Macrotyloma uniflorum (Lam.) Verdc. (Leguminosae) A Note on Chromosomal Studies

Sandip Halder, Animesh K. Datta*, Aninda Mandal and Benoy K. Ghosh

Department of Botany, Cytogenetics, Genetics and Plant Breeding Section, University of Kalyani, Kalyani-741235, West Bengal, India

Received May 31, 2012; accepted October 12, 2012

Summary Karyotype analysis of Macrotyloma uniflorum (Lam.) Verdc. (Family: Leguminosae; Sub-family: Faboideae; annual herb, potential usesnutraceuticals, forage and food for humans) re-vealed 5 (2n＝20; 4Am＋2Bsc

sm＋2Csm＋2Dsm＋10Em) morphologically distinct chromosome types. The karyotype showed a prevalence of chromosomes with median primary constrictions. Satellites were associated with the short arm of 2B-type chromosomes. Chromosome length in the comple-ment varies from 1.26 to 2.41 µm. The karyotype was symmetric in nature (TF%＝43.11). The mei-ocytes had 2n＝20 chromosomes always with an average of 9.96II＋0.08I per cell at diakinesis and metaphase I. Formation of 10 II was predominant (95.83%). All anaphase I cells were cytogeneti-cally balanced (10/10). About 66.67% diakinesis and MI cells showed secondary association of chromosomes and the chromosomes assorted themselves into variable groups of 4 (4.69%), 5 (6.25%), 6 (23.44%), 7 (64.06%) and 8 (1.56%). Out of 24 different types of associations among the group classes, 2II(3)＋1II(4)46.88% (7 group class) was the most frequent. Secondary polyploidy has been attributed as the possible cause of secondary association of chromosomes and the basic chromosome number for the species is suggested to be x＝7.

Keywords: Basic chromosome number, Karyomorphology, Macrotyloma uniflorum, Meiosis, Secondary polyploidy.

The chromosome number in Macrotyloma uniflorum (Lam.) Verdc. (Synonym: Dolichos biflo-rus L.; common name: horse gram; Family: Leguminosae; Sub-family: Faboideae; annual herb, po-tential usesnutraceuticals, forage and food for humansMorris 2008) has been reported variably (2n＝20Pritchard and Gould 1964, Maréchal and Otoul 1965, 1966; 2n＝20, 22, 24Lackey 1979, 1980, Cook et al. 2005); however, chromosome number alone does not provide any cytoge-netical criteria which aiding to efficient breeding. The present communication reports on the karyo-type analysis and meiotic chromosome behavior of M. uniflorum, which were previously unde-scribed, with the objective to collect cytogenetical information as a major step to enable improvement of the plant.

Materials and methods

Plant materialSeed samples of Macrotyloma uniflorum (Lam.) Verdc. were collected from the Medicinal

Plant Garden, Narendrapur, Ramkrishna Mission, Govt. of West Bengal, India (accession number not provided) and plants were raised in the Experimental Garden, University of Kalyani. A voucher specimen has been deposited in the Herbarium, Department of Botany, University of Kalyani.

© 2012 The Japan Mendel Society Cytologia 77(4): 447–451

* Corresponding author, e-mail: dattaanimesh@gmail.comDOI: 10.1508/cytologia.77.447

448 S. Halder et al. Cytologia 77(4)

Karyotype analysisRoot tips were pretreated for 4h in aqueous solution of para-dichlorobenzene (PDB) and aes-

culine mixture at 16±1°C, fixed in 1 : 3 propiono–alcohol (v/v) overnight and stained in orcein-1N HCl mixture (9 : 1). Slides were prepared using the squash technique (45% acetic acid). Suitable metaphase plates (5 properly and uniformly condensed) were scored and measurements of the chro-mosomes were taken from camera lucida drawings at a magnification of 15x×100.

The chromosomes were classified as median (m) and sub-median (sm) according to the posi-tion of the centromere (Hirahara and Tatsuno 1967) and on the basis of chromosome length (long: >2.25 µm, medium: 2 µm to <2.25 µm and small: <2.00 µm), centromeric position and the presence of secondary constriction (sc), the chromosomes were morphologically graded into the following types: type Along median chromosomes, type Blong sub-median chromosomes with satellites, type Csub-median chromosomes of medium sizes, type Dsmall sub-median chromosomes and type Esmall median chromosomes. Total haploid chromatin length, TF% (proportion of the short arm in the total chromatin length) and S% (relative length of shortest chromosome compared to the longest) were also analyzed. Karyomorphological data was used to compute karyotype formula.

Meiotic analysisFlower buds of suitable sizes from 5 randomly selected plants were fixed (6 to 7 a.m.) for 72 h

in Carnoy’s solution at 22±1°C (2 changes were given in the fixative at the intervals of 24 h) and preserved in 70% alcohol under refrigeration. Pollen and pollen mother cells (PMCs) were assessed from anther squash preparations in 2% propinocarmine solution. Fully stained pollen grains were considered fertile in accordance with Marks (1954). Meiotic data recorded at diplotene-diakinesis, metaphase I (MI) and anaphase I (AI) were pooled over the plants and represented in the text.

Photomicrographs for both mitotic and meiotic chromosomes were taken from temporary squash preparations and subsequently magnified.

Results and discussion

Mitotic analysisKaryomorphological studies revealed 5 (2n＝20; 4Am＋2Bsc

sm＋2Csm＋2Dsm＋10Em) morpho-logically distinct chromosome types (Table 1, Figs. 1–2). The karyotype showed preponderance of chromosomes with median (F%＝42.67 to 48.78) primary constrictions although there were 6 sub-median (F%＝36.00 to 38.56) chromosomes as well. Chromosome length in the complement ranged from 1.26 to 2.41 µm. Two chromosomes in B-type were with satellites and the satellites were associated with the short arm of the chromosomes. Total haploid chromosome length and S% in the species were 26.62 µm±1.6 and 52.28% respectively. The karyotype was symmetric in nature (TF%＝43.11).

Table 1. Karyotype details in M. uniflorum

Chromosome type

Number of chromosome Length (μm) F% Centromeric

nature

A 4 2.39–2.41 42.67–45.64 mB* 2 2.28 37.71 smC 2 2.23 38.56 smD 2 1.75 36.00 smE 10 1.26–1.73 44.44–48.78 m

* Chromosomes with satellites.

2012 Macrotyloma uniflorum (Lam.) Verdc. (Leguminosae)A Note on Chromosomal Studies 449

Meiotic analysisPMC squashes showed 2n＝20 chromosomes always. Out of 192 meiocytes analyzed at diaki-

nesis and MI, 95.83% cells were with 10II; while, the rest had 9II＋2I. The average chromosome association recorded per cell was 9.96II＋0.08I. All AI cells (79 cells observed) were cytologically balanced (10/10). Pollen fertility recorded in the species was 85.93% (1144 pollen grains assessed). Pollen grains were large (59.13 μm±1.11×55.50 μm±1.08); exine surface smooth and tuberculate; triporate; prolate-spheroidal in accordance with Erdtman (1952).

It is interesting to note that bivalents and univalents in the species lie in close proximity, with-out any distinct material connection forming groups of 4 (4.69%), 5 (6.25%), 6 (23.44%), 7 (64.06%) and 8 (1.56%). Out of 192 meiocytes analysed, 66.67% cells were clearly showing group-ings. A total of 24 different types of associations were scored among the group classes and the pre-dominant associations were 4II(1)＋3II(1)＋2II(1)＋1II(1) under 4 group clasFig. 3 (3.13%), 3II(1)＋2II(3)＋1II(1) under 5 group class (3.13%), 2II(4)＋1II(2)-(7.03%) and 3II(1)＋2II(2)＋1II(3) under 6 group classFig. 4 (9.38%), 2II(3)＋1II(4) under 7 group classFigs. 5–7

Figs. 1–2. Mitosis. 1) 2n＝20 chromosomes at metaphase (scale bar＝5 µm). 2) Idiogram showing the chromosomes types (scale bar＝1 µm).

Figs. 3–8. Diplotene (Fig. 5) and metaphase I configurations showing secondary groupings of chromo-somes. 3) 4 group class. 4) 6 group class. 5–7) 7 group class. 8) 8 group class. Scale bar＝10 µm.

450 S. Halder et al. Cytologia 77(4)

(46.88%) and 2II(2)＋1II(6) under 8 group classFig. 8 (1.56%).The χ2-test analysis computed among the group classes revealed heterogeneity (χ2＝173.82,

DF 4, p<0.001), thereby suggesting that the chromosomes assort themselves more preferentially into group of certain numbers (predominantly 7 group class) than they do into others much against random distribution. Further, cell frequency of 7 group class was significantly higher than expected and it was verified against the pooled frequency of rest of the group classes (observed: 7 group class82, rest46; expected: 7 group class64, rest64, total 128, χ2＝10.12, DF 1, p<0.001).

The persistent occurrence of secondary associations of chromosomes has suggested secondary polyploidy nature of the species, while the analysis of cytological data has revealed that the proba-ble basic chromosome number is 7 (selective doubling of the chromosomes possibly as x＝7, n＝10). The species possibly underwent cytological diplodization in the course of evolution to form diploid-like meiotic behavior. The total lack of multivalent formation in the meiocytes and occur-rence of variable number of associations possibly suggest allopolyploid lineage of the species. Lackey (1979) suggested x＝10 in M. uniflorum. The cytological approach involving the observa-tion of meiotic cells is conventional and has been used to ascertain the basic chromosome number and secondary polyploidy nature of different plant species (Sengupta and Datta 2003, Mukherjee and Datta 2005, Iqbal and Datta 2007, Bhattacharya and Datta 2010, Mandal and Datta 2011). Thus, cytogenetical information obtained may be utilized and explored for the crop improvement.

Acknowledgements

The authors are thankful to DST-PURSE programme, University of Kalyani for financial as-sistance.

References

Bhattacharya, A. and Datta, A. K. 2010. Secondary chromosome association in Uraria picta (Jacq.) DC (Family: Leguminosae). Cytologia 75: 37–40.

Cook, B. G., Pengelly, B. C., Brown, S. D., Donnelly, J. L., Eagles, D. A., Franco, M. A., Hanson, J., Mullen, B. F., Partridge, I. J., Peters, M. and Schultze-Kraft, R. 2005. Tropical Forages: an interactive selection tool. [CD-ROM], CSIRO, DPI&F(Qld), CIAT and ILRI, Brisbane.

Erdtman, G. 1952. Pollen Morphology and Plant Taxonomy: Angiosperms (An Introduction to Palynology I). Almqvist and Wicksell, Stockholm.

Hirahara, S. and Tatsuno, S. 1967. Cytological studies on Narcissus. I. Karyotype and nucleolinus of Narcissus jonquilla. Cytologia 32: 553–559.

Iqbal, M. and Datta, A. K. 2007. Cytogenetic studies in Withania somnifera (L.) Dun. (Solanaceae). Cytologia 72: 43–47.Lackey, J. A. 1979. A chromosome atlas of the Phaseoleae (Leguminosae-Papilionoideae). Iselya 1: 86–114. 1980. Chromosome numbers in the Phaseoleae (Fabaceae-Fboideae) and their relation to taxonomy. Am. J. Bot. 64: 595–

602.Mandal, A. and Datta, A. K. 2011. Secondary chromosome associations and cytomixis in Corchorus spp. Cytologia 76:

337–343.Maréchal, R. and Otoul, E. 1965. Contribution a l’etude cytotaxonomique des Papilionaceae-Phaseoleae-Phaseolinae. I.

Observations sur quatre especes de Dolichos L. et sur Haydonia triphylla R. Wilczek. Bulletin du Jardin Botanique de l’État à Bruxelles 35: 73–84.

and 1966. Contribution a 1’étude cytotaxonomique des Papilionaceae-Phaseoleae-Phaseolinae. III.-comparaison des chromosomes somatiques de trois especies de Doliches. Bull. Jard. Bot. Etat. 36: 325–333.

Marks, G. E. 1954. An aceto-carmine glycerol jelly for use in pollen-fertility counts. Biotech. Histochem. 29: 277–278.Morris, J. B. 2008. Macrotyloma axillare and M. uniflorum: descriptor analysis, anthocyanin indexes, and potential uses.

Genet. Resour. Crop Evol. 55: 5–8.Mukherjee, M. and Datta, A. K. 2005. Secondary chromosome associations in Ocimum basilicum L. and Ocimum tenuiflo-

rum L. Cytologia 70: 149–152.Pritchard, A. J. and Gould, K. F. 1964. Chromosome numbers in some introduced and indigenous legumes and grasses.

Division of Tropical Pastures Technical Paper No. 2 Commonwealth Scientific and Industrial Research

2012 Macrotyloma uniflorum (Lam.) Verdc. (Leguminosae)A Note on Chromosomal Studies 451

Organization, Camberra.Sengupta, S. and Datta, A. K. 2003. Secondary chromosome associations in control and mutant plant types of Sesamum indi-

cum L. Cytologia 68: 141–145.