Nuclear DNA Contents of Phalaenopsis Sp. and Doritis Pulcherrima
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J. AMER. SOC. HORT. SCI. 126(2):195–199. 2001. Nuclear DNA Contents of Phalaenopsis sp. and Doritis pulcherrima Sandy Lin and Hsiao-Ching Lee Department of Life Science, National Tsing Hua University, Hsinchu, 30043, Taiwan, Republic of China Wen-Huei Chen Department of Horticulture, Taiwan Sugar Research Institute, Tainan, 701, Taiwan, Republic of China Chi-Chang Chen and Yen-Yu Kao Department of Botany, National Taiwan University, Taipei, 10764, Taiwan, Republic of China Yan-Ming Fu and Yao-Huang Chen Department of Horticulture, Taiwan Sugar Research Institute, Tainan, 701, Taiwan, Republic of China Tsai-Yun Lin1 Department of Life Science, National Tsing Hua University, Hsinchu, 30043, Taiwan, Republic of China ADDITIONAL INDEX WORDS. Orchidaceae, endoreduplication, flow cytometry, genome size ABSTRACT. Nuclear DNA contents were estimated by flow cytometry in 18 Phalaenopsis Blume species and Doritis pulcherrima Lindl. DNA amounts differed 6.07-fold, from 2.74 pg/diploid nuclear DNA content (2C) in P. sanderiana Rchb.f. to 16.61 pg/2C in P. parishii Rchb.f. Nuclear DNA contents of P. aphrodite Rchb.f. clones, W01-38 (2n = 2x = 38), W01-41 (2n = 3x = 57), and W01-22 (2n = 4x = 76), displayed a linear relationship with their chromosome numbers, indicating the accuracy of flow cytometry. Our results also suggest that the 2C-values of the Phalaenopsis sp. correlate with their chromosome sizes. The comparative analyses of DNA contents may provide information to molecular geneticists and systematists for genome analysis in Phalaenopsis. Endoreduplication was found in various tissues of P. equestris at different levels. The highest degree of endoreduplication in P. equestris was detected in leaves. Phalaenopsis Blume sp. are among the most beautiful of all 1992). A better understanding of karyotypes and DNA contents of orchids. The genera Phalaenopsis and Doritis Sw. belong to the Phalaenopsis will aid in the development of new cultivars that will Orchidaceae, subfamily Epidendroideae, tribe Vandeae, subtribe improve the level of production. Haploid nuclear DNA contents of Aeridinae. Phalaenopsis comprises over 45 species in nine sections angiosperms are highly variable, differing over 600-fold (Bennett indigenous throughout most of Asia (Sweet, 1980). These species and Leitch, 1995; Bennett et al., 1982). Although intraspecific are distributed from India, south China, Thailand, Malaysia and variation in genome size occurs due to environmental influences, Indonesia to the Philippines, New Guinea, and Australia. All species common chromosome polymorphisms, and spontaneous aberra- of Phalaenopsis have the same chromosome number (2n = 38) with tions, DNA content per genome is fairly constant, both between cells chromosome sizes ranging from 1.5 to 3.5 µm (Arends, 1970). of an individual and between different individuals of the same Phalaenopsis sp. can be divided into large, medium and small species (Greilhuber, 1998; Greilhuber and Obermayer, 1997). Ac- chromosome groups, according to their chromosome sizes. curate determination of genome size provides basic information for Phalaenopsis, occurring in the Philippines, have smaller symmetric breeders and molecular geneticists. Comparisons of amounts of chromosomes, while those found outside of the Philippines, have nuclear DNA are also useful in cytotaxonomic and evolutionary larger asymmetric chromosomes (Shindo and Kamemoto, 1963). studies. The rapid technique of flow cytometry is widely used in Species of the genus Doritis have a very wide distribution, ranging studies of cell cycle analysis, chromosome ploidy, and nuclear DNA from the Himalayas of northern India, through Thailand and Burma, content in biological systems (Jones et al., 1998; Lysák et al., 1999; to Malaysia and Indonesia (Teuscher, 1977). Doritis pulcherrima Ormerod, 1994). In this study we examined the nuclear DNA Lindl. is synonymous with P. pulcherrima (Lindl.) J. J. Smith contents in 18 Phalaenopsis sp. and D. pulcherrima using a flow (Christenson, 1995; Sweet, 1980) and has been used extensively in cytometer. breeding with Phalaenopsis to produce Doritaenopsis such as Endoreduplication is widespread in large numbers of higher Doritaenopsis Asahi obtained by Baron Toshita Iwasaki in 1923 plants (Cebolla et al., 1999; Gendreau, et al., 1999; Grafi and from D. pulcherrima and P. lindenii Loher. Larkins, 1995; Sun et al., 1999). Sturdier flowers and better forms Interspecific hybridization and chromosome doubling are tech- may accompany an increase in ploidy in orchids. Although chromo- niques often applied to produce new cultivars of orchids (Arditti, some doubling is a technique for breeders to obtain new cultivars, little is known regarding the mechanism of endoreduplication in orchids. Use of flow cytometry in determining polyploidy is fast and Received for publication 11 Apr. 2000. Accepted for publication 12 Sept. 2000. We thank Cindy Buschena, Albert H. Markhart III, and Cheng-Teh Wang for makes determining polyploidy easier. We also demonstrate the insightful discussions, Anne Chao and Rouh-Jane Chou for statistical advice, and occurrence of endoreduplication in different tissues of P. equestris. the National Science Council and the Taiwan Sugar Corporation, Republic of China for grants NSC 87-TSC-B-007-001 and NSC 88-TSC-B-007-001. The cost Materials and Methods of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertise- ment solely to indicate this fact. PLANT MATERIAL. Samples of young leaves or other organs of 1Professor and corresponding author; e-mail: [email protected]. 18 nursery bred Phalaenopsis sp. and D. pulcherrima were obtained from the Taiwan Sugar Research Institute (TSRI). SE of the peak, which was calculated from the SD. Phalaenopsis aphrodite used in the experiments is native in Taiwan. DETERMINATION OF CHROMOSOME NUMBER. Root tips 0.8 cm in Plants were maintained in a growth chamber at 14 h days/10 h nights length were severed and pretreated in 2 mM 8-hydroxyquinoline of 26/25 °C with a 14-h photoperiod of 300 µmol·m–2 ·s–1 provided for 4 h at 18 to 20 ºC. After fixing in a 3:1 mixture of 95% ethanol by fluorescent light. Day/night relative humidity was 80% /90%. and glacial acetic acid overnight, samples were washed twice Plants were watered every 2 days and fertilized monthly with with sterile distilled water and incubated in 1 M HCl at 60 ºC for Hoagland’s solution. 7 min. Samples were washed again and stained with 0.5% basic PREPARATION OF NUCLEI SUSPENSION. Suspensions of nuclei fuchsin for 2 h in the dark, and then treated with 1% pectinase for were prepared according to the protocol of Arumuganathan and 40 min. Subsequently, the root tips were squashed and stained Earle (1991) with some modifications. Phalaenopsis sp. contain with 1% acetic orcein to determine chromosome numbers. large amounts of calcium oxalate crystals in their cells. The presence of crystals decreases the yield of intact nuclei. Conse- Results quently, a relatively high level of underlying debris and chemical residue that might fluoresce contaminate the samples. Therefore, NUCLEAR DNA CONTENTS OF Phalaenopsis SP. AND Doritis pulcherrima. a cotton filter was designed to remove the crystals. Cosmetic Flow cytometric analysis of isolated nuclei resulted in histograms of cotton was cut into 5 × 1-cm pieces, wetted with MgSO4 buffer the relative nuclear DNA content containing peaks corresponding to containing 10 mM MgSO4•7H2O, 50 mM KCl, 5 mM Hepes (pH 8), the G0/G1 nuclei of leaves of the internal reference standard P. and rolled up loosely. The cotton ball was inserted into a 5 mL sativum (Fig. 1A) and P. aphrodite W01-38 (Fig. 1B), W01-41 (Fig. pipette tip and rinsed with 6 mL MgSO4 buffer to remove floating 1C), and W01-22 (Fig. 1D) as illustrated in Fig. 1. Systematically, fibers. One hundred fifty milligrams of tissue from healthy plants three peaks were found in Phalaenopsis, corresponding to 2C, 4C, was cut into 0.5 mm pieces using a scalpel in 3 mL of solution A and 8C nuclei (Fig. 1B). Genome sizes of 18 Phalaenopsis sp. and in a petri dish (35 × 10 mm) on ice. Solution A contained MgSO4 D. pulcherrima are listed in Table 1. Chromosome numbers for all buffer with 6.5 mM dithiothreitol, 0.25% Triton X-100 and 0.1 the orchids examined and stained by basic fuchsin and acetic orcein mg·mL–1 propidium iodide (Calbiochem, La Jolla, Calif.). The were confirmed to be 2n = 2x = 38 except for the P. aphrodite W01- homogenate was filtered through the cotton column into a 15 mL 41 (2n = 3x = 57) and W01-22 (2n = 4x = 76). There was a 6.07–fold centrifuge tube. Four milliliters of solution A was added to the variation in genome size within 18 Phalaenopsis species, ranging cotton filter to elute the nuclei. The eluted nuclei were centrifuged from 2.74 pg/2C for P. sanderiana to 16.61 pg/2C for P. parishii. at 100 gn for 10 min and the supernatant was discarded. The pellet The genome size is small for P. aphrodite (2.80 pg/2C), P. sanderiana was resuspended in 400 µL solution B that is solution A with 1.25 (2.74 pg/2C), and P. stuartiana (3.13 pg/2C) in section Phalaenopsis µg·mL–1 RNase (DNase-free) and incubated for 15 min at 37 °C. and P. equestris (3.37 pg/2C) in section Stauroglottis. The nuclear Nuclei isolated from Pisum sativum L. ‘Minerva Maple’ were DNA content of section Amboinenses ranged from 5.28 pg/2C for used as an internal reference standard (IRS) for each sample P. gigantea to 14.50 pg/2C for P. amboinensis W25-08. Among the (Johnston et al., 1999). Seeds of P. sativum were kindly provided eight species in section Zebrinae, the variation is 2.92-fold, ranging by the Royal Botanic Gardens, Kew, United Kingdom.