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Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 06 (2018) Journal homepage: http://www.ijcmas.com Review Article https://doi.org/10.20546/ijcmas.2018.706.134 Review on Characterization of Cowpea Germplasm in Terms of Distinctness, Uniformity, Stability and Novelty for Morphological, Quality and Yield Attributing Parameters Sanjeev K. Deshpande1*, B.R. Mani2, S.A. Desai3, T.K. Nagarathna3 and R.R. Hanchinal3 1 2 G.P.B., University of Agricultural Sciences, Dharwad, Karnataka-580 005, India SRF, Cowpea DUS, University of Agricultural Sciences, Dharwad, Karnataka-580 005, India 3 PPV & FRA, IARI, New Delhi-110 012, India *Corresponding author email id: sanjeevgpb@gmail.com ABSTRACT Keywords DUS test, Novelty, germplasm, Pulses, Characteristics Article Info Accepted: 06 May 2018 Available Online: 10 June 2018 Cowpea (Vigna unguiculata L.) is the most important grain legume crop grown in tropical and subtropical regions of the world. In India cowpea is grown as sole, mix-crop, intercrop systems. Cowpea is however, the only pulse suited to both arid and semi-arid regions and adapted better than any other pulses in high rainfall regions. As a good source of proteins, calcium and iron, it is recognized for many routine uses of food, feed, forage, fodder, vegetable and a number of dishes prepared from its grains. India being a secondary centre of diversity for cowpea, possess huge germplasm and wild relatives of cowpea thus, it supplements to the genetic resources of worldwide cowpea collection. Characterization of germplasm became mandatory for varietal registration and to apply for a plant breeder right. The characterization data on various morpho-physiological and agronomic traits is essential to maintain genetic data base at seed banks. In this context, a review has been conducted for the literature regarding DUS characterization, its importance and method of experimentation, various descriptors and documentation of data. The data available in the literature has been utilized in classification of cowpea plant characters into ten distinct categories, considering the seedling, stem, leaf, flower, pod, seed and plant morphological traits. Further, under each category, distinguishable, unique and stable traits were also been formulated as DUS parameters of cowpea. The present review may help the researchers in understanding the scope of DUS test and methodology of experimentation moreover, its present day importance with respect to germplasm conservation and patents. Introduction The cowpea (Vigna unguiculata) is an annual herbaceous legume of the genus Vigna. Due to its tolerance for sandy soil and low rainfall it is an important crop in the semi-arid regions across India and other countries. It requires very few inputs, as the plants root nodules are able to fix atmospheric nitrogen, making it a valuable crop for resource poor farmers and well-suited to intercropping with other crops. The whole plant is used as forage for animals, with its use as cattle feed likely responsible for its name. Four subspecies of cowpea are recognized, of which three are cultivated. There is a high level of morphological 1124 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 diversity found within the species with large variations in the size, shape and structure of the plant. Cowpeas can be erect, semi erect (trailing) or climbing. The crop is mainly grown for its seeds, which are extremely high in protein, although the leaves and immature seed pods can also be consumed. Cultivated cowpeas are known by the common names black-eyed pea, southern pea, yard long bean, catjang, and crowder pea. They were domesticated in Africa and are one of the oldest crops to be farmed. A second domestication event probably occurred in Asia, before they spread into Europe and the Americas. The seeds are usually cooked and made into stews and curries, or ground into flour or paste. The Indian subcontinent is very rich in biological diversity, harboring around 49,000 species of plants, including about 17,500 species of higher plants. The Indian gene centre holds a prominent position among the 12 mega-gene centers of the world. It is also one of the Vavilovian centers of origin and diversity of crop plants. Two out of the 25 global hotspots of biodiversity, namely the Indo-Burma and Western Ghats are located here. India possesses about 12 per cent of world flora with 5725 endemic species of higher plants belonging to about 141 endemic genera and over 47 families. About 166 species of crops including 25 major and minor crops have originated and/or developed diversity in this part of the world. Further, 320 species of wild relatives of crop plants are also known to occur here. Presently, the Indian diversity is composed of rich genetic wealth of native as well as introduced types. It is also a secondary centre of diversity for African crops like finger millet, pearl millet, sorghum, cowpea, cluster bean, okra, sesame, niger and safflower Crop description There is a large morphological diversity found within the crop, and the growth conditions and grower preferences for each variety vary from region to region (Padulosil, 1997). However, as the plant is primarily self-pollinating its genetic diversity within varieties is relatively low. Cowpeas can either be short and bushy (as short as 20 cm) or act like a vine by climbing supports or trailing along the ground (to a height of 2 m) (Sheahan, 2012) and National Research Council (2006). The tap root can penetrate to a depth of 2.4 m after eight weeks of sowing (Egbadzor et al., 2014). The size and shape of the leaves varies greatly, making this an important feature for classifying and distinguishing cowpea varieties (Pottorff, et al., 2012). Another distinguishing feature of cowpeas is the long 20–50 cm peduncles which hold the flowers and seed pods. One peduncle can support four or more seed pods (Davis, et al., 1991). Flower color varies through different shades of purple, pink, yellow and white and blue (National Research Council, 2006). Seeds and seed pods from wild cowpeas are very small (Davis et al., 1991). While, cultivated varieties can have pods between 10 and 110 cm long (Rawal and Kanti 1975). A pod can contain 6– 13 seeds that are usually kidney shaped, although the seeds become more spherical the more restricted they are within the pod (Davis, et al., 1991). Their texture and color is very diverse. They can have a smooth or rough coat, and be speckled, mottled or blotchy. Colors include white, cream, green, red, brown and black or various combinations (Davis, et al., 1991). Classification of cowpea varieties based on its bean shape, size and color (Source: India Pulses and Grains Association, www.nutritiondata.self.com, www.nda.agric.za) 1125 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 Black-eyed or pink-eyed/purple hull peas These white colored seeds have black, pink or purple eye like shape around the hilum. Seeds are not tightly packed or crowded in the pod and are kidney shaped or oblong. cannot be created artificially except to a limited extent through mutation breeding or genetic engineering. Fortunately, a large reservoir of genetic diversity existed until recently in old „land races‟ or farmers‟ varieties; a great deal of useful variation also occurs in related wild species. Brown-eyed peas These types of cowpeas have green to lavender colored pods. When immature seeds of this type are cooked, it has very tender and a delicate flavor. Crowder peas As the name suggests, this type of cowpeas are “crowded” in the pod and have seed of black, speckled and brown eyed. Cream peas Seeds are cream colored. This type of seeds looks like intermediate between the blackeyed and crowder types. Modern agricultural technology and the introduction of new high-yielding varieties are largely eliminating the wide range of crop genetic diversity that has evolved during the five to ten thousand years since food plants were first domesticated. Related wild species are also on the decline because of new land use policies. These gene pools (or what is left of them) are generally spoken of as genetic resources, and are vitally needed in the creation of new crop varieties by plant breeders. Wild species and land races often furnish genes conferring resistance to diseases and pests and adaptation to environmental stresses which cannot be found in the modern crop varieties. DUS characterization studies in cowpea White acre type Seeds are kidney-shaped with a blunt end, semi crowded and generally tan in color. Clay types These older varieties are medium to dark brown in color and kidney- shaped, but are rarely grown Importance of germplasm resources in crop improvement In order to breed qualities of resistance, adaptation and better nutritive value into crops, breeders need sources of genetic diversity to draw upon when required. This diversity is generally spoken of as „genetic resources‟ or „gene pools‟. These gene pools To sustain high production and productivity of cowpea, a considerable number of varieties have been developed. In addition, a number of germplasm collections are available in the gene pool for use in plant breeding programmes. However, there is lack of compilation of key diagnostic characters of these varieties and germplasm collections, which is essential to carryout scientific seed production and certification, endorse proper quality control, DUS testing and utilization of these characteristics in further breeding programmes. The variety characterization and purity assessment are very important for maintenance, multiplication, seed certification and quality control of released varieties. The characterization is also essential for 1126 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 registration and protection of varieties. Traditionally varieties were identified on the basis of their morphological characters, either in the form of seed, seedlings or growing plant characteristics. virus, seed crowding in pod, texta structure, eye color, pod shape, pod attachment to peduncle, terminal leaflet shape, twining tendency and flower pigmentation, respectively. In this context, Sarutayophat et al., (2007), characterized 13 cowpea accessions based on growth habit, days to 50 per cent flowering, pod color, pod length, number of pods per plant, seed yield per plant. Similarly, Basavaraj, et al., 2013, conducted an experiment to characterize 35 cowpea genotypes using different quantitative descriptors at plant level. Highly significant differences were obtained among the genotypes for the characters studied. All characters were found stable and reliable for classification except for characters number of pods per peduncle and seed yield per hectare. Hence, it‟s reliable to consider these stable characters in future breeding programmes of cowpea crop and quality seed production and also in protection in plant variety. DUS characterization studies in related pulse (Green gram) Further, Stoilova and Pereira (2013), had used 24 different morphological descriptors in order to identify accessions with specific behavior that could be exploited by plant breeders and they found that descriptors like pod length, number of seeds per pod, seed thickness and 100 seed weight were the most stable traits and they concluded these characters can be used in characterization. Sunil Kumar et al., (2015), characterized 20 forage cowpea genotypes on the basis of various morphological characters which were mentioned in cowpea germplasm catalogue of IITA, Nigeria. All genotypes showed indeterminate type of main stem and raceme type of layer in canopy; whereas genotypes viz., KBC 2, IC 249141, HC 46, CO 4, EC 3941-1, EC 101980, Kohinoor and CO 5 showed unique state of expression among nine characters viz., incidence of cowpea mosaic The literature pertaining to DUS characterization in Cowpea is limited, a review is also been made by considering the green gram, a crop relative of cowpea. Rupinder et al., (2017) characterized five lines released by PAU, Ludhiyana (PAU911, SML668, ML818, ML613 and SML832) using morphological and molecular markers. Phenotypically these varieties showed variation for growth habit, leaf and flower characters, pod color, position and length, plant height, seed coat lusture and seed size during different growth stages of the crop. Plant morphology characters being polygenic in nature are liable to be influenced by the environment. Hence there is a need to use alternate descriptors which are rapid, accurate and less affected by environment. Methodology of DUS testing The principles and methods on various aspects of DUS testing of new crop varieties have been documented as National Test Guidelines (NTG). NTG has been developed for 35 crops by the National Core Committee constituted by ICAR. NTG contains details on plant material required, conduct of tests, methods and observations, grouping of varieties, characteristics and symbols, table of characteristics, literature and technical questionnaire. Usually the DUS examination requires at least two independent growing cycles. Not less than two centers have been identified for each crop for conduction of the tests. 1127 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 Distinct-Uniform-Stable (DUS) Morphological characterization Distinct means a variety should be clearly distinguishable by one or more essential characteristics from any other existing variety. The variety is deemed Uniform if it is sufficiently uniform in its relevant characteristics, subject to variation that may be expected from the particular features of its population. This is based on botanical or morphological descriptors or characters of the plant or plant part. Qualitative characteristics: Truly qualitative characteristics show discrete discontinuous states and are stable, heritable and uniformly expressed in all environments (shape, flower color, etc). The basis of assessment is normally the number of off-types in the variety, judged on the basis of a population standard and an acceptable probability fixed in the corresponding species. To identify off-types in a population, generally visual observation on characteristics may suffice. However, in a few crops, it may be necessary to make measurements of each plant to apply statistics to decide or not whether a plant is an off-type. In most of the crops, acceptance probability of 95% has been suggested. The variety is said to be Stable if its relevant characteristics remain unchanged after repeated propagation. Though it is not usually possible to assess stability with in a period of 2 or 3 years, the variety can be considered stable if is shown to be uniform. Pseudo-qualitative characteristics: Here, the range of expression is at least partly continuous varying in more than one dimension. In some cases, intermediate states of expression such as „weakly expressed‟ are included between „absent‟ and „strongly expressed‟ (Pubescence, Pigmentation, etc.). Qualitative characteristics are assessed visually while quantitative characteristics are usually measured. The following types of assessments are recommended: VG: Visual assessment by a single observation of a group of plants or parts of plant. VS: Visual assessment by observation of individual plants or parts or plants. MG: Measurement by a single observation of a group of plants or parts of plant. MS: Measurement of a number of individual plants or parts or plant. Characterization The requirement of distinctness, uniformity and stability are assessed on the basis of characteristics. The characteristics are a feature of whole plant or part of plant. Such characteristics may be morphological, biochemical, molecular or any other nature. The table of characteristics chosen by experts forms the main part of test guidelines and of DUS testing. In Genetic resources, the term „characteristic‟ is known as descriptors (with descriptor states) and describing a plant based on such descriptor is known as „characterization‟. Grouping characteristics can be universally used, either individually or collectively, for grouping the similar varieties. These characteristics are considered to be most reliable in distinguishing or discriminating varieties. Biochemical characterization: Isozyme based descriptors have been widely used for identification of crop varieties because of their reliability (Smith and Smith, 1992; Cooke, 1995). 1128 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 Table.1 Classification of DUS traits associated with cowpea S. N0 01 02 03 04 05 06 07 08 09 10 DUS traits associated with Frequency Seedling stage (Anthocynin coloration) 01 Stem 02 flower 07 Plant 10 Leaf 13 Pod 18 Seed 23 Response to abiotic stress 04 Response to biotic stress (Insect Pests) 05 Response to biotic stress (Diseases) 09 Total No. DUS traits Proposed 92.00 Percentage 1.08 2.16 7.61 10.86 14.44 19.56 25.00 4.35 5.43 9.78 100.00 Table.2 Proposed cowpea crop descriptors S. No 1 2 3 4 5 6 7 8 9 Characters Status Seedling Anthocyanin Absent coloration Present Stem color Purple Semi purple Green Stem hairiness Non-hairy Sparsely hairy Hairy Days to first flowering Early (<35) Medium (35-40) Late (>40) Days to 50% flowering Early (48) Medium (48-52) Late (>52) Plant height (height of Short (<55) main stem when Medium (55-65) supported) Long (>65) No. days to maturity Early (<75) Medium (75-80) Late (>80) Plant pigmentation Moderate on base and tip of petioles Extensive None or Very pale Almost Solid Plant growth habit Prostrate Semi prostrate 1129 Code 0 1 1 2 3 3 5 7 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 4 1 2 Adopted from IITA, Nigeria IITA, Nigeria IITA, Nigeria Cowpea catalogue, NBPGR, New Delhi, India Cowpea catalogue, NBPGR, New Delhi, India IITA, Nigeria IITA, Nigeria IITA, Nigeria Cowpea catalogue, NBPGR, New Delhi, India Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 10 11 S. No 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Intermediate Semi erect Erect Acute erect Climbing Twining tendency None Moderate Pronounced Vigor index Vigorous - Height < 37.5cm and Width < 75cm Intermediate - Height > 37.5cm or Width > 75cm Vigorous - Height > 37.5cm and Width > 75cm Very Vigorous - Height > 50cm and Width > 1m Characters Status Determinacy Determinant In determinant Primary branches per Less (<4) plant Medium (4-6) High (>6) No. of nodes on main stem Green fodder yield per plant Leaf to stem ratio Leaf texture Cariaceous Intermediate Membranous Stipule length (mm) Ryuuu Stipule width (mm) Rydrudu Leafiness Vigorously leafy Leafy Intermediate Sparse, leaf size average or above Sparse, leaf size small Leaf color Light green Green Dark green Leaf surface Glabrous Pubescent Intensity of leaf green Light color Medium Dark Petiole length Short Medium Long Terminal leaflet shape Sub-hastate 1130 3 4 5 6 7 1 2 3 1 Cowpea catalogue, NBPGR, New Delhi, India IITA, Nigeria 2 3 4 Code 1 2 1 2 3 1 2 3 1 2 3 4 5 1 2 3 1 2 1 2 3 1 2 3 1 Adopted from Cowpea catalogue, NBPGR, New Delhi, India Descriptors for Cowpea, IPGRI, Rome IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria Cowpea catalogue, NBPGR, New Delhi, India Cowpea catalogue, NBPGR, New Delhi, India IITA, Nigeria IITA, Nigeria Cowpea catalogue, NBPGR, New Delhi, India IITA, Nigeria Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 26 Terminal leaflet length 27 Terminal leaflet width 28 Leaf marking (Presence or absence of V mark on leaflet) Characters Number of flushing S. No 29 30 Flower pigmentation 31 Raceme position 32 Flower bud color 33 Flower color 34 35 First pod bearing node Days to first ripe pod 36 Pod pigmentation 37 Peduncle length Sub-Globose 2 Hastate (Lanceolate) 3 Globose 4 Short (<8) Medium (8-10) Long (>10) Narrow (<6) Medium (6-8) Broad (>8) Absent Present 1 2 3 1 2 3 0 1 Status Code Completely Pigmented Wing and standard petal has pigmented margins Wing pigmented petal has light V-shape pattern None Wing has pigmented upper margin; standard petal pigmented Wing is pigmented ; Petal is lightly pigmented Mostly above canopy Throughout canopy In upper canopy Yellowish Light green Medium green Purple Light purple White 1 2 IITA, Nigeria IITA, Nigeria IITA, Nigeria Adopted from Cowpea catalogue, NBPGR, New Delhi, India IITA, Nigeria 3 4 5 6 1 2 3 1 2 3 1 2 3 Cowpea catalogue, NBPGR, New Delhi, India IITA, Nigeria IITA, Nigeria IITA, Nigeria None Pigmented tips Pigmented suture Pigmented valves with green sutures Splashed Uniformly pigmented Short (<5) Medium Long (>5) 1131 1 2 3 4 5 6 1 2 3 Cowpea catalogue, NBPGR, New Delhi, India Cowpea catalogue, NBPGR, New Delhi, India Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 38 No. of pods per Peduncle 39 No. pods per plant 40 Pod attachment to peduncle S. No 41 Characters Number of Clusters per plant Less (<2) Moderate (2-3) High (>4) Low (<15) Medium (15-20) High (>20) Erect Angle of 30-90 o down from erect Pendent Status High (>11) Medium (9-11) low (<9) Crescent Erect coiled Long (>10) Medium (8-10) Short (<10) 42 Pod shape 43 Pod length 44 45 46 Pod width Number of locules per pod Pod curvature Straight Curved Slightly curved Coiled Immature pod color Green Dark green Light green Purple green Pod color at maturity Yellow Brown Pod twisting Absent Present Texture of pod surface Smooth Moderately rough Very rough Extent of seed crowding Non Crowder in pod Semi Crowder Extreme Crowder Number of seeds per Medium (14-18), Low, (<14), pod high (>18) Pod wall thickness Thin Intermediate Thick Eye pattern Watson group Holstein group 47 48 49 50 51 52 53 54 1132 1 2 3 1 2 3 1 2 3 Code 1 2 3 1 2 3 1 2 3 1 2 3 4 1 2 3 4 1 2 0 1 1 2 3 0 1 2 1,2,3 3 5 7 1 2 IITA, Nigeria IITA, Nigeria Cowpea catalogue, NBPGR, New Delhi, India Adopted from IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria Descriptors for Cowpea, IPGRI, Rome IITA, Nigeria IITA, Nigeria Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 1124-1139 55 S. No 56 Eye color Characters Testa texture 57 Seed size 58 Seed length 59 Seed thickness 60 Seed Shape 61 Seed color (Main colorcolor of largest area of seed) 62 Seed Secondary color 63 Seed secondary color 64 Pattern of seed secondary color Small eye Narrow eye Kabba eye Tan and Brown Red white, cream, brown splash gray Status Smooth Wrinkled 1 (810mg/10 seeds) 2(1400mg/10 seeds) 3(472mg/10 seeds) 4(2150mg/10 seeds) 5 (48mg/10 seeds) 6 (189mg/10 seeds) 7 (2880mg/10 seeds) 8 (117mg/10 seeds) 9 (3370mg/10 seeds) Very short Short Intermediate Long Very long Thick Flat Very flat Kidney Ovoid Crowdege Globose Rhomboid White Light yellow Brown Reddish brown Purplish brown Black Absent Present Brown Reddish brown Purplish brown Black Around hilum In veins Mottled on part of seed 1133 3 1 2 3 4 5 6 Code 1 2 1 2 3 4 5 6 7 8 9 1 2 3 4 5 1 2 3 1 2 3 4 5 1 2 3 4 5 6 0 1 1 2 3 4 1 2 3 IITA, Nigeria Adopted from IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria Cowpea catalogue, NBPGR, New Delhi IITA, Nigeria IITA, Nigeria IITA, Nigeria IITA, Nigeria