If the size is a little more, put extra paper pads to avoid damage due to movement. ‘Annotation’ or ‘determinavit (det.) slip’ (small paper slip fixed on the herbarium sheet) to indicate name changes/correct identity of a plant by person annotating should put on the herbarium sheet with his name and signature with date along with institutional affiliation. Indexing and documentation After mounting, labelling and identification, specimens are given a Herbarium Accession Number to organize collection (Fig. 6). Types specimens are housed separately with special care and are not handled routinely. A system of different coloured genus folders for different geographic regions is used to facilitate quick review of general distribution of a species. Duplicate collections are properly numbered and placed separately and used in case of exchange or otherwise. Due to changes in nomenclature, specimen might be placed in another location which can be noted by placing a dummy folder in the appropriate place. Bulky collection associated with herbarium may be refer to additions of special collections, viz., wood samples, cones, capsules or any shattering plant material (which can be stored in boxes with proper indexing). Illustrations, photographs, drawings, maps etc. may be mounted on sheets or placed on envelopes; reprints and additional material may be kept at the end of species/ genus. Any unidentified material may be kept at the last of each taxon section with label “Dubia” or “Unidentified”. Incoming material should be indexed as soon as received (Appendix III). In recent years, to facilitate fast access (and avoid mishandling of dried specimens), digital scans/ images are being made available for use (www.kew.org/data/herb_digitisation.html;http://www.efloras.org/object _page.aspx?object_id=60285&flora_id=2). For more information refer to Chapter 11. Storage Classification based on family wise, alphabetic order, or uses as crop- groups such as cereals, millets, oilseeds, pulses, ornamentals, etc. and then alphabetic listing of genus and species (within the genus) are simple methods of storage of herbarium suiting to non-taxonomists and agriculturists. If working in diverse plant groups, arrangement of specimens through alphabetic listing of families (genus, species, and forma) as followed in NHCP, may be helpful. Collection from different localities/ regions can be demarcated in separate files with distinct colour codes (Appendix III). Regardless of filing system used, specimen

Methods for Management of Herbarium (Anjula Pandey et al.) should be arranged in an order that they will match to the label pasted adjacent to herbarium case. ‘Type specimens’ should be kept in separate place from the main collections and should be treated with much care. For an established herbarium all the above activities are to be taken up in routine processes. Herbarium specimens in NHCP: methodology Success of any system is based on quality of material handled and maintained. Herbarium specimens in the NHCP are processed in routine way as discussed above. In special case microwave drying methodology has been standardized and found suitable for crop taxa and fleshy material. This method is fast, efficient, viable and eco-friendly. Diverse forms of material received, processed and represented here needs case by case treatment. Some suggested methods are discussed as given below: Diversity in cultivars: Spectrum of diversity in cultivars, to represent the ideal specimens complementary material of cultivars, elite types, breeding lines should be represented in form of diversity charts, folders etc. and crossed referred to the representative material (with herbarium identity number). For example specimens of cultivars of cereals may be represented by few ideal specimens with variability in panicle/heads depicted in form of display material which may be mounted or kept in the herbarium cabinets at the end. In maize since the plants are tall, cobs are bulky, representative specimens with portions of tilt roots with underground roots and other aerial parts could be included; material representing shanks, silk colour and kernel colour are to be displayed together, besides variability of photographs to complement with main herbarium collection. Different cultivars of okra are distinct in fruit morphology and can best be represented as dried mature fruits. Large specimens: pruning needs to be done to fold to the required size; give turn to form M, V or N shape. Specimens with big leaves/ parts like Musa, papaya, leafy brassicas, Araceae and Arecaceae are accommodated in standard herbarium sheet. Selected specimen may be split into two or three parts and pressed separately (put same number, e.g. NH232304a, b, c, d, etc.). Plants with large leaves can be represented by cutting the portion of leaf tip, base and striped from middle. Diagnostic plant parts- spathe of banana, ligule of bamboos, bark of trees, etc. are significant for identification and can be mounted either on full sheet with the main herbarium with free-outline drawn on label or on separated sheet (if size is big). Rhizomatous/tuberous/bulbous taxa: vegetatively propagated material/ bulky underground parts like potato, Dioscorea, Amorphophalus, taros etc. that lose their identity on drying may be

Methods for Management of Herbarium (Anjula Pandey et al.) depicted by photographs and outlines. Small sections of the peels, transverse section of succulent tuber/root can be dried and mounted on herbarium sheet. In bulbous plants, Allium with bulb coat, portion of bulb cut open in longitudinal section, also the leaf if hollow/ flat. In rhizomatous taxa, Curcuma, Zingiber, Canna, Xanthosoma, leaf with petiole, rhizome cut in section and inflorescence (if available) can be represented. Root material like carrot, radish, sugar beet, turnip etc can be represented as sections giving outline of the root shape and peel mount of the surface. Aquatic plants: need to be collected along with water, transfer in muslin cloth and sheets (many fold) replace quickly to avoid any infection. Special drying with heavy blotters for Trapa bispinosa, Euryale ferox, others can help quality processing. Artificial heat treatment for fast drying followed by repeated change (4-5 hrs) of heavy blotters (5-6 sheets in one plant) may yield best results. If the plants are very large specimens can be cut into parts and dried in the same way. Extra care has to be taken to avoid any fungal infection. Fleshy material: mature fruit with longitudinal slit, horizontal slit opening the cavity to expose seeds are helpful in identification. A peel section with fruit surface showing texture of fruit wall, any ornamentation, etc. can be included in the mature-young stage and dried separately with same number and mounted with original material. Citrus fruits has distinct fruit rind characters for identification and can be photographed or outline of fruit drawn on herbarium sheet. Cucurbit fruits have distinct characters of peel, fibres, etc. and dried portion of these can be pasted with herbarium specimen e.g. Luffa, Momordica, Cucumis (sativus/melo group). Identification markers: Vigna species can be identified at the flowering or early fruiting with characters of ovary that can be represented on herbarium sheet (with flower open, peel of fruit etc.). Flower can be split open longitudinally and pressed with corolla spread out show androecium and gynoecium; corolla to be pressed separately in tissue paper on tepals/ non absorbent tissue paper. Some of the fully mature flowers, buds can be separately dried and put in the pouch for later study (detaching plant part to be studied, soak in 5-1 per cent salt water in ambient temperature for 8-10 hours, spread on mounting glass and put under dissection microscope). Delicate and small flowers of species of Cucumis, Luffa, and Sesame can be pressed using tissue paper and handled carefully till they dry. Dried roots, bulbils, underground bulbils, flowers, scape, and capsule can be put in a pouch; diagramme of individual flower can be drawn on sheet. In tree taxa, characters of

Methods for Management of Herbarium (Anjula Pandey et al.) wood/bark are unique and need to be sampled for identification. Photographs could be the best option in all these. Problem taxa: processing of certain taxa is difficult due to the problem of leaf or floral parts fall during drying. Citrus, many legumes, moringa, etc. during drying can be treated using microwave heat in the begging of processing to avoid formation of abscission layer and improve the quality of processed specimen (Fuller and Barber 1981). Artificial sudden heat (electric iron/press) at 60 degree C or microwave drying followed by routine drying process can be applied. Specimens that tend to drop leaves due to abscission layer formation need boiling water treatment for one minute or more before pressing to kill the cells and prevent dropping of leaves. In NHCP, use of heavy weight iron (press) for this purpose drying specimens which are otherwise difficult to dry and leaf/ flower drop takes place on processing. Treating specimens with formaldehyde before drying or preserving in 5-10% formaldehyde or various formalin- alcohol solutions with small amount of glycerine, extra blotters, applying salting ensures quick drying of difficult-to-dry specimens, such as succulents, Basella, radish, etc. In NHCP we are preserving a subset of herbarium material untreated for biosystematics study. Methods for extracting DNA for molecular or biochemical study of herbarium material several are in practice (Rogers and Bendich 1985; Drábková et al. 2002). Methodology of plant herbarium voucher preparation without involvement of chemicals and harsh environmental conditions is available with best results (Seshagirirao et al. 2016). Herbarium preparation using the “Plant Specimen Preparation Kit” (https://www.oshibana.com/herbarium/en/index.php) can be used for study of analysis of chemical, biochemical and molecular biology (Fig. 6).

Figure 6. Plant specimen preparation kit” (https://www.oshibana.com/herbarium/en/index.php)

Methods for Management of Herbarium (Anjula Pandey et al.)

Protocols on processing of cultivated plants need to be routinely worked out. Material representation in physical form is essentially needed for PGR and teaching programmes. For this efforts are in progress for developing eco-friendly methods with low budgetary requirement and labour cost. References/selected readings Davis PH and VH Heywood (1963) Principles in Angiosperm Taxonomy. University of Edinburgh Press, Great Britain. Drábková L, J Kirschner and C Vlcek (2002) Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of Juncaceae. Plant Mol Biol Rep 20:161-175. Fuller TC and Barber GD (1981) A Micro-Wave Oven Method for Drying Succulent Plant Specimens. Taxon 30: 867. Jain SK and RR Rao (1977) A Handbook of Field and Herbarium Methods. Today and Tomorrow Printers and Publishers, New Delhi. Pandey Anjula, ER Nayar and Rita Gupta (2006) An Efficient Methodology for Processing of Herbarium Specimens of Cultivated Plants. Indian J Plant Genet Resour 19(1): 47-49. Pandey A, ER Nayar, K Pradheep and R Gupta (2013) Preparation of Herbarium Specimens of Cultivated Plants. In: Jacob et al. (eds.) Training Manual on Management of Plant Genetic Resources (eds. Jacob et al.) National Bureau of Plant Genetic Resources, New Delhi, India, pp14-20. Rao RR and BD Sharma (1990) A Manual for Herbarium Collections. Botanical Survey of India, Calcutta. Rogers SO and AJ Bendich (1985) Extraction of DNA from Milligram Amounts of Fresh, Herbarium and Mummified Plant Tissues. Plant Mole Biol 5: 69-76. Saldanha CJ and DH Nicolson (1976) Flora of Hasan District, Karnataka, India. Amerind Publishing Co. Pvt. Ltd., New Delhi. Seshagirirao K, L Harikrishnanaik, K Venumadhav, B Nanibabu, K Jamir, BK Ratnamma, R Jena and DK Babarao (2016) Preparation of Herbarium Specimen for Plant Identification and Voucher Number. Roxburghia 6(1-4):111-119.

Methods for Management of Herbarium (Anjula Pandey et al.)

Appendix I

Notes on Herbarium Preparation and Field Observation (Bulbous Group) Herbarium preparation  Habitat/ecological aspects: record habitat details using standard IPGRI passport (http://www.ecpgr.cgiar.org/fileadmin/bioversity/publications/pdfs/728_D escriptors_for_Allium__Allium_spp._.pdf) with niche-specific information, distinct habitat (locality), altitude, soil type, moisture, topography (slope condition (steep/ shallow; rocky, crevices, cliffs), exposure/ sun exposure) and if endemic.  Life form: perennials/ annuals, perennating organs- bulb/rhizome, leaf, scape visible at the time of visit.  Texture and colour of plant parts: flower colour (use colour chart), glaucous, texture, shininess; outer coat if membranous, coriaceous or fibrous, if fibrous loosely or finely knitted, colour of coat on harvest, roots fleshy, leaf thick, thin, erect or pendent, bulb shape, size colour coat; inflorescence as seen fresh (flower opening pattern, spathe valves in fresh; compact/ spreading, shape seen as 3-d in fresh but 2-d in press are lost in drying process).  Stage at which observations recorded (bulb and leaf): prepare herbarium specimen of vegetative/ reproductive plant over various growth stages. Determine the standard stage for taxa at which the collection is to be done. Bulbils if present should be represented with mother plant.  Flower character: pattern of opening of flowers, perianth orientation- horizontal or erect, filaments longer/ shorter than perianth, striations on the dorsal side of perianth, colour and length of stamen, etc. to be noted in fresh. Opened flower can be dried with care and mounted in cotton strip/ kept in pouch. Field/hand held microscope can be used for micro- characters.  Phenology: early/ late flowering, length of flowering time, pollinator specificity if any.  Extreme variants: variants with respect to good taxonomic characters within population (field and herbarium based study); species with high ecological amplitude (A. carolinianum).  Illustrations: drawings/cross sections of parts, leaves/ scape (hollow or solid), sketches/ line diagrammes to show details  Supplementary information: photographs of field/ population; close up of the leaf, bulb, flower, anthers; etymology, local use, ethnobotanical information/ local name etc. may help in identification.

Methods for Management of Herbarium (Anjula Pandey et al.)

 Equipments: humidity recording equipment, relative humidity meter, soil Ph meter, GIS (location data recording); camera, telescope, field lens, herbarium kits, seed packets. Field observation  Tall plant: to be bent into parts/small parts: cultivated (e.g. Allium ampeloprasum, A. cepa) groups with bigger upper, lower leaf surfaces, bending long scape; basal, middle and tip of leaf portion (in case of leaf).  Bulb: bulb membrane exposing outer and inner coat characters; bulb in full or section (poured with salt/ disinfectant on the cut), vertical section to show arrangement of basal leaves; membrane from completely dry or mature bulb can be preserved during processing of herbarium specimen; collect and dry it separately in pouch.  Leaf: cross-section, leaf insert (cut/ oblique) with coloured stick/strip to show hollowness; longitudinal section of leaf from bottom to tip length-wise; scape; trim some leaves (only in dense condition) leaving only 5-6 cm basal part to show density/ arrangement.  Pseudostem: length (size), arrangement of leaves; one should not try to alter orientation/arrangement of leaf.  Inflorescence: take measurements of diameter and draw line diagrammes to depict the shape; spathe splitting, pattern of flower opening (centripetal/ centrifugal vs. irregular) (depicted pictorially or with photograph), compact vs. loose arrangement can be shown with line diagram; data to be recorded in label or notes.  Flower: colour of flower, perianth shape, perianth orientation in fully wide open flower (lost while drying); anthers exerted or included, stamen colour, orientation of anthers, micro-characteristics of stamen (base of filament, toothed or not). in a small piece of blotter after opening/ slitting it wide to show all parts.  Illustration: prepare on spot/ free hand drawing to show the unique part (anther, fibrous nature of membrane; photos in close view from the top of the flower, longitudinal view of the inflorescences to show anther viz. tepals ratio (anthers included or peeping out).  Treatments: apply artificial heat, alcohol or formalin to kill the tissue- bulb/rhizome to kill the tissue. Fleshy parts can be dried slowly through blotters/ corrugated sheets; use dry salt powder facilitates fast drying and no fungal/ microbial growth on tissue.

Methods for Management of Herbarium (Anjula Pandey et al.)

Appendix II

Some Dos’ and Don’ts’ for NHCP users

Dos  Read the herbarium guidelines before the visit to a herbarium; define your purpose of visit.  Use only the visitors’ area designated for study in NHCP; observe silence while working  Enter your name and address in visitors’ book before starting the work  Handle the specimen with utmost care as they are the most precious vouchers for genetic resource study  Immediately inform the curator on finding any discrepancy/ damage or misplacement of a specimen  Put the ‘Determinavet Slip’, if sure of the wrong identity of herbarium specimen.  Handle the specimens after wearing mask and gloves especially while working with treated material; wash hands carefully after use  Close the storage chambers/ compactors immediately after use  Ensure to have emergency numbers/ contact numbers while working inside the herbarium; contact herbarium staff during emergency  Keep your valuables in safe custody while working Don’ts  Do not entre herbarium without permission of the curator  Do not bring any raw material/wet material/ live plants/ other sources into the herbarium  Do not bring any eatables/ food material inside the herbarium  Do not bring any fire/ hazardous material inside the herbarium  Do not removed any specimen or part of the specimen during study; if broken keep it in pouch and inform the curator  Do not bring your own herbarium specimen during visit or consultation of a herbarium; if you bring inform the curator and get it recorded.  Do not stack herbarium folders one on the other or upside down  Do not take photographs without the permission from the authorities  Do not try to place back the specimens back; it may tend to be in wrong place without consultation with herbarium staff

Methods for Management of Herbarium (Anjula Pandey et al.)

Appendix III

Herbarium Record, NHCP, NBPGR, New Delhi Botanical Name Family Local Name Loc. (Place, Town, Dist. State) Date of Collection Collector’s Name and No. Field Collection/Grown in experimental Conditions: Identified by Nature of Specimen (Adult Plant /Seedling, Vegetative/Flowering/Fruiting etc.) No. of Specimens Additional material supplied (Seed, fruit, Economic Product) users, if any, in locality of collection Notes Herb. Specimen No.: 1 2 3 4 (copy enclosed) Index Card

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.)

Taxonomic Literature: Role in Plant Systematics Study

Anjula Pandey and K Pradheep Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources, New Delhi-110 012, India Introduction Plant taxonomic literature is one of the oldest and comprehensive pieces of literature of science published and documented. It appears in various forms such as flora, monographs, books, illustrations, indexes, and bibliographic references, guides, journals and other supportive documents. Taxonomic literature is written in leading languages such as English, German and Russian, though original descriptions are quite often written in Latin. Information about the newly described plants, their names and classification are continuously published in research journals, books and monographs. Taxonomic knowledge appears in descriptive and diagnostic form can be attained from taxonomic literature and related bibliographic aids which are the essential instruments for scientific identification, nomenclature and classification of plants. This chapter is to illustrate the use of taxonomic literature in locating the relevant scientific information on a taxonomic group (Appendix I, II). Types of taxonomic literature Comprehensive taxonomic works in the form of floras, monographs, revisions, descriptions, illustrations and identification keys are useful for proper identification of unknown plants. Some of the historical works on literature in taxonomy are the works of Theophrastus, Pliny, Dioscorides, Albertus Magnus, Brunfels, Cesalpino, the Bauhins, Ray, Tournefort and Carolus Linnaeus, who is regarded as the father of taxonomy. Indexes to plant names Index of plant names is an alphabetical listing of taxa with reference to their publication. It provides references to the original publication of names and the placement of a particular plant in a classification system to quickly locate the source of original publication of a name, to learn if

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.) a particular name has been applied to a plant or to what order, family, subfamily or tribe, a plant of a given name may belong. Details on some indices are given (Table 1) below: Table 1. Some information on important indices/lists

Index Contents and other details Authority/linkage (abbreviated form)

Index Generic and binomial names of seed Royal Botanic Kewensis (IK) plants of the whole world given in an Gardens, Kew alphabetical order.

Gray Index (GI) All new names/new combinations of Gray Herbarium, names of vascular plants from the Harvard University New World (America)

Australian All information on scientific plant Centre for Australian Plant Names names of Australia, author, original National Biodiversity Index (APNI) publication details (protologue), Research scientific literature, typification details, distribution (in Australia); cultivars derived from the Australian flora

Index Filicum The original publication of generic and Williams & Norgate, species names of ferns (included in London the Index Kewensis)

Index Vascular Plants (1753-1935) used in Royal Botanic Londinensis Index Londinensis (with the Gardens, Kew illustrations)

Index Generic names for plants covered in International Nominum International Code of Botanical Association for Plant Genericorum Nomenclature Taxonomy (IAPT) and (ING) the Smithsonian Institution

The Plant List Provides theAccepted Latin Name for Royal Botanic plant species, with links to Gardens, Kew and allSynonyms known for the taxon; Missouri Botanical combining multiple checklist data sets Garden

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.)

The A database of the names and The Royal Botanic International associated basic bibliographical Gardens, Kew,The Plant Names details of seed plants, ferns and Harvard University Index (IPNI) lycophytes with the goal to eliminate Herbaria, and repeated reference to primary sources theAustralian for basic bibliographic information National Herbarium about plant names

Other publications: This group of publications include, Biological Abstracts (a bi-weekly publication listing abstracts of papers appearing in scientific journals); Regnum Vegetabile (selective guide to older bibliographical details of botanical literature for type material, priority of names, dates of publication); Kew Records of Taxonomic Literature (Royal Botanic Gardens, Kew, listing all taxonomic literature published in periodicals, books and papers); Index to American Botanical Literature (Consortium for Educational Communication Bulletin Torrey Botanical Club listings arranged alphabetically by author under subject groups); Index to Author of Plant Names (of the authors of plant scientific names of angiosperms, gymnosperms, pteridophytes, bryophytes, algae, fungi and fossil plants); Index to Plant Chromosome Numbers (IPCN- original plant chromosome numbers of naturally occurring and cultivated plants); and Index Holmensis (plant distribution maps). Out of these only selected one as discussed below: Index Herbariorum (IH): IH is a directory of major herbaria of the world and associated staff. For any herbarium to make entry into the IH, it must have a physical location, web address, contents (e.g., total number specimens including types), history, names, contact information and areas of expertise of the associated staff. For new registration permanent in the scientific repositories usually minimum of 5,000 specimens are desired. Each institution is assigned a permanent unique identifier number with a single-digit code or four-eight letter code, a practice that dates from the founding of IH in 1935. For example, for the herbarium located in the ICAR-National Bureau of Plant Genetic Resources, New Delhi, the National Herbarium of Crop Plants the code assigned by IH is ‘NHCP’. There are over 3,000 active herbaria in the world, containing 387,007,790 specimens located in 176 countries (Thiers 2017;sweetgum.nybg.org/science/ih/). Floras, monographs and revisions Flora is a systematic arrangement of the species of a given area or a particular region, usually restricted to a major segment of the plant kingdom (flowering plants etc.), with keys and descriptions and often

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.) illustrations. The uses may determine the names and characteristics of the wild plants of the area using these treaties. Based on the coverage, a flora may pertain to a country, a section of a country, a state, a valley, a desert, or a vicinity of a city. A taxonomic monograph is a treatise including all significant information based on the analysis and synthesis of existing taxonomic knowledge of that taxon of the group (family, tribe, or genus). Taxonomic revision differs from the monograph in its degree of scope and contents. It may often account for whole family or genus, or a section of a genus representing a particular geographical region/area. In order to make revisions, comprehensive works from already attempted areas need to be studied in detail (refer Chapter 8). Other supporting literature The above referred taxonomic treaties cover the wider scope and have technical contents and take longer time to be completed. For other supporting sources of taxonomic literature, published in short duration are discussed below: Journals: the scientific journals (hard copy version and e-version) dealing with the systematic research are published periodically; they are highly specialized documents updating progress on new fields relating to taxonomic and systematic research, plants identification, new aspects studied. The research papers on taxonomy can be on the extended distribution of a taxon, new record of a species, notes on new findings, nomenclatural changes, etc. Field inventory: field inventory is also called the ‘botanical inventory’. The field inventory list the species occurring in an area. Field inventories need updating by repeatedly adding information and composition of plant species varying with growing seasons. The inventories are developed for a specific goal, for example, field inventories on: (i) floristic diversity; (ii) crop plant taxa; (iii) wild relatives of crops; and iv) weedy species of a region. The most common types of field inventories in use are simple and advanced inventories. Simple inventories (for routine use) may be prepared based on familiarity with the area as well as the plant species available. Simple inventory can be prepared to place an unidentified species in a larger taxonomic group (family or genus) and then use taxonomic keys and laboratory equipment to complete the identification (Fig.1). Less familiar species may either be new to the area or may occur in a different season and have been overlooked during the earlier study. In contrast to the simple inventory, the advanced inventories provide comprehensive

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.) information on the mapping of plant diversity, location data and additional details pertaining to the botanical study of an area. Field record books, data books, field guides, and modern equipment (polycorders for recording tree data, GIS- Global Information System, GPS-Global Positioning Systems) help in attaining precision.

Figure 1. Inventorization of crop plants: selecting material for study in farmer’s field; recording of information on plant use (photo: Drs DP Semwal and KC Bhatt, ICAR- NBPGR) Catalogues: catalogues account for the books of special libraries rich in botanical titles, and are of special value in taxonomic studies. It is necessary to include information on the full name of a particular author, the unabridged and exact title of a work, date of publication/edition issued. Periodicals: periodicals appear at regular intervals (biweekly, monthly, or quarterly) and a volume usually comprises the issues of a calendar year. An abstract with a brief factual summary of a paper, frequently prepared by its author, whereas a review is an often critical appraisal and evaluation of the paper, and is by a person other than the original author. Collectively these numbers or fascicles comprise a volume. Scientific periodicals usually are sponsored either by a scientific organization, learned society, or an educational or non-profit research institution, viz. a university or museum. Dictionaries and glossaries: A botanical dictionary may list and describe all known genera of certain plant groups e.g. A Dictionary of Flowering, Plants and Ferns (by JC Willis); Plant-Book: A Portable Dictionary of Plants, their Classifications and Uses (by DJ Mabberley), etc. A glossary is an alphabetical list of difficult terms with their interpretations. Almost all modern manuals and many floras include a glossary of the botanical terms. Most botanical dictionaries are of plant

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.) names and are sources for the etymology of Latin or vernacular names, for biographical data of persons for whom plants have been named, and for vernacular names in various languages. Legislation: Since 1867 botanists have met somewhat regularly at the international level and agreed upon legislation in plant names. These rules are subjected to revision at each Botanical Congress and a new edition prepared. International Code of Nomenclature for Algae, Fungi, and Plants (ICN), which in July 2011 replaced the International Code of Botanical Nomenclature (ICBN) and the earlier International Rules of Botanical Nomenclature. Illustrations/icons: these are compiled separately and serve as a useful tool for plant identification. They are graphical details of the plant/parts with the text in the Floras; they illustrate distinctive characters of the natural plants are the ready reckoner of plant species of that area or region or part or state or country. Examples: Hooker’s and Wight’s Icones Others: besides, some significant publications-Prodromus Systematis Naturalis Regni Vegetabilis, Genera Plantarum, Genera Filicum, Die Naturlichen Pflanzenfamilien, Genera of Flowering Plants, Key to the Families of the Flowering Plants of the World and System et Phylogenia Magnoliophytorum are also of great value to our understanding in taxonomic literature. Conclusions Centuries-old taxonomic data can be used to reconstruct the past climate history which can throw light on various issues as climate change. Different taxonomic literature -indices, floras, monographs, journals and keys, dictionaries, guides and illustrations serve as handy identification tools during the field survey and collection trips. References/ selected readings Bentham G and JD Hooker (1862-1883) Genera Plantarum. 3 volumes, London, UK. Davis PH and VH Heywood (1963) Principles in Angiosperm Taxonomy. University of Edinburgh Press, Great Britain. de Candolle A and de Candolle C (1879-91) Monographiae Phanerogamarum. 7 Volumes, Paris, France. de Candolle A and C de Candolle (1824-73) Prodromus Systematis Naturalist Regni Vegetabilis. 17 volumes, Paris, France. Engler A and K Prantl (1887-1960) Die Naturlichen Pflanzenfamilien. 23 volumes, Leipzig, Germany.

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.)

Englier A and L Diels (1936) Syllabus der Pflanzenfamilien, 11th ed. berlin, Germany. Engler A (1900-1937) Das Pflanzenreich. regni Vegetablis Conspectus. Lepzig, Germany. Lawrence GHS (1951) Taxonomy of Flowering Plants. MacPublishing Co. Linnaeus C (1753) Species Planturum, 2 vols. Mabberley DJ (2008) Mabberley's Plant-Book: A Portable Dictionary of Plants, their Classifications and Uses (3rd edition) Cambridge University Press, Cambridge. Thiers BM (ed) (2017) The World’s Herbaria: A Summary Report Based on Data from Index Herbariorum. Index Herbariorum 2.1 (published January 5, 2018). Appendix I Nomenclature/taxonomy databases Grin Taxonomy www.ars-grin.gov/cgi-bin/npgs/html/index.pl): Includes accepted names, distribution, economic importance and bibliography for each plant. Searchable by scientific and common name. International Code of Nomenclature for algae, fungi, and plants (http://www.iapt-taxon.org/nomen/main.php): An electronic version of the code adopted by the Eighteenth International Botanical Congress Melbourne, Australia, July 2011. International Plant Names Index (www.ipni.org): A searchable database of names included in the Index Kewensis (IK), the Gray Card Index (GCI) and the Australian Plant Names Index (APNI). Includes names that have been published to date, but is not a source for verifying currently accepted names. ITIS - Integrated Taxonomic Information System www.itis.gov: Taxonomic database created through a partnership of U.S. federal and international agencies. Entries include authority, taxonomic rank, synonyms and common names, taxonomic serial number, and data source. It is one of the sources for verifying currently accepted names but not complete like “The Plant List”. The Plant List http://www.theplantlist.org: The Plant List provides theAccepted Latin name for most plant species, with links to allSynonymsby which that species has been known. Tropicos

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.) http://www.tropicos.org: Missouri Botanical Garden's VAST (VAScular Tropicos) nomenclature database and authority files. Searchable by scientific name. Source for currently accepted names for selected vascular plants. Appendix II

Additional links, source of information/literature for identification and use of herbarium resources Weblinks  http://apps.kew.org/wcsp/prepareChecklist.do?checklist = selected_families%40%40204220720081409914(for synonyms, distribution)  http://www.kew.org/data/grasses-syn.html(for grass synonymy)  http://www.iucnredlist.org/(for threatened plants)  http://www.efloras.org/(Pakistan, China, Nepal, North America)  http://www.ildis.org(for legumes)  http://envfor.nic.in/bsi/research.html(for threatened plants of India)  http://mobot.mobot.org/W3T/Search/ipcn.html(for chromosome numbers)  http://www.unep-wcmc.org/(for threatened plants)  Flora of India (recent initiative)http://efloraindia.nic.in/efloraindia/homePage.action Virtual herbarium  JSTOR Global Plants  Kew herbarium catalogue (K)  Edinburgh herbarium catalogue (E)  Paris herbarium (P)  Chinese Virtual Herbarium (PE)  Harvard Herbarium (GH)  Linnean specimens typification project  IPK Gatersleben (GAT)- also botanical images Indian online herbaria  JCB, Bengaluru  IIIM, Jammu  RPRC, Bhubaneswar  KFRI, Thrissur  NBRI, Lucknow  HIFT, Puducherry Protologue/old literature  Biodiversity Heritage Library (its prototype was originally developed at the MOBOT as Botanicus) – 2005- ten major institutes have collaborated in bringing this to present form  Botanico-Periodicum-Huntianum (worldwide bibliography of periodicals published between 1665 and 1966 that included any botanical content

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.)

and covered more than 12,000 titles published in more than 45 languages)  JSTOR by Ithaka Harbors, Inc. Journals relating to plant taxonomy and systematics  Bangladesh Journal of Botany  Blumea  Botanical Journal of The Linnean Society  Botanical Review  Botanical Studies (Botanical Bulletin of Academia Sinica)  Brittonia  Genetic Resources and Crop Evolution  Journal of Threatened Taxa  Journal of the Torrey Botanical Society  Nordic Journal of Botany  Novon  Pakistan Journal of Botany  Silvae Genetica  Taxon  The Indian Forester  Annals of Missouri Botanical Garden  Rheedea  Bulletin of Botanical Survey of India (Nelumbo)  Phytomorphology  Journal of Economic and Taxonomic Botany  NEBios  Kew Bulletin  Garden’s Bulletin (Singapore) Online journal and the links  American Journal of Botany(http://intl.amjbot.org) (http://www.amjbot.org/)  Annals of the Missouri Botanical Garden(http://www.botanicus.org/bibliography/b12973130)  Kew Bulletin (http://link.springer.com/journal/12225)  Plant Systematics and Evolution(http://link.springer.com/journal/606)  Systematic Botany (http://www.sysbot.org/)(http://www.bioone.org/loi/sbot);  Taxon (http://www.botanik.univie.ac.at/iapt/)  Rheedea (http://www.iaat.org.in/#)  PhytoKeys (http://phytokeys.pensoft.net/). Databases  Solanaceae Source  Gymnosperm Database

Taxonomic Literature: Role in Plant Systematics Study (Anjula Pandey et al.)

 Dipterocarpaceae Database  Gesneriaceae Database  Convolvulaceae Unlimited  BrassiBase  PALMweb  Grassbase-Kew  Index Herbariorum (http://sweetgum.nybg.org/science/ih/)  PGR Abstracts  ANGIOSPERM PHYLOGENY WEBSITE, version 13.  IAPT-APG

The Relevance of Field and Ecological Studies in Herbarium (E. Roshini Nayar)

The Relevance of Field and Ecological Studies in Herbarium E. Roshini Nayar Former Principal Scientist and Scientist Emeritus (ICAR) ICAR-National Bureau of Plant Genetic Resources, New Delhi 110012, India Introduction The herbarium is a repository of information- on morphology from the specimen mounted on the sheet, and data noted on the herbarium label from the collector’s passport data book; on the other hand, herbarium resources are a tool in both the representation of diversity and the study of species diversity. Over time, methods and measures have been standardized for ensuring representation of patterns and processes observed in the field as herbarium specimens; and subsequently, using these herbarium resources, collected over time and space, identified and classified, for present and future field survey, the study of diversity patterns and changes therein. Value of well-prepared and well-documented herbarium holdings from its natural habitats and also substantiated by further experimental study of change, adaptation to new habitats and realizing its evolutionary potential has been enumerated (Funk 2003). Any study, whether morphological, biosystematics or molecular profiling, when accompanied by a parallel representation of sample specimens (‘Vouchers’) in a herbarium, makes it a record that can be referred to by later workers, thereby increasing its authenticity. Descriptions of plant species based on observed morphological characters during collection and available in floras and monographs, notes on variation and variants that are a clue to identifying new variants/ species, biometric analysis of population characters, etc. are some of the valuable data better understanding the morphological diversity and ecological processes affecting the species diversity, and future field studies. Whereas the herbarium is part and parcel of the study of botany, it is a significant resource in plant genetic resources programmes. While describing plants for PGR studies the glossary of terms commonly used terms- taxon, genus, subgenus, race, PGR, collecting, taxonomy, biosystematics, crop, wild, weed, genepool, etc.

The Relevance of Field and Ecological Studies in Herbarium (E. Roshini Nayar) may be used appropriately so as to convey proper understanding (ICAR- NBPGR 2016; Nayar et al. 2003). The role of the herbarium in PGR, methods of build-up, management, and its basic use are being considered in various other lectures. The contents of this chapter are therefore confined to the specific significance of the field and ecological study on the build-up of herbarium and vice-versa using specific examples. Examples have been drawn of taxa of PGR importance, either of the Indian region and that has been the subject of taxonomic and systematic investigation, or those of more widespread species but of relevance in introduction and exchange of PGR. Some concepts dealt with in this presentation are as follows: a) in relation to field study- description of a species, notes associated with descriptions in a floristic work, as a source of information on new taxa, occurrence of inter-specific/ inter-generic hybrids, search for missing species also referred to as the ‘missing link’ between ‘domesticated’ and ‘wild species’, observations of development and plant maturation relevant to conservation; and b) related to ecological study- population characters of species vis-à-vis its ecology, sympatric occurrence of species, ecological study of habitat conditions associated with high variation, habitats and areas of record of hybridization, ecotypic forms of species, adaptation and evolution of species. Some examples of specific crop taxa A) Vigna (Family Leguminosae, Tribe Phaseolineae, Subtribe Phaseoleae): Over 200 species; a complex of related genera- Phaseolus-Vigna and others (Marechal, Mascherpa and Stainier 1978). Biogeographically, Phaseolus belongs to the New World, and Vigna belongs to the Old World. Phaseolus well investigated through field study, eco-morphological survey, substantiated by molecular studies, and phylogenetic analysis. Close parallels in ecological and evolutionary patterns in Phaseolus and Vigna. Genus Vigna too consists of various biogeographically differentiated subgenera; the Asian sub-genus is subgenus Ceratotropis. Vigna subgenus Ceratotropis: specialized group with uniform characters of habit, leaf, stipule, flower, pod and seed; over 16 wild species in the Indian region, and with more survey, collection and study more species described; closely similar and with minor distinguishing characters of size and shape of stipule, leaf, flower, pod and seed, hairiness of parts, position and shape of pods. Domesticated species- South Asia,

The Relevance of Field and Ecological Studies in Herbarium (E. Roshini Nayar)

Southeast Asia and Far East area, the centres of origin of cultivated and wild species; mung bean has maximum widespread cultivation with areas of diversity identified in South Asia and West Asia (Sangsiri et al. 2007). Examples of the field- herbarium study- ecological parameters: 1. Description of species with emphasis on characters such as undulations of a leaf, the relative softness of hairs, clearly noted sizes of flower and seed, help to correctly identify the species. 2. Minor variations among species of the Indian region, and areas of occurrence of newly described species. 3. Intermediate forms among major species pairs. B) Cajanus-Atylosia (Family Leguminosae): Over 35 species of tropical and subtropical regions; over 50 per cent of species in the Asian region (Indian, Chinese and Indo-Chinese regions, 18) and a secondary build-up of species (14) in the Australian region; habit ranging from erect forms to shrubby and creeping forms. Cajanus cajan (Bengal gram, pigeon pea), the cultivated species (van der Maesen 1985). Examples: 1. Species diversity in an area as an indication of the centre of diversity. 2. Widespread species and occurrence of regional variants. 3. Centre of diversity for more than one species in a region vis-à-vis Vavilov’s centres of diversity. C) Eragrostis (Family Poaceae): a complex group characterized by at least three different subgroups distinguished on the basis of characters of inflorescence, distribution of spikelets in inflorescence, the pattern of development and maturation of spikelets and florets within a spikelet. One domesticated species, E. tef (teff’s grass) of Ethiopian origin. Diversity centre in the northern and central, especially the western parts in India. Examples: 1. Notes from field observations as an aid in later study and collection of new variants. 2. Observations on developmental and maturation patterns in species related to germination in wild species, and determining methods for collection and conservation of germplasm samples.

The Relevance of Field and Ecological Studies in Herbarium (E. Roshini Nayar)

D) Triticum-Aegilops and related genera (Family Poaceae): Approximately 25 species; Aegilops tauschii, the primary centre of diversity, south of Caspian Sea, and Transcaucasia, extending to East Turkey, Crimea and Caucasus to the west and Pakistan, Kashmir and Tibet, from sea level to high altitudes (1800m and above). Highly variable species; anther size small to long in autogamous and allogamous ecotypes, respectively. In Iran, introgression between the ssp. strangulata and ssp. tauschii, higher than in Transcaucasia; intermediate populations of Aegilops tauschii (between S and T pools) near Ramsar, Iran (Wang et al. 2013) where diversity in resistance to rust high; Caspian Iran probable area of spontaneous hybridization with cultivated tetraploid emmer wheat, to produce hexaploid wheat. Examples: 1. Survey and collection of diversity within A. tauchii-farmer’s practices in the area of diversity/ centre of diversity- intermediates between A. tauchii variants. 2. Introgression between other genera of Triticeae as a source of wild germplasm for resistance breeding. D) Oryza (Family Poaceae): 22 species; AA genome species available crossable with crop species in all major continents, resulting in crop- weed-wild complexes/ hybrid swarms. Examples: 1. Habitat-specific forms/ taxa with the mandatory requirement of recording ecological features in herbarium material for identification and study. 2. Habitat conditions to define experimental conditions for raising plants out of the wild. F) Sorghum (Family Poaceae): 22 species approximately; predominantly African and crop genepool characterized by considerable inter- specific and infra-specific crossing. Examples: 1. Hybrid swarms and break-down of barriers among crop and closely related wild species. 2. Races of the crops/species and build-up of diversity with the spread in cultivation and user preferences. 3. Diversity in use related to the area of origin/ diversity.

The Relevance of Field and Ecological Studies in Herbarium (E. Roshini Nayar)

G) Meconopsis (Family Papaveraceae): nearly 50 species, over 40 of these species in the Himalaya; many with local use as medicine, but significantly important as ornamentals. Examples: 1. Co-occurring species- common and rare ones, methods for raising them 2. Niche specificity Specialized habitats/regions The specialized habitats/regions included megacentre of diversity viz. Indian region. Example: 1) Species distribution over its range of occurrence; 2) Distinguishing features of megacentres of diversity, and 3) Analysis of diversity. Observations made in the field on a set of randomly collected specimens are briefed below (Table 1). Table 1. Parameters for use of herbarium data in the field and ecological study in PGR Herbarium Observations from Field/ ecological studies the field study

Description: from larger  confirmation of the  ‘Key’ characters for to smaller part, from identity of the distinguishing known outer to inner organs- taxon species habit; vegetative parts- root, stem and leaves;  areas with a wider  Finding new taxa reproductive parts- range of variability inflorescence/ flower;  Planning collecting in fruit; seeds areas for a survey for assembling a collection

Sizes and number of  reproductive  Number of plants parts recorded as a potential (ratio of needed for sufficient range of values flowers produced to seeds for conservation fruit set)

Reference specimen no.  Type’ specimen  Type’ locality

Phenology  date of collection,  Planning collecting trip area of collection (for herbarium and  period of maximum germplasm collection flowering and together or separately) fruiting

The Relevance of Field and Ecological Studies in Herbarium (E. Roshini Nayar)

Habitat: special  forest-forest edges,  Indicator of germination observations on farmer’s field, field requirements of seeds population edges- wastelands; near moist-dry  Habitat conditions for areas; shady areas; raising plants in soil type: scattered experimental conditions plants, a large number of plants per sq. ft.

Plant characters:  anthers exserted/  Out-crossing/self- distinct traits included, change of compatibility, related to colour of flowers; the number of seeds for pollinators were collection seen- probable reproductive system  spines, hairs on young vs. old plants- probable dispersal mechanisms

Notes: variants  distinct types, in  Areas of spontaneous population; hybridization; genepool intermediate types/ and success of probable hybrids hybridization

Notes: resistance/  infection,  uninfected types among infestation infestation- the population selected probable resistance for field further trials to pests & diseases  correlating presence of spines, hairs on aerial parts with diseases and pests occurrence

Notes: associated  associated plants-  co-occurrence as an flora/plants aid to identify indicator of inter- habitats of dependence/ecology probable availability of species

The Relevance of Field and Ecological Studies in Herbarium (E. Roshini Nayar)

Conclusions To sum-up, the examples listed above are illustrative of the importance of the field and ecological study in making the herbarium an invaluable tool, for botanical study in general, and PGR in particular in reference mainly to the grasses and legumes. Similar trends are available in other families and crops/ crop genepools. References Funk Vicki A (2003)100 Uses for a Herbarium: well at least 72. Am Soc Plant Taxonomists Newslet 17 (2): 17-19. ICAR-NBPGR (2016) Guidelines for Management of Plant Genetic Resources in India. ICAR-National Bureau of Plant Genetic Resources, New Delhi, 142 + xxiv p. Maréchal R, J-M Mascherpa and F Stainier (1978) Etude taxonomique d’un groupecomplexed’espèces des genres Phaseolus et Vigna (Papilionaceae) sur la base de donnéesmorphologiques et polliniques, traitéesparl’analyseinformatique. Boissiera 28: 1-273. Nayar ER, APandey, KVenkateswaran, R Gupta and BS Dhillon (2003) Crop Plants of India: A Check-list of Scientific Names. ICAR- National Bureau of Plant Genetic Resources, New Delhi. Sangsiri C, A Kaga, N Tomooka, D Vaughn and P Srinives (2007) Genetic Diversity of the Mung Bean (Vigna radiata, Leguminosae) Genepool on the Basis of Microsatellite Analysis. Austr J Bot: 55(8). van der Maesen LJG (1985) Cajanus DC. and Atylosia W.& A. (Leguminosae) A Revision of All Taxa Closely Related to the Pigeon Pea, with Notes on Other Related Genera Within the Subtribe Cajaninae. Papers 85-4, Agricultural University, Wageningen, The Netherlands. Wang J, M-C Luo, Z Chen, FM You, Y Wei, Y Zheng and J Dvorak (2013) Aegilops tauschii Single Nucleotide Polymorphisms Shed Light on the Origins of Wheat D Genome Genetic Diversity and Pinpoint the Geographic Origin of Hexaploid Wheat. New Phytol 198: 925-937. Some common links: https://historyofknowledge.net/2018/05/26/a-few-plain-instructions/ http://www.theplantlist.org/tpl1.1/search?q=ThePlantList http://www.tropicos.org/

Modern Tools in Plant Taxonomy (S Rajkumar)

Modern Tools in Plant Taxonomy S Rajkumar Division of Genomic Resources ICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012, India Introduction Plant taxonomy is an ancient discipline nowadays facing many new challenges with the availability of a vast array of molecular approaches allowing reliable genealogy-based classiﬁcations. Although the primary focus of plant taxonomy is on the delimitation of species, molecular approaches also provide a better understanding of evolutionary processes, a particularly important issue for some taxonomic complex groups. One of the advantages of molecular techniques for plant taxonomy is that analyses can be performed at early developmental stages, from living plant material as well as from voucher herbarium specimens. This allows an “integrative” approach combining modern molecular data with a taxonomic description of reference species. This chapter deals with some modern tools- serotaxonomy, cytotaxonomy and molecular taxonomy in detail. Important procedures used in the molecular taxonomy are given in brief in Appendix I. Serotaxonomy Serology is defined as that branch of biology, which is concerned with the nature and interactions of antigenic material and antibodies. When foreign cells or particles (antigens) are introduced into an organism, antibodies are produced in the blood (antiserum). Smith (1976) defined it as “the study of the origins and properties of antisera.” The substance capable of stimulating the formation of an antibody is called antigen and the highly specific protein molecule produced by plasma cells in the immune system in response to the antigen is called antibody. Proteins most widely used as antigens in serotaxonomy are those, which carry useful taxonomic information and are easy to handle. Both structural and reserve proteins can be used in the field of systematics, as long as they belong to the same group and the same organs are always compared. Generally, storage proteins are most amenable for taxonomic studies followed by pollen proteins. Stem tubers, algal cells, fern spores, fruits and leaves can also be employed as satisfactory antigenic material for systematic investigations. Phytoserology, which deals with

Modern Tools in Plant Taxonomy (S Rajkumar) immunochemical reactions, between serum antibodies and antigens, has also established itself as a valid method in systematics because it helps to detect homologous proteins. It uses the specific properties of antisera produced by animals against plant proteins as characters to assess plant relationships. Serotaxonomy developed and became popular in Germany, which has been an active centre since the beginning of this century. Nuttal was the first biologist to compare the immunochemical specificity of serum proteins for systematic purposes. Kowarski, Bertarelli and Magnus were the other early notable serologists, who compared proteins from various grass and legume species, showing similarities and differences. Their experiments gave phytoserology a hopeful start, but unfortunately due to inadequate methods and extraordinary claims of the Konigsberg school, founded in 1914 and headed by Gohlke, spoiled this momentum. Based largely on serological studies, Mez and Ziegenspeck produced a “Stammbaum” or “family tree” for the whole plant kingdom. But the results of the Koenigsberg school was unaccepted by another school, that of Gilg and Schurhoff, in Berlin. As a result of this conflict, there was a decline of serology in Germany. Later, Otto Moritz gave a critical new start to phytoserology in the 1950s, and plant serology has now been established as a valid method in systematics. Rives, Nelson and Biskeland and Moritz are some of the other early workers, who have made a significant contribution to the application of serology to systematics. The role of the school of serology at Rutgers University, New Jersey, has also been very significant in the theoretical and technical progress of serotaxonomy in recent years. Methods used in serotaxonomy In this method, a crude protein extract of a particular plant taxon (antigen), is injected into the bloodstream of an experimental animal, usually a rabbit or a rat, to develop antibodies. In response to the specific antigen injected, a specific antibody is produced in the blood of the animal. The serum (termed the antiserum) containing the antibody is then collected and made to react in vitro with the antigenic proteins as well as proteins from other related taxa, of which the affinities are in question. Serological reactions between antibodies and antigenic material results in the formation of a precipitate. This is called a precipitin reaction. The degree of protein homology is determined by the amount of precipitation and hence is taken as a phylogenetic marker and taxonomic character. The crude protein extracts contain a large number of proteins, which stimulates the production of a vast range of antibodies, which differ in

Modern Tools in Plant Taxonomy (S Rajkumar) their specificity and reactivity. Some are produced in abundance while others are hardly detectable. Further, each protein, in turn, carries a number of antigenic determinant sites (the portion of the antibody molecule that reacts with a portion of the antigenic material) or epitopes, each of which is capable of eliciting the production of distinct antibody molecules with a specific antibody determinant site or paratope, specific to that epitope. To overcome these problems and to make highly refined serotaxonomic comparisons possible, a novel method of pre-absorption i.e., an antibody system induced by immunization with a crude protein extract (the antigen system) of one species is ‘pre-absorbed’ by the antigen system of a second species and then tested with the antigen systems of other species, was developed. The recent development of powerful analytical techniques and instrumentation has also made possible the use of monoclonal antibodies, i.e. single kinds or species of antibody directed against single epitopes, although such techniques may not be of great importance in taxonomic studies. a. Immuno-diffusion in agarose gels: In this method, the antigen- antibody reaction is carried out in gels, mostly of agarose, in petri- dishes. The antiserum containing antibodies is filled in a well at the centre of the gel and the antigens from related taxa are placed in outer or radial wells. The antigen and antibody react to produce the insoluble antigen-antibody complex, forming a thin immobile band of precipitin (protein) at equilibrium, which can be visualized either directly or after protein staining for interpretation. Immuno-diffusion in gels can be further of two types: i. Single radial immune-diffusion: In this technique, the antigen is usually allowed to diffuse into the gel containing the antiserum. ii. Ouchterlony-double immune-diffusion: In this method, both the antigen and antibody are allowed to diffuse into the gel and meet each other. b. Rocket immune-electrophoresis: It is a simple, rapid and reliable method in which, rocket-like immune-precipitate is formed when the desired protein (antigen) is electrophoresed in an agarose gel containing its mono-specific antiserum. A comparison of the height of the peaks of the unknown and standard samples also allows the unknown protein concentration to be determined Enzyme-Linked immunosorbent assay (ELISA) This method is generally used for quantitative estimation of a particular protein in a mixture, but can also be used to study the antigen-antibody

Modern Tools in Plant Taxonomy (S Rajkumar) reaction. The antibodies against a particular antigen are adsorbed to a solid support, in most cases a polystyrene microtiter plate. The support after coating with antibody is washed and then the antigen is added, which binds to the adsorbed antibodies. An enzyme-linked antibody molecule called the conjugate is then added, which also binds to the antigen, which is followed by a chromogenic substrate for the enzyme. The coloured product generated is observed for confirmation of antigen- antibody reaction as well as measured for quantitative estimation. The intensity of the colour is proportional to the bound enzyme and thus to the amount of the bound antigen. Cytotaxonomy Chromosome number is the karyotype feature most commonly used in cytotaxonomical analyses. The chromosome number can be a plesiomorphic characteristic of a large clade or a recurrent trait which arose independently in two or more clades. Some concepts regarding chromosome number variation, such as base number, aneuploidy, paleopolyploidy and neopolyploidy have been used by different authors in quite different ways. Therefore, its use in cytotaxonomy and karyotype evolution deserves much attention Chromosome number: The number of chromosomes in a species is usually constant and this makes it an important taxonomic character. However, there are exceptions where chromosome number varies. These changes usually occur in chromosomes during the process of division, and these changes may affect the gene sequence, their number or even there may be loss of chromosomes themselves. The perpetuation of this slow process results in the evolution of new chromosomal races. The chromosome number shows a wide range in vascular plants. The lowest chromosome number in flowering plants is recorded in Haplopappus gracilis (Asteraceae) [2n = 4] and the highest in Poa litorosa (Poaceae) [2n = 265]. Ophioglossum species (Pteridophyte) has the highest number of chromosomes in the plant kingdom (2n = 1240). This great diversity of chromosome numbers and their relative constancy within populations and species provide an important character for taxonomic groupings of a large number of plants. i. Constant number: in certain groups of vascular plants, the chromosome number is constant throughout the whole group, e.g., Quercus and other members of the Fagaceae have the same basic number, n = 12. In such cases, chromosome number is not of any help in distinguishing various taxa within the group.

Modern Tools in Plant Taxonomy (S Rajkumar) ii. Euploidy: when chromosome numbers in various members of a taxon are in the proportion of exact multiples, the series is described as euploidy. For example in Malvaceae, the somatic numbers in various species range from 10, 15, 20, 25, 40; from 12, 18, 24 to 30; from 14, 28, 42, 56 to 84 and so on. a. Basic number: in a euploid series, the various members may be unified by a basic number (x) which is the gametic number of a diploid species. As in the above example of Malvaceae, the basic number x = 5. The other species in the series are described as triploids (3x), tetraploids (4x), hexaploids (6x) --polyploids (nx). The basic number is usually constant for a genus or higher taxa and has proved useful in supra- specific studies. b. Primary and secondary basic numbers: in many cases, more than one basic number can be present in a group. For example, in the living species of Chlorophytum (Liliaceae), the chromosome numbers vary from 14 to 28, 42, 56, 84, etc. and also from 16 to 32, which means that Chlorophytum has two basic numbers x = 7 and x = 8. In such cases, the inferred base numbers ranging between 2 and 13 may be referred to as the primary basic numbers in the absence of living diploid members, while the remaining are termed secondary basic numbers. In case of Chlorophytum however, Naik (1976 ) from his detailed analysis of the meiotic behaviour of chromosomes in one of the species, C. laxum, has shown that the two base numbers 7 and 8 should be considered as secondary, most probably derived from the primary basic number x = 4. c. Polyploid pairs: closely related species in certain groups of plants may be cytologically distinct i.e., one may be diploid while the other a tetraploid. Such related pairs are termed polyploid pairs. For example, Cardamine hirsuta (2n = 16) and C. flexuosa (2n = 32) of the Brassicaceae/Cruciferae is a polyploid pair. d. Dibasic polyploidy: it is now a well-known fact that hybridization has a dominant role in the evolution and such hybridization may involve the crossing of any two genetically unlike individuals, which have different base numbers of chromosomes. Such hybrids undergo polyploidy since each chromosome is doubled as the pairing at meiosis is restored, and this type of polyploidy is termed as dibasic polyploidy. An artificially synthesized Raphanobrassica, which is a hybrid between Brassica oleracea (2n = 18) and Raphanus sativus (2n = 20) and has 2n = 38 chromosomes. iii. Aneuploidy: if the chromosome numbers in the different members within a group bear no simple numerical relationship to each other, then the series is termed as an aneuploid series or simply aneuploidy.

Modern Tools in Plant Taxonomy (S Rajkumar)

Various species of Carex (Cyperaceae) show a wide range of chromosome numbers from n = 6 to 112 with multiples of 5,6, 7, and 8 thus exhibiting aneuploidy. Aneuploidy may result due to either of the following: (i) Change in the basic number: an increase or decrease in the number of chromosomes may take place, whereby the same genetic material becomes distributed in a different number, leading to a change in the basic number. This phenomenon is important from the taxonomic and evolutionary point of view, as a change in the basic number results in new variations and recombination’s, leading to the evolution of new varieties and ultimately to new species. The changes in the basic number of chromosomes can be brought about by a process called polysomaly. This is very common and results in increased sets of genes wherein there is a duplication of one or a pair of chromosomes. This means that anyone pair may undergo polyploidy. Due to this some of them can afford to lose one or two chromosomes and get stabilized in nature with this new chromosome number. This naturally brings about a different basic number. For example, most of the species of Dahlia (Asteraceae), have x=8, but D. merckii is regarded as a polyploid, with n= 18, resulting by addition of two pairs of chromosomes. Polyploidy has also been reported in Datura, Nicotiana, etc. Polysomics can be of various types: a. Trisomies: plants containing one extra chromosome are known as trisomies i.e., 2n + 1. In trisomies, the extra chromosome produces a certain amount of unbalance and thus limiting their taxonomic significance. b. Tetrasomics: plants containing two extra chromosomes are known as tetrasomics i.e., 2n + 2, etc. c. Monosomies: plants with one chromosome less are known as monosomies i.e., 2n – 1. Normally diploid monosomies are inviable. d. Nullisomics: plants with two chromosomes less are known as nullisomics i.e., 2n – 2, etc. Polysomics are usually unstable and as they are not isolated genetically from their relatives under natural conditions, they would lose their identity through crossing with normal plants followed by selection for more viable, genetically balanced normal disomic types. ii) Basic number remaining unaltered: The basic number may remain unaltered, but the genetic material present may be changed due to the addition or loss of chromosomes. This phenomenon is less important from the evolutionary point of view as the genetic make-up of a taxon

Modern Tools in Plant Taxonomy (S Rajkumar) becomes unbalanced due to loss or addition, resulting in an unstable condition, which cannot be perpetuated and thus cannot give rise to well-differentiated novelties. This type of aneuploid alteration of the basic number has been reported in Crepis (Asteraceae) and its relatives. This loss of chromosome/s, fragmentation or misdivision of centromere can take place as irregularities during the cell division even in the diploid species. (iii) B-Chromosomes: they are one or more accessory or supernumerary chromosomes, in addition to normal chromosomes and have been detected in a large number of plants. In plants, when compared to the other members of the chromosome complement, they are generally of a much smaller size and are of unknown origin. They reduce fertility or increase the vigour of plants. It has been found that they perpetuate in certain natural populations and may have some evolutionary significance. Chromosome size: the individual chromosomes of some taxa show marked differences in shape and size at mitotic metaphase. The size of the chromosome varies greatly in different families and also amongst members of the same family. The monocotyledons usually have larger chromosomes than the dicotyledons. In general, woody plants have smaller chromosomes than their herbaceous relatives. Chromosome size is not related to the phylogeny of angiosperms in general but is characteristic of only certain groups and families. Chromosome morphology: apart from the number and size of the chromosomes of many genera and families of flowering plants, conspicuous differences in appearances of the karyotype of the chromosomes have also been found in species having the same chromosome number. The chromosomes are best discernible at mitotic metaphase. The karyotype of the chromosomes can be characterized on the following basis: a. Relative length of the arms of chromosomes; b. Position of the centromere; and c. Presence of satellites. Accordingly, the chromosomes can be characterized as the following types: Symmetrical: a karyotype consisting of chromosomes all essentially similar to each other in size and with median or sub-median centromeres and with two equal arms are termed as symmetrical chromosomes. (i) Metacentric or V-shaped- Chromosomes with median centromere. (ii) Sub-metacentric or L-shaped- Chromosomes with sub-median centromere.

Modern Tools in Plant Taxonomy (S Rajkumar)

Karyotypes of this nature are the most common ones and considered to be the generalized types. For example, the chromosomes in the karyotype of the primitive genus Helleborus (Ranunculaceae), differ little from each other in size and most of them are V-shaped with median or submedian centromeres. II. Asymmetrical: this type of karyotype possesses many chromosomes with sub-terminal or terminal centromeres, or great differences in size between the largest and the smallest chromosomes, or both. Depending on the position of the centromere they may be further of two types (i) Acrocentric or J-shaped- Chromosomes with sub-terminal centromere. (ii) Telocentric or I-shaped- Chromosomes with terminal centromere. Karyotypes of this nature are considered as specialized types. For example, in the advanced genera Aconitum and Delphinium (Ranunculaceae), the flowers have the largest number of J-shaped chromosomes. III. Secondary constrictions and satellites: the karyotypes can also be differentiated on the basis of secondary constrictions, which are small bead-like appendages. Occasionally they occur at the terminal ends of one or more pairs of chromosomes in many species and are known as satellites. These structures are widely distributed in the plant kingdom, which shows that they are a valuable, if not essential, part of the chromosomal complement. However, very little is known about the evolutionary changes in the satellites and nucleoli. Generally, the asymmetrical karyotypes are most common in plants, which are usually specialized morphologically, while symmetrical ones are found in more or less generalized plants, but also occur in morphologically specialized ones too. The reason for this increasing asymmetry in karyotype evolution is not known. However, Stebbins (1950) suggested that unequal translocations and inversions involving the centromere seem to be responsible for this. Chromosome behaviour at meiosis A study of chromosome behaviour at meiosis can provide some valuable information about the relationship between populations and species. Pairing behaviour at meiosis is mostly determined by chromosome number and chromosome homology. 1. Show whether hybridization has occurred – T\the degree of chromosome homology, in hybrids, is an indication of the degree of relationship of the parental species.

Modern Tools in Plant Taxonomy (S Rajkumar)

2. Indicate structural differences in the parental chromosomes – meiotic pairing behaviour in hybrids can also point to structural differences in the chromosomes of its parents. a. Translocations- a chromosomal segment is removed from one place and reinserted somewhere else in the genome, either in the same or in some other chromosome. b. Inversion of segments- a segment of a chromosome becomes reinserted in the same chromosome but the opposite way around. It can be further of two types: (i) Paracentric- inversions involving only one chromosomal arm; (ii) Pericentric- inversion incorporating the centromere i.e., involving both chromosomal arms. a. Deletions- an interstitial or terminal chromosomal segment is lost. b. Duplications- when a segment of the chromosome is represented two or more times in a chromosome of a homologous pair. c. Patterns of variations of populations Causes of sterility: a study of the process of meiosis in hybrids yields information of great evolutionary significance. The fertility of a hybrid depends upon the degree of homology between the chromosomes of its parents. When this process is irregular in hybrids, it results in disharmony between the genetic systems of the parents involved. The more the number of non-homologous segments in the hybrid, the more is the incidence of irregularities, the greater the degree of sterility and the greater the magnitude of evolutionary distance between the parents. Thus, whether the hybrid is vigorous or weak, whether it produces viable or in-viable pollen, and whether it is capable of producing a greater or lesser amount of seed, helps in estimating the degree of homology of the genomes involved, which ultimately reflects a measure of species relationships i.e., degree of pairing is proportional to the degree of homology of the genomes. Molecular taxonomy Genomic approaches make use of the diversity among DNA sequences to identify organisms. These sequences being embedded in every cell are considered as genetic ‘barcodes’. Barcodes have only four choices of nucleotides at each position but the string of sites is huge. It should show a higher level of variation among the species and should be conserved enough to show no variation within the species. The barcode should contain enough phylogenetic information to assign the species to a particular taxonomic group. The region should have highly conserved

Modern Tools in Plant Taxonomy (S Rajkumar) primer binding sites for the case of amplification and sequencing. The sequence should be short enough to amplify even from degraded DNA. The insertions, deletions and substitutions at nucleotide level of the barcodes are the characteristics of the evolutionary path which makes barcoding technique as a valuable tool to classify even cryptic species. The overall components of the barcoding technology consist of sample to be barcoded, laboratory technique including the use of universal primers to amplify barcodes from the sample and online databases that contain the sequences of standard barcodes most of the species. The success of barcoding depends on the construction of an online library that contains the standard sequences of barcodes of almost all species. The Consortia for Barcode of Life (CBOL) has recommended the use of ITS, matK and rbcL as universal barcode loci for land plants. The internally transcribed spacers (ITS) 700bp long sequence, present in the rRNA genes of all eukaryotes serves as a barcoding region. The ITS 1 and ITS 2 on either side of 5.8s rRNA gene shows variation at length and nucleotide level which is useful to assign an organism into a specific taxon/family. As ITS 1 and 2 are flanked by conserved rRNA genes universal primers can be synthesized for the amplification in PCR. The rbcL is present in the chloroplast genome codes for larger sub unit of the enzyme ribulose -1,5- bisphosphate carboxylase which is an important enzyme in the process of photosynthesis, is used as a universal barcode for land plants. The variation due to substitutions of nucleotides in this gene infers to the amino acid sequence of the enzyme. This characteristic of rbcL gene favours the use of this region in classifying plants. The length of the locus is 1380bp. As a barcode should be short enough for ease of amplification and sequencing, this property of rbcL may be a drawback in the use of barcoding. Even though, the high substation rates and evolutionarily informative sites of this gene makes it as a standard barcode. The group II intron named trnk (lysine tRNA) codes for the enzyme ‘maturase’ that serves as a putative protein in the RNA splicing process is used as an universal barcode for plants as the maturase activity on splicing depend upon the intron of a particular species, the matk shows variation at molecular level to diverge species. References/ suggested readings Besse P (2014) Molecular Plant Taxonomy- Methods and Protocols. In: Methods in Molecular Biology, Walker JM (ed.). Springer (Humana Press).

Modern Tools in Plant Taxonomy (S Rajkumar)

Naik VN (1976) Chromosomal Behaviour and Evolutionary Trends in Chlorophytum (Liliaceae). Bot J Linn Soc 72: 45-50. Smith PM (1976) The Chemotaxonomy of Plants. Kdward Arnold Limited, London. Stebbins GL Jr (1950) Variation and Evolution in Plants. Columbia University Press, New York. Quicke DLJ (1993) Principle and Techniques of Contemporary Taxonomy. Springer Science Buisness Media. Appendix I Procedutes in molecular taxonomy Materials 1. Authenticate plant materials (a group of species of a genus/genera/family) 2. Thermal Cycler 3. Electrophoresis unit with power pack 4. Microcentrifuge 5. Micropipettes 6. Microcentrifuge tubes 7. PCR tubes 8. Tips 9. Gloves Laboratory regents 1. Plant genomic DNA (20-30 ng/ul) 2. PCR reaction components: a. Taq polymerase (5U/ul) (Cat no. TQ252 Geneaid) b. Taq buffer (10X) (Cat no. TQ252 Geneaid) c. dNTP’s (10mM) (Cat no. DN4400 Geneaid) d. Nuclease free water (Cat. No.P1193 Promega e. PCR primers (custom synthesized Eurofins Bangalore) 3. Electrophoresis reagents a. Agarose (0.8%) (Cat. No.A2132 Biomatik) b. TAE Buffer (50X) (Cat. No. B49 Fermentas) c. Loading dye(1x) (Cat. No.DM010-R500 Biobasic) d. 100 bp DNA ladder (Cat. No.DM010-R500 Biobasic)

Modern Tools in Plant Taxonomy (S Rajkumar)

e. Ethidium bromide (Cat. No. 2512 Biomatik) 4. PCR purification kit (Hipur A Cat.No.MB512 Himedia) 5. DNA sequencing (Eurofins Bangalore) PCR and Sequencing primer map for ITS, matK and rbcL loci ITS1 CTT GGT CAT TTA GAG GAA GTA A ITS2 GCT GCG TTC TTC ATC GAT GC

ITS3 GCA TCG ATG AAG AAC GCA GC ITS4 TCC TCC GCT TAT TGA TAT GC

trnK570F TCC AAA ATC AAA AGA GCG ATT GG matK80F CTA TAC CCA CTT ATC TTT CGG GAG T matK390F CGA TCT ATT CAT TCA ATA TTT C matK800F CAT GCA TTA TGT TAG ATA TCA AGG matk1200F GAY TCT GAT ATT ATC AAC CGA TTT G matK190R ATT CGA GTA ATT AAA CGT TTT ACA A matK530R GTT CCA ATT CCA ATA CTC GTG AAG

matK950R AAA ATM ACA TTG ACA TAA ATT GAC AAM G matK1300R CGA AGT ATA TAY TTY ATT CGA TAC A trnK1710R GCT TGC ATT TTT CAT TGC ACA CG

rbcL 1F ATG TCA CCA CAA ACA GAA ACT AAA GC rbcL 560F GTT TAT GAA TGT CTT CGT rbcL 724F GCT ACT GCA GGT ACA TG rbcL 724R CAT GTA CCT GCA GTA GC

rbcL 1352R CTT CAC AAG CAG CAG CTA GTT C

trnK570F TCC AAA ATC AAA AGA GCG ATT GG

matK80F CTA TAC CCA CTT ATC TTT CGG GAG T

matK390F CGA TCT ATT CAT TCA ATA TTT C

matK800F CAT GCA TTA TGT TAG ATA TCA AGG

Modern Tools in Plant Taxonomy (S Rajkumar)

matk1200F GAY TCT GAT ATT ATC AAC CGA TTT G

matK190R ATT CGA GTA ATT AAA CGT TTT ACA A

matK530R GTT CCA ATT CCA ATA CTC GTG AAG

matK950R AAA ATM ACA TTG ACA TAA ATT GAC AAM G

matK1300R CGA AGT ATA TAY TTY ATT CGA TAC A

trnK1710R GCT TGC ATT TTT CAT TGC ACA CG

rbcL 1F ATG TCA CCA CAA ACA GAA ACT AAA GC

rbcL 560F GTT TAT GAA TGT CTT CGT

rbcL 724F GCT ACT GCA GGT ACA TG

rbcL 724R CAT GTA CCT GCA GTA GC

rbcL 1352R CTT CAC AAG CAG CAG CTA GTT C

ITS1 CTT GGT CAT TTA GAG GAA GTA A

ITS2 GCT GCG TTC TTC ATC GAT GC

ITS3 GCA TCG ATG AAG AAC GCA GC

ITS4 TCC TCC GCT TAT TGA TAT GC

Composition of PCR amplification mix

S.no Component Volume (µl)

1. Taq polymerase (5U/ul) 0.3

Modern Tools in Plant Taxonomy (S Rajkumar)

2. Taq buffer (10X) 5

3. Mgcl2 (15mM) 5

4. dNTPs (15mM) 2.5

5. Forward primer (5 pmoles) 1

6. Reverse primer (5 pmoles) 1

7. Template (20-30ng) 2

8. Nuclease free water 33.2

Total 50

PCR amplification condition for ITS Step 1.Initial denaturation : 94 C for 3 minutes Step 2. Cycle Denaturation : 94 C for 30 seconds Step 3. Annealing : 50 C for 1 minute Step 4. Cycle Extension : 72 C for 1 minute Step 5. Final Extension : 72 C for 5 minutes Step 6. Hold : 4 C for 30 cycles of PCR was carried out from Steps 2 to 4. PCR amplification condition for matK Step 1.Initial denaturation : 94 C for 3 minutes Step 2. Cycle Denaturation : 94 C for 30 seconds Step 3. Annealing : 47 C for 1 minute Step 4. Cycle Extension : 74 C for 1 minute Step 5. Final Extension : 74 C for 5 minutes Step 6. Hold : 4 C for 10 minutes 35 cycles were carried out from step 2 to 4. PCR amplification condition for rbcL

Modern Tools in Plant Taxonomy (S Rajkumar)

Step 1.Initial denaturation : 94 C for 3 minutes Step 2. Cycle Denaturation : 94 C for 30 seconds Step 3. Annealing : 52 C for 1 minute Step 4. Cycle Extension : 74 C for 2 minutes Step 5. Final Extension : 74 C for 10 minutes Step 6. Hold : 4 C for 10 minutes 35 cycles were carried out from step 2 to 4. Preparation 0.8% Agarose gel (earlier chapter): Load 5 ul above PCR reaction to check the amplification and determining the purity and size of the amplified fragment for further analysis. PCR purification (Kit based): DNA is bound on a silica membrane within the spin column. The bound DNA is washed and the clean, concentrated DNA is eluted. This is to be done using Hi Pura (HI MEDIA)PCR purification kit. 1. Add 5 volumes of Binding Solution to 1 volume of the PCR reaction and mix. For example, add 500 µlof Binding Solution to 100 µl of the PCR reaction. Transfer the solution into the binding column. Centrifuge the column at maximum speed (12,000-16,000 Xg) for 1 minute. Discard the eluate, but retain the collection tube. 2. Replace the binding column into the collection tube. Apply 0.5 ml of diluted Wash Solution to the column and centrifuge at maximum speed for 1 minute Discard the eluate, but retain the collection tube. 3. Replace the column into the collection tube. Centrifuge the column at maximum speed for 2 minutes, without any additional wash solution, to remove excess ethanol. Discard any residual eluate as well as the collection tube. 4. Transfer the column to a fresh 2 ml collection tube. Apply 30 µl of Elution Solution to the centre of each column. Incubate at room temperature for 1 minute. 5. To elute the DNA, centrifuge the column at maximum speed for 1 minute. The PCR amplification product is now present in the elute. The purification product can be further checked by running it on 0.8% Agarose gel and used for sequencing using sequencing primers.

Chemotaxonomy - A Tool for Taxonomic Delineation and Identification of Plant Genetic Resources (Poonam Suneja et al.)

Chemotaxonomy - A Tool for Taxonomic Delineation and Identification of Plant Genetic Resources Poonam Suneja, Rakesh Bhardwaj* and Anjula Pandey * Division of Germplasm Evaluation (Biochemistry Unit) ICAR-National Bureau of Plant Genetic Resources New Delhi 110 012, India * corresponding author Introduction Many traditional approaches - morphological, anatomical, cytogenetical and palaeobotanical have facilitated taxonomic identification and classifications of plant taxa. Data derived from the external morphological study alone is not sufficient and therefore other branches of science are of considerable value in assessment of taxonomy and its phylogenetic status. The concept of specificity of the chemical structure of the secondary metabolites and biosynthetic pathways has led to resolving taxonomic issues. ‘Chemotaxonomy’ is advanced approach chemistry used for classification of plants on the basic structure of primary and secondary metabolites. The concept of ‘Chemotaxonomy’ has been elaborated in the past century. De Candolle identified that chemical characteristics of plants will serve as a valuable aid in plant taxonomy and useful guide to search for new industrial and medicinal plants (de Candolle 1816). The contributions of chemotaxonomy have supported the ideas of classification and phylogeny (Vidita et al. 2013; Ram Singh 2016). The findings of chemotaxonomic studies are helpful to taxonomists, phytochemists and pharmacologists to solve selected taxonomical problems. Chemotaxonomy involves the study of chemical variation at a level of genera/species/subspecies in plants and microbes. It is a useful tool for the investigation of the distribution of chemical compounds or groups of biosynthetically related compounds in a series of related genera. In plants, primary metabolites (amino acids, fatty acids, oligosaccharides and organic acids) provides insight for distinguishing at genera/species level whereas secondary metabolites such as phenols/flavones, essential oils provides more specific details. Any form of the sample (as fresh, dried or crushed) can be used in

Chemotaxonomy - A Tool for Taxonomic Delineation and Identification of Plant Genetic Resources (Poonam Suneja et al.) chemotaxonomic analysis and thereby making it more advantageous than other methods. Chemotaxonomy- some case studies In general chemotaxonomy in plants is more commonly applied for medicinal and aromatic plants as compared to the food crops; especially for checking adulteration. In angiosperms, some families that have been studied through chemotaxonomic methods are- Malvaceae, Ranunculaceae, Magnoliaceae, Polygonaceae and Solanaceae (Sivarajan 1991). Pulse crops moth bean, cowpea, black gram, green gram and rice bran can be distinguished chemically on the basis of primary metabolites. Rice bean has stachyose >4.5% and negligible verbascos and raffinose, while black gram and green gram have >1% Verbascose which is not present in other Vigna pulses. Moth bean and cowpea have stachyose 1-2% and a significant proportion of raffinose. Rice bean has a very high content of insoluble oxalates >300mg/100g, while cowpea and green gram have <5 mg/100g; moth bean and black gram have 10-30 mg/100g. Secondary metabolites viz. profile of phenols gives a clear distinction between cowpea and green gram. Cowpea seeds have caffeic acids, chlorogenic acid, daidzein and genistein while green gram has vanillic acid, apigenin, kaempferol and isorhamnetin which is absent in cowpea (Longvah et al. 2017) A ‘Chemotype’ denotes a group of organisms usually plants and micro-organism, belonging to the same species that produce distinctly different sets of secondary metabolites. In aromatic plants, ‘Chemotype’ were traditionally characterized by the difference in single dominating constituent. The taxonomy of aromatic plants of different genera from diverse geographical locations is complicated among species, varieties and cultivars without significant differences in morphology. Taxonomic identification of medicinal and aromatic is possible due to the essential oils and chemical constituents which is directly linked to their identity (Stanko et al. 2011; Sobti et al. 1982; Labra et al. 2004). The genus Ocimum L. comprises of more than 30 species is found in tropical and subtropical regions. The oil extracted from its leave is used in aromatherapy, perfumes and cosmetics. Therefore, the determination of essential oil content and composition of basil varieties is very important and can serve as a useful classification tool (Chemotaxonomy) of the various Ocimum/basil plants from different regions. A system of classification of essential oil chemotypes has been proposed as a simpler and easy method to identify different basil species (Ahmed et al. 2017).

Chemotaxonomy - A Tool for Taxonomic Delineation and Identification of Plant Genetic Resources (Poonam Suneja et al.)

Basil is known to have several chemotypes based on variation in essential oil composition. The most commonly found species of Ocimum basilicum var. basilicum showed four chemotypes: linalool rich, methyl chavicol rich, (E)-methyl cinnamate and mixed type having both linalool and methyl chavicol type. Among the O. basilicum varieties, linalool-rich chemotype was identified in thyrsiflora, difforme, purpurascens and glabratum, whereas methyl chavicol-rich chemotype was found in pilosum. Ocimum species/varieties were classified into seven different chemotypes. Ocimum x citriodorum showed two different chemotypes rich in geranial/neral and methyl chavicol. O. kilimandscharicum showed camphor-rich chemotype (43.21%), while O. viride was found to be eugenol-rich chemotype (77.86%) (Raina et al. 2018). The essential oil extracted from species of Cymbopogon yield palmarosa oil, citronella oil and lemongrass oil documented for various pharmacological uses as anti-diarrheal, antifungal, anti-amoebic, antibacterial and anti-inflammatory properties (Subramaniaum et al. 2015). These oils are rich in citral, geraniol, citronellol, citronellal, linalool, elemol, 1,8-cineole, limonene, b-carophyllene, methyl heptenone, geranyl acetate and geranyl formate. The essential oil of lemongrass (Cymbopogoncitratus) yields 1-2% on a dry weight basis (mainly citral); of palmarosa (C. martinii) with geraniol (90%) with rose like odor, has geranyl acetate (Padalia et al. 2011) In the literature, the majority of C. citratus analyzed showed a remarkably high percentage of neral and geranial. Analysis of C. citratus species from Brazil, India, West and Eastern Africa and Asia showed the high value of neral and geranial chemotypes. The most distinguishing feature between C. citratus of African origin is the high amount of myrcene observed. High occurrence of piperitone characterizes the oils of C. parkeri and C. olivieri from Iran. Jiroveltz et al. (2007) reported a significant presence of cis-p-mentha-1(7), 8-dien-2-ol and its isomer trans-p-mentha-1(7), 8-dien-2-ol in the oils of C. giganteus from Cameroon. Predominant components observed in other Cymbopogon species essential oils from around the world include δ-2-carene (30) in C. proximus (ex Cameroon), linalool in C. nardus (ex Malaysia), limonene in C. schoenanthus (ex Tunisia) and C. giganteus (ex Burkina Faso) and elemicin from the oils of C. pendulus (ex India). In C. winterianus two major chemotypes have been identified based on the amount of geraniol and citronellal (ex Brazil) (Shah et al. 2011). The study was undertaken by Ganjewala and Gupta (2013) on essential oil compositions of eight lemongrass cultivars revealed that seven of the eight essential oils had citral (75-85%) as a major constituent, while only one cultivar GRL-1 had geraniol (90%) as a major 82

Chemotaxonomy - A Tool for Taxonomic Delineation and Identification of Plant Genetic Resources (Poonam Suneja et al.) component. Khanuja et al. (2005) have also reported similar variation in the essential oil content and composition in 19 taxa of Cymbopogon and discerned phylogenetic relationship among these taxa. The essential oil of C. confortiflorus and C. nardus var. confortiflorus were very rich in geraniol (68% and 46%) whereas C. nardus var. nardus and C. winterianus had a very little amount of geraniol. The essential oil of C. pendulus, C. flexuosus and C. citratus mainly consisted of citral with 80- 84% of the total monoterpene content. The essential oil of C. giganteus showed a distinct composition due to the presence of cis- and trans-p - 1(7), 8- menthadien-2-ol (19.9% and 22.3%), cis- and trans-p -2,8- menthadien- 1-ol (10.1% and 14.3%). Similarly, C. schoenanthus from Tunisia also has a distinguished EO composition due to the presence of limonene (10.5-27.3%), β-phellandrene (8.2–16.3%), d -terpinene (4.3– 21.2%) and α -terpineol (6.8-11.0%) (Ganjewala 2009). The essential oil of palmarosa (C. martini) has revealed the presence of geraniol (65%) and geranyl acetate (20%) as major constituents and citronella oil (C. winterainus) is mainly comprised of geraniol (40.06%), citronellal (27.44%) and citronellol (10.45). Essential oil composition of C. nardus has been found identical to citronella oil with the dominance of the geraniol, citronellal, and citronellol. However, essential oil of C. parkeri from Iran has a unique composition with presence of piperitone (81%) as major component and other minor constituents such as germacrene-D (5%), santolinyl acetate (2.1%) and a -eudesmol (2.1%). Chemical composition of artemisia oils has been investigated in many species of Artemisia for chemotaxonomic reasons. Camphor, 1,8- cineole, camphene, terpinen-4-ol and a-terpineol are the main oil components of A. sieberi (Weyerstahl et al. 1993), A. hololeuca, A. gmelinii and A. pontica (Bodrug et al. 1987), whereas artemisia ketone made up 94% of the total oil of A. alba (Bodrug et al. 1987). Out of three chemotypes identified in rosemary oil the main component in chemotype 2 and chemotype 3 contained 1,8-cineole (49.8% and 40.0%, respectively); followed by α-pinene (18.7% and 23.5%, respectively) and camphor (12.6% and 13.2%, respectively (Mouhssen and Berrada 2003). Oil originating from Rabat represented an α-pinene chemotype with 34.0% of the total oil in addition to borneol (16.9%) and camphor (11.7%). Chemotaxonomic approaches There are several chemical constituents used as a tool for distinguishing chemo-types viz. glycosides, glucosinolates, alkaloids, polyphenols, carotenoids, phytosterols (stigmasterols, saponins, etc.), essential oils (terpenoids), organic acids, sugars, oligosaccharides, amino acids, fatty

Chemotaxonomy - A Tool for Taxonomic Delineation and Identification of Plant Genetic Resources (Poonam Suneja et al.) acids, starch granule structure and more. Selection of a specific compound or a group of the compound for chemo-taxonomic purpose is based on the distribution of targeted compounds in the genera/species under study. Less frequent distribution of a particular compound enables easy distinction whereas if the compound is widely distributed is of little use. However, in certain cases pattern of distribution or ratio of compounds also provides clear insights about chemotypes such as ratios of different fatty acids in Brassica sp. The methods used for chemotaxonomic classification are now much better and advanced than classical one be it plant sample extraction or quantitative & qualitative estimation of metabolites. Latest analytical chemistry tools such as for extraction- supercritical fluid extraction, pressurized liquid extraction, sonication/microwave-assisted extraction enables rapid and complete extraction with little loss/degradation. Similarly for estimation using UPLC/HPLC/HPTLC/GC coupled with versatile and powerful detectors like triple quadrupole mass spectroscopic detectors enables identification and analysis of trace quantities of µg/ng/pg level, and even the absence of standard reference compounds. There are reports where IR/NIR/ Raman spectroscopy-based vibrational methods are used for distinction/classification of citrus oils, basil, Oreganum, fennel (Steuer et al. 2001; Schulz et al. 2003; Branska et. al. 2005; Gudi et al. 2014). The advantage with these methods is that they are rapid, non- destructive, highly cost-effective and does not require much technical skill as is the case when the actual chemical analysis is performed. Conclusions Due to advancement in analytical techniques and use of sophisticated instruments, identification and classification using methods like chemotaxonomy have become popular and easier to operate. The phytochemical data along with morphological data are proving important in interpreting taxonomy of the plants. It may be useful in understanding phylogenetic relationships, especially considering that species of some genera are not easily identified using traditional methods. References/ selected readings Ahmed AH, HB Khaled, HA Abdulkarem, MI Hassan and A Lebnane (2017) Chemotaxonomy and Spectral Analysis (GC/MS and FT-IR) of Essential Oil Composition of Two Ocimum basilicum L. Varieties and Their Morphological Characterization. Jordan J Chem 12 (3): 147-160. Bodrug MV, IP Dragalin and PF Vlad (1987) Introduction of Some Artemisia Species into Moldavia: Characterization and Chemical

Chemotaxonomy - A Tool for Taxonomic Delineation and Identification of Plant Genetic Resources (Poonam Suneja et al.)

Composition of Their Essential Oils. Izu. Akad. Nauk Mold. SSR, Ser. Biol Khim Nauki 2:14-16. Baranska M, EH Schulz, ER Kru¨ger and ER Quilitzsch (2005) Chemotaxonomy of Aromatic Plants of the Genus Origanum via vibrational Spectroscopy. Anal Bioanal Chem 381: 1241-1247. de Candolle AP (1816) Essaisur les propridtdsmédicales des plantes, cornparees avec le urs forms extérieuresetleur classification naturelle (second edn.), Paris. Ganjewala D (2009) Cymbopogon Essential Oils: Chemical Composition and Bioactivities. Intern J Essent Oil Therap 3: 56-65. Gudi G, AKrähmer, HKrüger, LHennig and H Schulz (2014) Discrimination of Fennel Chemotypes Applying IR and Raman Spectroscopy: Discovery of A New γ-asarone Chemotype. J Agric Food Chem 62(16): 3537-3547. Jirovetz L, G Buchbauer, G Eller, MB Ngassoum and PM Maponmetsem (2007) Composition and Antimicrobial Activity of Cymbopogon Giganteus (Hochst.) Chiov. Flower, Leaf and Stem Oils from Cameroon. J Ess Oil Res 19: 485-489. Khanuja SPS, AK Shasany, A Pawar, RK Lal, MP Darokar and AA Naqvi (2005) Essential Oil Constituents and RAPD Markers to Establish Species Relationship in Cymbopogon Spreng. (Poaceae). Biochem Syst Ecol 33:171-86. Labra M, M Liele, B Ledda, F Grassi, M Mazzei and F Sala (2004) Morphological Characterization, Essential Oil Composition DNA Genotyping Ocimum basilicum L. Cultivars. Plant Sci 167: 725-731. Mouhssen Lahlou and R Berrada (2003) Composition and Niticidal Activity of Essential Oils of Three Chemotypes of Rosmarinus officinalis L. Acclimatized in Morocco Flav Fragr J 18:124-127. Subramaniaum P, Che Wan Imanina, Che wan Takwa, Nurul Emelia and Ahmad Zubair (2015) Chemical Composition and Antibacterial Activity of Essential Oil of Cymbopogon citratus and Cymbopogon nardus Against Enterococus faecalis. Intern J Biosci 6(9): 9-17. Longvah T, R Ananthan, K Bhaskarachary and K Venkaiah (2017) Indian Food Composition Tables 2017, ICMR-National Institute of Nutrition, Hyderabad, India. Padalia RC, RS Verma, SC Chandan and A Yadav (2011) Chemical Fingerprinting of the Fragrant Volatiles of Nineteen Indian Cultivars of Cymbopogon Spreng. (Poaceae) Rec Nat Prod 5(4): 290-299.

Chemotaxonomy - A Tool for Taxonomic Delineation and Identification of Plant Genetic Resources (Poonam Suneja et al.)

Raina AP and V Gupta (2018) Chemotypic Characterization of Diversity in Essential Oil Composition of Ocimum Species and Varieties from India. J Essen Oil Res 30 (6): 444-456. Ram Singh (2016) Chemotaxonomy: A Tool for Plant Classification. J Med Plants 4 (2): 90-93. Schulz H, BSchrader, RQuilitzsch, RSPfeffer and HKrüger (2003) Rapid Classification of Basil Chemotypes by Various Vibrational Spectroscopy Methods. J Agric Food Chem 51 (9): 2475-2481 Shah G, Richa Shri, V Panchal, Narender Sharma, BharpurSingh and AS Mann (2011) Scientific Basis for the Therapeutic Use of Cymbopogon citratus Stapf (Lemongrass). J Adv Pharma Tech Res 2(1): 3-8. Stanko KC, Z Liber, O Politeo, F Strikic, I Kolak, M Milos and Z Satovic (2011) Molecular and Chemical Characterization of the Most Widespread Ocimum species. Plant Syst Evol 294: 253-262. Steuer B, H Schulz and ELäger (2001) Classification and Analysis of Citrus Oils by NIR Spectroscopy. Food Chem 72(1): 113-117. Sivarajan VV (1991) Introduction to the Principles of Plant Taxonomy. Cambridge University Press, Cambridge. Sobti SN and P Pushpangadan (1982) Studies in the Genus Ocimum: Cytogenetics, Breeding and Utilization of Aromatic Plants. In: Atal CK and BM Kapur (eds) Regional Research Laboratory, Jammu- Tawi, India, pp 457-472. Vidita V, Bhargava, SC Patel and SD Kruti (2013) Importance of Terpenoids and Essential Oils in Chemotaxonomic Approach. Intern J Herb Med 1: 14-21. Weyerstahl S, S Schneider, H Marschall and A Rustaiyan (1993) The Essential Oil of Artemisia sieberi Bess. Flav and Frag J 8: 139-145.

Applications of Geographic Information Systems (GIS) Tools in Harbarium Studies (DP Semwal et al.)

Applications of Geographic Information Systems (GIS) Tools in Harbarium Studies DP Semwal, Anjula Pandey, Rita Gupta and NS Panwar Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources New Delhi 110 012, India Introduction Eco-geographic data associated with a herbarium collections (dried plant specimens, properly processed using a standard system of classification) are useful source of information in many fields of science including climatic analogues. With their linkage with Geographic Information System (GIS) tools, it becomes a powerful method to analyse, verifying, improving, refining and add information to herbarium databases especially in location mapping of conservation sites, gap analysis, and studies of endangered/rare species. Through application in herbarium databases processing such as overlay of collection site data with natural resource, geographic and thematic maps, diversity rich area could be identified. Additionally, spatial queries that a herbarium database alone cannot perform (such as identifying new areas for rare species based on the occurrence of habitat characterizations and matching of known collection sites) are possible through this tool. This chapter presents some examples of upgradation in the herbarium data sets and thus highlighting its application in improving the quality of data for higher achievements using the herbarium specimens. Herbarium data mapping using GIS application Geographic Information System helps database consisting of collection/herbarium specimens on spatially distributed features, which are definable in space as points, lines and polygons (Burrough 1986; Heywood et al. 2002). Herbarium specimen locality data can be visualized and analyzed using (GIS) tools for different purposes. Herbarium specimens located in different herbaria normally have associate locality descriptions (place, district, state), with mappable geo- coordinates. Pre-processing of data included data standardization (for example of district and state name) and other locality information. Data preparation, the specific methods used depended on the type of

Applications of Geographic Information Systems (GIS) Tools in Harbarium Studies (DP Semwal et al.) information available in the locality area. For the majority of specimens, coordinates were assigned by associating with a geographic feature found in an electronic gazetteer/survey map. Herbarium specimens collected during earlier times had location data that only had either district or state level information or sometimes directions. Only here comes the role of this tool to fine tune for its application. The Google earth geo-referenced map/electronic gazetteer can be used to locate actual sites of herbarium collection. The data analysis can be done by using DIVA-GIS software customized for the purpose, in which gazetteer localities could be displayed with districts, state boundaries and topographic maps (Hijmans et al. 2001). For example, a place name is used more than once within a state, the GIS could be used to quickly find the location with which the specimen is best matched, provided that the specimen label has the location information necessary to distinguish among options. Name changes, missing names, and spelling variations required investigation. Specimen collection dates are available in addition to location, Spatio-temporal analyses and mapping can be carried out. Therefore year of collection was extracted from the field in the original data which held date information exactly as written on the specimen. In order to make some conclusions about sampling effectiveness and distributions of numbers of species, a grid with cells 55 km by 55 km was superimposed on the resulting data. The number of specimens and of species was calculated for each grid cell and divided by the area of the land circumscribed by the grid cell. Locations of major herbarium specimens as having been sources of specimens examined were also superimposed on the data, using the relevant GPS coordinates. With the availability of digital platform and advanced GIS tools, the advantages of automating herbarium specimen records have become increasingly evident. GIS systems offer improved access to large quantities of herbarium data as well as expanded analytical potential. As data are automated it is important to address the way location information is recorded in order to maximize the potential for spatial analysis of herbarium data using contemporary tools such as GIS. In a GIS system, data output as maps as well as in tabular form. Maps can reveal patterns or trends within the data which would be difficult to distinguish in tabular presentations (Burrough 1986). However, to realize the maximum potential of a GIS and to avoid misinterpretation, the data must have well-defined locality information along with correct geo-coordinates.

Applications of Geographic Information Systems (GIS) Tools in Harbarium Studies (DP Semwal et al.)

Diversity mapping of herbarium data- case study Analysis of collected diversity using the passport databases vis-à-vis their areas of availability was taken up as a priority activity in planning and execution of exploration programmes across the country. In the present demonstration herbarium specimens (17 genera, 26 species) at NHCP collected from three districts of Andhra Pradesh (Table 1) were without any geo-coordinates of the sites. Location data were searched for geocoordinates of that particular site and entered into herbarium database for diversity mapping (Table 2). Map clearly depicted that genera collected from East Godavari covers large area as compared to West Godavari and Vishakhapatnam districts of Andhra Pradesh (Fig. 1). Diversity mapping for gap analysis was done for many crops. Herbarium data always can display as point data format linking with geo- coordinates of specimens collection sites, which later can used in grid mapping for diversity analysis. Although geo-referenced data is currently limited in ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), the potential benefits of applying GIS tools to herbarium studies and management of genetic resources are providing strong incentives for curators, explorers and collection users to improve the quality and quantity of information in herbarium database. Table 1. Herbarium specimens lacking the geo-coordinates data

Hs no. Bot. name Fam name District Locality Lat. & Long. 23162 Toddalia Rutaceae West Godawari Chidipi (Kovvur) - asiatica 23163 Trichosanth Cucurbitaceae West Godawari Chipurugudem - es bracteata (Kavuluru) 23164 Kedrostis Cucurbitaceae West Godawari Kurukuru - foetidissima (Devarappalli) 23165 Solanum Solanaceae West Godawari Kurukuru - trilobatum (Devarappalli) 23166 Begonia Begoniaceae Visakhapatnam 5 Km. before - picta Sileru from Chintapalli 23167 Phyllanthus Euphorbiaceae Visakhapatnam RARS farm, - fraternus Chintapalli 23168 Vigna Fabaceae Visakhapatnam 2 Km before - mungo Darakonda from Chintapalli 23169 Dumasia Fabaceae Visakhapatnam 5 Km. before - villosa Sileru from Chintapalli 23170 Ziziphus Rhamnaceae East Godawari Maredumilli - rugosa

Applications of Geographic Information Systems (GIS) Tools in Harbarium Studies (DP Semwal et al.)

23171 Ampelocissu Vitaceae East Godawari Maredumilli - s sp. 23172 Solanum Solanaceae Visakhapatnam 10 Km. before - erianthum Darakonda from Chintapalli 23173 Trichosanth Cucurbitaceae Visakhapatnam Darakonda (G. - es bracteata K. Veedhi) 23174 Ischaemum Poaceae Visakhapatnam 2 Km. before - rugosum Darakonda from Chintapalli 23175 Phaseolus Fabaceae Visakhapatnam Borramamida - lunatus (Araku) 23176 Cajanus Fabaceae Visakhapatnam Gurra panghu - albicans (Paderu) 23177 Trichosanth Cucurbitaceae Visakhapatnam Gurra panghu - es bracteata (Paderu) 23178 Luffa Cucurbitaceae East Godawari Rajavaram, near - aegyptiaca Kothapalli (Rampa- Chodavaram) 23179 Momordica Cucurbitaceae East Godawari Bedhasantra - dioica 23180 Momordica Cucurbitaceae East Godawari Garulapadu - dioica 23181 Momordica Cucurbitaceae East Godawari Adateegala - dioica 23182 Amaranthus Amaranthaceae East Godawari Peddaddapalli - dubius (Gangavaram) 23183 Amaranthus Amaranthaceae East Godawari Peddaddapalli - tristis (Gangavaram) 23184 Cucumis Cucurbitaceae East Godawari Peddaddapalli - melo (Gangavaram) 23185 Trichosanth Cucurbitaceae East Godawari Konalova - es bracteata 23186 Trichosanth Cucurbitaceae Visakhapatnam Lemmasinghi, - es bracteata Chintapalli - : data missing Data accuracy Spatial analysis of herbarium data must have locational information (place name) that can be related to a point on a map. The point might represent the location of a village (or farmland), a city (or other administrative setup) within the metropolitan region, or the exact collection site recorded with geographic coordinates (latitude, longitude and altitude). A herbarium database which contains location information at each of these three levels of scale could be used to generate many different maps to show specimens information in a variety of ways. For example, data could be mapped by place name or area, the map presents equally accurate, but more generalized information. When location is recorded using precise measurements a variety of maps can be created according to how data are to be used.

Applications of Geographic Information Systems (GIS) Tools in Harbarium Studies (DP Semwal et al.)

Sensitive data can be masked by aggregating the point data into increasingly generalized levels of scale. However, data gathered only at a low scale (e.g. a state) cannot be 'degeneralized' to show a more highly defined location. Table 2. Herbarium specimens data along with geo-coordinates information upgraded

Hs no Bot. name Fam name District Lat. Long. In decimal degree 23162 Toddalia asiatica Rutaceae West Godawari 16.90 81.36 23163 Trichosanthes Cucurbitaceae West Godawari 16.72 81.44 bracteata 23164 Kedrostis Cucurbitaceae West Godawari 16.86 81.28 foetidissima 23165 Solanum Solanaceae West Godawari 16.86 81.28 trilobatum 23166 Begonia picta Begoniaceae Visakhapatnam 17.87 82.34 23167 Phyllanthus Euphorbiaceae Visakhapatnam 17.86 82.35 fraternus 23168 Vigna mungo Fabaceae Visakhapatnam 17.86 82.36 23169 Dumasia villosa Fabaceae Visakhapatnam 17.87 82.34 23170 Ziziphus rugosa Rhamnaceae East Godawari 17.16 82.05 23171 Ampelocissus sp. Vitaceae East Godawari 17.16 82.05 23172 Solanum Solanaceae Visakhapatnam 17.86 82.31 erianthum 23173 Trichosanthes Cucurbitaceae Visakhapatnam 17.79 82.34 bracteata 23174 Ischaemum Poaceae Visakhapatnam 17.86 82.32 rugosum 23175 Phaseolus lunatus Fabaceae Visakhapatnam 18.01 82.49 23176 Cajanus albicans Fabaceae Visakhapatnam 17.96 82.40 23177 Trichosanthes Cucurbitaceae Visakhapatnam 17.96 82.40 bracteata 23178 Luffa aegyptiaca Cucurbitaceae East Godawari 17.44 81.77 23179 Momordica dioica Cucurbitaceae East Godawari 17.16 82.05 23180 Momordica dioica Cucurbitaceae East Godawari 17.16 82.06 23181 Momordica dioica Cucurbitaceae East Godawari 17.47 82.02 23182 Amaranthus Amaranthaceae East Godawari 16.73 82.04 dubius 23183 Amaranthus tristis Amaranthaceae East Godawari 16.73 82.04 23184 Cucumis melo Cucurbitaceae East Godawari 16.73 82.04 23185 Trichosanthes Cucurbitaceae East Godawari 17.15 82.04 bracteata 23186 Trichosanthes Cucurbitaceae Visakhapatnam 17.87 82.34 bracteata

Applications of Geographic Information Systems (GIS) Tools in Harbarium Studies (DP Semwal et al.)

Figure 1. Map generated form the herbarium specimens data (shown by dots) Conclusions At present there is a hidden wealth of herbarium data in databases which cannot be easily analysed using GIS because of highly variable location information. Much of these data may never be suitable for detailed spatial analysis. However, by applying this tool, it can be moderately operated through resolution and scale of individual records, and appropriate uses can be determined. At the NHCP intends to use GIS application in future for utility of this tool and to locate occurrence/distribution of species using data of herbarium specimens.

References Burrough PA (1986) Principals of Geographic Information Systems, a Monographs, Clarendon Press, Oxford, UK. Heywood I, S Cornilius and S Carver (2002) An Introduction to Geographic Information Systems, Amazon Co., UK. Hijmans Robert J., Luigi Guarino, and Prem Mathur (2012) DIVA-GIS Manual Version 7.5, online publications. Semwal DP (2009) Geographic Information System (GIS): A Tool for the Ecological Modelling. The Botanica 57: 42-45. Semwal DP and SP Ahlawat (2016) Applications of Geoinformatics in Plant Genetic Resources Studies, e-publication link http:// www. nbpgr. ernet.in /Download file.aspx?EntryId=7271.

Illustrative Field Aids for Identification of Plants (Anjula Pandey et al.)

8 Illustrative Field Aids for Identification of Plants Anjula Pandey, S Nivedhitha and Pavan Malav Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012, India Introduction In field of taxonomy insufficient knowledge on correct identification and assessment of plant diversity creates a problem. It can be tackled by imparting training on the use of different field aids. Field guides for identification of plants are one of the best taxonomic aids designed for use in the 'field' or ‘local area’ to facilitate distinguishing/ identifying plant species with visual perception. Use of the traditional identification methods is difficult due to highly specialized technical terms used in description and procedures used for identification (Lawrence 1951; Davis and Heywood 1963; Jain and Rao 1977). To facilitate this it is highly desirable to develop illustrative field aids for identification and species delineation. It is therefore desired to develop user-friendly aids for identification that not only enhance the efficiency by fast access but also develops interest among the users especially the PGR workers, and agriculturists and others with little background in taxonomy. Illustrations included in the field guides make them user-friendly and efficient for identification. Illustrative guides (hard form/electronic guides) have images, illustrated keys and apps devices. Illustrated keys or guides show taxonomic variation in characters/ character states, use of multiple images that allows easier taxon identification and facilitates the understanding of characters. Technological advances in publishing and computer technology have made illustrated guides more prevalent. Many modern identification tools, both written and computer-based, include multiple illustrations and/or high-quality photographs with written descriptions to reduce ambiguity in identifications (Hawthorne 2006). In addition to these most modern guides include an introduction and/or glossary; thekey and glossary may be illustrated, but images are most commonly found in the descriptive section and introduction. This chapter besides illustrating use of field guides in identification of plants also highlights the creation of simple guides by the user.

Illustrative Field Aids for Identification of Plants (Anjula Pandey et al.)

How to start? The schematic drawings illustrate the use of a single image to gain experience with a typical example, terms used, but do not provide sufficient experience to form a concept. Concept formation requires experience with the diversity of forms (Fig. 1). Prior to proceeding in the field for collecting plant genetic resources, it is mandatory to know about target species well in advance. Consultation to local and national herbaria to study the diversity represented helps in gaining knowledge on plant morphology, areas/spot location, habitats/ niche specificity (if any), the period of flowering and fruit maturity, etc. If possible the digital images can be captured in-camera, mobile or computer-based devices. Distribution data needs to be gathered from reports of earlier field visits, records from floristic literature/monographs, field genebanks and other repositories holding live material. This work can help the collector to gather precise information needed for exploration.

Figure1. Source: https://www.pinterest.com/pin/119063983873981065/: Illustrations from Lance (2004) Illustrations for plant identification Plant identification tools for field use could be very simple as photo album in hard prints in small book form/pocket guides [with labelled

Illustrative Field Aids for Identification of Plants (Anjula Pandey et al.) botanical names, close-up view of parts] of the target species, or images of species stored in some electronic devices (camera, video, tablet, etc.).

Figure 2. Digital library of Allium ampeloprasum with scans of plant/ parts at different growth stages (top - left to right: plant in field; bulb with underground bulbils; bulbils in close view; bottom row (from left- right: inflorescence; herbarium specimen mounted on sheet; seedling raised from aerial bulbils; aerial bulbils) One of the traditional ways is to use the field guides with illustrations drawn by the artist (Maheshwari 1966) highlighting the most significant character with a short description. In the electronic era, printed version of illustrated field guides are not only cumbersome to use in the field but also time-consuming. One can develop small booklet containing the most identifiable parts (grass booklet, flowers/inflorescence mounted on booklet), in the form of illustrative diagrammes of species, seed/ herbarium collection, photo-library with identity or with floristic records (Polunin and Stainton 1987; Yen et al. 1996; Vincent et al. 2013; Yadav et al. 2014).

Illustrative Field Aids for Identification of Plants (Anjula Pandey et al.)

Digital library: digital library holds a collection of images of plants at different stages and other details on availability time of the species, noted distribution, etc. Digital scans with zooming facilitate identification of characters in a plant intended to be collected and can be used to access family representative well arranged (Fig. 2). Through family characters of plant one can refer to the genus and species for identification. Field guides: a field guide contains more elaborated contents for the purpose of identification including images (pictures/ photos of plant and close-up view of identifiable part(s). Keeping photographs of live plants is generally a better and most preferred option as it brings more familiarity to the species. The data for plant species provide a description for each species highlighting key features that distinguish the target species from its close relatives in that region and provide additional information like phenology, habitat and altitude range and a suggested seed collecting technique to ensure high-quality collections. It should have a pictorial representation of species with most important recognizable part and preferably with photos of fruit and seed. Display on locations of species (herbarium collections with a cross-reference to collections represented in seed banks and other ex situ holdings) facilitates finding gaps in collections of each species. Some elaborated illustrated field guides are available on the genus Vigna, Cucumis and Abelmoschus (Fig. 3a). The size of the field guide should preferably be small (pocket size), with short diagnostic characters descriptions and photos that can be seen without the fine equipment (avoid the use of micro-characters in the description). Images from herbarium specimens though maybe less attractive than those of the live plants but are authentic source for identification. The rare and threatened plants of an area are rarely available as photographs/pictures; for such purposes, the images of herbarium specimens (validated from expert/ herbarium resource) could be used.

Figure 3a Some field guides

Illustrative Field Aids for Identification of Plants (Anjula Pandey et al.)

Figure 3b. Field guides on grasses identification (Yadav et al. 2010) Checklist: a check-list of plants with notes on probable sites of availability area of visit (shown on a map with dots), commonness/ rarity of use (if any) in the area can be appended at the end of a field guide; this is always helpful and enhances the utility of guide. Depending on the resources as well as the purpose of use of the field guide, printed or digital versions can be developed. An identification key to the species may be put at the beginning or end of the treatment. Horticultural field guides: to facilitate identification of most common horticultural species, especially popular flowers guides have been prepared with coloured pictures (Flowers of the Himalaya 1984 by Polunin and Stainton; Trees of Delhi 2006 by Pradip Krishen).

Field identification keys: field keys are based on the same principle as that of the taxonomic “Identification Keys” but are practically presented

Illustrative Field Aids for Identification of Plants (Anjula Pandey et al.) as a shorter and reduced version of the expanded keys that are available in floras/ monographs. These keys can be developed based on the purpose of use; may have photographs/illustrations on traits of fruit, seed, leaf, bark, canopy structure etc. For example, field keys for seedling identification should be based on seed and seedling characters (Yen et al. 1986). For PGR collectors the preferable traits could be the mature pods, seeds, bulb rather than leaves, flower, inflorescence characters which are seldom available at the time of germplasm collection. Keys based on mature fruit and seed characters are helpful to delimit related wild species (Pandey et al. 2005; 2014). Seed identification handbook: seed identification aids are generally in the form of seed atlas (region, state-wise, or other order) or identification handbook. Taxa could be arranged species-wise, family-wise, geographic region-wise (alphabetically) on the basis of use which may facilitate the user in the identification process (Fig. 4). Such aids are used by seed testing personnel, weed identification experts or those handling the germplasm of PGR especially conservationists dealing with wild taxa and crop wild relatives. While collecting or handling the germplasm some characters of seed may be lost. The seed to be identified should be healthy, mature, and complete without any damage. To reach the correct conclusion one needs to examine the maximum of five seed samples (from 5 lots). Scientist continuously involved in identification works can create his own collection and developing field- aids. In case of doubt on identity, he may seek the expert opinion or rely on the grow-out test to have confirmatory identification. This aid can help in identification more at family and genus level. Developing field illustrative guides depends on the purpose of use, the funds available and target users. To prepare the guides with information and data on characters, there should be: a) regular visits to the area of occurrence of the species (different seasons); b) study and observation on special features of flower, fruit, seed, and underground parts and capturing photos using close-up lens in fresh form so as to help correct identification (Annexures I, II); records of good taxonomic characters based on which key to be made, micro-characters (small seeded taxa, grasses); d) observations on habitat and photos with associated information on soil, flora etc.; and e) comparative study with related species to identify a new variant.

Illustrative Field Aids for Identification of Plants (Anjula Pandey et al.)

Figure 4. Seed identification aids with digital images Some modern tools based on signal based image processing technique can be used. Plant structures (leaf, flower, seed testa structure) are crucial parts in determining the characteristics of a taxon. Such tools based on image analysis based approach for distinguishing species (Bhattacharyya et al. 2011) and use of ‘Machine Vision for character Identification’ at IARI have been attempted out minute differences in cultivars/variants. Digital herbarium: digital herbarium or virtual herbarium are good resource for handy use and more easy to consult for identification. Well prepared herbarium specimens available in digital form are the aids for identification of taxa, or variants. These resources can also be referred for advanced study (biosystematics, chemotaxonomy, or molecular work). These images can be stored in tablet, digital album for use in the field to work as handy resource for identification. One should make use the ‘Type Images’ with experts ‘Determinavit slips’ or ‘Comments’ readily available in the web link as authentic source for identification and after matching can confirm the identity (refer Chapter 2).

Illustrative Field Aids for Identification of Plants (Anjula Pandey et al.)

Figure 5. Seed data base on medicinal plants (http://firoz-pc/MDP/) Advantages and disadvantages of using aids illustrative guides Each one of the field aid has its own advantages and disadvantages. These aids may represent only a part of the diversity represented. For seed identification only grow-out are the ultimate methods for correct identity especially in case unknown taxa. Herbarium resources tend to lose characters on drying. Field guides and filed libraries with micro characters can give the confirmation only after the microscopic study. Preparation of these aids takes a long experience and time consuming exercise and needs to be upgrade/update. For using all these, a basic knowledge on identification up to the family level will give best utility. Conclusions Field identification aids are in different forms but their basic purpose is not only strengthening the knowledge, scientific and technological skill but also carry wider scope in terms of PGR value. Process of developing these aids is an ever evolving and demanding area that needs routine input from experienced field workers. Tools and methods for field surveys and predictive characterization for collection and conservation of PGR can make identification aids functional through data software in more scientific way. References/selected readings Bhattacharyya D, Kim Tai-hoon and Lee Gang-soo (2011) Leaf Image Analysis Towards Plant Identification. Communications in Computer and Inform Sci 260: 113-125.

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Davis PH and VH Heywood (1963) Principles in Angiosperm Taxonomy. University of Edinburgh Press, Great Britain. Jain SK and RR Rao (1977) A Handbook of Field and Herbarium Methods. Today and Tomorrow Printers and Publishers, New Delhi. Lawrence GHS (1951) Taxonomy of Flowering Plants. Macmillan Publishing Co., Inc., New York. Leggett R and BK Kirchoff (2011) Image Use in Field Guides and Identification Keys: Review and Recommendations. AoBPlants. 1 (https://doi.org/10.1093/aobpla/plr004) Maheshwari JK (1966) Flora of Delhi- An Illustrated Guide. Council for Scientific and Industrial Research, New Delhi. Pandey A and R Pandey (2005) Wild Useful Species of Allium in India- Key to Identification. Indian J Plant Genet Resour 18(2): 180-182. Pandey A, K Pradheep and DP Semwal (2014) Notes on Luffa (Cucurbitaceae) Genetic Resources in India: Diversity Distribution, Germplasm Collection, Morphology and Use. Indian J Plant Genet Resour 27: 47-53. Polunin O and A Stainton (1987) Flowers of the Himalaya. Oxford University Press, New Delhi. Krishen Pradip (2006) Trees of Delhi: A Field Guide. Penguin Books, India. Vincent H et al. (2013) A Prioritized Crop Wild Relative Inventory to Help Underpin Global Food Security. Biol Conserv 167:265-275. Yadav SR, MM Aitawade, S Shrikant, KV Bhat, M Latha, Joseph K John, SKMalik, S Umdale and SR Rao (2014) Genus Vigna Savi In India- An Illustrated Guide for Species Identification. National Bureau of Plant Genetic Resources, New Delhi. Yadav SR, KV Bhat, MLatha, Joseph K John, SK Malik, MM Aitawade, SRRao, S Scariah, M Nissar, S Umdale, P Patil, G Krishnan and R Khedsana (2014) An Illustrated Guide for Species Identification of Vigna Savi, Cucumis L. and Abelmoschus Medik.- Species in India. National Bureau of Plant Genetic Resources, New Delhi. Yen M, H Yuasa and K Kyoda (1986) Identification of the Food Legumes Based on the Seed Morphology. J Agric Assoc China (repr version), New Series no. 133.

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Floras, Monographs, Taxonomic Revisions and Identification Keys Anjula Pandey Division of Plant Exploration and Germplasm Collection National Bureau of Plant Genetic Resources, New Delhi-110 012, India Introduction The conventional taxonomic and systematic documentation in the form of floras and monographs include multi-dimensional research information. The information contained is very useful in the formulation of plant diversity in a region and are helpful in the planning of local projects. They help the environmentalists, conservationists, students and local botanists in scientific research. For example, a project on conservation of threatened plant species of Narmada Dam Basin in Madhya Pradesh would be facilitated by information given in Flora of Narmada region. Many of the modern floras are now available as an e- resource. For example Flora of China (www.efloras.org/flora_page.aspx?flora_id=2); Flora of Pakistan (www.efloras.org/flora_page.aspx?flora_id=5). Very old floras can also be made available as archival documents (http://www.biodiversitylibrary.org/item/13814#page/3/mode/1up). Taxonomic work for identification of plants, especially when these are unknown indigenous plants, they are identified usually by the aid of a manual or flora of an area. Key (an important component of a flora) is an artificial analytical device useful in identifying an unknown plant species. In a key a choice is provided between two contradictory propositions resulting in the acceptance of one and rejection of the other. Specimens somewhat similar but distinct in other respect need to be further referred to treaties such as the floras, taxonomy books, illustrations, etc. to work out distinctiveness as type or a species (new variant). In this chapter documentation in the form of treaties like floras, monographs, taxonomic revisions and identification keys are discussed in detail.

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Floras The main component of flora includes keys, usually descriptions of families, genera and species, and often illustrations. They may include a citation to original publications of taxonomic groups, complete synonymy, illustrations and sometimes information on plant uses or ethnobotanical notes. Keys to all taxa, full synonymies, citations of specimens examined, distribution maps, habitat descriptions; a taxonomic treatment which constitutes the author’s classification of all taxa in this group; and a discussion of phylogenetic relationships. Flora is an inventory of plants of a defined geographical region. It deals with the systematic arrangement of the species of a given area/ region, usually restricted to a major segment of the plant kingdom (flowering plants etc.). Some floras of India and links are: Flora of British India: Hooker, J.D.,Flora of Andhra Pradesh by Sharfudding Khan; Flora of Andhra Pradesh: Flora of Andhra Pradesh by RD Reddy and Flora of Delhi by JK Maheshwari. Floras are the strongest tool that can help in reaching the conclusion and identifying a species. In addition to the large regional floras, local floras are also published. These included treatments on smaller areas as districts, geographical regions (Flora of Himalayan region, Flora of Cold Arid Regions of Himalaya, District Flora of Bhopal, etc.). Latest floras or check-list of particular region/ area are the best referable literature before preparing keys. Most floras of a state, region, country lack keys and description. They have an index to the plant known for the locality, prominent distributional data (rare/ common). The floras may be classified into four major groups based on the coverage of contents: a) Local flora (limited to geographical area, usually a district, city, a valley, mountain range or an islands, e.g. Flora of Bhopal, Flora of Delhi, Flora of Western Ghats); b) Regional Flora (wider geographical area, Upper Gangetic Plains; phytogeographical region. e. g. Flora USSR, Flora Iranica); c) Country Flora (countries plant records- The Flora of British India, Flora of India, Flora of China, Flora of Pakistan); and d) Continental flora (with coverage of the entire continent- Flora Europaea, Flora Australiensis, Flora of North America). While using floras for identification, one should preferably use the flora from the lower region to higher. The floras covering a smaller region are helpful in quick identification of taxa. The floras follow the system of plant arrangement according to one or another classification systems (Benthem and Hooker, Engler and Prantle, Bessey, Hutchinson, etc.). The world floras (Bentham and Hooker’s Genera Plantarum, Engler and Prantl’s Die naturalichen Pflanzenfamilien), do not treat units below the category of the genus, and some of them do not account for units below the category of family.

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Monographs and taxonomic revisions A monograph or taxonomic monograph is a treatise including all significant information of a morphologic or taxonomic coverage of the group (family or genus). It is a comprehensive taxonomic treatment of a group of plants, usually genus or family. It provides all the available taxonomic data about a particular group of plants. A monograph is usually global in its geographical scope because without including the taxonomic details of all the known members of a taxon (i.e. genera, species, subspecies), it is impossible to propose a sound classification and phylogeny for that taxon. They contain detailed studies extracted from various sources and attempt to bring the data available on taxa or group; they include detailed keys, citation to original publications, list of synonymy, list of specimens studied, etc. Some classical examples of monographs are: The Genus Nicotiana by Goodspeed (1955), The Genus Datura by Avery et al. (1959); Biosystematics Monograph of the Genus Cucumis (Cucurbitaceae): Botanical Identification of Cucumbers and Melons byJoseph H Kirkbride(1993).Monograph on Crotalaria in India by AA Ansari (http://www.abebooks.co.uk/Crotalaria-L-India-Ansari-A.A- Bishen/1170289873/bd) (Ansari 2008) gives details of taxonomic treatments, synonymy, and illustrations in this genus in India, with information on pollen structures, cytology, etc. A taxonomic monograph is a complete account made at a given time of anyone family, tribe, or genus, considering all necessary knowledge available then. They also interpret the phylogeny of the group in relation to data available from different fields of biology such as morphology, anatomy, embryology, cytology, palynology, chemistry, molecular biology, palaeobotany, ecology and phytogeography. Monographs are more specialized documents in a group of the plant with deep information on taxonomy and bring out supplementary information to floras and manuals or are published as book, booklet and comprehensive work in a research journal. While writing a monograph, extensive literature review on detailed reports on the original research work done by the author, nomenclatural information including designated type specimens, exhaustive descriptions and experience gained in the specific field the monographer specialises in the area, group or taxon. All elements of the treatise are accounted for by dichotomous keys, full synonymies, complete descriptions, precise designations of types, together with notes as to where the types are deposited, citations of specimens examined,

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Floras, Monographs, Taxonomic Revisions and Identification Keys (Anjula Pandey) distributional ranges (supplemented by maps), notes on habitats, and discussions of taxonomic and nomenclatorial considerations. Taxonomic revision: a taxonomic revision is comparatively less detailed than a monograph, and differs from the latter primarily in its degree of scope and completeness. Often it accounts for only a section of a genus or for the elements as restricted to a continent or smaller geographical area. In order to make revisions, comprehensive work on morphology, anatomy, cytology/cytotaxonomy, genetics, ecology, and herbarium studies are needed. It includes complete synonymy, keys to identify the taxa within the group, diagnostic descriptions, distribution maps and the classification scheme. Identification keys An identification key is an artificial analytical device useful in identifying an ‘unknown plant species’. In a key, a choice is provided between two contradictory propositions resulting in the acceptance of one and rejection of the other. Knowing the identity of the group to which the given specimen belongs we depend on existing material to compare. Diagnostic keys, field identification keys, other aids are available for reaching at correct recognition to plant identity. Field keys are very simple to use the broadest characters visible without any minute details, may have pictures to diagnose a species. The keys available in flora, manual, guide, handbook, monograph or revisionary works can be consulted for identification of the above group. If the locality of the unknown plant is known, select a flora, guide or manual treating plants of that geographic area. If the family or a genus is recognized, one may refer to a monograph or revisionary work. If the locality is unknown select a general work. If the material to be identified is a cultivated taxon select a manual dealing with a cultivated plant of a region/ country. While dealing with crop(s) and crop wild relative(s), we must consider the following points: Floras/ keys: cultivated species or crop wild relatives to be identified using one treating cannot be done as most floras do not include cultivated plants unless they are naturalized. Stage desired: (germplasm collected/received as vegetative part (tuber/cuttings, etc.), or as a fruit/seed/economic product). The identification generally requires a ‘grow out test’ (the plant has to be grown in an experimental area for a complete study of the characters.). In floristic keys, these characters are not sufficient enough to establish

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Floras, Monographs, Taxonomic Revisions and Identification Keys (Anjula Pandey) correct identity. Such material may be identified after analyzing the characters from fully grown plants. Cultivars of crop taxa: cultivars are distinguishable by some specific characters only, and the standard herbarium specimens of such material are seldom available as reference. However, identification keys may be developed only for selected taxa. Using an identification key Key is a printed or computer-aided device that aids the identification ofplants. Traditionally, identification keys have single. These work by offering a fixed sequence of identification steps, each with multiple alternatives, the choice of which determines the next step. If each step has only two alternatives, the key is said to be dichotomous, else it is polytomous. A key is prepared for identification only after a complete study of all species for which key is to be prepared. Recording of the morphological characters is done for most visible, identifiable and clear cut features from a live sample of the group. For characters of the size of plant, leaf shape, size and other such details examine the whole range of variation. Avoid overlap of character range to avoid any confusion. A dichotomous taxonomic key contains information useful in identifying similar kinds of organisms. It is a list of paired statements (each statement is called a lead) that guides you to the identification of a specimen. The paired leads (the pair of leads together is called a couplet) are contrasting descriptions of certain characteristics. In a good key, couplets are written in such a way one or the other of the leads match with the specimen to be identified. Couplet leads should always be mutually exclusive. Modern multi-access or interactive keys allow the user to freely choose the identification steps and their order. At each step, the user must answer a question about one or more characters of the entity to be identified. For example, a step in a botanical key may ask about the colour of flowers or the disposition of the leaves along the stems (http://www.botanicalgarden.ubc.ca/links/plant-identification-key- making-tools). There are two major types of keys in use: indented or yorked key: most commonly used key in taxonomic literature such as floras, manual, etc. The other is bracketed or parallel key: no indenting but couplets are placed at equal distance and at the end plant identification or a number refereeing further down below and this process keeps on till plant name is designated. How to prepare keys

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 Key to be strictly dichotomous  First word of each couplet to be indented; should be identical, ie, if it starts with leaf character, second should also start with leaf character.  The two leads of one couplet should be contradictory to one another so that in each couplet of the two prepositions one of which will follow and the second will not apply  Do not use the average, range or overlapping data for plant characters; Plant height 2-3 m and another plant up to 3.5m  Plant part must be mentioned first followed by its description  Separate keys for flowering and fruiting specimen/ male-female specimens may be prepared when necessary.  Use of micromorphological data, cytological data, distributional data to be avoided as they may not give the comparative data. Suggestions for use of keys  Gather as much information as possible about the characters of the unknown plant before starting identification with the help of a key. Attempts to use the key may fail if a specimen is incomplete (consists of a flower or leaf only).  Select keys appropriate for the plant material and for the geographic area where the plant was collected.  Read the introduction to the key to understand the abbreviations and other details. Consult a dictionary or glossary to know the correct meaning of new terms which you may come across in the key.  Always read both choices carefully, observing punctuation. Sometimes, the first statement appears to be correct with reference to the unknown plant specimen but the second statement may be more appropriate and could lead to proper identification of the unknown specimen. If both choices seem possible, try going both ways.  Confirm your choices by reading a description of the identified plant.  Verify your identity by comparing the specimen with an authentic illustration or with an authentic herbarium specimen.  If the specimen does not seem to fit the key and all choices are unlikely, you probably made a mistake. Retrace your steps. Field identification keys: field identification keys are modified versions of the traditional keys and developed for convenience for field use. Illustrated guides for identification of taxa can serve a good purpose and are also user-friendly (Yadav et al. 2014). (Refer to chapter 8 for details).

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Based on traits of fruit, bulb, seedling etc., keys have been designed for easy identification (Pandey and Pandey 2005; Pandey et al. 2014). While working in groups, obtain one specimen of each species, and construct a dichotomous key to the specimens. Limit the use of colour as a character. Flower or leaf colour may fade in preserved specimens and may vary among individuals in different growing conditions. Morphological characters such as the overall shape of the canopy (tree), stipule/ spine, leaf shape, flower characters are used. One should draw structures so that it can be used in the key. Using taxonomic literature/ manuals determine the family to which this plant belongs. Artificial keys are used for identification of plants family; using the key to genera. Knowing the name of the family one turns to the section where it is treated. Using the key to genera, repeat the procedure to reach to the generic name; and repeat the process. There are problems in usage of taxonomic keys: 1) as they cannot be applicable to variant form or for part/ flower/ seed only; 2) do not include diverse forms/ taxa that have been poorly characterized; 3) they excluded specimen size, colour variant from perception, the magnification, point; 4) they excludes newly described taxa and ; 5) language is technical and difficult to understand. Key for identification of Allium species in India 1. Roots thick, sometimes sub-tuberous; bulb not developed/ formed, scape usually angled 2. Perianth white 3. Root elongate; leaves linear to broad, shorter than or sub- equal to scape, midvein distinct; filament adnate to perianth segments A. hookeri 3. Roots short, tuberous/fasciculate; leaves linear or lorate to lorate- oblanceolate, usually longer than scape, midvein obscure; filament subulate A. fasciculatum 2. Perianth coloured (pink, red, purple) 4. Pedicels straight, perianth red, purple or blackish purple, stellate, reflexed after anthesis, inner and outer segments similar, A. wallichii 4. Pedicels nodding at apex, perianth pink, campanulate, not reflexed after anthesis, inner segments somewhat longer and narrower than outer ones, A. macranthum

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1. Roots thin, fibrous, bulbs well developed, scape usually cylindrical or solid 5. Leaf slender, flat, linear 6. Bulbs cylindrical, leaves arising from rhizomatous base A. tuberosum 6. Bulbs globose to ovate, leaves arising from the bulb/bulblets A. sativum 5. Leaf fistular 7. Bulb oval-elliptic, generally large, single or two A. cepa 7. Bulb narrowly ovoid, in clusters A. chinense (Source: modified from Pandey and Pandey 2005) Other methods of identification Among the other methods, multi-access keys are largely designed to serve the same purpose as single-access (dichotomous or polytomous) keys, but the advantages they have are in especially in the form of computer-aided interactive keys. The user of an interactive key may select or enter information about an unidentified specimen in any order, allowing the computer to interactively rule out possible identifications of the entity. Interactive keys may be equipped with images, audio, video, supplemental text, much-simplified language and hyperlinks to assist the user for better understanding. The problem of not having a reliable and complete description to compare a specimen with is alleviated by full-service interactive keys that include or link to such descriptions and authoritatively identified images. In case of paper-based dichotomous keys, with the discovery of a new species the key turns incomplete; however, interactive keys are easily updated by adding information for newly discovered species and reposting computer files on the internet. Many different computer programmes for interactive keys are currently available. Popular full-featured free software packages are DELTA, Discover Life and Xper2. An example of an open-source package is SLIKS. Computerized identification Therefore, in today’s IT-driven world, plant taxonomy has progressed with the computerized identification of plants. Special software has been prepared for providing easy identification of plants. A very important requirement of using the GPS/Google guide while travelling is “proper mapping of the area”. In the same manner, it is necessary to remember that a thorough botanical knowledge is the most important requirement for preparing the appropriate computer programmes which can be used for the identification of plants. Thus, as indicated in the introduction, a

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Floras, Monographs, Taxonomic Revisions and Identification Keys (Anjula Pandey) detailed characterization of the plants will play a very prominent role in providing the basic data even for computer programmes used in identification. It becomes imperative for taxonomists to analyze a large number of plants to recognize the distinct characters which can be used in generating computerized identification. As in identification by dichotomous keys, the identification of plants by polyclaves or computers requires the confirmation of the identification by reading a detailed description and comparison with an authentic specimen. Interactive keys Interactive keys (also called multi-access keys) work as single-access (dichotomous or polytomous) keys, but have many advantages, especially when computer-aided interactive keys are used. The computer-aided interactive keys, allow the user to freely choose the identification steps and the sequence in which they may be used. At each step, the user must answer a question about one or more features (characters) of the entity to be identified. For example, in a key one may ask the symmetry of flowers, stem structure (whether solid or fistular) or the feature of leaf surface (whether glabrous, i.e. without any hairs or pubescent - having hairs, etc.) or texture (whether membranous or chartaceous, etc.). The identifier using an interactive key may enter information about an unidentified specimen in any order, allowing the computer to interactively rule out possible identifications of the entity. Interactive keys are generally equipped with images, audio, video, supplementary text, much-simplified language and hyperlinks to facilitate the identification process. In traditional published dichotomous keys, the new plant descriptions/ new species are not included. Interactive keys are easily updated by adding information on newly discovered species and reposting computer files on the internet. Some of the popular programmes for interactive keys are Lucid, DELTA, Discover Life and Xper, etc. The DELTA format (DEscription Language for TAxonomy) is a powerful method of recording taxonomic descriptions for computer processing. It was adopted as a standard for data exchange by Biodiversity Information Standards (TDWG) (http://delta- intkey.com/www/overview.htm). The system was developed in the Division of Entomology, CSIRO, Australia. The facilities available include the generation and typesetting of descriptions and conventional keys, conversion of DELTA data for use by classification programmes, and the construction of Intkey packages for interactive identification and information retrieval. The DELTA System can produce high-quality

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Floras, Monographs, Taxonomic Revisions and Identification Keys (Anjula Pandey) printed descriptions. The book: ‘The Grass Genera of the World’ (CABI International: Wallingford), generated automatically from a DELTA database, contains descriptions of about 800 genera in terms of more than 500 characters. Conclusions Different taxonomic treaties in the form of flora, monographs, revision, keys etc. have their own advantages for use in plant systematic research. The steps to construct keys will facilitate in understanding the character and character states of a taxon. References/ selected readings Ansari AA (2008) Crotalaria in India. Bishen Singh Mahendra Pal Singh, Dehra Dun, India. Avery AG, S Satina and J Ritsems (1959) The Genus Datura. In: Blakeslee AF (ed) Chronica Botanica, No. 20. Ronald Press, New York, USA. Chakravarty HL (1982) Cucurbitaceae. Fascicles of Flora of India: Fascicle 11, Botanical Survey of India, Calcutta. Davis PH and VH Heywood (1963) Principles in Angiosperm Taxonomy. University of Edinburgh Press, Great Britain. Goodspeed TH (1955) The Genus Nicotiana. Chronica Botanica Company, Waltham, Mass., and Stechert Hafner, Inc., New York. Hajra PK and M Sanjappa (ed) (1996) Leguminosae. Fascicles of Flora of India: Fascicle no. 22, Botanical Survey of India, Calcutta. Kirkbride Jr JH (1993) Biosystematic Monograph of the Genus Cucumis (Cucurbitaceae): Botanical Identification of Cucumbers and Melons. Parkway Publishers, North Carolina, USA Lawrence GHS (1951) Taxonomy of Flowering Plants. Macmillan, New York. Pandey Anjula and R Pandey (2005) Wild Useful Species of Allium in India-Key to Identification. Indian J Plant Genet Resour 18(2):180- 182. Pandey A, Pradheep K and Semwal DP (2014) Notes on Luffa (Cucurbitaceae) Genetic Resources in India: Diversity Distribution, Germplasm Collection, Morphology and Use. Indian J Plant Genet Resour 27: 47-53. Pullaiah T (1998) Taxonomy of Angiosperms. Regency Publications, New Delhi.

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Saldanha CJ and CK Rao (1975) A Punched Card Key to the Dicot Families of South India. Centre for Taxonomic Studies, Bangalore. Singh G (2011) E-flora: the future of floristic documentation. In: Gupta R (ed) Plant taxonomy: past, present and future. The Energy and Resources Institute, TERI Press, New Delhi, pp329-350. Singh P and Singh V (2001) Monograph on Indian Subtribe Cassiinae (Caesalpiniaceae). Scientific Publishers, Jodhpur, India. Yadav SR, MM Aitawade, S Shrikant, KV Bhat, MLatha, Joseph KJohn, SKMalik, SUmdale and SR Rao (2014)Genus Vigna Savi in India- An Illustrated Guide for Species Identification. National Bureau of Plant Genetic Resources, New Delhi. Yen M, H Yuasa and K Kyoda (1986) Identification of the Food Legumes Based on the Seed Morphology. J Agric Assoc China (repr version), New Series no. 133. Some selected links http://www.efloras.org/florataxon.aspx?flora_id=2&taxon_id=101086 http://www.efloras.org/florataxon.aspx?flora_id=5&taxon_id=101086 http://www.efloras.org/florataxon.aspx?flora_id=1&taxon_id=101086 http://linnean-online.org/view/collection/linnean=5Fherbarium/Allium.html http://www.botanicalgarden.ubc.ca/links/plant-identification-key-making- tools

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Biosystematic Studies on Crop Plant Taxa Anjula Pandey and K Pradheep Division of Plant Exploration and Germplasm Collection National Bureau of Plant Genetic Resources New Delhi-110 012, India Introduction In the Indian gene centre, some of the major crops like brinjal, cucumber, melon, sesame, okra, etc. and their crop wild relatives(CWR) have unresolved issues on the origin and diversity. Issues on identity with respect to allied/related species can be resolved using evidence from morphology, cytology, cytogenetics, and molecular tools. The term ‘Biosystematics’ deals with the evaluation of phenotypic expression taking evidence from fields of morphology, ecology and phytogeography, supplemented from cytogenetics and cytotaxonomy and molecular evolution of genes in determining the taxonomic hierarchy. The term was first coined as 'Biosystematy' (by Camp and Gilly 1943) which later changed to Biosystematics. Biosystematics in a broader sense covers “the study of biodiversity and its origins”. Biosystematics is also called population systematics and new systematic. Tools such as predictive characterization models can facilitate in better understanding of useful diversity in genetic resources programme including issues relating to origin and diversity through the use of biosystematics. The classical systematics is mainly based on the study of mor•phological characters of few specimens with supporting evidence from other fields. It strives to bring out evolutionary relationships amongst organisms. In taxonomic literature, besides this term, the term ‘Taxonomy’ and ‘Systematics’ are invariably and interchangeably used. These two use data from morphological, behavioural, genetical, and the biochemical observations; taxonomy mainly differs from the systematics in the classification and naming of organisms whereas the latter deals with the determination of evolutionary relationships among taxa (some think taxonomy as a subset of systematics). These can help in resolving nomenclatural disputes, delimitation of taxa by establishing systematics relationship among domesticated and wild taxa and use of modern tools. This chapter briefly discusses about the biosystematics, taxonomy and systematics of selected taxa studied by the authors at ICAR-NBPGR

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Biosystematic Studies on Crop Plant Taxa (Anjula Pandey et al.) under the institute project. The systematics study of selected taxa- Allium (representing diversity rich region) and Trichosanthes, Luffa and leafy Amaranthus (Indian centre of origin and diversity) were taken up with primary data from field/ herbarium study and experimental work. Biosystematics study on selected taxa Since the inception of the ICAR-NBPGR, biosystematics work has always been a high priority more particularly during the 1980s when taxa like Asiatic Vigna, Macrotyloma, and other legumes were collected and studied. Sub-project on biosystematics of the genera Vigna, Cucumis, Abelmoschus was taken up by ICAR-NBPGR [in collaboration with Shivaji University, Kolhapur, North Eastern Hill University, Shillong (NEHU) under National Agricultural Innovative Project (NAIP)] with prime objective to resolve issues on the lines of biosystematics using tools such as micro- morphology, crossability study, wide hybridization, conservation of wide crosses, meiotic study in inter-specific hybrids, molecular cytology and molecular taxonomy was completed. During 2013-18 some of the prioritised taxa i.e. Allium, Trichosanthes, Amaranthus and Luffa were studied through the field, experimental and herbarium work under the institute projects with the following objectives (Fig. 1):  Delineate taxon boundaries, update on the taxonomic status and ecogeography of wild and cultivated taxa  Trace the morphological relationship among various taxa with an emphasis to closest relatives of cultivated taxa  Develop field diagnostic keys (mainly using characters of fruit, seed and vegetative parts) for species identification and use by the PGR personnel. Samples of the population belonging to the same taxonomic category (species, sub-species) from different environments were grown in a common experimental plot/controlled environmental conditions. Selection of crop taxa: a selection of taxa depends on the level of priority to which a crop may fall in national priority and research priority; however the rare/threatened and endemic taxa always attain top priority.

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Biosystematic Studies on Crop Plant Taxa (Anjula Pandey et al.)

Figure 1. Flow chart on activities on biosystematics studies on taxa of PGR importance In Trichosanthes in addition to rich species diversity, there are identity and nomenclature-related issues. In Allium wild taxa related to cultivated and less-known useful types were taken up. Field and herbarium studies were related and characters were recorded during explorations in areas of availability of the species. Usually ten plants were used to record data in field/on-site; additionally, 3-5 herbarium specimens were collected. Taxonomically good characters were noted especially for micro-characters (bulb membrane, the pattern of flower opening, tepals and stamens) in Allium and characters of probract, sepal, fruit and seedin Trichosanthes, flower and seed in Amaranthus and flower, pericarp, seed, and fibre in the fruit of Luffa.

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Need-based herbarium studies were undertaken to understand character variation with respect to eco-geography and distribution range. Rare/endangered species (e.g. Allium gilgiticum), not observed from the area of distribution was studied from herbarium material). The images of seed morphology in Allium and wild Luffa were captured using Image Analyzer; in some cases where the seed coat morphology did not show the clear distinction on the pattern of testa, the scanning electron microscopic images were taken (300dpi). The vouchers of field-collected and experimental material was prepared as herbarium specimens and deposited in recognized herbaria such as NHCP. Case study Allium Genus Allium represents tremendous genetic diversity in the Indian region. Of about 35-40 species reported from the Indian region, species diversity concentrated in alpine and temperate zones of Himalaya. Wild taxa of Allium occur as a part of natural flora, as less-known wild species and sporadic cultivation in backyards. The utility of wild species as donors of genes to widen the base of crop plants has earned wide acceptability. The accomplishment of inter-specific crosses between Allium cepa, A. sativum and A. roylei (Indian wild species) has indicated the possibility of achieving this goal. Transferring resistance trait from A. roylei to A. cepa for powdery mildew (Peronospora destructor) and for leaf blight (Botrytis squamosa) (de Vries et al. 1992) has opened a new avenue for using the Indian species. A. atrosanguineum subsp. atrosanguineum, A. atrosanguineum subsp. fedtschenkoanum, and A. semenovii have been prioritized as they are distant relatives of the cultivated Allium (onion and garlic). A. roylei (wild species that belongs to different subgenus) has been used in onion breeding and shares similarity with cultivated onion based on molecular study needs further study. Also many wild Allium taxa under-protected habitats or grown in home gardens (in Himalaya) without much changes in characters from the wild types need to be studied for change in character under domestication. Flower characters were compared among newly collected variants of A. tuberosum (from Leh, Jammu & Kashmir) and five accessions collected earlier from north-eastern hill region and western Himalaya (Uttarakhand). Preliminary studies showed that pink-flowered forms from Jammu & Kashmir were apparently similar to white-flowered forms. Phylogenetic and molecular evolutionary analyses have confirmed that Allium tuberosum is distinct from A. ramosum (EC328498) on the basis of the sequence of ITS region amplification. Field identification key was

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Biosystematic Studies on Crop Plant Taxa (Anjula Pandey et al.) developed for Indian alliums for sect. Cepa and Bromatorrhiza. Species of subg. Amerallium, section Bromatorrhiza - A. hookeri, A. fasciculatum, A. wallichi and A. macranthum were well distinguishable based on bulb and root characters. Allium hookeri (two variants) were studied for selected characters in field genebank at Bhowali showed variability in plant height, leaf and inflorescence characters. Endemic species of Allium viz. A. kachrooi, A. rosenbachianum and A. oreoprasum, and A. farctum Wendelbo, and A. rhabdotum using herbarium specimens at University of Kashmir, Srinagar. Distinct identity of Himalayan species, often confused/merged with allied ones such as A. przewalskianum Regel vs (A. stoliczkii Regel), A. fedshenkoanum Regel (vs. A. semenovii Regel), A. prattii C.H.Wright (vs. A. victorialis L.) and A. fasciculatum Rendle (vs. A. hookeri Thwaites) was confirmed through this study. While characters such as tunic texture, leaf cross-section and filament vs tepal length are important to distinguish subg. Cepa from other subgenera, bulb shape, leaf vs scape length, nature of scape (hollow/ solid), tunic colour, presence of bulbils and perianth colour are useful in delimiting taxa within it. The molecular systematic study confirmed the distinctly-shaped (clavate) onion (locally called ‘dunna’), cultivated sporadically in Himalaya and NEH region as one of the morpho-types of A. cepa var. aggregatum, based on matK sequences. Assessment of diversity: Western Himalaya recorded maximum concentration (over 85%) of wild species followed by Eastern Himalaya (6%); others from both the region (9%). Vertical distribution of species was more confined to the alpine-sub-temperate region (2500-4500m; 29 species), followed by subalpine-alpine region (4500m and above; 5 species) and temperate region (1500-2500m; 4 species). A. fasciculatum and A. stracheyi were confirmed as species under domestication. Based on diversity and availability of the material in the field genebank, priority for uncollected diversity was worked out. A set of newly collected materials was maintained at field genebank by the ICAR- NBPGR RS Bhowali. During study, a total of 31 accessions (7 taxa) from field-collected material were raised in experimental conditions at Delhi and Bhowali. Field and herbarium study: live material maintained in the field genebank at Bhowali and raised in the experimental area in ICAR-NBPGR, New Delhi were used for the study of vegetative and reproductive morphological traits. Observations were recorded through IPGRI descriptor list for vegetative and flowering details: bulb (shape, size), membrane (colour), leaf (in section), scape (hollow/ round, angular), flower (anther structure), odour (onion-like-/ garlic-like) and seed characters (shape, surface). : Morphological study and grow out of aerial

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Biosystematic Studies on Crop Plant Taxa (Anjula Pandey et al.) bulbils of an unidentified Allium species collected from Himachal Pradesh, which was initially confused with unreported species from India i.e. A. vineale, was identified as Allium ampeloprasum. Herbarium based work involved study of over 350 herbarium specimens (700 digital scans) housed in herbaria of Botanical Survey of India (Dehradun and Shillong) and Forest Research Institute (Dehradun). Some of the neglected key characters studied included- plant odour (mild, strong, onion/garlic type), bulb membrane (texture), length of pseudo-stem, the pattern of flower opening in an inflorescence, stamen character (filament toothed/not toothed) and seed characters. Seed morphological study: seed morphological study was done in 31 samples (20 taxa maintained in NHCP) for seed shape, size, seed coat colour and ornamentation. Seeds were studied for micro-morphological characters (seed structure, size, shape, the ornamentation of testa, etc.) and seed shape. The images of seed morphology (11 species) were captured using Image Analyzer. Seeds of wild species were black- coloured, width ranging from 1 mm (A. tuberosum (IC353524; A. carolinianum VDV/AK/37) to 3 mm. A. fistulosum (IC353547) had the smallest, and A. tuberosum (IC353524)and A. ramosum (EC328498) hadthe largest-sized seeds; seed varied in shape (ovate, round- and showed only slight difference), and seed coat pattern (variable from convex, granulate to verrucose). Seeds of A. victorialis and A. carolinianum, could be identified on the basis of seed shape (using hand lens), while that of taxa like A. stracheyi, A. roylei and A. ramosum and A. tuberosum needed use of microscopic aids (high-resolution images) for delineating their identity. Developing identification key: identification key was prepared based on characters- bulb, leaves, scape and spathe characters, shape/size and compactness of inflorescence, the structure of capsules, seed/seedlings and quality traits as taste/odour, flower fragrance etc. These keys include characters over and above given in the available floras/ taxonomic works/published literature. Trichosanthes Field study: explorations/field-trips were carried out across the country mainly in parts of Assam, Andaman, Madhya Pradesh, Uttar Pradesh, Western Ghats, West Bengal, Odisha, Eastern Himalaya, Manipur and Meghalaya, and Rajasthan in order to understand the diversity in native habitat across diverse area of distribution of various species and to collect germplasm for study/conservation. Field notes such as place of collection and habitat particulars, habit, sexual condition and those characters lost on drying the herbarium specimens were taken. About

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200 herbarium material augmented during the study were submitted to NHCP. Accessions showing interesting/unique traits were further grown to corroborate the findings. Augmentation of material and experimental study: Accessions augmented/collected during field study and also augmented through various sources such as genebank collections at national and regional level formed the material for study at ICAR-NBPGR, New Delhi (in pots) and at ICAR-NBPGR Base Centre, Cuttack (in experimental field). About 5-10 plants/accession were maintained and observations were taken on 65 characters (using a number of character states; Appendix II). Herbarium study: herbarium study involved about 1,200 specimens from NHCP, Central National Herbarium, Kolkata, Port Blair, Coimbatore, Jodhpur, Gangtok and two Regional Centres Botanical Survey of India (Dehradun and Shillong), Forest Research Institute (Dehradun) and North-Eastern Hill University (Shillong). Botanical notes were taken in the prescribed format, covering details on herbarium no., collector no., place and date of the collection along with remarks. In addition to it, the on-line digital herbarium of Royal Botanic Gardens, Kew (K), Royal Botanic Gardens, Edinburgh (E), Chinese Academy of Sciences Herbarium (PE), muséum national d'histoire naturelle, Paris (P); and of type specimens were also consulted. Two distinct forms of Trichosanthes bracteata, and distribution of T. himalensis in Eastern Ghats of Andhra Pradesh were first time recorded during field study of the region. Herbarium studies revealed that the specimen (M.S. Ramaswami 1398), claiming the presence of Gymnopetalum chinense in Andhra Pradesh, turned out to be Solena; also studies indicated the occurrence of (true) T. tricuspidata in Andaman Nicobar Islands. Type specimen (in Kew and Harvard Herbarium) study revealed that the type (♀ and ♂) of T. khasiana in fact represent two different species, and confirmed the occurrence of T. subrosea in India. A field study of Eastern Himalayan endemic species T. dicoelosperma C.B.Clarke was carried out; this possesses red pulp (akin to snake gourd) and seeds are tridentate on distal end (central one prominent). Herbarium studies indicated the extended distribution of T. cucumerina L. subsp. sublobata (Kundu) K.Pradheep, D.R.Pani & K.C.Bhatt in Gujarat and T. cordata Roxb. in terai region of Upper Gangetic Plains. Analyses of the rbcL sequence amplified in 71 accessions comprising of three species of Trichosanthes (T. cucumerina, T. dioica and T. nervifolia) and two species of Gymnopetalum (G. chinense and G. wightii) revealed that this locus has grouped almost all the accessions as per the species they belong to.

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Identification keys were developed for valid taxa using all the information obtained from the above studies, spot-characters enabling quick identification of various species in the field have prepared (Appendix II). The outcome of the work suggested modification in the key in the light of observations recorded and knowledge generated in the range of some continuous characters and overlooked underestimated or misinterpreted characters. Four taxa have been recognised as accepted ones namely T. cucumerina L. subsp. cucumerina (syn. T. lobata Roxb.); T. cucumerina L. subsp. cucumerina ‘Anguina’ (L.) K.Pradheep, D.R.Pani & K.C.Bhatt; T. cucumerina L. subsp. villosula (Cogn.) K.Pradheep, D.R.Pani & K.C.Bhatt (syn. T. perrottetiana Cogn.); T. cucumerina L. subsp. sublobata (Kundu) K.Pradheep, D.R.Pani & K.C.Bhatt;and their diagnostic keys worked out. Amaranthus Gap analysis revealed no collection of wild species - Amaranthuspolygonoides, A. x caturus, A. tenuifolius and different subspecies of A. graecizans, and poor representation of A. graecizans subsp. graecizans, A. blitum and A. dubius indicating the need for proper collection and identification. A. graecizans (4 accn.) and A. blitum (8) have been studied in detail; in A. blitum, characters like sub-erect plant habit and retuse leaf nature were observed to be stable, whereas ‘lanceolate’ bract shape as reported was validated as ‘linear’ in all the accessions. Study of types of intended taxa in virtual herbaria revealed a high level of variation in A. tricolor complex. Field studies revealed variation among cultivated and wild/weedy types in A. graecizans L. (in Rajasthan), A. tricolor L. (in Tamil Nadu and Jharkhand) and A. dubius Mart. & Thell. (in Jharkhand and Arunachal Pradesh), besides fast spread of A. palmeri S.Watson in North India. Amaranthus blitum, A. dubius, A. tricolor, A. graecizans and A. powellii were augmented from Assam, Jammu & Kashmir, Kerala, Nagaland and Tamil Nadu. During field study species distribution /cultivation/ naturalisation status, interspecific variation in relation to eco-geography and field distinguishing traits at taxon level was also observed. Experimental study: in A. tricolor complex (86 acc.) revealed some mistaken taxonomic identity in about one-third of the studied material. Morphological study for selected characters in this complex identified some stable characters (branching pattern of the lower stem, length of petiole vs lamina, leaf shape) and some linked characters. Diagnostic characters: bract vs tepal length, bract shape, tepal shape, branching of terminal inflorescence and filament length were unreliable. Amaranthus tricolor var.tristis is traditionally distinguished from var. tricolor by

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Biosystematic Studies on Crop Plant Taxa (Anjula Pandey et al.) branching from the base of the stem (vs branching above middle). Besides var. tristis is further different from the typical variety by stem being yellowish-green, petiole as long as lamina, leaves ovate with subcordate to obtuse base. A. mangostanus, presently subsumed under A. tricolor, deserves distinct identity at an infraspecific level due to semi- perennial, decumbent-ascending habit, rooting at nodes, small rhombic- ovate leaves. Herbarium study: studies made in Botanical Survey of India (PBL, Port Blair, CAL, DD, BSD, MH, Coimbatore) and PCM, Chennai and at Rapinat Herbarium, Tiruchirappalli indicated differences between wild and cultivated forms of A. tricolor and variation in spiny nature of Amaranthus spinosus under different habitats. Study of type specimen revealed that A. parganensis Saubhik Dasand A. bengalensis Saubhik Das & Iamonico are not sufficiently distinct from A. tricolor var. tricolor and A. mangostanus, respectively. Good taxonomic /field characters were identified for all taxa in leafy amaranth. Distribution of unfamiliar taxa − A. polygonoides L., A. tenuifolius Willd., A. x caturus B.Heyne ex Hook.f. was worked out. Meagre herbarium holdings of A. dubius indicated recent naturalisation of this weedy species in the country, supported by its exclusion in past floristic literature. Luffa Field and experimental studies: experimental study undertaken using 48 accessions (all 5 taxa) revealed that 18 out of 44 characters were of taxonomic value- leaf pubescence, stem angularity, probract shape, sex form, flower bud shape, flower colour, time of flower opening, calyx and petal colour, stamen shape (anther thecae), stigma length (vs stamen), fruit surface, fruit distal tip, flesh colour, sponge/fibre texture, seed shape, colour and surface. Some linked characters identified were: fruit ridges and seed surface (in L. acutangula); pericarp fibre netting pattern in wild vs cultivated taxa, and fruit shape and seed orientation. At infra- specific level, leaf shape, flower opening time, flower colour, stamen shape and fruit/seed surface were found to be important for classification. In contrast to the usual treatment of L. hermaphrodita Singh & Bhandari under L. acutangula (L.) Roxb. (on the basis of crossability and molecular evidences), there is a need to place this cultigen infra- specifically under ridge gourd owing to distinguishing characters such as short plant stature, earliness, bisexuality, small and less-fleshy fruit, loose fibre and small seeds. Grouping was evident based on data on fruit and seed characters in L. acutangula and a closely related cluster-fruit bearing L. hermaphrodita and also its solitary-fruited type. Luffa

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Biosystematic Studies on Crop Plant Taxa (Anjula Pandey et al.) acutangula var. amara from Uttarakhand and Rajasthan showed variation among fruit and seed characters especially mottled pattern. In L. echinata (at infra-specific level) characters studied in material from Haryana and Uttarakhand and plants grown under experimental conditions showed good variation. Experimental studies: gap analysis revealed the need for augmenting germplasm collections in L. echinata and L. graveolens across the distribution range especially north and eastern India (Bihar, Uttar Pradesh and parts of Madhya Pradesh). Poor growth of Luffa hermaphrodita (no healthy fruit formation), and L. graveolens in New Delhi indicated habitat specificity in these taxa. Herbarium studies: revealed the occurrence of L. echinata Roxb. in Andhra Pradesh, and the rarity status of L. graveolens Roxb. Luffa echinata studied in herbarium revealed that var. echinata as much variable in shape and size of fruit as well as echines; var. longistylis not deserve a varietal status (only available as type). Conclusions Biosystematics studies can help in resolving issues on identity and nomenclature. Based on prioritization, the focus is currently on a collection of not-yet-collected/niche-specific taxa/species and employing cytological and molecular tools to validate the research results obtained from the project. Micro-morphological investigation for seed coat texture, trichome, epidermal characters and pollen studies and other often neglected characters need to be studied to correlate the status among the related groups. References Camp WH and CL Gilly (1943) The Structure and Origin of Species. Brittonia 4: 323-385. de Vries JN, WA Wietsma, and de T Vries (1992) Introgression of leaf blight resistance from Allium roylei Stearn into onion (A. cepa L.) Euphytica 62: 127-133. Duyfjes BE and K Pruesapan (2004) The Genus Trichosanthes L. (Cucurbitaceae) in Thailand. Thai For Bull (Botany) 32: 76-109. Negi KS, A Pandey, AJ Gupta, JK Singh and B Lepcha (2018) Notes on the Distribution of a Rare and Little Known Species: Allium fasciculatum Rendle from Sikkim and West Bengal Indian. J Plant Genet Resour 31(1): 97-100.

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Pandey A, K Pradheep and R Gupta (2014a) Chinese Chives (Allium Tuberosum Rottler ex Sprengel): A Home Garden Species or A Commercial Crop in India. Genet Resour Crop Evol 61:1433-1440. Pandey A, K Pradheep and R Gupta (2014b) Systematic Study Based on Morphological and Distribution of Allium (Alliaceae) in India. In: Proc. National Symposium on Crop Improvement for Inclusive Sustainable Development, PAU, Ludhiana, Punjab, pp 878-879. Pandey Anjula, P Malav, K Pradheep, KS Negi and Ruchira Pandey (2018) An Underutilized Species Allium fasciculatum Rendle ‘‘Shanu Dungdunge’’ (subgenus Amerallium, section Bromatorrhiza) from North Eastern India: Study on Variability, Eco-geography and Domestication Trends. Genet Resour Crop Evol 65: 1049-1058 Pradheep K, DR Pani and KC Bhatt (2015) Taxonomic Notes on the Trichosanthes Cucumerina Group (Cucurbitaceae) from India. Novon 24(1): 39-45. Rugayah (1999) Trichosanthes (Cucurbitaceae) in Malesia. Dissertation. Institute Pertanian Bogor, Indonesia. Appendix I

Key Vegetative and Reproductive Characters Used for Study of Different Species of Allium

S. No. Plant part Character(s)

1. Storage organ bulb, rhizomes, swollen roots (shape, size, longitudinal/ transverse section)

2. Bulb not developed/well-developed, single/ clustered, shape (cylindrical, oval, etc.), size, bulb colour, the texture of outer tunic (membranous, fibrous), bulblets (if present exterior to the main bulb); not developed (A. hookerii, A. fasciculatum)

3. Roots normal, swollen (storage roots)

4. Pseudo-stem if well developed (length, diameter)

5. Leaf fistular/ flat, erectness; flat leaves not folded lengthwise (A. tuberosum), keeled (A. ampeloprasum, leek); fistular (circular outline in section; triquetrous in section)

6. Scape enclosed in bulb, shorter/equal, longer than pseudo-stem, tapering at tip, straight/ coiled,

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hollow/ solid (full or partly)

7. Inflorescence spathe size (LxB), shape (globular, oval, elliptical), tailed when in bud, if persistent; size of the inflorescence, number of flowers, compactness

8. Bulbils presence of bulbils

9. Flower size, colour, shape of flower when completely opened

10. Perianth shape, colour, markings and colour of mid-vein

11. Stamens equal/ unequal sized, exserted/ not exserted, anthers and filament colour, position in relation to stigma

12. Pistil tip, colour, shape/ size

13. Fruit capsule with or without persistent spathe

14. Seed colour, shape, size (LXB), seeds coat texture (rough/ shiny), 100 seed weight

15. Seedling cotyledonary leaf (coiled/not coiled)

Other characters

16. Anatomy of leaf structure of mesophyll cells and stomata, stomata index

17. Pollen shape (oblate, super-oblate, elliptical), ornamentation

18. Odour garlic or onion type (strong/lighter odour)

Appendix II

Spot Characters for Field-Level Identification of Some Taxaof Trichosanthes Trichosanthes -genus  White fringed flowers (night flowering)  Smooth & red fruits (round to oblong) T. cucumerina subsp. cucumerina  Disturbed habitat  Annual climber  Leaves soft, 3-7 lobed

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 No probracts, bracts  Fruits ovate-oblong, white striped  Seed edges toothed T. cucumerina subsp. cucumerina ‘Anguina’  Cultigen  Plant parts- larger (than subsp. cucumerina)  prominent peduncle merging with snake like fruits T. himalensis  Mid-hill forests of north eastern India  Perennial, tuberous-rooted  Leaves shiny, rarely lobed  Probract absent, bract small  Seeds turgid, drum-shaped T. dioica  Cultigen  Dioecious  Scabrous leaves  Male flower solitary  Fruit yellow-orange  Seed round T. bracteata (syn. T. palmata)  Frequent  Liana  Scabrous leaves  Large ovate bracts  Calyx entire/dentate  Round-ovate fruits  Seeds marginated T. khasiana  Forests in north-eastern India  Leaves big, deeply lobed, membranous  Calyx segments entire  Fruits always oblong  Seed with raised central line, ± angular T. truncata  Mid-hill forests in north eastern India  Liana along with Hodgsonia  Leaves entire, glabrous, ovate, 3-prominent nerved  Fruit big, round-oblong, greenish-yellow at maturity  Seed size 2 x 1.2 cm

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Digitizing Specimens in the Herbarium: Procedures and Approaches Anjula Pandey, Sunil Archak*, Rita Gupta and ER Nayar Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources New Delhi 110 012, India *Division of Genomic Resources Introduction In earlier times the specialists used to get specimens on loans or to travel the herbaria to study collections. Since the earlier times, the plant specimens as herbarium resources housed in herbaria across different regions of the world are valuable collections mainly for taxonomic use (Chapman 2005) but their potential was realized as a valuable resource. Herbarium specimens have been used for documenting distributions of biodiversity (Baird 2010), plant viruses (Malmstrom et al. 2007), seeds viability study (Godefroid et al. 2011), ecology and climate change (Hovenden et al. 2008), evolutionary biology and invasive species (Marsico et al. 2010), and phylogenetic studies (Soltis and Soltis 2016). Technological innovations in imaging, molecular biology, and genetics, associated data through databases herbaria have enhanced their utility (Soltis 2017; Sweeney et al. 2018). The tremendous underutilized data available with the herbaria can address pressing issues on climate change, food security and conservation, biodiversity informatics, information technology, geography, and education and various scientific studies (Funk 2003;Chapman 2005; Boyd 2008). Digital herbarium (also called e-herbarium or virtual herbarium) have associated data and an emphasis on wide accessibility and broadly available to wide users including scientific community through internet access. Greater efficiency in compiling information for floristic studies and other resources for undertaking botanical work is achieved by online digital resources (Allen 1993; Schmull et al. 2005). Need for digital documentation of the herbaria Digital herbarium provides an image of specimen and documentation of associated data. A resolution digital image of traditional herbarium sheets, additional information such as the passport data and notes on taxonomic identity is attached to every specimen. With a gradual

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Digitizing Specimens in the Herbarium: Procedures and Approaches (Anjula Pandey et al.) decrease in interest towards taxonomy, the practicing taxonomists are a rare breed. Due to many technical problems in herbarium management (labour, funds, and staff) institutions, housing herbaria are giving low priority to related activities. Digitization of taxonomic information and use of technologies particularly in terms of omnipresent internet access through mobile devices has increased a number of users including non- specialists. Constructing an online facility of carefully curated herbarium information will, therefore, be widely accessible and can be used on-demand. In this chapter, we intend to facilitate understanding about the functioning of virtual herbarium and guide medium-smaller herbaria to develop their own digital resources and develop linkages to other herbaria. The methodology followed in NHCP serves as a model to create digital herbarium for herbarium collections with limited funding resources. Purpose of digitization The general process of digitization is well documented in the literature. It is necessary to identify the digitization system, the task of imaging in herbarium specimens depending, on the purpose, aim and objectives. The contents of digital herbarium may be based on: a) Location of herbarium and target users (students, taxonomists); b) close-up of plant parts-flower, fruit, seed, and live plants as the identifiable parts not can be shown in specimens; c) allied and supplementary material: seed images, shape, size, testa characters etc.; d) plant products: bark samples, commercial products; and e) seedlings and seed images for genebank and forestry use. The added advantages of digitizing specimens are more in rare and endangered taxa and type material where we cannot afford to mishandle and damage to the specimens. Digitization of herbarium holdings was by the larger herbaria with the objective to maintain their databases and web site. The medium- sized and smaller herbaria which are generally providing academic support (such as teaching, advising students and researchers) are attached to the educational institutions. Curators and collections managers of these herbaria due to lack of funding support, modality, staff training are not initiating digitizing their collections to initiate work of digitalization of collections (iDigBio 2013). Out of about 800 active global herbaria maintaining an estimated number of 90 million specimens, two-third of the collections are contributed from academic institutions (Barkworth and Murrell 2012).

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Kew’s herbarium which is one of the largest and the most representative one in the world, however, has only 10% of these collections currently are available for study. Procedures for developing virtual herbarium The digitization of herbaria process consists of pre-digitization curation, imaging specimens, databasing and identifying information, and dereferencing locality information (Nelson et al. 2012.). Each step takes a considerable amount of time to accomplish the final output. Digitizing process required six steps: (1) organizing all specimens in the cabinets using updated taxonomy and nomenclature including many new collections, (2) setting up an imaging station, (3) developing efficient imaging protocol and imaging each specimen individually, (4) choosing the database template to fit to our need, (5) updating card/label information and entering in the database, and (6) collating all data with images, and (7) exporting to web through an online database for use (Fig. 1). Predigitization 1) Prioritized curation: Sort out specimens based on priority, clean and organize them using updated taxonomy and nomenclature. Examining specimens provides the curator or collection manager to determine the best digitization workflow for the collection. Annotating a collection, adding the notes, is a step of verifying identifications which is most important and would considerably increase the time before digitization can begin. This step may be skipped if the goal is to make collection information more easily available to taxonomy specialists who can verify identifications from online image and database information. Combining the nomenclatural review and imaging steps slow both of these steps in the process of the workflow. The approach of updating nomenclature invests more time that could have been spent imaging and databasing label information. After the specimens were organized and nomenclatural verifications were nearly completed, each person verifying nomenclature is trained to print and use annotation labels. This ensures updated information in a readable and clearly documented form. The users verifying nomenclature have little taxonomic training, so specimens that are previously misidentified remained misidentified after the nomenclatural updates. Two-three technical personnel in a chain together resulted in a slightly faster rate of imaging as compared to one person. Unidentified, wrongly identified specimens are not included and stored at the end of family or taxon.

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Digitizing Specimens in the Herbarium: Procedures and Approaches (Anjula Pandey et al.)

Figure 1. Flow chart for digitizing process Some curators may eliminate pre-digitization curation steps from the workflow to fasten the process of digitisation. Many curators may feel that they cannot begin digitizing process of their collections until the nomenclature has been verified and the collection has been properly organized. Beginning with nomenclatural verification will give the curator an idea of the content of holdings and time needed. In NHCP pre-digitization curation was done before the actual workflow. Prioritise the herbarium specimens to be digitised: some major strategies worked out for digitization of herbarium specimens are listed: i) ‘spot’ characters for use in digitization, include those that can be observed on the herbarium specimen/ field grown plants with a hand lens or (with a close-up, up to x 200 magnification or elsewhere micromorphological characters are essential for identification of specimens viz. in grasses ii)use of images of live material in the field helped to complement the specimen in selected cases to avoid misinterpretation of characters lost during processing, viz. spreading panicles of the perennial wild rice versus the non-spreading branches of the annual types of wild rice. Identity of specimens was checked for confirmed identity (the process of authentication) with characters delineating taxa for digitization and data/information on the web link. For correct naming of taxa internationally accepted nomenclature was used to the extent possible.

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Specimens (1 or more) were selected for inclusion in digital herbarium collection. Adding specimens of the value of the herbarium viz. type specimen, RET taxa, an heirloom variety of crops, cultivars/ wild relatives/ potential domesticates used in breeding programmes, be given priority. Herbarium holdings may include many specimens collected before the Global Positioning System (GPS) era and therefore reference for the location of collections made are to be validated. In many instances, where the label data provide addresses, directions, and township, range, etc., data can be used to approximate GPS coordinates using geo- referencing software. NHCP has initiated the geo-referencing of herbarium specimens and intends to do for the whole holdings. 2) Setting up of imaging station: while setting up of image station, make purchases related to the equipment needed. If equipment required are already available, the work of digitization can be started. Imaging area is identified along the cabinets of accessioned collections to be digitized and computer workstation so as to shorten the time of taking out the specimens to be digitized, non-mixing, no damage and no misplacement of the specimen. 3) Imaging: the image of each specimen is individually developed through efficient imaging protocols. The work area includes: (1) a scanner, (2) a stable fixed area with illumination and an aerated light source that minimizes shadows, (3) a computer, (5) an external hard drive, and (6) a colour ruler to be placed alongside the specimens. The efficiency of work-flow increases many folds with 2-3 persons working continuously in a flow as compared to when single person imaging work. Digitization work is interrupted due to several reasons like visitors, non- continuity in flow, irregular power supply, which may result into poor efficiency and quality of virtual images captured. At NHCP barcode labels for naming image files and linking images to database records were not used, but its use in the herbarium is strongly recommend for unique identification. Databasing and dissemination 4) Databasing: data basing work is divided into two: card data and label data. This allowed creating an easy-to-use transcribed datasheets in individual lines similar to an Excel Spreadsheet, with corresponding images attached to each. We recommend having a single person (preferably curator or herbarium manager) responsible for uploading data into the database to efficiently allow specimens to be checked before they are uploaded into the main database.

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Figure 2. Digital imaging station at NHCP. Digital scans (jpeg images) with good resolution (300 dpi for close up of parts-seed, trichomes, and 600-1200 dpi for micro details of trichomes, hard extensions on plant surface) Data recorded for various attributes for each taxon included ‘spot’ characters for identification of species, distributional records of taxa vis- à-vis herbarium specimens represented in NHCP, use of the germplasm in PGR, and the reference herbarium specimen(s) which have been digitized, and provide the link to the digital image. Data records were entered up to family level in the able to sync with the taxon tree upon upload. Taxon records represent all individual names (i.e., collectors, annotators, catalogers; locality and habitat data). 5) Data storage: captured data (images and data sheets) should be stored at least two backups in different locations. We take the back up of our data on AKMU data server as well as an external hard drive. Having a barcoding system and globally unique identifiers (GUIDs) can help prevent duplicated records. Images may represent a significant amount of data and a hard drive with a minimum capacity of one terabyte will be needed to store them. If the host institution grants the server space, images and database can be placed on secure server. Storing data in multiple locations in both physical and cloud-based systems is suggested. 6) Collate data: all data to make it accessible to the scientific community and public through an online database. Status: NHCP virtual herbarium The approximately 24,000 herbarium specimens in the NHCP, selected 8,000 herbarium sheets (over 4,500 taxa) of different categories plant

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Digitizing Specimens in the Herbarium: Procedures and Approaches (Anjula Pandey et al.) specimens have been imaged, database and available electronically on the herbarium’s website. Crop taxa are represented by nearly 500 crop species and other minor locally useful taxa in the NHCP; digitization of these taxa, as source material for identification and study was taken up under project mode. Diversity in crop species represented as: a) cultivars and landraces as herbarium specimens and genebank samples, b) cultivar- groups, marked by distinctive traits related to locality, use and cultivation, c) germplasm with unique identity number, either indigenously collected (with IC or equivalent number) or introduced into India (with EC number). The Checklist of Crop species of India, published by the ICAR-NBPGR in 2003 (Nayar et al. 2012) was used as the basis for selecting taxa for creating taxon sheets (major crop categories-cereals, grain legumes, vegetable crops, fruit crops and oilseeds). The digitization work was started with aim to digitizing of whole herbarium and its networking for wide users. The digitization work was initiated with DST-funded project on “Digitization of the National Herbarium of Cultivated Plants” operative from 2011-2014. Data of a subset of taxa (450 species) with the digital images of vouchers specimens of plant germplasm introduced into India represented in NHCP (Nayar et al. 2012; Nayar et al. 2014) were used for digitization work. Supplementary images of seed samples/fruit images, and live plants with close up of flowers were also added in selected cases. This web based application was used for developing NHCP herbarium specimens available for research study purpose and required hardware and software specification. Contents were provided by NHCP after proper curation. Developed image resources through web-based software with linked data have been provided as a database through the AKMU (Agricultural Knowledge Management Unit) of the ICAR-NBPGR. The web-interface was developed following three-tier system. The database was placed at bottom layer and was developed by using MSSQL Server 2012. The server side layer was developed by using C# and the client side application layer was developed by using HTML, CSS, Bootstrap and Microsoft Visual Studio 2010 with ASP.NET version 4.0. The entire application was hosted on Windows Server 2008 version 6.1 with IIS version 7.5.7600.16385 at URL http://pgrinformatics.nbpgr.ernet.in/nhcp. (Appendix I). The web-based interactive digital system of NHCP is currently intending to provide access to the valuable information on identities of

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Digitizing Specimens in the Herbarium: Procedures and Approaches (Anjula Pandey et al.) over 8,000 taxon sheets of genetic resource value including the crop wild relatives and exotic taxa. Future initiatives Digitization of herbarium specimens and linking it to database is a continuing process. Need based nomenclature check and geo-referencing of data need routine updating. High-quality digital photographs are important source of documentation of diversity for larger number of users (James et al. 2015). At the NHCP, adding digital field photographs as supplementary material for conventional herbarium specimens particularly for ‘difficult to make as herbarium’ will be initiated to serve as identification tool. Geo- referencing of herbarium data enhances the value of collection records and enables searches more relevant to some research questions than searches across taxa (de Smith et al. 2015). Digital images of herbarium suffer some limitations as compared to the conventional scientific specimens and thus they cannot replace traditional herbarium specimens. However, tremendous data available with them and instant use as a tool for various scientific studies is worth appreciating. Acknowledgements: We acknowledge the DST for funding support for execution of the project on digitization. We also thank all contributors for depositing their valuable material to make this facility richer than ever before. Selected readings/references/links Allen WH (1993) The Rise of the Botanical Database: Computerized Information Offers Increased Efficiency, Access to Unpublished Records and New Ways Of Asking Questions. BioScience 43: 274- 279. Baird R (2010) Leveraging the Fullest Potential of Scientific Collections through Digitization. Biodiversity Informatics 7: 130-136. Barkworth ME and ZE Murrell (2012) The US Virtual Herbarium: Working With Individual Herbaria to Build A National Resource. ZooKeys 209: 55-73. Boyd A (2008) Collecting for Education: Herbaria at Small Liberal Arts Colleges. Plant Science Bulletin 54: 86-91. Chapman AD (2005) Uses of Primary Species-Occurrence Data. Report for the Global Biodiversity Information Facility, Copenhagen, Denmark. (http://www.niobioinformatics.in/books/Uses%20of%20Primary%2 0Data.pdf [accessed 21 March 2017]).

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Digitizing Specimens in the Herbarium: Procedures and Approaches (Anjula Pandey et al.) de Smith MJ, MF Goodchild and PA Longley (2015) Geospatial Analysis: A Comprehensive Guide to Principles, Techniques and Software Tools. (5th edn) Winchelsea Press, Winchelsea, United Kingdom. Funk V (2003) 100 Uses of a Herbarium (well at least 72). US National Herbarium, Smithsonian Institution. (http://botany.si.edu/documents/2003_Funk_100Uses.pdf [accessed 13 September 2016]). Funk VA, PC Hoch, LA Prather and WL Wagner (2005) The Importance of Vouchers. Taxon 54: 27-129. Godefroid S, A Van De Vyver, P Stoffelen, E Riobbercht and T Vanderborght (2011) Testing the Viability of Seeds from Old Herbarium Specimens for Conservation Purposes. Taxon 60: 565- 569. Hovenden MJ, KE Wills, JK Vander Schoor, AL Williams and PCD Newton (2008) Flowering Phenology in A Species- Rich Temperate Grassland is Sensitive to Warming But Not Elevated CO2. New Phytol 178: 815-822. iDigBio (2013) Mobilizing Small Herbaria Workshop. 10-11 December 2013. (https://www.idigbio.org/content/mobilizing-small-herbaria- workshop [accessed 13 September 2016]). James V La Frankie Jr and Anna I Chua (2015) Application of Digital Field Photographs as Documents for Tropical Plant Inventory. Appl Plant Sci. 3(5): apps.1400116. Johnson KG, SJ Brooks, PB Fenberg, AG Glover, KE James, AM Lister, E Michel, M Spencer, JA Todd, E Valsami-Jones, JR Young and JR Stewart (2011) Climate Change and Biosphere Response: Unlocking the Collections Vault. Biosci 61: 147-153. Malmstrom CM, R Shu, EW Linton, LA Newton and MA Cook (2007) Barley Yellow Dwarf Viruses (Bydvs) Preserved in Herbarium Specimens Illuminate Historical Disease Ecology of Invasive and Native Grasses. J Ecol 95: 1153-1166. Marsico TD, JW Burt, EK Espelend, GW Gilchrist, MA Jamieson, L Lindström and GK Roderick (2010) Underutilized Resources for Studying The Evolution of Invasive Species during Their Introduction, Establishment, and Lag Phases. Evol Appln 3: 203- 219. Nayar ER, A Pandey, K Pradheep, R Gupta, D Upadhyay and U Jaiswal (2014) Crop Species and their Digitisation in the National Herbarium of Cultivated Plants (NHCP). National Symposium on

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‘Crop Improvement for Inclusive Sustainable Development, November 7-9, 2014, Punjab Agricultural University, Ludhiana, India. Nayar ER, Anjula Pandey and K Pradheep (2012) Digitisation of the NHCP: Technical Guidelines and Procedures. Indian J Plant Genet Resour 27(2): 163-170. Nelson G, D Paul, G Riccardi and AR Mast (2012) Five Task Clusters That Enable Efficient and Effective Digitization of Biological Collections. ZooKeys 209: 19-45. Nelson G, P Sweeney, LE Wallace, RK Rabeler, D Allard, H Brown and JR Carter (2015) Digitization Workflows for Flat Sheets and Packets of Plants, Algae and Fungi. App Plant Sci 3: 1500065. Schmull M, J Heinrichs, R Baier, D Ullrich, G Wagenitz, H Groth, S Hourticolon and SR Gradstein (2005) The Type Database at Gottingen (GOET): A Virtual Herbarium Online. Taxon 54: 251-254. Soltis DE and PS Soltis (2016) Mobilizing and Integrating Big Data in Studies of Spatial and Phylogenetic Patterns of Biodiversity. Plant Diversity 38:264-270. Soltis PS (2017) Digitization of Herbaria Enables Novel Research. Am J Bot 104(9): 1281-1284. Sweeney WP, B Starly, PJ Morris, Yiming Xu, A Jones, S Radhakrishnan, J Christopher and Charles C Davis (2018) Large- scale Digitization of Herbarium Specimens: Development and Usage of An Automated, High-Throughput Conveyor System. Taxon 67(1): 165-178. Thiers B (2012) Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff. (http://sciweb.nybg.org/science2/IndexHerbariorum.asp). Appendix I For user home PC and workstation  Hardware PC with higher configuration  50 GB free space on HDD  4 GB RAM  Internet connection  Software  MS Windows /2000/NT/XP or higher  MS IE, Netscape or Opera browsers with IIS support For Server

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 Hardware: PC with higher configuration; 16 GB RAM or higher; 80 Gbytes free space on HDD  Software: MS SQL server 2008/12 and MS Access (Database server) and MS Excel; MS Internet Information Server (Web server); Visual Studio 2010 or higher; Development environment - C# programming language With.Net Framework Appendix II

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Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures (SP Ahlawat et al.)

Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures SP Ahlawat and Anjula Pandey Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources New Delhi 110 012, India Introduction Plant genetic resources for food and agriculture are vital to human beings and other animals. They are a source of food, fodder, fuel, fibre and several items of life support. Plant genetic resources consist of the diversity of crops and their wild relatives, contributing to agricultural production. Over 7,000 plant species in the world that are edible, but only 4 species: rice, wheat, maize and potato provide over 50% of our plant-derived calories. Approximately 940 species of cultivated plants are threatened globally (Khoshbakht and Hammer 2007). In recent largest survey of 330,000 seed-bearing plant species extinctions published on 10th June, 2019, the world’s plant species have been disappearing at a rate of nearly 3 species a year since 1900, which is up to 500 times higher than would be expected as a result of natural forces alone (Humphreys et al. 2019). Ever since the establishment of modern genebanks, conservation of PGR and their flows within and across borders have been tremendous. These collections are made through augmenting germplasm for traditional breeding and modern biotechnological programmes. Augmentation of germplasm is the first and foremost activity in the PGR management system, therefore meticulous planning and exploration following scientific principles governing the diversity distribution are crucial. Sporadic collections of indigenous crop germplasm were made during the earlier part of the 20th century (Howard and Howard 1910), for jute and few crops (Burkill and Finlow 1907). Dr BP Pal in his classic paper on ‘Search for New Genes” emphasized on the importance of germplasm augmentation in crop improvement programmes (Pal 1937). Significant progress has been made subsequently, in collecting and conservation of indigenous and exotic plant genetic resources (PGR) of food and agriculture by several countries like USA, China, India, United Kingdom, Germany, Japan and

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Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures (SP Ahlawat et al.) international centers of CGIAR like, CYMMET, IRRI, ICARDA, ICRISAT, CIAT and IITA. Since major part of the herbarium build-up at the NHCP is contributed through various exploration related activities, this in this chapter the outlines on the standard methods followed for undertaking plant genetic resources explorations and collection of germplasm and preparation of herbarium in the field in brief are discussed. Experiences shared here are of the authors while collecting germplasm in the country. Collecting plant genetic resources germplasm and herbarium specimens Germplasm collection is a prerequisite to utilize the valuable resources in crop improvement and analyze temporal changes. Besides, the voucher herbarium specimens are collected, pressed, plant sample deposited for future reference and to supports research the identity of the specific plant. Besides their use in taxonomic correction/validation, herbarium specimens data is commonly used in the fields of ecology, plant anatomy and morphology, conservation biology, biogeography, ethnobotany and palaeobotany. The herbarium sheets provide bio- geographic information that can be used to document the historic ranges of plants, to locate rare or endangered species, or to trace the expeditions of explorers and plant collectors. Physically, the specimens are important sources of genetic material for DNA analyses and of pollen for palynological studies. For details on the NHCP refer to chapter 1).

Collection mission for germplasm sample and herbarium specimen requires almost similar preparations including finding the target species and populations to capturing a maximum number of species, diversity for the amount of material collected and resources invested (Guarino et al. 1995). In a vast and diverse country like India and particularly tropics, great specific and intra-specific variation is expected in various agro-ecological systems and forests. Conducting an eco-geographical survey has been a canonical method for such a preparation (Maxted et al. 1995). Such surveys help in increasing the emphasis on localized floristic and germplasm collecting with a focus on specific traits. To get a fair idea on the extant of floristic and genetic diversity, their distribution across disparate geographical and ecological niches needs to be searched prior to actual collection expeditions. Step- wise activities of exploration mission are depicted by a flow chart (Fig.1) Significance of the ecogeographic survey

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Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures (SP Ahlawat et al.)

An eco-geographic survey is defined as an information gathering and synthesis process on geographical, ecological, taxonomic and genetic diversity data (Maxted et al. 1995; Castañeda Álvarez et al. 2011). Eco- geographic survey provides diagnostic information on PGR to infer on the history of their evolution and adaptation, to assess the status of conservation and to prioritize areas for conservation. The survey generally consists of collating information from herbarium specimens, genebank accessions, PGR databases, in addition to published as well as informal literature, etc. The outcome of such a survey is predictive and can be used in the formulation of collection priorities. Generally, an eco-geographic survey and analysis result in: i) delineation of priority crop species, ii) identification of areas for germplasm, herbarium collecting and in situ conservation, iii) identify populations of cultivated and CWR species which are not conserved in the genebank, iv) to plan and execute collection missions. Data have also been employed to define core collections in crop species (Frankel and Brown 1984) and identify gaps in collections (Shehadeh et al. 2013). Such substitution is possible because, eco-geographic representativeness indirectly reﬂect genetic representativeness, due to the relationship that exists between the eco-geographic characteristics of a location and the genetic characteristics of the species and populations occurring at that site through natural selection and local adaptation (Greene and Hart 1999). Planning exploration mission Since this activity is being undertaken by organizations with different mandates, the guidelines have been framed to help the explorers in maintaining standard methods and procedures in collecting PGR (Arora 1981; Gautam et al. 1998; Guriano et al. 1995; Hawkes 1976, 1980; Jain and Rao 1977; Annexure I). An excerpt of guidelines made by the Division of Plant Exploration and Germplasm Collection at ICAR- NBPGR are given here for training purpose. 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 the database of National Genebank/Field Genebank. However, for foreign explorations guidelines of the agricultural research department/ministry in the country needs to be followed to finalize the mission. The explorer should be well-versed with the nature and extent of diversity and breeding behaviour of crop/species to be collected and plan well in advance to facilitate preparations of proposed missions

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Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures (SP Ahlawat et al.) except those to be carried out under special situations like rescue collecting. Visit to herbaria should be made to study the range of distribution, localities, diversity pattern and period of collection, particularly for wild species. Floras of targeted area, R&D institutions and experts in the area should be consulted. Collaborator(s) should be identified and communicated to join the mission well in advance with details of preparations, if required, in case of vegetatively propagated and recalcitrant material. Phytosanitary regulations should be followed in case the material is transported from a foreign country. 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 itinerary of collecting mission. Besides, explorers should establish local contacts, especially at the 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; while fine-grid survey is carried out to build-up more collections for a specific trait(s) known to exist in identified pockets in previously explored areas. Multi-crop/ crop-specific explorations: multi-crop exploration is carried out to collect the diversity in general of a given region (also referred to as region-specific exploration) or in unexplored areas. Crop-specific exploration is undertaken to collect the variability in particular crop and its genepool. The samples collected must be representative of the diversity that exists within each crop/ crop groups in a given area. Permission of collection from protected/restricted areas: prior permission should be obtained from the concerned authorities for exploring and collecting in protected (biosphere reserves, sanctuaries, national parks) and restricted areas (international border) and particularly for foreign missions. Period of collection: for systematic study and herbarium collection, the flowering season of species is best. For germplasm collection of 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

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Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures (SP Ahlawat et al.) the 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 the appropriate time. Period of explorations being organized within the country should be of at least 10-15 working days (excluding journey period) and more than a month when organized in foreign countries. Team composition: the exploration and collecting team should consist a taxonomist and breeder who are familiar with the basics of agriculture/plant genetic resources to meet the objective of the mission. The team consisting of 2-3 members including a collaborator and need- based local-aid may be formed preferably a botanist/ breeder as the team leader. Area and route of exploration: this should be fine-tuned in consultation with the subject experts of local bodies, the staff of forest, agriculture departments, as soon as the team reaches to the starting point keeping in view the targeted species and areas of the proposed mission. Items and types of equipment 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, several items and types of equipment are required (listed in chapter 10). Herbarium press, secateurs and large size polybags (1.5x2 m) are essential items required for herbarium specimen collection (refer to Chapter 13 for details). Domestic quarantine: all precautions including need-based international/ domestic quarantine should be followed for pest-free collection and its transportation. Collection methodology Sampling sites: inaccessible areas of valleys, isolated hills, villages at the edge of deserts, forests, mountains and isolated coastal belts may hold rich genetic diversity, potential/ trait-specific germplasm and wild relatives. For cultivated species, sampling sites in order of preference should be farmers’ field, backyard/ kitchen garden, threshing yard, farm store, local village market, etc. Collecting mission should be started first from warm, drier tracts (vs. humid), un-irrigated areas (vs. irrigated), valleys (vs. hills) to capture maximum available diversity in a planned manner. The crops often vary with ethnic diversity and different array of materials may be collected even from contiguous belts occupied by different tribes.

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Why collect For utilization, germplasm in danger of extinction or erosion (wild species), gap filling in existing ex-situ collections , rescue collecting, loss of genetic diversity due to overexploitation, for future use: the material not considered useful today may turn out to be vital tomorrow particularly in changed climatic condition, to address issues of IPR and CBD

Exploration Aims at Capture prevailing genetic diversity; search of novel/unique genetic material

Planning of Exploration Missions National Exploration Plan (Five years) Annual Exploration Plan (at the national level) Based on the need of breeders, conservation for posterity, gaps in collections, taxonomic and phylogenetic study on prioritized species Gathering information on areas of diversity, unique traits, landraces, availability, crop maturity, visit twice for disease/pests tolerance

Exploration Missions (Type) Specific (trait-specific, biotic stress, quality) Broad-based (multi-crop/Region specific) Rescue Mission (during a calamity or land-use change)

Planning of exploration including logistic arrangements Team composition, liaison and involving crop breeder/collaborator, area and route of exploration, collection time, exploration items and equipment (transport, instruments, GPS, chemicals, miscellaneous items, published material, medical items, other items)

Exploration and Collection Methodology Method: Coarse grid and fine grid survey in targeted areas/fields Sampling Techniques: Random sampling, biased sampling, bulk sampling Plant Parts Collected: Based on the reproductive nature of crop such as seed, pollen, vegetative dormant bud, rhizome, tuber, stolen, etc. Sample Size: 2000/4000 seeds of self/cross-pollinated crops

Recording Passport data, ITK information

Germplasm: Processing, multiplication and conservation in the genebank

Germplasm: Characterization and evaluation

Germplasm: Supply, Pre-breeding and Use

Figure 1. Flow chart of exploration for germplasm and herbarium collection activities Sites having stress situations viz. saline habitat, un-irrigated/ drought conditions, desert (cold and hot), flood-prone areas should be identified as target areas for collection of respective trait-specific

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Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures (SP Ahlawat et al.) material. In such cases, selective sampling of promising genotypes should be done. For biotic stress tolerant material, hot-spot areas should be visited to collect healthy plants in fields where severe pest damage is evident. The frequency of sampling (number of samples per site) should be decided based on on-the-spot observations on the variability available. In general, more sites per target area are preferred to sample the targeted species rather than sampling from a few sites. Sampling method (self, cross-pollinated and vegetative propagated material): while collecting the seed, the explorer should keep in mind the required quantity of material to be sampled for long term conservation (2,000 and 4,000 seeds for self and cross-pollinated crops, respectively) besides meeting the requirement of characterization, evaluation and related studies. The optimum sample size per site would be the number of plants required to obtain, with 95 per cent certainty, all the alleles at a random locus occurring in the target population with a frequency greater than 0.05 (Hawkes 1976; Marshall and Brown 1975). In case of species with extremely small-sized seeds, low seed-set, asynchronous maturity and low seed viability, care should be taken to collect adequate sample size. In case of extremely variable populations, one can either make larger samples (bulking), or take as sub-samples if observed interesting variants, and be given separate collecting numbers. In general, random sampling should be made by collecting single spike/panicle or fruit/berry/pod from at least 50 plants along a number of transects throughout the field (Hawkes 1976; 1980) to obtain a representative and adequate sample. Plant population at the border of the field should be avoided. In a situation when wild population with few individuals occurs, one should better collect from all the plants so as to make the representative sample from that site. In case of certain wild and semi- domesticated species occurring in the small pocket with scattered populations (treated as sampling site) having specific use/traits, the seed should be bulked. However, one should not deplete the populations of farmers’ planting stocks or wild species, or remove significant genetic variation. In case of large tubers, only a portion, e.g. head or proximal ends in yams, crown or tuber in taro and other aroids should be collected (Pandey et al. 2019). Since vegetative propagules are subject to rapid

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Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures (SP Ahlawat et al.) deterioration after harvest and damage during transportation care should be taken while sampling and in transportation. In case of scion collection for budding and grafting the sample size will depend upon the number of rootstocks available but not less than ten per sample so that at least eight grafts may survive. In case of cuttings and rooted suckers (e.g. grapes, ornamentals, passion fruits, black pepper, beetle vine, banana, cardamom, etc.) 15-20 cuttings may be sufficient. Establishing taxonomic identity Material with dubious identity, unidentified material and only vernacular name should be collected along with herbarium specimen and photographs for authentication. In case, when herbarium specimens are not available, efforts should be made by the explorer to raise plants to establish its correct identity. Normally 4-5 individual plants/parts having a representation of all parts especially flowers, or fruits or both should be collected for preparing herbarium specimen. Locality, date of collection and field notes should be clearly recorded. Characters which are lost on drying, or which may not be represented in the herbarium specimen (plant height), flower colour, leaflets (which may be shed on drying) should be mentioned in field notes. The detailed guidelines for preparation and processing herbarium specimen (refer Chapter 2 for details). Transportation In case of vegetative propagules, if required, the explorer should make prior arrangements for the en-route transportation of collected material to the place of its establishment/ maintenance to avoid deterioration. Daily checking of collected material, change of blotting papers of herbarium sheets, room drying of collected material after reaching a place of halt is essential. Recording information Passport data: passport data are an important source for database, documentation, enhanced utilization of PGR and studying the variation in distributional pattern with respect to ecological and socio-economic factors. It is advisable to record information on both the essential and optional fields in the passport datasheet or field book at the site of the collection itself by the explorer. For contents of passport data book of ICAR-NBPGR, New Delhi refer Chapter 13. However, in any circumstances, the explorer should not leave the information blank on essential fields namely sample labeling (name of organization(s) and collectors, collectors’ no., date and type of material); sample

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Exploration and Germplasm Collection of Plant Genetic Resources: Prospects and Procedures (SP Ahlawat et al.) identification (botanical identity, vernacular name, its biological status); sampling information (sampling type, method and source) and collecting site localization (state, district, village, latitude, longitude and altitude). This information is important for herbarium specimens and recorded in field books. Related information: information on genetic erosion should be gathered from aged-farmers particularly on the depletion of landraces cultivated over time and the reasons for their loss in general and crops of Indian origin in particular. Indigenous traditional knowledge on plants, their use, agricultural technologies etc. are also asked and recorded. Meaning of the name of landraces and their properties should be asked from farmers and recorded. Observations on the distribution pattern and frequency status of crop wild relatives, rare, endangered and threatened species of PGR importance should be recorded for their sustainable management. Post-collection handling Seed extraction, cleaning/drying: the extraction and cleaning of seed should be done preferably on the same day or immediately after completing the expedition and process for their drying under shade/ sun/ controlled conditions. The seeds with short longevity should be processed at the earliest and care should be taken during threshing/ cleaning to avoid damage. In a situation, when a delay in processing is anticipated, all precautions should be taken to maintain its viability. The observations on variability parameters on fruit/ pod/ seed should be recorded along with photographs for report writing, documentation and publication. Packaging and labelling (for sharing, accessioning, multiplication and conservation): the clean and dried material should be kept in the envelopes with a proper label specifying its botanical name and collector number. One set of the material along with passport data should be sent for accessioning, conservation (LTS/MTS) and another set be sent to the collaborating institute for initial seed increase (if required), maintenance, characterization and evaluation. Establishment/maintenance for vegetatively propagated material: the vegetatively propagated material should be sent for establishment/ maintenance in field genebank or at a suitable site. The material for in vitro and cryo-genebank should be handed over to the concerned curators. The elite material, if any, should be studied in detail to generate supporting information as well as for validation of the known trait(s) for its registration with the concerned agency.

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Report writing and publication

After completing the mission of herbarium and germplasm collection from a target area and processing the collected material, it is important to write a comprehensive report to fulfill the mission’s objectives. This helps in follow-up collecting(s) and also the users to know the availability of the germplasm as well as in publications. The information on the samples collected can be entered into a database for its access to users. The report on the exploration and collection should broadly include:  Name of the organisation(s)  Name of the scientist(s)/person(s) involved  Collaborating organisation(s)  Objectives of the collecting mission  A description of the environment, flora, people of the target area  An account of the logistics and scientific planning  Details of the execution of the mission (timing, itinerary, sampling strategy and collecting techniques)  A summary of the results (areas surveyed along with route maps, germplasm and herbarium specimens collected, indigenous knowledge documented and extent and magnitude of diversity collected with elite germplasm, if any)  Role of women in the conservation of diversity  Details of sharing germplasm and information  Photographs  An account on the loss of germplasm and IK (indigenous knowledge), if any  Difficulties encountered during collecting mission  Recommendations for follow-up action(s) Conclusions Success of a collection mission depends on the least gaps between planning and execution. While collecting the germplasm, wherever possible the efforts should be made to collect the herbarium specimens (possibly for all variants of a taxon), economic products with recorded local use and documented information. References/selected readings Arora RK (1981) Plant Genetic Resources Exploration and Collection: Planning and Logistics. In: KL Mehra, RK Arora and SR Wadhi (eds) Plant Exploration and Collection. NBPGR Sci. Monograph 3. ICAR- National Bureau of Plant Genetic Resources, New Delhi, pp 46-54.

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Burkill IH and RS Finlow (1907) Races of Jute, Agric Ledger 14: 41-137. CastañedaÁlvarez NP, HA Vincent, SP Kell, RJ Eastwood and N Maxted (2011) Ecogeographic surveys. In: Guarino L, V Ramanatha Rao, E Goldberg (eds) Collecting Plant Genetic Diversity: Technical Guidelines. Bioversity International, Rome. FAO (1993) International Code of Conduct for Germplasm Collecting and Transfer. FAO, Rome. (www.fao.org/nr/cgrfa/cgrfa-global/cgrfacodes/en) FAO (1997) The state of the world’s plant genetic resources for food and agriculture. FAO, Rome. pp 444 (http://www.fao.org/3/a- w7324e.pdf) Frankel OH and Brown AHD (1984) Plant Genetic Resources Today: A Critical Appraisal. In: Holden JHW and JT Williams (eds) Crop Genetic Resources: Conservation and Evaluation. Allen and Unwin, Winchester, pp 249-257. Gautam PL, BS Dabas, U Srivastava and SS Duhoon (1998) Plant Germplasm Collecting: Principles and Procedures. ICAR-National Bureau of Plant Genetic Resources, New Delhi, 218 p. Greene SL and TC Hart (1999) Implementing A Geographic Analysis in Germplasm Conservation. In: Greene SL, L Guarino (eds) Linking Genetic Resources and Geography: Emerging Strategies for Conserving and Using Crop Biodiversity. American Society of Agronomy; Crop Science Society of America, Madison, pp 25-38. Guarino L, V Ramanatha Rao and R Reid (1995) A Brief History of Plant Germplasm Collecting. In: Guarino L, V Ramanatha Rao and R Reid (eds) Collecting Plant Genetic Diversity. Technical Guidelines. CAB International, Wallingford, UK, pp 1-11. Guarino L, V Ramanatha Rao and R Reid (1995) Collecting Plant Genetic Diversity: Technical Guidelines. International Plant Genetic Resources Institute (IPGRI), Rome, Italy; Plant Production and Protection Division, FAO, Rome, Italy; World Conservation Union (IUCN), Gland, Switzerland; CABI, Wallingford, UK, 748 p. Hawkes JG (1976) Manual for Field Collectors (Seed Crops).International Board for Plant Genetic Resources, FAO, Rome, Italy. Hawkes JG (1980) Crop Genetic Resources - A Field Collection Manual. IBPGR/EUCARPIA, University of Birmingham, UK. Howard A and GLC Howard (1910) Wheat in India, its Production, Varieties and Improvement. Thacker Spinn & Co., Calcutta.

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Humphreys Aelys M, RafaëlGovaerts, Sarah Z Ficinski, Eimear NicLughadha and Maria S Vorontsova (2019) Global Dataset Shows Geography and Life Form Predict Modern Plant Extinction and Rediscovery. Nature Ecol Evol June 10 (http://doi.org/gf3szp). IBPGR (1985) Ecogeogrpahic Surveying and in situ Conservation of Crop Relatives. Report of an IBPGR Task Force Meeting held at Washington, IBPGR, Rome, 33 p. Khoshbakht and Hammer (2007) Threatened and Rare Ornamental Plants. Journal of Agriculture and Rural Development in the Tropics and Subtropics 108(1):19-39. Marshall DR and AHD Brown (1975) Optimum Sampling Strategies in Genetic Conservation. In: OH Frankel and JG Hawkes (eds) Genetic Resources for Today and Tomorrow Cambridge Univ. Press, Cambridge, pp 53-80. Maxted N, Van Slageren, JR Rihan (1995) Ecogeographic Surveys. In: Guarino L, Ramanatha Rao V, Reid R (eds) Collecting Plant Genetic Diversity. CABI International, Wallingford, UK, pp 255-285. Nayar E Roshini, Anjula Pandey, K Pradheep, Rita Gupta, DC Bhandari and KC Bansal (2011) National Herbarium of Cultivated Plants. National Bureau of Plant Genetic Resources, New Delhi, 20 p. Pal BP (1937) The Search for New Genes. Agriculture and Livestock 7(5): 573-578. Pandey A, NS Panwar, R Singh and SP Ahlawat (2019) Vegetable Genetic Resources- Collection and Conservation. ICAR-National Bureau of Plant Genetic Resources, New Delhi. Shehadeh A, A Amri and N Maxted (2013) Ecogeographic Survey and Gap Analysis of Lathyrus L. Species. Genet Resour Crop Evol 60: 2101-2113.

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Appendix I Do's and Don’ts

In addition to above guidelines for exploration, germplasm and herbarium collection, the collector(s) should observe a well-defined code of conduct as well as take necessary precautionary measures in its smooth execution as mentioned below: Do's

 Get acquainted with the International Code of Conduct for Plant Germplasm Collecting and Transfer developed by FAO (1993).  Always keep a route map of the target area with list of important places and the distance covered during travel to facilitate report writing.

 Before entering into a forest take the help of forest guards to have forehand knowledge of possible dangers in the target area. If needed, the help of a gunman is taken during a survey in a dense forest.

 Explain the purpose and get consent from the farmers for collecting germplasm.

 Keep important telephone numbers of concerned officers including district authorities, hospitals, dispensaries and police station.

 Keep your identity card and a certificate from Head of Organization for the proposed mission.

 Honour social customs of local inhabitants of the target area.

 While talking and discussing with ladies, be polite and respectful to them.

 After day's collection and before retiring to bed, have a glance at your equipment, passport data and collected material for need-based updating. Don’ts

 Do not provide a lift to strangers in your vehicle under any pretext.

 Do not indulge in unnecessary discussion related to politics, religion and local beliefs with the local people.

 Do not make false promises with donors.

 Do not plan the expedition during important festivals and peak election campaign in the target area.

 Do not enter any house for seed collection in the absence of male members of the family.

 Do not eat unknown wild fruits since some of them may be toxic or internally infected.

 Do not collect the seed in large quantities from any household if the farmers wish so.

 Over-collecting of the genetic diversity with similar attributes should be avoided to save time and energy in collection and evaluation and to save space in the genebank.

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Field Visits: Guidelines for Programme Execution KC Bhatt, RS Rathi and Anjula Pandey Division of Plant Exploration and Germplasm Collection ICAR-National Bureau of Plant Genetic Resources New Delhi 110 012, India Introduction

Field visits are an integral part of the plant genetic resources programmes and may be organized for plant/germplasm collection, documentation of indigenous knowledge/ethnobotanical information, monitoring of disease outbreak, teaching/ education, demonstrating and preparing inventory or monographs/ flora. They are undertaken by a group of scientists under the leadership of an experienced person(s) for study pertaining to germplasm collection or education, non-experimental research or for field experiences. Field trips not only do provide alternative educational opportunities but serve as the interface between theoretical and practical knowledge; they are the living laboratories for students in plant sciences. Field visits also provide experience in hands-on learning and take a break from their normal table research/routine activities. Besides, they facilitate the first-hand understanding of a subject or concept in the programme. While selecting field sites one may choose farmer’s field, natural area (Biosphere reserves, National parks and Sanctuaries), biodiversity parks, botanical gardens, community parks, science musea and demonstration plots. The duration of the field visit may depend on the purpose of the visit. For example for exploration for germplasm collection of cultivated/wild species (10- 15 days); locating and tagging of identified material (1-2 days); herbarium specimens (4-5 days or more) and for the educational purpose (2-3 days or more) the duration may be decided by the coordinating institute/ organizer(s). (For more details on the technical aspects, refer to chapter 12). Field visits in the plant genetic resources perspective The pre-field trip may include activities as: delivering lectures and demonstrations on biodiversity, collecting of plants/germplasm and herbarium processing methods. Field visits in relation to taxonomy are desired to understand: a) plant characters (canopy, bark, leaf fall, orientation, actual colour etc.) as visible in live material vs herbarium and, b) understanding of allied fields- ecology, plant biodiversity, population biology, etc. Planning of field visits may vary with respect to the objectives of the visits. If the visit is planned only for survey and collection of germplasm, appropriate procedures may be followed (Annexure I). If collectors are visiting an area for germplasm collecting he may assemble the

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Field Visits: Guidelines for Execution (Bhatt et al.) herbarium also. The appropriate herbarium collection time is 15 days to one month prior to maturity (at full bloom stage). Herbarium specimens should be representative of the population including the range of variation. In a single trip, the collector may find it difficult to assemble the germplasm and herbarium hence, two trips may be planned by the same team or else two different teams can visit to meet different objectives. For seed-producing crops/species, exploration should be undertaken when crops/species are physiologically mature and ready to harvest. In species with shattering nature (crop wild relatives/wild species) usually at maturity, the material is not available to the collector on plants (though in some cases collecting from the ground may be possible) (Refer chapter 12 for details). Preparatory steps before the trip  Discuss the purpose of the field trip and its relation to the present study.  Overview on the field trip schedule.  Assigning role of the "specialists" with respect to the study.  Collect literature and data on area, plant species likely to occur and germplasm conserved.  View route map and photographs of the site to be visited. The field visit planned for the purpose of education, teaching and knowledge dissemination needs to be organized and well-planned tour itinerary chalked out with the involvement of the teachers/experts/explorers. Such trips can also create interest among the students about the knowledge of germplasm collecting/taxonomy/genetic resource science. Teaching faculty may be organized such trips by involving students from schools/colleges and research institutes. Areas of visit: the areas of the mission are selected based on breeders’ requirements, crops/species to be targeted. For example, herbarium collection is exclusively made in those areas which are rich in species to be targeted. When an explorer visits the areas as per his target, priority needs to be given for germplasm collecting in the areas which remained unexplored and inadequately covered like remote/inaccessible localities, tribal-dominated areas, coastal areas, mountainous ranges, biosphere reserves/National parks/sanctuaries. The explorer should have adequate knowledge about nature and extent of diversity and breeding behaviour of the crop/species to be collected. Well in advance planning is required to facilitate the preparations of the proposed missions except those to be carried out under special situations like rescue collecting. Information on topography, climatic conditions, vegetation, crops in

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Field Visits: Guidelines for Execution (Bhatt et al.) cultivation and their maturity time, etc. need to be gathered to finalize the itinerary of collecting mission. Agricultural fields with crops associated weeds/ crop wild relatives and other floristic components are the important sites for making visits for germplasm collecting and assembling herbarium specimens, hence may be targeted for collecting rabi and kharif crop germplasm after assessing the gaps. Open forested areas may be visited for wild/ weedy plants. Literature survey, the study of previous collection reports, published information, expert’s help is very much required for working out gaps in collecting germplasm. While conducting trips for the educational/teaching programmes, the visits are generally planned to expose the students about habitats and to inculcate the knowledge on the diversity for creating interest in their mind. Areas like public parks, botanical parks/ gardens have well- labelled plants, which attract the attention of students to a larger extent. In gardens, biodiversity parks, public/educational parks, local/ plant names are labelled, or information is with curators/ in-charges. Else a well-informed taxonomist with knowledge on plants should accompany. Punjabi Bagh Herbal Garden (Delhi), Aravali Biodiversity Park (Haryana), Botanical Garden (Noida, Uttar Pradesh) are some of the sites in Delhi and National Capital Region which have been identified for educational trips. Field equipment/tools: while moving in the field visits, one has to be well equipped with the tools, material essential for conducting study tour or to collect germplasm (Annexure I). While regional/ national flora, digital herbarium, lap-top and accessories, list of local names of plants, road- map, vegetation/climate maps, list of rest-houses/ lodges, hotels, resting/ stay places and list of local ex-situ conservation of genetic resources of native crops, crop wild relatives and locally important potential species. Photography: field photography is an essential tool to support various activities during field trips and facilitates in capturing the entire picture of the area and plant diversity. Shots of closer parts of the plant are helpful in identification. Photographs/information on associated wild plants, soil type, tree canopy, standing crop area is also important. Moving to fields When moving in the field for collection, always wears shoes to protect the foot. Resources- botanical garden includes a herbarium, research laboratories and collection of photographs, paintings, illustrations, reprints, etc. One should keep recording information and additional data

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(Annexure II). Plants must be carefully uprooted to ensure maximum characters available on genetic diversity and to retain some on site. Well-planned field visit may support activities:  Research and development: field learning on plant taxonomy; enhancing indigenous, to encourage the sustainable use of plant resources for sustainable development.  Education: strengthening the understanding of the importance of plants, visiting diverse habitats and create knowledge and intimacy with plants.  Linking: plants with the well-being of people; organizations involved in collaborative field visit to the team members. Collecting methodology for germplasm and herbarium specimens are given in chapters 2/3. Keep a careful record of collection data and field observations in a field notebook which can be used later for the herbarium label or for preparing a collection report. Do not collect species that are rare or endangered. Take a photograph of the plant and make a well-written description and/or sketch. Take several close-up shots showing the plant and parts necessary for positive identification. Collectors can combine information gathered by visual observation onto data sheets with GPS readings and photographs of locality, site, plants and plant parts. Recording of information as per passport data sheet may help in the planning of future collection of germplasm at the right season. Because, each accession is accompanied with a minimum set of information (the passport data) which is useful for any future reference like a place of collection, period/ time of collection, unique attributes, ethnobotanical uses, etc. Post-visit exercise: after the field visit is over, the activities that need to be undertaken is to assess if the goals of the visit are met. For germplasm collecting visit sorting and preparing germplasm samples; completing and editing the collecting data; distributing the germplasm samples, and writing a report on the collecting mission and ensuring all data that was collected is entered into the appropriate database. Hence, taking care of the collected material/herbarium specimens after completing the mission is absolutely essential. The passport information along with material collected is sent for assigning national identity number and then the same is to be sent for characterization and deposition in the genebank. Herbarium specimens from the field to the facility need extra care (information discussed in chapter 3). For an educational trip, the participants are asked to prepare a report on a field visit and their experience of learning.

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Conclusions Field visits are an integral part of taxonomy, exploration and economic botany curricula. Planning and organizing for a successful programme need is essential and can add much value to the gathered material as well as information after the completion of the visit. Selected readings/references Arora RK (1991) Plant Diversity in Indian Gene Centre. In: RS Paroda and RK Arora (eds) Plant Genetic Resources Conservation and Management. International Board for Plant Genetic Resources, New Delhi, India, pp 25-54. Bhandari DC, ER Nayar, Anjula Pandey, R Srivastava, KC Bhatt, AK Singh and SK Sharma (2009) Exploration and Germplasm Collection. National Bureau of Plant Genetic Resources, New Delhi, 12 p. FAO (1993) International Code of Conduct for Germplasm Collecting and Transfer. FAO.www.fao.org/nr/cgrfa/cgrfa-global/cgrfacodes/en/. Guarino L, V Ramanatha Rao and R Reid (1995) Collecting Plant Genetic Diversity: Technical Guidelines. International Plant Genetic Resources Institute (IPGRI), Rome, Italy; Plant Production and Protection Division, FAO, Rome, Italy; World Conservation Union (IUCN), Gland, Switzerland; CABI, Wallingford, UK. Guarino L, V Ramanatha Rao and E Goldberg (eds) (2011) Collecting Plant Genetic Diversity: Technical Guidelines. Bioversity International, Rome, Italy. (online:http://cropgenebank.sgrp.cgiar.org/index.php?option=com_ content&view=article&id=390&Itemid=557) Havens K, P Vitt, M Maunder, EO Guerrant and K Dixon (2006) Ex-situ Plant Conservation and Beyond. Biosci 56: 525-531. Maunder M, S Higgens and A Culham (2001) The Effectiveness of Botanic Garden Collections in Supporting Plant Conservation: A European Case Study. Biodivers Conserv 10: 383-401. Nayar ER, A Pandey, K Pradheep, R Gupta, DC Bhandari and KC Bansal (2011) National Herbarium of Cultivated Plants. National Bureau of Plant Genetic Resources, New Delhi. Pareek SK, IS Bisht, KC Bhatt, Ashok Kumar, MN Koppar, PN Gupta, SK Mithal and Rakesh Singh (2000) Manual on Exploration and Collection of Plant Genetic Resources and Related Indigenous Knowledge. Agro-biodiversity (PGR)-5. Jai Vigyan National Science & Technology Mission on Conservation of Agro-biodiversity (Plant

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Genetic Resources), National Bureau of Plant Genetic Resources, New Delhi. Prescott-Allen R and C Prescott-Allen (1981) In-situ Conservation of Crop Genetic Resources: A Report. IBPGR, Rome, Italy. Wyman D (1970) How to Establish An Arboretum or Botanic Garden. Arbori Bot Gard Bull 4(52): 52-60. Annexure I List of items and equipment* Survey/  Global Positioning System (GPS), digital camera with an a collecting additional memory card, binocular, magnifying glasses, items handheld microscope, digital vernier calliper and portable balance.  Haversack/ kitbag, seed envelopes, cloth bags, polythene bags, aluminium and tag labels, drying sheets, old newspapers, plant press, moss, rubber bands, packing tape, sutli (thick and thin), secateurs, scissors, knife, digger, torchlight, measuring tape, passport data book, field notebook, pencil, ballpoint pen and permanent marker.  Others: Stapler, candle, matchbox, water bottle, formaldehyde, hunter shoes, hand gloves, waist pouch, rain suit (shirts, trousers), rucksacks, sunglasses, etc.

Reference  Regional/ national flora, digital herbarium, lap-top and material accessories, list of local names of plants, road-map, vegetation/climate map, list of rest-houses/ lodges, hotels, resting/ stay places and list of local contacts (phone, fax, e- mail).

First aid  Anti-malaria tablets, anti-allergen tablets, pain killers, anti- box amoebic and anti-diarrhoeal tablets, mosquito repellent, antifungal/ antibacterial/ antiseptic creams or lotions, cotton-packs, band-aid, anti-bacterial/Dettol, dressing gauze, water-purifying tablets, etc.

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Annexure II NATIONAL BUREAU OF PLANT GENETIC RESOURCES, NEW DELHI -110 012 Passport Data Sheet (*) Date...... Collector’s No...... Accession No...... Botanical Name...... Common Name(English)...... Crop/Vernacular Name...... Cultivar name...... Region Explored...... Village/Block...... District...... State...... Latitude………………………0N Longitude……………………..0E Altitude…………………m Temp------Rainfall ------COLLECTION SITE 1. Natural wild 2. Disturbed wild 3. Farmer’s field 4. Threshing yard 5.Fallow 6. Farm store 7. Market 8. Garden 9. Institute 10...... BIOLOGICAL STATUS 1. Wild 2.Weed 3.Landrace 4.Primitive cultivar 5. Breeder’s line FREQUENCY 1. Abundant 2. Frequent 3.Occassional 4. Rare MATERIAL 1. Seeds 2.Fruits 3.Inflorescence 4.Roots 5.Tubers 6.Rhizomes 7.Suckers 8.Live plants 9. Herbarium 10………….. BREEDING SYSTEM 1.Self-pollinated 2.Cross-pollinated 3.Vegetatively propagated SAMPLE TYPE 1.Population 2.Pure line 3.Individual plant SAMPLE METHOD 1.Bulk 2.Random 3.Selective (non-random) HABITAT 1. Cultivated 2. Disturbed 3.Partly disturbed 4. Rangeland 5……. DISEASE SYMPTOMS 1. Susceptible 2. Mildly susceptible 3. Tolerant 4. Resistant INSECT/ PEST/ 5. Immune NEMATODE 1. Mild 2.Moderate 3. High INFECTION CULTURAL PRACTICE 1. Irrigated 2. Rainfed 3. Arid 4. Wet 5...... SEASON 1. Kharif 2. Rabi 3. Spring-summer 4.Perennial type ASSOCIATED FLORA 1.Sole 2.Mixed with……………………… SOIL COLOUR 1. Black 2.Yellow 3.Red 4.Brown 5………….. SOIL TEXTURE 1.Sandy 2.Sandy loam 3.Loam 4.Silt loam 5.Clay 6.Silt TOPOGRAPHY 1.Swamp 2.Flood plain 3.Level 4.Undulating 5.Hilly dissected6.Steeply dissected 7.Mountainous 8.Valley AGRONOMIC SCORE 1. Very poor 2.Poor 3.Average 4.Good 5.Very good

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ETHNOBOTANICAL USES PART(S) 1. Stem 2. Leaf 3.Root 4. Fruit 5.Flower 6.Whole plant 7.Seed 8. Others KIND 1. Food 2.Medicine 3.Fibre 4. Timber 5. Fodder 6. Fuel 7. Insecticide/Pesticide 8. Others HOW USED ...... INFORMANT(S) 1. Local Vaidya 2.Housewife 3.Old folk 4. Graziers /Shepherds 5.Others PHOTOGRAPH 1. Colour/ Black and white/ Transparency FARMER’S/DONOR’S NAME...... ETHNIC GROUP...... ADDRESS ...... PLANT ...... CHARACTERISTICS/ ...... USES ADDL. NOTES ...... *: Arora (1991 with modifications)

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ICAR-National Bureau of Plant Genetic Resources, New Delhi, India Training Programme on “Herbarium Management” (Tentative Programme)

Date Time Topics/Activity Resource Person(s) 15.07.2019 09:30 - 10:30 AM Registration &Introduction of Course Co- (Monday) the Trainees Ordinator’s 11:00 - 12:30 PM ICAR-NBPGR Documentary Neeta Singh, DGC, Film NBPGR 12:30 - 01:30 PM Visit to the National SmitaLenka Jain, Genebank DGC, NBPGR 02:30 - 04:30 PM Visit to ICAR-NBPGR AD Sharma/ NS Facilities Panwar, NBPGR 16.07.2019 09:30 - 10:45 AM Herbarium management- role Anjula Pandey, (Tuesday) in genetic resources study DPEGC, NBPGR 11:00 - 12:15 PM Field to herbarium- SP Ahlawat, prospects and procedures DPEGC, NBPGR 12:15 - 01:30 PM The relevance of field and ERoshni Nayar, ecological studies in Ex- NBPGR herbarium management

02:30 - 04:30 PM Practical: Herbarium Anjula Pandey and methodology and its Rita Gupta, applicability DPEGC, NBPGR 17.07.2019 09:30 - 10:45 AM Digitization of herbarium Anjula Pandey, (Wednesday) DPEGC, NBPGR 11:00 - 12:15 PM Modern tools: phylogenetic Roshni R Mathur. approach in species DU identification 12:15 - 01:30 PM Systematics of crop plants- KV Bhat, NBPGR case studies and demonstration 02:30 - 04:30 PM Practical: Herbarium tools: DP Semwal, NS geo-informatics in the PanwarandRita mapping of species diversity Gupta, distribution, key DPEGC, NBPGR construction and use 18.07.2019 09:30 - 10.45 AM Modern tools in herbarium S Rajkumar, DGR, (Thursday) management: cytomolecular NBPGR approach 11:00 -12.15 PM Modern tools: biochemical R Bhardwaj, DGE, methods - case studies in NBPGR crop taxa 12:15 - 01:30 PM Taxonomic literature and its K Pradheep, use in identification NBPGR, Thrissur 02:30 - 04:30 PM Practical: Modern tools in S Rajkumar, DGR, herbarium management NBPGR

19.07.2019 08:30 – 12.30 PM Field visit- significance and KC Bhatt, RS (Friday) method of collection of plant Rathi, Anjula for herbariumin Pandey, DPEGC, NBPGR 01.30 - 04.30 PM Field visit KC Bhatt, RS (Issappur&Jhajjar) Hands Rathi, Anjula on training on Method of Pandey, DPEGC, preprocessing plant material NBPGR for herbarium in the field 20.07.2019 09:30 - 11:00 AM Feedback Session Sherry R Jacob/ (Saturday) MsPadmavati G Gore, DGC. NBPGR 11:30 - 01:30 PM PGR-Indian Perspective Kuldeep Singh, Director, NBPGR 02:30 - 04:00 PM Valedictory Function

*Tea from 10.45 to 11.00 am and 3.30 to 3.45 pm daily; lunch from 1.30 to 2.30 pm daily

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List of the Participants-Herbarium Management

Name : Ikhlas Hussain Alwan

Designation : Assistant Chief of Researcher

Department : National Herbarium of Iraq/Plant Department

Organization : Directorate of Seed Testing and Certification

E-mail ID : [email protected]

Phone No. : +9647719220419

Name : Nagwan Dalaf Abbas

Designation : Senior Agronomist

Department : National Herbarium of Iraq/Plant Department

Organization : Directorate of Seed Testing and Certification

E-mail ID : [email protected]

Phone No. : +9647704084557

Name : Russul Saad Hamshkan

Designation : Biologist

Department : National Herbarium of Iraq/Plant Department

Organization : Directorate of Seed Testing and Certification

E-mail ID : [email protected]

Phone No. : +9647715191335

Faculty members and other contact persons involved in Training Programme

Name and affiliation E-mail id AD Sharma, Technical Officer, Conservation axma.sharma@@icar.gov.in Division, ICAR-NBPGR, New Delhi

Anjula Pandey, Pr Scientist, Exploration [email protected] Division, ICAR-NBPGR, New Delhi

DP Semwal, Pr Scientist, Exploration Division, dinesh.semwal@@icar.gov.in ICAR-NBPGR, New Delhi

ER Nayar, Former Scientist Emeritus (ICAR), [email protected] ICAR-NBPGR, New Delhi

K Pradheep, Pr Scientist, ICAR-NBPGR, RS [email protected] Thrissur, Kerala

KC Bhatt, Pr Scientist, Exploration Division, [email protected] ICAR-NBPGR, New Delhi Kuldeep Singh, Director, ICAR-NBPGR, New [email protected] Delhi

KV Bhat, Scientist Emeritus, ICAR-NBPGR, New [email protected] Delhi

MP Sharma, Prof. (Botany), Jamia Hamdard, [email protected]; Delhi [email protected]

Neeta Singh, Pr Scientist, ICAR-NBPGR, New [email protected] Delhi

NS Panwar, Assistant Chief Technical Officer, [email protected] Exploration Division, ICAR-NBPGR, New Delhi

OP Dhariwal, Technical Officer, Exploration [email protected] Division, ICAR-NBPGR, New Delhi

Poonam Suneja, Former Assistant Chief [email protected] Technical Officer, Germplasm Evaluation Division, ICAR-NBPGR, New Delhi

R Bhardwaj, Pr Scientist, Germplasm Evaluation [email protected] Division, ICAR-NBPGR, New Delhi

Rita Gupta, Sr. Technical Officer, Exploration [email protected] Division, ICAR-NBPGR, New Delhi

Roshni R Mathur, Assistant Professor, University [email protected] of Delhi, Delhi

RS Rathi, Pr Scientist, Exploration Division, [email protected]

ICAR-NBPGR, New Delhi

S Nivedhitha, Scientist, Exploration Division, [email protected] ICAR-NBPGR, New Delhi

S Rajkumar, Pr Scientist, Division of Genomic [email protected] Resources, ICAR-NBPGR, New Delhi

Shashi kant Sharma, Technical Officer, [email protected] Exploration Division, ICAR-NBPGR, New Delhi

Sherry R Jacob, Pr Scientist, Conservation [email protected] Division, ICAR-NBPGR, New Delhi

SmitaLenka Jain, Conservation Division, ICAR- [email protected] NBPGR, New Delhi

SP Ahlawat, Head of the Division & Pr Scientist, [email protected]; Exploration Division, Conservation Division, [email protected] ICAR-NBPGR, New Delhi

Sunil Archak, Pr Scientist &Incharge, [email protected] Agricultural Knowledge Management Unit, ICAR-NBPGR, New Delhi

Functional Committees to Organize the Training Programme

I . Core Committee 1. Dr Kuldeep Singh 2. Dr Kavita Gupta 3. Dr Veena Gupta 4. Dr Anjula Pandey 5. Dr SK Kaushik

II. Technical Committee 1. Dr Anjula Pandey 2. Dr Sushil Pandey 3. Dr S Rajkumar 4. Dr Sherry Jacob 5. Dr KC Bhatt 6. Dr RS Rathi 7. Ms S. Nivedhitha 8. Ms Rita Gupta 9. Sh Satyaprakash 10. Ms Anjali 11. Sh Abhay Sharma (photography)

III. Refreshment, Accommodation and Transport 1. Dr Chithra Pandey 2. Dr DP Semwal 3. Sh J Arvind 4. Dr AD Sharma 5. Dr SS Ranga 6. Dr NS Panwar 7. Sh Arun Sharma 8. Sh Shashi Kant Sharma

IV. Programme Support Committee 1. Sh Ganga Nand 2. Ms Urmila Singh 3. Sh OP Dhariwal 4. Smt Manju Devi 5. Smt Sharda Devi 6. Sh Hari Chand Paswan 7. Sh Surinder Kumar

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