Karyotype analysis of Santalum album L.

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1 CARYOLOGIA Vol. 63, no. 2: , 2010 Karyotype analysis of Santalum album L. Zhang 1 Xin-Hua, Jaime A. Teixeira da Silva 2 and Guo-Hua Ma 1, 1 South China Botanical Garden, the Chinese Academy of Sciences, Guangzhou, , P.R. China 2 Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken, , Japan. Abstract A karyomorphological study on Santalum album was conducted for the first time. The interphase nucleus was a simple chromocenter type while the prophase chromosomes were of the interstitial type. The shoot-tip meristems of some S. album individuals were found to be mixoploid, i.e. 2n = 2x = 20 and 2n = 4x = 40. Two different karyotypes, one diploid (2n = 2x = 20) and one tetraploid (2n = 4x = 40), were also present; their karyotypic formulae were 2n = 20 = 18m + 2sm and 2n = 40 = 32m (2SAT) + 8sm, respectively. The chromosomes of both karyotypes showed centromeres predominantly in a median position and a few submedian centromeres, of 2B type, of a primitive and symmetrical nature. Key words: Santalum album, karyomorphology, karyotype, mitosis, mixoploid. INTRODUCTION The genus Santalum belongs to the family Santalaceae, which consists of 15 extant species and approximately 14 varieties. These are distributed throughout India, Australia, and the Pacific Islands, and as far southeast as the Juan Fernandez Islands, only 600 miles off the coast of Chile, and as far northwest as the Bonin Islands, 600 miles south of Honshu, Japan (HARBAUGH and BALDWIN 2007). The most well known and commercially valuable Santalum species is the Indian sandalwood tree, Santalum album L. (AB- DUL AZEEZ et al. 2009). S. album is an evergreen and hemi-parasitic tree found in semi-arid areas from India to the South Pacific and the northern coast of Australia, besides the Hawaiin islands (HARBAUGH and BALDWIN 2007). The great value of this species is its heartwood and oil, which are widely used in cosmetic, perfume, medicine and aromatherapy industries (SRINIVASAN et al. 1992; SCARTEZZINI and SPERONI 2000; KIM et al. 2006). Polyploidy is widely acknowledged as one form of breeding in plants. Polyploid plants usually have larger cells and plants are often larger, with thicker shoots, larger leaves and fruits, and a higher nutritional content, etc. In order to increase oil content and economic benefits from S. album, we decided to induce polyploidy in this tree species. Even though some cytological data on S. album has been generated (RAO 1942; SRIMATHI and SREENIVASAYA 1962; BHATNA- GAR 1965; CARR 1978; GOLDBLATT and JOHNSON 2000; HARBAUGH 2008), there exists to date no literature on the karyotype analysis of S. album. Knowledge of karyotypic relationships is an important prerequisite for effective plant genetic and breeding studies. The aim of the present study was to investigate the karyotype of S. album, including the interphase nucleus and various stages of mitosis, and to provide valuable cytological information for breeding programs using this species. MATERIAL AND METHODS Corresponding author: magh@scib.ac.cn The seeds of sandalwood were obtained from Mysore in southern India. One hundred seeds

2 KARYOTYPE ANALYSIS OF SANTALUM ALBUM L. 143 were sown in a sand bed of the greenhouse in the South China Botanical Garden. The seeds were pretreated with a 500 mg/l GA 3 (gibberellic acid) solution for 24 h at room temperature. Since alot of santal oil was detected in root cells in preliminary experiments, only shoot tips were chosen and excised from 20 plantlets every 10 minutes from 10:30 to 11:30 am, the period in which shoot tips are most actively growing. For squashes, different preparative methods for metaphase chromosomes were evaluated using shoot tips (ZHANG and XIA 2007). The material was pretreated with M 8-hydroxyquinoline for 2-3 h, a saturated aqueous solution of paradichlorobenzene for 4-5 h, or an ice water mixture solution for 24 h at 4 C. The shoot tips were fixed with freshly prepared Carnoy fluid (ethanol alcohol: glacial acetic acid = 3:1). Another method was to fix shoot tips with freshly prepared Carnoy fluid immediately after collection for h at room temperature. Direct fixing of shoot tips without any pretreatment was optimum for detecting metaphase chromosomes. Incidentally, chromosome condensation was also better and centromeres were easily discerned. The fixed samples were hydrolyzed in 1 N HCl for 8 min at room temperature after washing with distilled water three times, then stained in 1% aceto-orcein for 15 min, and squashed on a glass slide after being heated slightly. The squashed accessions were observed under a microscope (Zeiss Axioplan 2), and photographs were taken automatically. The experiment was repeated three times. The karyomorphological classification of resting nuclei and mitotic prophase chromosomes followed TANAKA s categories (1977; 1971; 1980). The karyotypes were analyzed following LI and CHEN (1985). A karyogram for each genotype was constructed by arranging the chromosomes in homologous pairs by order of their length and arm ratio. The symbols for the description of chromosomes followed LEVAN et al. (1964). The symmetry of karyotypes was classified according to STEBBINS (1971). Mean length was calculated for each chromosome pair. These values were then used to calculate total chromatin length of each karyotype. Measurements of chromosome arms were based on at least five cells. Karyotypic asymmetry rate (As. k %), based on the long arm (LA) and on each chromosome length (CL) was calculated as follows: As. k% = 100ΣLAΣCL-1 (LI and CHEN 1985). RESULTS Mitosis - The interphase nucleus was a simple chromocenter type according to TANAKA s catalogue (1971; 1977). It was characterized by several darkly stained chromocenters with an irregularly protruding rough surface which gradually transformed into diffuse chromatin (Fig. 1A). At the mitotic prophase, hetero- and euchromatic segments were distinguishable, but their boundaries were not so clear, and the heterochromatic segments were distributed in the distal and interstitial regions as well as in the proximal regions (Fig. 1B). According to TANAKA (1980), prophase chromosomes belong to the interstitial type. The chromosomes gradually became short and thick as mitosis progressed, and individual TABLE 1 Chromosome parameters of S. album. Abbreviations: No.: chromosome pair number; RL: relative length; CL: chromosome length (µm); AR: arm ratio; PC: position of centromere; m: median chromosome; sm: submedian chromosome. No. 2n=40 CL RL AR PC RL CL AR PC 2n= = m = m = m = m = sm = m = m = m = m = sm = m = m = m = m = m = m = m = m = m = sm

3 144 ZHANG, DA SILVA and MA Fig. 1 The interphase nuclei and mitosis phases of S. album. A. the interphase nuclei; B. prophase chromosomes; C. premetaphase chromosomes; D, metaphase plate; E, F. anaphase chromosomes; G, H. telophase chromosomes. Bar: 5µm.

4 KARYOTYPE ANALYSIS OF SANTALUM ALBUM L. 145 TABLE 2 The parameters of mitotic metaphase chromosome of S. album. Karyotypic formula Percentage of chromosome with arm ratio >2 Longest chromosome/ shortest chromosome n=20=18m+2sm n=40=28m+12sm 2.27 Type Fundamental Index of karyotype number asymmetry (%) 2B B chromosomes became visible. The premetaphase chromosomes were long and curved, but some centromeres could not be easily discerned (Fig. 1C). The chromatids remained aligned between the poles of the cell during metaphase (Fig. 1D). Anaphase began when the pairs of sister chromatids separated; the separated chromatids then moved toward opposing poles of the cell (Fig. 1E, F). At the end of anaphase, two identical groups of single chromosomes congregated at opposite poles of the cell (Fig. 1G). In telophase, the final stage of mitosis, the chromosomes amassed at the poles, and a new nuclear membrane formed Fig. 2 Somatic metaphase chromosomes (A), karyogram (B), and idiogram (C) of the diploid S. album. White arrows and black arrows indicate secondary constrictions and supernumerary chromosome, respectively. Bar: 5µm. around each new group of chromosomes, whose cell plate was obviously visible (Fig. 1H). Chromosome numbers - The somatic chromosome number of S. album is 2n = 20 (Fig. 2). A mixoploid (2n = 2x = 20 and 2n = 4x = 40) was found in the shoot-tip meristems of some individuals, accounting for about 5% of the accessions (Fig. 3). Karyotype of 2n = 2x = 20 diploid - The metaphase chromosomes of the diploid S. album are presented in Fig. 2A. The chromosomes were short, ranging from 1.16 to 2.33 µm, and the ratio of the longest to shortest chromosome was 2.01 (Table 1). The arm ratios ranged from 1.13 to The karyotype was slightly bimodal and consisted of three larger and seven smaller pairs of chromosomes. The karyotypic formula was 2n = 2x = 20 = 18m + 2sm, consisting of 18 metacentric chromosomes and a pair of submetacentric chromosomes (Fig. 2B, C). Secondary constrictions were found in the long arm of the third pair of chromosomes (Fig. 2A, C). The karyotype was type 2B with an asymmetry index of (Table 2). Karyotype of 2n = 4x = 40 tetraploid - The metaphase chromosomes of the tetraploid S. album, which are presented in Fig. 3A, were arranged into 10 groups of four chromosomes each (Fig. 3B). The chromosomes in each group had a similar morphology. The median size of chromosomes ranged from 1.30 to 2.95 µm in length, and the ratio of the longest to the shortest chromosome was The arm ratios ranged from 1.13 to The karyotype was slightly bimodal and consisted of three larger and seven smaller groups of chromosomes. The karyotypic formula was 2n = 4x = 40 = 32m (2SAT) + 8sm, consisting of 32 median regions and eight submedian regions. Secondary constrictions were found in the long arm of the third chromosome and long and short arms of the eighth chromosome. Satel-

146 ZHANG, DA SILVA and MA lites were seen on the short arm of the ninth and tenth chromosomes (Fig. 3B). The karyotype was type 2B with an asymmetry index of 58.96 (Table 2).

5 146 ZHANG, DA SILVA and MA lites were seen on the short arm of the ninth and tenth chromosomes (Fig. 3B). The karyotype was type 2B with an asymmetry index of (Table 2). DISCUSSION Our study confirmed previous ones in which the somatic chromosome number of S. album was noted as 2n = 20 and with basic chromosome number x = 10 (RAO 1942; GOLDBLATT and JOHNSON 2000; HARBAUGH 2008). A mixoploid (2n = 2x = 20 and 2n = 4x = 40) was found for the first time for S. album in our study. The karyotype of S. album, reported for the first time in the present study for this hardwood species, showed a predominance of chromosomes with centromeres in a median position and a few submedian centromeres. Moreover, the arm ratios of only a few chromosome pairs exceeded 2.0, while most others ranged from 1.13 to This indicated that the intrachromosomal symmetry in S. album was very high. STEBBINS (1971) pointed out that karyotype evolution is generally from symmetry to asymmetry in higher plants. In other words, primitive plants have symmetric karyotypes whereas asymmetric karyotypes occur in more evolutionary advanced taxa. Karyotypic analysis indicates that S. album was a more primitive taxon. In the tetraploid (2n = 40) accessions analyzed, the chromosomes were relatively larger in size than those of the diploids (2n = 20). This was consistent with a report by SRIMATHI and SREENIVASAYA (1962) in which there was a 2- to 5-fold increase in the size of the chromosomes of the haustoria. There were two groups of submetacentric chromosomes in the tetraploids; however, only one such pair was found in diploids. Satellites occurred in the tetraploids. We believe that the tetraploids were autotetraploids since they consisted of three larger and seven smaller pairs or groups of chromosomes, and since the chromosomes of each group in all 10 groups had a similar morphology. Mixoploidy is induced by natural, artificial or chemical factors. To date, many mixoploids have been documented (CARDI et al. 1993; CHEN and WAN; MARTIN and WIDHOLM 1996; HAN et al. 1997; SU et al. 2000; OCKENDON 2008). The majority were found in anther culture regenerants. This phenomenon was thought to be caused by the application of plant growth regulators such as kinetin, indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), TDZ and BAP (DOLEZEL and NOVÁK 1984; RODRIGUES et al. 2004; JIN et al. 2008). WANG et al. (2008) claimed that the occurrence of tetraploidy in Rubus parvifolius L. (Rosaceae) was related to abnormal mitosis in some diploid cells. PALIWAL (1956) reported cytomixis in the anthers of S. al- Fig. 3 Somatic metaphase chromosomes (A) and karyogram (B) of the tetraploid S. album. White arrows and black arrows indicate satellite chromosomes and secondary constrictions, respectively. Bar: 5µm.

6 KARYOTYPE ANALYSIS OF SANTALUM ALBUM L. 147 bum, i.e. two microspore mother cells lying near each other became connected through a narrow projection forming a sort of conjunction tube, and this resulted in the mixing of cytoplasm and nuclear contents of the two cells. It occurred in those cells showing the presence of restitutional nuclei or some other similar irregular division. He also frequently observed that only three cells were organized in S. album and that one of the microspores was fairly large with two nuclei while the other two were of normal size and uninucleate. Polyspory was reported in pollen meiosis of S. album (PALIWAL 1956; BHATNAGAR 1965). SRIMATHI and SREENIVASAYA (1962) observed that many S. album haustorium cells had 40 chromosomes, possibly endopolyploidy, which might be related to physiological traits of the haustoria, such as the presence of IAA and other related plant growth regulators. In the present study, we found a mixoploid (2x + 4x) in shoot-tip meristems of sandalwood seedlings, but the ratio was small, accounting for about 5% of the accessions analyzed. This might have been caused by abnormal mitosis or meiosis induced by a doubling in chromosome number. The genus Santalum distributed throughout India, Indonesia, and Australia is diploid (GOLDBLATT and JOHNSON 2000) although two Hawaiian clades are tetraploid (CARR 1978). By studying data from flow cytometric analysis of Pacific island sandalwoods, HARBAUGH (2008) reported that Santalum appeared to include four ploidies, ranging from diploid (n = 10) to octoploid (n = 40). The majority were diploid and tetraploid, and S. album was diploid among all the accessions investigated. Even the ploidy levels of taxa within the same section in the genus were not completely consistent. For example, S. freycinetianum was related to S. album and they were all clustered into the same section, even though S. freycinetianum is tetraploid. There is evidence that Santalum polyploids have already colonized the Pacific islands (HARBAUGH 2008). In the present study, about 95% of shoot tips of S. album accessions analyzed were diploid, with the exception of 5%, which were 2x + 4x mixoploids. The specimens collected were from sandalwood distributed in South India. This indicates that the ploidy of S. album is slightly different depending on the geographical region. The occurrence of polyploidy and mixoploidy in S. album deserves further investigation in combination with the species geographical distribution. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant No , ), and Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, CAS (Grant No ). REFERENCES ABDUL AZEEZ S., NELSON R., PRASADBABU A. and SRINI- VAS RAO M., 2009 Genetic diversity of Santalum album using random amplified polymorphic DNAs. African Journal of Biotechnology, 8: BHATNAGAR S.P., 1965 Microsporgenesis and male gametophyte in Santalum L. Proceedings of the Indian National Science Academy. Part B, Biological Sciences, 32: CARDI T., IANNAMICO V., D AMBROSIO F., FILIPPONE E. and LURQUIN P.F., 1993 In vitro regeneration and cytological characterization of shoots from leaf explants of three accessions of Solanum commersonii. Plant Cell, Tissue and Organ Culture, 34(1): CARR G.D., 1978 Chromosome numbers of Hawaiian flowering plants and the significance of cytology in selected taxa. American Journal of Botany, 65: CHEN L.S. and WAN S.Y., 1995 Studies on cytology and embryology of Guilin-Liangfeng seedless ponkan (Citrus reticulata Blanco). Acta Horticulturae, 43: CHEN L.S. and WAN S.Y., 1996 Cytological identifications on Guilin Liangfeng seedless Ponkan (Citrus reticulata Blanco) A new type mutant of Ponkan. Journal of Wuhan Botanical Research, 14 (1): 1-5. DOLEZEL J. and NOVAK FJ., 1984 Cytogenetic effect of plant tissue culture medium with certain growth substances on Allium sativum L. meristem root tip cells. Biologia Plantarum, 26(4): GOLDBLATT P. and JOHNSON D.E., 2000 Index to plant chromosome numbers Missouri Botanical Garden. HAN D.S., NIIMI Y. and NAKANO M., 1997 Regeneration of haploid plants from anther cultures of the Asiatic hybrid lily Connecticut King. Plant Cell, Tissue and Organ Culture, 47: HARBAUGH D.T. and BALDWIN B.G., 2007 Phylogeny and biogeography of the sandalwoods (Santalum, Santalaceae): repeated dispersals throughout the Pacific. American Journal of Botany, 94: HARBAUGH D.T., 2008 Polyploid and hybrid origins of pacific island sandalwoods (Santalum, santalaceae) inferred from low-copy nuclear and flow cytometry data. International Journal of Plant Sciences, 169: IYENGER G.S., 1937 Life history of Santalum album L. Journal of Indian Botanical Society, 16:

7 148 ZHANG, DA SILVA and MA JIN S., MUSHKE R., ZHU H., TU L., LIN Z., ZHANG Y. and ZHANG X., 2008 Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers. Plant Cell Reports, 27(8): KIM T.H., ITO H., HAYASHI K., HASEGAWA T., MACHIGUCHI T. and YOSHIDA T., 2006 New antitumor sesquiterpenoids from Santalum album of Indian origin. Tetrahedron, 62: LEVAN A., FREDGE K. and SANDBERG A.A., 1964 Nomenclature for centromeric position on chromosomes. Hereditas, 52: LI M.X. and CHEN R.Y., 1985 A suggestion on the standardization of karyotype analysis in plants. Journal of Wuhan Botanical Research, 3: MARTIN B. and WIDHOLM J.M., 1996 Ploidy of small individual embryo-like structures from maize anther cultures treated with chromosome doubling agents and calli derived from them. Plant Cell Reports, 15: OCKENDON D.J., 2008 The ploidy of plants obtained from anther culture of cauliflowers (Brassica oleracea var. botrytis). Annals of Applied Biology, 113(2): PALIWAL R.L., 1956 Morphological and embryological studies in some Santalaceae. Agra University Journal of Research, 5: RAO L.N., 1942 Studies in the Santalaceae. Annals of Botany, 6: RODRIGUES I.R., FORTE B.C., OLIVEIRA J.M.S., MARIA- TH J.E.A. and BODANESE-ZANETTINI M.H., 2004 Effects of light conditions and 2,4-D concentration in soybean anther culture. Plant Growth Regulation, 44: SCARTEZZINI P. and SPERONI E., 2000 Review on some plants of Indian traditional medicine with antioxidant activity. Journal of Ethnopharmacology, 71: SRIMATHI R.A. and SREENIVASAYA M., 1962 Occurrence of endopolyploidy in the haustorium of Santalum album Linn. Current Science, 31: SRINIVASAN V.V., SIVARAMAKRISHNAN V.R., RANGASWAMY C.R., ANANTHAPA-DMANABHA H.S. and SHANKARA NARAYANA M.H., 1992 Sandal (Santalum album L.), Institute of Wood Science and Technology (ICFRE), Bangalore, India, pp STEBBINS G.L., 1971 Chromosomal evolution in higher plants. Edward Arnold Ltd., London. SU C., SU L.H., ZHU G.Y. and HAN M.Z., 2000 The ways of bringing about and classifications of mixoploid in Morus L. North Sericulture, 21: TANAKA R., 1971 Types of resting nuclei in Orchidaceae. Botanical Magazine Tokyo, 84: TANAKA R., 1977 Recent karyotype studies. In: K. Ogawa, et al. (Eds), Plant Cytology, Asakura Shoten, Tokyo. TANAKA R., 1980 The karyotype. In: K. Yamashita, (Ed.), Plant Genetics, pp Shokabo, Tokyo. WANG X.R., TANG H.R., DUAN J. and LI L., 2008 A comparative study on karyotypes of 28 taxa in Rubus sect. Idaeobatus and sect. Malachobatus (Rosaceae) from China. Journal of Systematics and Evolution, 46: ZHANG X.H. and XIA N.H., 2007 A karyomorphological study of the genera Michelia and Manglietia (Magnoliaceae). Caryologia, 60: Received June 11 th 2009; accepted June 4 th 2010

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