C2000

The Japan Mendel Society

Cytologia

65: 443-446,

2000

Karyotype
Sheikh

Analysis
Alam*
of Botany,

of Three
and
University Accepted

Corchorus

Species
Bin Rahman

Shamimul
Department

A. N. M. Rubaiyath
of Dhaka, Dhaka-1000,

Bangladesh

October

16, 2000

Summary trilocularis interphase Moreover, make their prophase, of stable length

The fluorescent showed 14 more nuclei revealed the nucleoli interphase

banded

somatic

karyotypes

of Corchorus surrounded satellites

capsularis,

C. olitorius

and C. stained zone.

or less equal sized metacentric of a big nucleolus with a pair Only 2 CMA-stained

chromosomes dots. All were

each. The orcein of these

the presence unique.

by a clear non-staining

were associated nucleus

of prominent

characteristics

found in the interphase,

prometaphase and metaphase plates of these 3 species. These bands indicate the presence GC-rich sequences at the satellited region of these species. The size of satellites and the chromosomes contained C. olitorius with CMA were found nearly one interstitial band and C. trilocularis was and DAPI. banding, same in these species. had 2 DAPI-negative in C. capsularis. banding ancestor. Two chromosomes of chrohowever, CMA-positive found band in each. The 2 satellited These similarity

of the satellited

C. capsularis, mosomes ed regions. could mosome

of C. capsularis, One interstitial

bands at the satellitchromosomes of their chro-

DAPI-positive

easily be identified suggests Karyotype,

The base specific

that their genomes Fluorescent

may derive from a common Corchorus.

Key words

The genus Corchorus L. belongs to the family Tiliaceae. There were 170 Corchorus species listed in Index Kewensis. Out of these, only 50 to 60 are recognized as valied species distributed throughout the warm regions of the world (Edmonds 1990). Corchorus is a fibre yielding genus commercially known as jute and next to cotton in importance. In Bangladesh, jute assumes high socio-economic importance because it constitutes a major source of foreign exchange earning. The wild species of Corchorus are known to contain a lot of variations. It is necessary to explore the possibility of using these in future breeding programme. In this view, the International Jute Organization (IJO) has been collecting jute germplasms from Bangladesh and neighbouring countries and these are being stored in the Gene Bank of the Bangladesh Jute Research Institute (BJRI). The germplasms are mainly collected and identified on the basis of plant morphology. Due to phenotypic plasticity, plants belonging to the same taxa may show different morphology. In such a situation, identification of germplasms may be very difficult. Since the identification of genome is more reliable, attempts should be made to characterize the germplasms genomically. Although a number of reports are available regarding the morphological and cytological studies of both wild and cultivated species of jute (Arangzeb 1980, Sobhan 1992), their actual genetic make up is scarcely known. Karyotype analysis by conventional techniques of Corchorus spp. presents a number of problems, these are (i) most of the Corchorus spp. contain the same somatic chromosome number (2n=14), (ii) no sharp chromosome difference exists among the karyotypes of the different species and (iii) satellites, an useful marker are not always detectable. Keeping these in mind, an attempt has been made here to characterize the karyotypes of 2 cultivars (C. capsularis, C. trilocularis) and a wild species (C. trilocularis) by (i) conventional orcein staining and (ii) chromomycin A3 (CMA) and 4'-6 diamidino-2-phenyl indole (DAPI) staining to
* Corresponding author, e-mail:baptc@bd.drik.net, botany@du.bangla.net

444

Sk. S. Alam

and R. B. Rahman

Cytologia

65

provide more informations for furture breeding programme. Materials and methods The seeds of 2 cultivars and a wild species of jute were collected from the Gene Bank of BJRI. Seeds were germinated on moist filter paper in Petri dishes at 28C. About 2.0 cm primary roots were cut by a clean blade. The root tips (RTs) were pretreated with 0.002 M 8-hydroxyquinoline for 1.45 h at room temperature followed by 15min fixation in 45% acetic acid at 4C. The pretreated RTs were hydrolysed for 13s for C. capsularis Linn. and C. olitorius Linn. and 7 s for C. trilocularis Linn. at 60C in a mixture of 1 N HC1 and 45% acetic acid (2 : 1). It was then squashed in 2% aceto-orcein. For CMA and DAPI-staining the methods proposed by Alam and Kondo (1995) were followed with a slight modification. Results and discussion The orcein stained property of interphase nuclei showed 3 unique features. It was common to all these 3 species. These were: (i) it was very difficult to recognize the nucleus in the cytoplasm. In all cells, a big nucleolus was observed occupying nearly 1/4th of the nuclear space (Fig. 1). A hollow like clear zone appeared around each nucleolus. (ii) a pair of minute but prominent dots (arrows in Fig. 1) was found associated with the nucleolus. The reasons for the appearance of a clear non-staining zone around each nucleolus was not clear. More than 200 nuclei for each species were examined. Therefore, the characters were very stable. Moreover, the 2 minute dots present in each nucleolus were stable and found in almost every nucleolus of these 3 species. These dots seemed to be connected to the nucleolar organizing regions (NORs). In interphase, a pair of prominent CMA-positive bands was found in the non-staining region (nucleolar region) of each nucleus in these 3 species (Fig. 2). The area of a CMA-positive band was about 0.50 ,um2.The size of these bands was same in every species. The 3 species possessed a terminal CMA-positive band of same size in 2 different chromosomes (as observed in interphase). In C. capsularis, however, 2 distinct CMA-positive satellites were observed in a few cells (Fig. 4). The later observation made us careful in determining the actual nature of the terminal bands found in the other 2 species. It was observed that 2 CMA-bands were located far away from the respective prophase and prometaphase chromosomes (Fig. 3). It clearly indicated that the banded regions were actual satellites. It appears that to determine whether a satellite is present or not, it is not sufficient to depend only on metaphase plates. Few slides of C. capsularis could be counterstained with DAPI fluorochrome. In these slides, 2 clear DAPI-negative bands were observed at exactly the same regions where CMA-positive satellited bands had appeared (arrows in Figs. 4, 7). In C. trilocularis and C. olitorius, 2 DAPI-negative bands were observed at the satellited regions of 2 chromosomes (arrows in Figs. 8, 9). The observed reversible banding pattern of the satellites revealed that these portions were fully made of GC-rich base sequences. The GC-rich satellites were observed in plants by Hizume et al. (1992), Alam and Kondo (1995) and in insect by Suja et al. (1993). Schweizer (1976) reported that the NORs, where the rDNA is located are generally GC-rich and therefore, CMA-positive. Thus the results of the present investigation correspond with their findings. Two additional CMA-positive bands were found at the interstitial regions of 2 chromosomes in C. capsularis (big arrow in Fig. 4). When counterstained only one DAPI-positive band was ob-

2000

Karyotype

Analysis

of Corchorus

spp.

445

Figs. nucleus

1-9. of C.

1) Orcein capsularis,

stained

interphase

nucleus

of Corchorus of C.

capsularis, olitorius,

2) CMA-stained 4) CMA-stained metaphase of C. of C. olitorius,

interphase metaphase trilocularis, 9) DAPIof

3) CMA-stained metaphase of stained C. capsularis,

prometaphase of C. olitorius, 8)

C. capsularis, 7) DAPI-stained

5) CMA-stained metaphase

6) CMA-stained metaphase scale =10 m.

DAPI-stained

metaphase

of C. trilocularis.

446

Sk. S. Alam

and R. B. Rahman

Cytologia

65

served at the same place where CMA positive band has found (big arrow in Fig. 7). This feature suggested the existance of GC- and AT-richbase sequences together at the same location. Haque and Ahmed (1972) reported 4 satellites in C. capsularis as well as in C. olitorius. They found 6 satellites in C. trilocularis. More than 2 satellites were reported by earlier workers in these 3 speices (Datta et al. 1975, Arangzeb 1980). Mukai et al. (1991) stated that satellite was the most useful marker for the karyotype analysis in common wheat. In this investigation by using orcein, CMA and DAPI, only 2 satellites could be found in these 3 species. Therefore, the 3 species are characterized by the presence of 2 satellited chromosomes. In conclusion, it may be said that 3 Corchorus species showed marked similarities such as: i) all the 14 chromosomes of the somatic complements are metacentric, ii) out of these, only 2 chromosomes are satellited, iii) the satellited chromosomes are of same size and characteristics, iv) the size of the satellited regions is almost equal, v) satellites are GC-rich, vi) satellited GC-rich areas are stable and keeping their domain intact throughout the cell cycle. All these features indicate that these 3 species may be evolved from a common ancestor. Acknowledgement We express our gratitude to Prof. M. Akhtaruzzaman, Department of_Botany, University of Dhaka for his constructive criticisms and inspiration throughout the course of study. We also express out sincere thanks to Dr. M. A. Sobhan, Principal Scientific Officer, Bangladesh Jute Research Institute, Dhaka, Bangladesh for providing us the germplasms. The research was partially funded by the Biotechnology Research Centre, Dhaka University.
References Alam, Sk. S. and Kondo, K. 1995. Differential staining with orcein, Giemsa, CMA and DAPI for comparative chromosome study of 12 species of Australian Drosera (Droseraceae). Amer. J. Bot. 82: 1278-1286. Arangzeb, S. 1980. Giemsa banding pattern among cultivars of Corchorus capsularis and C. olitorius. Bangladesh J. Bot. 9: 50-53. Datta, R. M., Mukhopodhaya, D., Panda, B. S. and Sasmal, P. K. 1975. Cytotaxonomic studies of different Corchorus (Jute) species II. Cytologia 40: 645-692. Edmonds, J. M. 1990. Herberium survey of African Corchorus L. species. Systematic and ecogeographic studies on crop genepools 4, IBPGR, Rome. Haque, A. and Ahmed, Q. N. 1972. Observations on the chromosomes of some Crochorus species. Bangladesh J. Bot. 1: 185-198. Hizume, M., Ishida, F. and Kondo, K. 1992. Differential staining and in situ hybridization of nucleolus organizers and centromeres in Cycas revoluta chromosomes. Jpn. J. Genet. 67: 381-387. Mukai, Y., Endo, T. R. and Gill, B. S. 1991. Physical mapping of the 18S, 26S, rRNA multigene family in common wheat identification of new locus. Chromosoma 100: 71-78. Schweizer, D. 1976. Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58: 307-324. Sobhan, M. A. 1992. Diversity of Corchorus and Hibiscus accross the world. Proceedings of the IJO/BJRI training course on specialized techniques in jute and kenaf breedings BJRI, 20-29 July 1992, Dhaka. Bangladesh. pp. 35-43. Suja, J. A., Antonio, C., Gonzalez-Garcia, J. M. and Rufas, J. S. 1993. Supernumery heterochromatic segments associated with the nucleolar chromosomes of Pyrgomopha conita (Orthoptera) contain methylated rDNA sequences. Chromosoma 102: 491-499.