© 2006 The Japan Mendel Society
Cytologia 71(1): 1–4, 2006
Karyotyping of Nigella sativa L. (Black Cumin) and Nigella damascena L. (Love-in-a-mist) by Image Analyzing System Anindita Ghosh and Animesh K. Datta* Department of Botany, Cytogenetics and Plant Breeding Section, Kalyani University, Kalyani–741235, West Bengal, India Received October 11, 2005; accepted October 24, 2005
Summary Karyotyping through Image Analyzing System in two species of Nigella (family: Ranunculaceae) revealed 4 (N. sativa: 2n124A4B2C2D; karyotype formula: 2Lsc1sm 2L1m2Lsm2Lm2Mm2St) and 3 (N. damascena: 2n126B4C2D; karyotype formula: 4Lsc m 2Lm4Mm2St) morphologically distinct chromosome types (L1very long 15.0 m m, Llong 13.0 to 15.0 m m, Mmedium 7.0 to 13.0 m m, Sshort 7.0 m m, mmetacentric, smsub-metacentric, ttelocentric and scsatellites). The somatic chromosome complements in the species formed graded karyotypes which were symmetric in nature (TF%: N. sativa 42.90, N. damascena 40.62). Total haploid chromatin length was noted to be 78.622 m m in N. sativa and 70.136 m m in N. damascena. Key words
Imaging techniques, Karyotypes, Nigella sativa, N. damascena.
Nigella sativa L. (black cumin; spice of commerce) and Nigella damascena L. (love-in-a-mist; garden ornamental), members of the family Ranunculaceae have been karyotyped manually and the species were considered to be good materials for cytological studies as they have relatively low numbers of chromosomes (2n12) of suitable sizes with good stainability and a marker telocentric (Datta and Biswas 1983, Saha and Datta 2002, Datta and Saha 2003). Such cytological novelty can be used to explore cytogenetical behaviour of the species. However, manual karyotyping may yield erroneous results due to biased judgements. As karyotyping through Image Analyzing system allows accurate numerical data to be obtained from the chromosome images (Fukui 1985, 1986, 1988), this paper documents karyotype analysis of N. sativa and N. damascena by Imaging Techniques. Materials and methods For karotype analysis, seeds of Nigella sativa L. (mother stock: cultivated variety Persian Jewels, Sutton and Sons’, Kolkata) and N. damascena L. (mother stock: Royal Botanic Garden, Kew, London—accession no. 0016287) were presoaked in distilled water for 12 h and allowed to germinate (181°C) in petriplates lined with moist filter papers. Healthy roots of proper sizes (2–3 mm, collected at 12 pm to 12:30 pm) were pretreated (PDB aesculine for 3 to 4 h at 16°C), fixed (1 : 3 propiono-alcohol for overnight) and stained in 2% aceto-orcein: 1 N HCl mixture for 2 to 3 h and the root tips were squashed in 45% acetic acid. Scattered metaphase plates (properly condensed chromosomes) prepared for the species were analyzed through Image Analyzing System. Metaphase plates observed under the microscope (Olympus CH 40) were photographed using * Corresponding author, e-mail:
[email protected]
Anindita Ghosh and Animesh K. Datta
2 Table 1.
Cytologia 71(1)
Karyotype details in N. sativa and N. damascena Chromosome pairs
Plant types
N. sativa
N. damascena
1
2
3
4
5
6
A1A1* 15.987 0.595 32.89 100.00 sm B2B2* 13.961 0.933 41.64 100.00 m
A2A2 15.188 0.944 47.27 95.00 m B3B3 13.615 0.930 46.46 97.52 m
B1B1 14.101 0.680 39.42 88.20 sm B4B4* 13.069 0.973 40.04 93.61 m
B3B3 13.600 0.780 42.53 85.07 m C1C1 11.781 0.858 44.93 84.39 m
CC 12.888 0.782 43.36 80.61 m C2C2 11.183 0.843 43.54 80.10 m
DD 6.858 0.186 15.57 42.90 t DD 6.527 0.175 14.59 46.75 t
1st line: somatic chromosome pairs; 2nd line: absolute length in m m; 3rd line: arm ratio (short arm/long arm); 4th line: F value in percent; 5th line: relative length in per cent; 6th line: centromeric nature of the chromosomes (mmetacentric, smsub-metacentric, ttelocentric); * Chromosomes with secondary constriction.
Olympus C-5060 Wide Zoom Camera and those photographs were transferred directly to computer and the chromosome images were analyzed through Micro ImageTM Lite Software (Version 4.0 for windows, 47N40155 2000 0515 MAN VG MIX). Chromosome images were measured from 3 plates (in each species) in pixel unit and were converted to micrometer with reference to a standard scale (stage micrometer). Photoplate karyogram of either species was prepared using Adobe Photoshop 7.0 version. Karyotyping details have been worked out as suggested by Saha and Datta (2002). Results and discussion On the basis of chromosome length, F% (Hirahara and Tatuno 1967) and presence or absence of secondary constrictions, the chromosomes of either species were morphologically classified into following types: type A, very long (L115.0 m m) chromosomes possessing sub-metacentric (sm) primary constrictions with satellites (A1A1) and metacentric (m) chromosomes without satellites (A2A2); type B, long (L13.0 to 15.0 m m) sub-metacentric (B1B1) and metacentric (B2B2–B4B4) chromosomes with satellites (sc) on B2B2 and B4B4; type C, medium (M7.0 to 13.0 m m) metacentric chromosomes; and type D, small (S7.0 m m) telocentric (t) chromosomes. Among the chromosome types, somatic chromosome pairs were graded according to chromosome length. Karyotyping details of N. sativa and N. damascena have been presented in Table 1. Karyotype analysis revealed 4 (N.sativa: 2n124A4B2C2D; Figs. 1–3; karyotype formula: sc 2L1sm 2L1m2Lsm2Lm2Mm2St) and 3 (N. damascena: 2n126B4C2D; Figs. 4–6; karyotype formula: 4Lscm2Lm4Mm2St) morphologically distinct chromosome types in the species. The somatic chromosome complement of N. sativa possessed 1 pair (A1A1) of chromosomes with secondary constrictions; while, N. damascena had satellites in 2 pairs (B2B2, B4B4). Thus, the longest pairs of chromosomes in both species were with secondary constrictions. Satellites were always associated with short arms. Based on manual karyotyping, Datta and Biswas (1983) reported 2 pairs of chromosomes with satellites in both species; whereas, Datta and Saha (2002) documented 1 and 2 pair(s) of chromosomes with secondary constriction in N. sativa and N. damascena respectively. In all the cases, however, the authors were of opinion that there were 4 chromosomes types (A–D) and satellites were not associated with the longest pairs. On the contrary, Bhattacharyaya (1958) was of opinion that 2 longest pairs in N. sativa were with satellites;
2006
3
Karyotyping by Imaging Techniques in Nigella
B1 B3 A2
B1
C C
A2
A1
B3
D D
1
A1 A1
A2 A2
2 A1
B1 B1
A2
B1
B3 B3
B3
C
C
C
D
DD
3 C2 B4
B3
C1 D
B3
B2
C1
C2 D
B2 B4
4
B2 B2
B3 B3
B4 B4
5 B2
B3
C1 C1
B4
C1
C2 C2
C2
D
D D
6 Figs. 1–6. Somatic chromosomes of Nigella species with their types marked. 1–3) N. sativa (2n12) with idiogram (2) representing haploid set of chromosomes and karyogram (3). 4–6) N. damascena (2n12) with idiogram (5), haploid set of chromosomes and karyogram (6). Bar: 9.142 m m in 1 and 3, 5.748 m m in 2; 6.198 m m in 4 and 6, 5.440 m m in 5.
while, Subramanian (1985) reported 1 pair of chromosomes with secondary constriction in N. damascena. In the present investigation, metacentric chromosomes are prevalent; while, a telocentric pair (DD) has been marked in both species. Two pairs of sub-metacentric chromosomes were noted in the somatic complement of N. sativa. The somatic chromosome complements in both species
4
Anindita Ghosh and Animesh K. Datta
Cytologia 71(1)
were graded and symmetric in nature as evidenced from relative chromosome lengths and TF% respectively (TF%: N. sativa 42.90, N. damascena 40.62). Absolute chromosome length in the species indicated that the chromosomes were relatively longer in N. sativa than in N. damascena. Total haploid chromatin length was assessed to be 78.622 m m (77.953 to 79.041 m m) in N. sativa and 70.136 m m (69.507 to 71.014 m m) in N. damascena. Thus, present findings based on Imaging Techniques confirm the number of chromosome types and the number of chromosomes with secondary constriction with their locations in the complements of the species. References Bhattacharyaya, N. K. 1958. Cytology of the two species of Nigella. Genet. Iber. 10: 179–190. Datta, A. K. and Biswas, A. K. 1983. Karyotype analysis in four Nigella species. Cell Chrom. Res. 6: 21–24. — and Saha, A. 2003. Cytomorphological studies and seed protein characterization of Nigella sativa L. and Nigella damascena L. Cytologia 68: 51–60. Fukui, K. 1985. Identification of plant chromosomes by image analysis method. The Cell (Tokyo) 17: 145–149 (in Japanese). — 1986. Standardization of karyotyping chromosomes by a newly developed chromosome image analyzing system (CHIAS). Theor. Appl. Genet. 72: 27–32. — 1988. Analysis and utility of chromosome information by using the chromosome image analyzing system. CHIAS. Bull. Natl. Inst. Agrobiol. Resour. 4: 153–176. Hirahara, S. and Tatuno, S. 1967. Cytological studies on Narcissus. I. Karyotype and nucleolinus of Narcissus jonquilla. Cytologia 32: 553–559. Saha, A. and Datta, A. K. 2002. Gamma-rays induced reciprocal translocation in black cumin (Nigella sativa L.). Cytologia 67: 389–396. Subramanian, D. 1985. Cytotaxonomical studies in South Indian Ranunculaceae. Cytologia 50: 759–768.
