Karyomorphological Study of Some Selected Cycads

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1 Karyomorphological Study of Some Selected Cycads Nitsri Sangduen, Mathanee Toahsakul and Vipa Hongtrakul Department of Genetics, Faculty of Science, Kasetsart University Bangkok, Thailand Abstract Karyotype and idiogram of three species of Cycas and Zamia from Nong Nooch Tropical Botanical Garden were obtained. All the three species of both Cycas and Zamia have an equal chromosome number in each species, namely 2n = 2x = 22 in Cycas and 2n = 2x = 16 in Zamia. The karyotype formulae of Cycas varied into 3 groups: 12 M + 8 SM + 2 A, 10 M + 8 SM + 2 A and 12 M + 8 SM + 2 T and of Zamia was 12 M + 4 SM. Of all three Cycas species studied, the karyotype of female and male plants could be distinguished. The Zamia evidence was complicated even all three species had the same chromosome number and karyotype pattern as well. Only Z. pumila could be differentiated between male and female plants. Keywords: Cytological technique, karyotype, chromosome morphology, satellite chromosome, Cycas, Zamia. Introduction Cycads are an important relic from the past and represent the oldest living seed plants (Brenner, et al. 2003). Cycas is considered the most primitive genus among the cycads (Stevenson 1990). Since the genus seems to generate a homoploid series with the chromosome number of 2n = 2x = 22 and to possess similar karyotypes in all species with only slight variation (Sax and Beal 1934; Kondo, et al. 1995). Among the genera of cycads (Cycadales), Zamia (Zamiaceae) is unique in showing interspecific karyotype variation (Marchant 1968; Norstog 1980 and Moretti 1990). Moreover, six species of Zamia have been reported to have intraspecific variation (Norstog 1980; Moretti and Sabato 1984 and Moretti 1990). Chromosome numbers known for the genus are 2n = 16, 18, 21, 22, 23, 24, 25, 26, 27 and 28. In dioecious gymnosperms, very little work has been reported about the cytology of sex determination. It may probably due to the fact that in many of the dioecious gymnosperms, the female and male individuals are identical in their external vegetative features, and as they normally take many years to become reproductively mature, one has to wait for a long period to discern the sex of a particular plant (Abraham and Mathew 1962). The first report of chromosome-based sex determination in plant was made by Allen (1917) on Sphaerocarpos - liverwort. Subsequently, chromosomes associated with sex have been discovered in a number of dioecious plants. Lee (1954) and Pollock (1957) reported evidence of association of satellite-based heteromorphic chromosome pair with sex. Similar observation was found in Cycas pectinata by Abraham and Mathew (1962). An XY type of sex mechanism has been assumed to be present in these plants. Toahsakul, et al. (2002) reported the difference between the male and female plant of Cycas wadei. In the female ten chromosomes of the somatic complement (pair IV, V, VI, VII and VIII) bore satellite, while in the male plant only one chromosome (pair IV) was found to be a satellite. Toahsakul, et al. (2004) presented comparative cytogenetic studies of sex determination in three species of Cycas, such as C. clivicola, C. edentata and C. pectinata Regular Paper 1

2 using conventional karyotyping and NORband. The work aims to study the general morphology of chromosomes in the male and female plants of Cycas and Zamia and also to make their karyotype and idiogram. Materials and Methods Three species of both Cycas and Zamia, collected from different geographical areas throughout the world and maintained at Nong Nooch Tropical Botanical Garden, were used (Table 1). Young curly leaflets were collected and chopped into small pieces ca. 2 mm. They were pretreated in 5.5 μmole/l isopropyl-nphenyl- carbamate for 24 h then fixed in a Carnoy s fluid (ethanol: glacial acetic acid: chloroform in the ratio 2:1:1) for 24 h. twice and incubated in 1 N HCl for 5 min. Chromosome preparation was made by enzyme maceration of 2% cellulose Onozuka R-10 (Yakult) and 1% pectolyase Y-23 (Kikkoman) in distilled water (W/V) at 37 C for 20 min, removed trichomes, stained and macerated in 2% aceto-orcein. For identification of the chromosomes, specimens with well-spread chromosomes were photographed. Chromosome lengths and arm ratios were then measured from the pictures taken and converted into actual lengths according to the magnification. The number and size of chromosomes and satellite chromosomes were recorded. Chromosome identification followed Levan, et al. (1964): Median-centromere chromosome (= m-chromosome; arm ratio = long arm/short arm = ), submetacentriccentromeric chromosome (= sm-chromosome; arm ratio = ), subterminal-centromeric chromosome (= st-chromosome; arm ratio = ), and terminal-centromeric chromosome, and t-chromosome means telocentric chromosome. Results and Discussion Three species of Cycas: Cycas elephantipes Lindstrom, C. siamensis Miquel, and C. tansachana K.D. Hill & S.L. Yang had the chromosome number of 2n = 2x = 22. It agrees with the previous finding of Toahsakul, et al. (2002). Chromosomes of the 1 st and 2 nd pairs were observed to possess secondary constriction in all the preparation. C. elephantipes Lindstrom & Hill, a new species from Thailand (Lindstrom and Hill 2002) had the karyotype comprised 12 m- chromosome, 8 sm-chromosome and 2 stchromosome. The satellite chromosome was located on the long arm of the 1 st, 4 th and 5 th chromosome in male plants (Fig.1 A, B and C). Two satellite chromosomes on the long arm of the 5 th chromosome were found in female plants (Fig.1 D, E and F). C. siamensis Miquel had the karyotype comprised 10 m-chromosome, 10 smchromosome and 2 st-chromosome. The satellite chromosome was found on the long arm of 1 st, 4 th, 5 th and 6 th chrom-osome in male plants (Fig. 1 G, H and I). Only two satellite chromosomes on the long arm of 3 rd chromosome were found in female plants (Fig.1 J, K and L). C. tansachana K.D. Hill & S.L. Yang had the karyotype comprised 12 m- chromosome 8 sm-chromosome and 2 stchromosome. Three satellite chromosomes were found on the long arm of 1 st and 8 th chromosome in male plants (Fig.1 M, N and O). Thus there were two-type gamete of male plants. Only 2 satellite chromosomes on the long arm of 8 th chromosome were found in female plants (Fig. 1 P, Q and R) The difference in sex determination in Cycas studied could be categorized in terms of number of satellite chromosomes, heteromorphic or homomorphic pattern as followed: C. elephantipes showed homomorphic satellite chromosome in both male and female plants but varied in number of satellite chromosomes (2 in female and 6 in male plants) C. siamensis and C. tansachana showed the heteromorphic chromosome on the 1 st chromosome in male plants. (7 satellite chromosomes). Homomorphic chromosome pattern was found in female plants of these 2 species. (2 satellite chromosomes). There was 7 and 3 satellite chromosomes in male plant of C Regular Paper 2

3 siamensis and C. tansachana, respectively. But these two Cycas species possess the same pattern. Three species of Zamia: Z. integrifolia L. fil in Aiton, Z. pumila L., and Z. pygmaea Sims. had the same chromome number 2n = 2x = 16. Z. integrifolia L. fil in Aiton had the karyotype comprised 12 m-chromosomes and 4 sm-chromosome. The satellite chromosome was not found in both male and female plant (Fig. 2 A, B, C, D, E and F). Z. pumila L. had the karyotype comprised 12 m-chromosome and 4 sm-chromosome (Moretti and Sabato 1984; Moretti 1990). There were 3- and 4-satellite chromosomes on the 1 st and 8 th chromosome were visible in male and female plants, respectively (Fig.2 G, H, I, J, K and L) Z. pygmaea Sims. is the smallest of all living cycads (Jones 1998). Its karyotype comprised 12 m-chromosome and 4 smchromosome. Two satellite chromosomes on the 8 th chromosome were observed in both male and female plants (Fig. 2 M, N, O, P, Q and R). The differences in sex determination in Zamia studied could be categorized in terms of number of satellite chromosomes, heteromorphic and/or homomorphic pattern as followed: 1. Satellite chromosome was not observed in both male and female plants. Thus there was no difference between male and female plants. Z. integrifolia was categorized in this pattern. So it required to develop other techniques for more detail such as chromosome banding, fluorescence in situ hybridization technique (FISH) and DNA markers. 2. Heteromorphic male and homomorphic female were observed in Z. pumila. 3. Homomorphic in both male and female plants was observed in Z. pygmaea. Of these three Zamia species, the difference between male and female plants was observed only in Z. pumila. It needs to work out other technique to identify sex in Z. integrifolia and Z. pygmaea. Even though they had the same chromosome number as well as karyotype pattern, satellite chromosomes might not be used for sex determination in all cycads. Acknowledgements This research was financially supported by Kasetsart University Research and Development Institute (KURDI) and Public Private Technology Transfer Center. The authors would like to thank Mr. Kampon Tansacha and Mr. Anders J. Lindstrom at Nong Nooch Tropical Botanical Garden for providing Cycas and Zamia specimens. References Allen, C.E A chromosome difference as correlated with sex difference in Sphaerocarpos. Science 46: Abraham, P.; and Mathew, P.M Cytological studies in the Cycads: Sex chromosome in Cycas. Ann. Bot. 26: Brenner, E.D.; Stevenson, D.W.; and Twigg, RW Cycads: Evolutionary innovations and the role of plant-derived neurotoxins. Trends in Plant Science 8 (9): Jones, D.L Cycads of the World. Reed New Holland, Sydney, Australia. Kondo, K.; Kokubugata, G.; Hizume, M.; Tanaka, R.; and Satake, T A karyomorphological study of five species and variety of Cycas. Cytologia 60: Lee, C.L Sex chromosome in Ginkgo biloba. Amer. J. Bot. 41: Lindstrom, A.J.; and Hill, K.D New species and a new record of Cycas (Cycadaceae) from Thailand. Brittonia 54: Marchant, C.J Chromosome pattern and nuclear phenomena in the cycad family Stangeriaceae and Zamiaceae. Chromosome 24: Moretti, A Cytotaxonomy of cycads. Mem. New York Bot. Gard. 57: Moretti, A.; and Sabato, S Karyotype evolution by centromeric fission in Zamia (Cycadales). Plant Syst. Evol. 146: Regular Paper 3

4 Norstog, K.J Chromosome number in Zamia (Cycadales). Caryologia 33: Pollock, E.G The sex chromosome of the maiden hair tree. J. Heredity 48: Sax, K.; and Beal, J. M Chromsome of the Cycadales. J. Arnold Arb. 15: Stevenson, D.W Chigua, a new genus in the Zamiaceae with comment on its biographic significance. Mem. New York Bot. Gard. 57: Toahsakul, M.; Hongtrakul, V.; and Sangduen, N Cytological studies of sex determination in Cycas. Proc.12 th Genetic Conf.: Gene Revolution Era, pp , March 28-30, Kasetsart University, Bangkok, Thailand. Toahsakul, M.; Hongtrakul, V.; and Sangduen, N Comparative cytogenetics studies between male and female of Cycas. Proc. 13 th Genetic Conf. pp : Genetics and Sustainable Development. June 5-4, Naresuan University, Pitsanulok, Thailand. Table 1. Three species of Cycas and Zamia used. Species C. elephantipes Lindstrom & Hill C. siamensis Miquel C. tansachana K. D. Hill & S.L. Yang Z. integrifolia Linnaeus fil in Aiton Z. pumila Linnaeus Z. pygmaea Sims Cultivation source Chaiyaphum, Thailand Kanchanaburi, Thailand Saraburi, Thailand Florida, USA Dominican Republic Cuba Chromosome number 2n=2x=22 2n=2x=22 2n=2x=22 2n=2x=16 2n=2x=16 2n=2x=16 Regular Paper 4

5 B. A. C. E. D. F. H. G. I. K. J. L. N. M. O. Q. P. R. Fig. 1. A. Metaphase cell of Cycas elephantipes (male). B-C. Karyotype and idiogram of C. elephantipes (male) D. Metaphase cell of C. elephantipes (female). E-F. Karyotype and idiogram of C. elephantipes (female). G. Metaphase cell of C. siamensis (male). H-I. Karyotype and idiogram of C. siamensis (male). J. Metaphase cell of C. siamensis (female). K-L. Karyotype and idiogram of C. siamensis (female). M. Metaphase cell of C. tansachana (male). N-O. Karyotype and idiogram of C. tansachana (male). P. Metaphase cell of C. tansachana (female). Q-R. Karyotype and idiogram of C. tansachana (female). ( =10 µm) Regular Paper 5

6 B. A. C. E. D. F. H. G. I. K. J. L. N. M. O. Q. P. R. Fig. 2. A. Metaphase cell of Zamia integrifolia (male). B-C. Karyotype and idiogram of Z. integrifolia (male) D. Metaphase cell of Z. integrifolia (female). E-F. Karyotype and idiogram of Z. integrifolia (female). G. Metaphase cell of Z. pumila (male). H-I. Karyotype and idiogram of Z. pumila (male). J. Metaphase cell of Z. pumila (female). K-L. Karyotype and idiogram of Z. pumila (female). M. Metaphase cell of Z. pygmaea (male). N-O. Karyotype and idiogram of Z. pygmaea (male). P. Metaphase cell of Z. pygmaea (female). Q-R. Karyotype and idiogram of Z. pygmaea (female). ( =10 µm) Regular Paper 6