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A High Polyploid Chromosome Complement of Ophioglossum Nudicaule L

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A High Polyploid Chromosome Complement of Ophioglossum Nudicaule L © 2007 The Japan Mendel Society Cytologia 72(2): 161–164, 2007 A High Polyploid Chromosome Complement of Ophioglossum nudicaule L. f. Marunnan Faseena and John Ernest Thoppil* Genetics and Plant breeding Division, Department of Botany, University of Calicut,Pin Code-673635, Kerala, India Received January 9, 2007; accepted January 31, 2007 Summary The diploid (2nϭ720) and haploid (2nϭ360) chromosome numbers were determined in Ophioglossum nudicaule. The somatic chromosome count was made on the plant for the first time. O. nudicaule is a very high polyploid plant, either exhibiting 48-ploidy, if the basic chromosome ϭ ϭ number is x2 15 or a 24-ploid, originating from the basic chromosome number of x3 30. Key words Ophioglossum nudicaule, Ophioglossaceae, Mitosis, Meiosis, Polyploidy, Basic chro- mosome number. Ophioglossum nudicaule L. f. (Slender Adder’s tongue) belongs to Ophioglossaceae, a family of primitive ferns. It is a tiny terrestrial herb found in dense patches on the thin soil cover over lat- erite boulders in open areas, roadside ditches and lawns. It is distributed throughout Mexico, West Indies, Central America, South America, Asia, Africa and Pacific Islands. In Kerala, it is common in hills and rocky areas all over the Malabar plains and Munnar (Kumar 1998). Previous studies re- port that homosporous ferns show extremely high chromosome numbers. In O. nudicaule different chromosome numbers, viz. nϭ120 (Ninan 1958), nϭ240 (Ninan 1956, Manickam 1984) and nϭ360 (Ghatak 1977) have been reported. So the present investigation is an attempt to find out the exact somatic and gametic chromosome numbers of O. nudicaule. Materials and methods In the present investigation, O. nudicaule that flourishes after the early part of the rainy season were collected from the Calicut University Campus and herbarized (CALI CU.11697). Healthy root tips were collected from young actively growing plants at the time of peak mitotic activity (9–9.30 a.m.). It was washed thoroughly with distilled water and pre-treated in ice-cold dis- tilled water with a trace of saponin. The pretreatment solution was initially chilled at 0–5°C for 5 min and then kept at 12–15°C for 2–3 h under refrigeration. After this, the root tips were washed thoroughly with distilled water and fixed in modified Carnoy’s fluid (1 acetic acid: 3 ethyl alcohol) for 24 h. The fixed and stored root tips were washed in distilled water and hydrolyzed with 1 N HCl for 15 min at 60°C. After thorough washing in distilled water the root tips were stained with modified staining techniques (Sharma and Sharma 1990). Young, developing sporangia from fertile fronds portion of the plants were collected and fixed in 1 : 3 mixture of glacial acetic acid and absolute ethyl alcohol for 48 h. Meiotic study was made by taking out sporangium, which were hydrolyzed in 1 N HCl for 10 min at 60°C. These hydrolyzed sporangia were smeared by conventional 2% aceto-orcein method (Sharma and Sharma 1990). * Corresponding author, e-mail: [email protected] 162 Marunnan Faseena and John Ernest Thoppil Cytologia 72(2) Fig. 1. Mitotic chromosomes in Ophioglossum nudicaule 2nϭ720. Barϭ10 mm. Fig. 2. Bivalents in meiotic metaphase I stage in O. nudicaule nϭ360. Barϭ10 mm. Photographs of well spread preparations were taken with an OLYMPUS CAMEDIA C-4000 (Zoom Digital Camera) attached to an OLYMPUS CX21 Binocular Research Microscope. Results and discussion Ophioglossum nudicaule possesses a large chromosome complement with numerous, homoge- neous chromosomes, 2nϭ720 (Fig. 1). Meiosis was found to be normal with nϭ360 bivalents at metaphase I stage (Fig. 2). The chromosomes of O. nudicaule were found to be small, with the primary constrictions faintly visible. It is difficult to distinguish chromosomes with secondary constrictions. Close exami- nation of the chromosome numbers reported by earlier workers revealed considerable difference in 2007 Chromosome complement of Ophioglossum nudicaule 163 Fig. 3. Phylogenetical scheme showing the probable evolution of polyploid chromosome number from the basic numbers in O. nudicaule. Barϭ10 mm. the chromosome number. A wide range of haploid chromosome numbers of O. nudicaule were re- ported viz. nϭ120 (Ninan 1958), nϭ240 (Ninan 1956, Manickam 1984) and nϭ360 (Ghatak 1977). So in the present investigation the diploid chromosome count of 2nϭ720 and the haploid count nϭ360 is confirmed in O. nudicaule. Base number of the genus Ophioglossum is controversial. Love et al. (1977) considered it to be xϭ15, whereas Khandelwal (1990) gave xϭ30. ϭ Grant (1981) proposes the original primary base numbers of Angiosperms range from x1 7–9. According to Fernandes and Leitao (1984) primary, secondary and tertiary basic chromosome num- bers exist in plants. So in the present investigation, the very high polyploid number in O. nudicaule ϭ ϭ might have originated from the primary basic chromosome numbers of x1 7 and x1 8. The sec- ϭ ondary basic number of x2 15 may arise either by amphiploidy or by ascending or descending dys- ϭ ploidy (Fig. 3). If we accept this secondary basic chromosome number of x2 15 as the original base complement, then the somatic chromosome number of 2nϭ720 seems to be a ‘48-ploid’. Dur- ϭ ing the course of evolution, this secondary basic chromosome number of x2 15 via proto-auto- ϭ ploidy forms the tertiary basic chromosome number of x3 30. Thus in the present investigation there is a probability that O. nudicaule may be a ‘24-ploid’, having evolved from a tertiary basic ϭ chromosome number of x3 30. Endomitosis seems to be one of the causes of the high level of polyploidy observed in O. nudi- caule. This polyploid fern seems to be thriving well with its original high polyploid chromosome complement without any variations. O. nudicaule seems to be a living example without much evo- lutionary changes. References Fernands, A. and Leitao, M. T. 1984. Contribution a 1Ј etude Cytotaxonomique Spermatophyta du Portugal XVII–Lami- aceae. Mem. Soc. Broteriana 27: 27–57. Ghatak, J. 1977. Biosystematic survey of Pteridophyta from Shevaroy hills, South India. The Nucleus 20: 105–108. Grant, V. 1981. Plant Speciation. Columbia University Press, New York. Khandelwal, S. 1990. Chromosome evolution in the genus Ophioglossum L. Biol. J. Linn. Soc. 10: 205–217. Kumar, M. 1998. Studies on the fern flora of Kerala with special reference to Sylvan Valley, Munnar. Research report No. 164 Marunnan Faseena and John Ernest Thoppil Cytologia 72(2) 145. Kerala Forest Research Institute, Peechi. Love, A., Love, A. and Pichi-Sermolli, R. E. G. 1977. Cytotaxonomical Atlas of the Pteridophyta. Vaduz: J. Cramer, Lon- don. Manickam, V. S. 1984. Cytology of thirty species of ferns from Palni hills (South India). Cytologia 49: 49–59. Ninan, C. A. 1956. Cytology of the Ophioglossaceae. Curr. Sci. 25: 161–162. — 1958. Studies on the cytology and phylogeny of the pteridophytes VI. Observations on the Ophioglossaceae. Cytologia 23: 291–316. Sharma, A. K. and Sharma A. 1990 . Chromosome Techniques: Theory and Practice. III Ed., Adithya Books, New Delhi..
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