Karyomorphological Studies in Mucuna of India

Karyomorphological studies in Mucuna of India

Mahammad Jaheer and N Sathyanarayana1

Department of Bio-Technology, Sir M Visvesvaraya Institute of Technology Bangalore 562157, India

1Author for correspondence: [email protected] Received July 12, 2010; accepted July 31, 2010

ABSTRACT. Mucuna Adans. is a large genus with more than 150 species of annual and perennial legumes of pantropical distribution. Despite considerable economic importance of some of its species, almost no work has been done on their cytogenetic characterization in any part of World. In this paper, we have presented a ﬁ rst report on karyomorphological analysis in four Indian species viz., Mucuna atropurpurea, Mucuna monosperma, Mucuna nigricans and Mucuna pruriens bringing out diploid number for M. atropurpurea and karyotypic formula for all the four species. Root tips were treated with 0.002 M 8-hydroxyquinoline and temporary preparations were made after squash in aceto-carmine. Karyomorphological description for each of the species has been detailed taking into account the following features: chromosome number, total length and relative length, arm ratio of each chromosome, haploid set length and degree of asymmetry. Mitotic metaphases in all the species showed 2n=22 chromosomes. Mucuna nigricans showed two pairs of sat-chromosomes. None of the species, however, revealed identical karyotypic formula indicating occurrence of rearrangements. The haploid set length of annual M. pruriens was found to be reduced in size when compared to perennial species pointing its evolution from the latter.

KEYWORDS: Cover crop, Karyomophological analysis, Mucuna, Velvetbean

Mucuna Adans. (Fabaceae-Papilionoideae-Phaseoleae) taxonomic ambiguities (St. Laurent 2002) very little efforts originated in southern Asia, possibly in Eastern India, have been made to draw evidences from chromosomal Malaysia or Southern China (Burkill 1966, Duke 1981, studies. Thus, so far, among Indian species only diploid Wilmot Dear 1984). The genus includes approximately numbers of 2n=22 has been reported in M. monosperma, 150 species of annual and perennial legumes of pantropical M. nigricans and M. pruriens and (Bairiganjan 1989; distribution. Recently there has been increased attention Kayna Agostini et al. 2009) and the basic numbers are on one of its species M. pruriens (velvetbean) for its available for M. pruriens var. pruriens, M. pruriens var. potential as cover crop, green manure, feed and food utilis and M. gigantea (Sastrapradja 1974). But none of (Capo chichi et al. 2003). All the members are also known these are with detailed karyotype analysis. Lack of such to produce very high levels of L-DOPA (L-3, 4- basic information, among others, has impended effective dihydorxyphenylalanine), a well-known non-protein utilization of Mucuna genetic resources in the country for amino acid that acts as precursor for the neurotransmitter constructive breeding programs. In view of this, a dopamine used in the treatment of Parkinson’s disease preliminary investigation has been carried out in four (Barbeau and McDowell 1970). One of its Sanskrit names, Indian taxa viz., M. atropurpurea, M. monosperma, M. atmagupta, (“Having hidden properties”), seemingly nigricans and M. pruriens so that we could validate their denotes its importance as medicinal plant. In addition, chromosome numbers and assess karyo-morphological many species of the genus are also traditionally used in characteristics. various other ways like dye (M. argyrophylla, Standl. 1946); treatment of pain, numbness of joints and irregular MATERIALS AND METHODS menstruation (M. birdwoodiana Tutcher, See Ding et al. Plant material. Four Mucuna species collected from 1991) and food (Mucuna calophyalla, M. macrocarpa diverse geographical regions in India viz., M. atropurpurea Wall., Wilmot-Dear 1984; Mucuna pruriens (L) DC. var. (Roxb.) DC. ex Wight & Arn, M. monosperma DC ex utilis (Wll. ex Wight). Wight, M. nigricans (Lour) Steud and M. pruriens (L) DC A review of the taxonomic status of the genus Mucuna were used in the present study (Table 1). Of these, fi rst reveals presence of nine species in Indian subcontinent three are perennials while the latter exhibit annual growth (Wilmod-Dear 1987). These are: M. atropurpurea and M. habit. The botanical description of each one of these is pruriens (var. hirsuta) endemic to Peninsular India; M. given below: imbricata, M. bracteata, M. macrocarpa, M. sempervirens and M. nigricans restricted to the North-Eastern parts and Mucuna atropurpurea is a large woody liana with dark M. pruriens (excluding var. hirsuta), M. monosperma and reddish bark and wrinkled stem. Leaves membranous and M. gigantea as widely distributed species all over. In a pale brown in colour. Infl orescence, often present in recent revision, P. R. Sur (2002) has raised some of the clusters, contains dark reddish-purple fl owers with earlier reported botanical varieties to an independent pedicels fl ashing adpressed reddish brown irritant hairs. species raising this number to 13. Despite widely reported They produce thick leathery, highly wrinkled fruits, 38 JAHEER AND SATHYANARAYANA

Table 1. Mucuna species used in the study Species Accession Locality Geographical ordinates Number M. atropurpurea 500114TN Azhagarkoil, Madurai, TN, India 9° 91’ N, 78° 12’ E M. monosperma 500187MH Kolhapur MH, India 16o41’N, 74o13’E M. nigricans 500198WB Gorumara National Park WB, India 26o7’N, 88o8’E M. pruriens 500120TN Ayurvedic Vendor, Madurai TN, India － covered with long red erect irritant deciduous bristles. with distilled water for 10 min. The meristematic region Seeds, usually 2-3 per pod are black in colour and swollen was squashed in a drop of aceto-carmine stain on a glass oblong shaped in outline. Distribution is endemic to slide by immobilizing the root tip with cover slip. Followed peninsular India. by gentle tapping, strong squashing was applied to spread the cells uniformly. The chromosomes were visualized, Mucuna nigricans is a large climber with slender branches photographed and archived using Olympus digital camera. and grayish-brown bark. Leaves are blackish green. Analysis of chromosome complement was based on Infl orescence axis and pedicels show short dense adpressed microphotographs. pale hairs which are often persistent in fruiting stage; Karyotype analysis and Idiogram preparation. Well corolla is dark purple in color. Pods, covered thickly with separated somatic chromosomes were selected for scattered reddish brown bristles, are winged with 2 to 4 karyotyping. The length of the short (S) or long arm (L) of seed and linear oblong in shape while seeds are oblong each chromosome was measured and the values were used fl attened in shape. Distribution is in Himalaya to Burma, to calculate the: total length of chromosome (TL = L + S), East central India and Sri Lanka. haploid set length (HSL), arm ratio (A = L/S) and relative lengths (RL = L + S × 100/HSL). Chromosomes were Mucuna monosperma is a perennial, moderately woody classifi ed based on arm ratio according to Levan et al. climber with prominent small dark lenticels on the bark. (1964). Karyotype symmetry and asymmetry was Stem and petioles covered with very dense, fi ne reddish measured by the criteria proposed by Stebbins (1958). adpressed hairs and sparse coarser hairs. Flowers large, grouped in globose infl orescence on a very short, branched RESULTS AND DISSCUSSION axis. Fruit broadly oblong to elliptic and covered with Chromosomes of Mucuna species, observed in the present very dense, golden reddish irritating deciduous bristles study fi t into ‘very small to small’ category as per the that covers single kidney shaped large reddish-brown chromosome size classifi cation described by Lima de seed. This species, distributed extensively in Eastern and Faria (1980). The data on karyotypic details of each of Western Ghats of Peninsular India and North Eastern parts this species is described below. blooms from September to January and fruits ripen from February to May. M. atropurpurea. Mitotic cell showed 2n = 22 (Fig. A). The karyotype consisted of 3 pairs of median point ‘M’ Mucuna pruriens comprises only annual members and chromosomes, 7 pairs of median region ‘m’ chromosomes shows extensive morphological diversity and ubiquitous and only 1 pair of submedian ‘sm’ chromosome. The distribution in Indian subcontinent. Stem, stipule, petiole chromosome lengths ranged from 1.65-0.6 μm and the and leafl ets are glabrous. Infl orescence of long drooping Haploid Set Length (HSL) was 10.6 μm. The idiogram is racemes contains 10-70 dark purple or yellowish-white given in the Fig. E. Thus, the taxon display karyotypic fl owers. Fruits are usually distinctly curved into ‘S’ shape formula 2n=22, n=11, 3M+7m+1sm. The karyotype is of with very dense covering of golden or silvery brown symmetrical type (Table 2). irritant bristles. Almost all parts of the plant d exhibits wide ranging diversity in size, shape, hairiness and color M. nigricans. Mitotic cell showed 2n = 22 (Fig. B). The and has been the matter of much taxonomical debate. karyotype consisted of three pairs of median point ‘M’ chromosomes and eight pairs of median region ‘m’ Chromosome preparation. For chromosome preparations, chromosomes. Small satellite was found connected to Kondo et al. (1992) method was employed with minor short arms of chromosome pairs I and IV. The chromosome modifi cations. The growing root tip were cut (1-1.5cm length ranged from 1.825-1 μm and the haploid set length long) and pre-treated with an aqueous solution of 0.002M (HSL) is 14.02 μm. The idiogram is given in the Fig. F. 8-hydroxyquinoline for 4 h at 4°C. Fixation was done in Thus, this taxon has a karyotypic formula 2n=22, n=11, 1:3 acetic-alcohol for 24h at 4°C. Maceration was done in 3M+8m. The karyotype is of symmetrical type (Table 2). HCl-acetic acid (2 parts of 1N HCl + 1 part of 45% glacial acetic acid) for 1 min at 60°C. The roots were then washed M. monosperma. Mitotic cell showed 2n=22 (Fig. C). KARYOMORPHOLOGICAL STUDIES IN MUCUNA OF INDIA 39

Fig. 1. Metaphase cell plates (1000X). (A) Mucuna atropurpurea,. (B) M. nigricans, (C) M. monosperma. (D) M. pruriens. Idiogram (magniﬁ cation=1000X). (E) M. atropurpurea. (F) M .nigricans. (G) M. monosperma. (H) M. pruriens. 40 JAHEER AND SATHYANARAYANA

Table 2. Measurements (μm) of somatic metaphase chromosomes Mucuna atropurpurea Mucuna nigricans Chromosome Arm Ratio Total Length Relative Centromere Arm Ratio Satellite Total Length Relative Centromere pair Numbers L/S TL=L+S Length in % Type L/S Length TL=L+S Length in % Type I 1.75 1.65 15.56 sm 1.591 0.4 1.825 13.01 m II 1.40 1.2 11.32 m 1 - 1.55 11.05 M III 1.444 1.1 10.37 m 1.443 - 1.5 10.69 m IV 1.333 1.05 9.9 m 1.2 0.4 1.5 10.69 m V 1.5 1 9.43 m 1.4 - 1.2 8.55 m VI 1.25 0.9 8.49 m 1 - 1.2 8.55 M VII 1 0.8 7.54 M 1.2 - 1.1 7.84 m VIII 1 0.8 7.54 M 1.2 - 1.1 7.84 m IX 1.667 0.8 7.54 m 1.1 1.05 7.48 m X 1.333 0.70 6.6 m 1.5 - 1 7.13 m XI 1 0.6 5.66 M 1 - 1 7.13 M * HSL = 10.6 HSL = 14.025 * The total haploid set length.

Table 3. Measurements (μm) of somatic metaphase chromosomes Mucuna monosperma Mucuna pruriens Chromosome Arm Ratio Total Length Relative Centromere Arm Ratio Total Length Relative Centromere pair numbers L/S TL=L+S Length in % Type L/S TL=L+S Length in % Type I 1.455 1.350 12.55 m 1.2 1.1 12.02 m II 0.474 1.175 10.93 m 1.5 1 10.98 m III 1.75 1.1 10.23 sm 1.5 1 10.98 m IV 1.2 1.1 10.23 m 1.25 0.9 9.89 m V 1.5 1 9.3 m 1.25 0.9 9.89 m VI 1 1 9.3 M 1 0.8 8.79 M VII 1.25 0.9 8.87 m 1.143 0.75 8.24 m VIII 1.176 0.925 8.6 m 1.3 0.7 7.69 m IX 1.667 0.8 7.44 m 1.3 0.7 7.69 m X 1.333 0.7 6.51 m 1.167 0.65 7.14 m XI 1.333 0.7 6.51 m 1 0.6 6.59 M * HSL = 10.75 HSL = 9.1 * The total Haploid Set Length.

The karyotype consisted of one pair of median point ‘M’ Phaseoleae display basic number of x=11. This includes, chromosome, nine pairs of median region ‘m’ chromosomes among others genuses, Phaseolus (Mercado-Ruaro and and only one pair of submedian ‘sm’ chromosome. The Delgado-Salinas 1998), Vigna (Sneff et al. 1992) and chromosome length ranged from 1.35-0.7 μm and the Rhynkosia (Biondo et al. 2003). Thus, the countings, haploid set length (HSL) is 10.75 μm. The idiogram is including that of M. atropurpurea (Fig. A and E) reported given in the Fig. G. Thus this taxon has a karyotypic for the fi rst time, corroborate with the trend for chromosome formula 2n=22, n=11, 1M+9m+1sm and the karyotype is number conservation in tribe Phaseoleae, with basic of symmetrical type (Table 3). number x=11. The chromosome lengths reported for different species ranged from 1.825 to 0.6 μm with average M. pruriens. Mitotic cell showed 2n=22 (Fig. D). The at 0.775 and 1.48 μm. This is agreement with the standard karyotype consisted of two pairs of median point ‘M’ chromosome lengths reported for the subfamily chromosome and nine pairs of median region ‘m’ Papilionoideae. Mercado-Ruaro and Delgado-Salinas chromosomes. The chromosome length ranged from 1.1- (1998) found averages ranging from 0.99 to 1.84 μm in 0.6 μm and the haploid set length (HSL) is 9.1 μm. The ten species of Phaseolus; Souza and Benko-Iseppon idiogram is given in the Fig. H. Thus this taxon has a (2004) have reported chromosome lengths ranging from karyotypic formula 2n=22, n=11, 2M+9m. The karyotype is 1.80 to 0.90 μm in Bowdichia nitida and 2.50 to 1.10 μm of symmetrical type (Table 3). The diploid number 2n=22 in Diocleia virgata. Signifi cant differences were observed confi rmed in three species of Mucuna such as M. in chromosomal size between perennial and annual species monosperma, M. nigricans and M. pruriens in the present in this investigation. Bigger chromosomes were seen in study is in consensus with earlier report (Bairiganjan perennial species (1.825-0.6 μm) when compared to 1989; Kayna Agostini et al. 2009). It has been noted that, annual ones (1.1-0.6 μm). The chromosomes sizes were in of the most frequent basic numbers of x=8, 11, 7 and 16 the order of M.nigricans > M. attropurpurea > M. described for the subfamily Papilionoideae (Souza and monosperma > M. pruriens respectively, which evidently Benko-Iseppon 2004), majority of the genera under tribe supported the theory of reduction of genome size that KARYOMORPHOLOGICAL STUDIES IN MUCUNA OF INDIA 41 accompanied the evolutionary change from perennial to and Sazima and M. urens (L.) Medikus. Phytomorphology annual species reported in angiosperm groups (Price and 59 (1 and 2): 61-69. Kondo, K., Tanaka, R., Ge, S., Hong, D. and Nakata, M. 1992. Bachmann 1976, Greilhuber and Ehrendorfer 1988). Cytogenetic studies on wild Chrysanthemum sensu lato in In conclusion, this fi rst report on karyomorphological China. IV. Karyomorphological characteristics of three studies in four Mucuna species of India has offered, in species of Ajania. Jpn. Journ. Bot. 67: 324-329. addition to establishing its usefulness, necessary Levan, A., Fredga, K. and Sandberg, A. A. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: information for undertaking detailed cytotaxonomic 201-220. studies in Mucuna. However, further investigation Lima De Faria, A. 1980. Classifi cation of genes, rearrangements involving large number of species is needed to gain actual and chromosomes according to the fi eld. Hereditas 93: 1-46. insight evolutionary relationship that framed this genus. Mercado-Ruaro, P. and Delgado-Salinas, A. 1998. Karyotypic studies on species of Phaseolus (Fabaceae: Phaseolinae). ACKNOWLEDGEMENTS. The authors thank Department of Am. Journ. Bot. 85: 1-9. Science and Technology, Government of India for the fi nancial Price, H. J. and Bachmann, K. 1976. DNA content in higher support and Sri Krishnadevaraya Educational Trust (Sri KET), plants. Botanical Review 46: 27-52. Bangalore, India for facilities. Sastrapradja, S., Sastrapradja, D., Aminah, S. H., Lubis, I. and Idris, S. 1974. Annals Bogorienses 5(4): 173-178. Senff, M. I., Hickenbick, C. M. and Paim, N. R. 1992. Cytogenetic LITERATURE CITED studies in species of the genus Vigna savi (Leguminosae- Barbeau, A. and McDowell, F. H. 1970. L-Dopa and Papilionoideae). Rev. Bras. Genet. 15: 406-418. Parkinsonism, F. A. Davis Co., Philadelphia, U.S.A. Souza, M. G. and Benko-Iseppon, A. M. 2004. Cytogenetics Bairiganjan, G. C. and Patnaik, S. N. 1989. Chromosome and chromosome banding patterns in Caesalpinioideae evolution in Fabaceae. Cytologia 54: 51-64. and Papilionioideae species of Pará, Amazonas, Brazil. Biondo, E., Nascimento, A. R. T., Miotto, S. T. S. and Schifi no- Bot. J. Linn. Soc. 144: 181-191. Wittmann, M. T. 2003. Primeiros estudos citotaxonômicos Standley, P. C. and Steyermark, J. A. 1946. Flora of Guatemala. de Rhynchosia naineckensis Fortunato (Leguminosae) In: Fieldian (ed.) Botany Vol. 24- V. para o estado de Goiás. Rodriguesia 54: 5-11. Stebbins, G. L. 1958. Longevity, habitat and release of genetic Burkill, I. H. 1966. A dictionary of the economic products of variability in higher plants. Cold Spring Harb. Symp. the Malay Peninsula. Kuala Lumpur, Malaysia, Quant. Biol. 23: 365-377. Government of Malaysia and Singapore. St-Laurent, L., Livsey, J. Arnason, and Bruneau, A. 2002. Capo-chichi, L. J. A., Weaver, D. B. and Morton, C. M. 2003. Variation in L-Dopa concentration in accessions of Mucuna The use of Molecular Markers in study of genetic diversity pruriens (L.) DC var. utilis (Wall. Ex Wight, Baker ex in Mucuna. Tropical and Subtropical Agro-ecosystems 1: Burck), and in Mucuna brachycarpa (Rech). pp: 352-373. 309-318. In Mucuna as a food and feed: Current Uses and the way Ding, Y., Kinjo, J., Yang, C. and Nohara, T. 1991. Triterpenes forward. Flores BM, Eilitta M, Myhraman R, Carew LB, from Mucuna birdwoddiana. Phytochemistry 30: 3703- Carsky RJ. Workshop held in April 26-29, 2000 in 3707 Tegucigalpa, Honduras. CIDICCO, Honduras Duke, J. A. 1981. Handbook of legumes of world economic Sur, P. R. 2002. Taxonomy and ethnobotany of the genus importance. Plenum Press. New York. Mucuna Adans in India. Journ. Economic and Taxonomic Greilhuber, J. and Ehrendorfer, F. 1988. Karyological approach Bot. 26: 494-498. to plant taxonomy. ISI Atlas of Science. Plant and Animal Wilmot-Dear, C. M. 1984. A revision of Mucuna (Leguminosae- Science 1: 289-297. Phaseoleae) in China and Japan. Kew Bull. 39: 23-65. Kayana, A., Marlies, S., Ana Maria Goulart de Azevedu Tozzi Wilmot-Dear, C. M. 1987. A revision of Mucuna (Leguminosae, and Eliana Regina Forni-Martins. 2009. Microsporogenesis Phaseoleae) in the Indian subcontinent and Burma. Kew and pollen morphology of Mucuna japira Azevedu, Agostini Bull. 42: 23-46.