Journal of Medicinal Plants Research Vol. 6(20), pp. 3681-3688, 30 May, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.345 ISSN 1996-0875©2012 Academic Journals
Full Length Research Paper
A preliminary study of leaf morphology and flow cytometry in the Vatica diospyroides Symington, endangered medicinal plant of Peninsular Thailand
Theera Srisawat1*, Sawitree Thipnetr1 and Charun Maknoi2
1Faculty of Science and Industrial Technology, Prince of Songkla University, Suratthani Campus, Muang, Suratthani 84000, Thailand. 2Queen Sirikit Botanic Garden, Maerim, Chiang Mai 50180, Thailand.
Accepted 17 April, 2012
In modern drug discovery program, Vatica diospyroides Symington consisted of chemical constituents that were found as anticancer in human. V. diospyroides was described as endangered species, and its tree is puzzled by means of leaf forms. Investigating the unique features was very important to support the program. To classify these features, leaf traits and C-DNA values were investigated. Leaf characters of twenty-eight trees of V. diospyroides, including herbarium specimens, were recorded using leaf morphometric study and subsequent analysis by cluster. The results revealed that lamina length, leaflet length and leaflet width were found to be significantly different characters. After being analyzed by cluster, the specimens were evaluated into: 1) small size (SS) and 2) large size (LS) forms using three correlated variables namely lamina length, leaflet length/width and petiole length. The 2C-DNA contents were estimated by using flow cytometric study in each form of seedlings. The results showed that 2C- DNA contents of SS and LS seedlings were 1.83 ± 0.27 and 2.21 ± 0.31 pg; consequently, one haploid genomes (1C-value) were therefore 895 and 1080 Mbp, respectively. It is thus accepted that leaf morphometric and flow cytometric studies were able to distinct V. diospyroides into two specific individuals. It is important to carry out classifications using DNA markers and phytochemical properties in further experiment.
Key words: 2C-DNA content, Chan Ka Pho, flow cytometry, genome size, leaf morphology, Vatica diospyroides.
INTRODUCTION
Vatica diospyroides Symington known in Thai as Chan heart health of Thai people. Moreover, since V. Ka Pho, is a species belonging to the family diospyroides is propagated by seed, there are a few Dipterocarpaceae and is distributed in Malaysia, seedlings in the field on the basis of limited number of Thailand; it is endemic in peninsular (Smitinand, 1966) pollinated flowers. V. diospyroides is therefore highly and Vietnam. In Thailand, there are two main usages of endangered and is considered to be at risk of complete V. diospyroides; firstly, flowers of V. diospyroides have loss (Artorn Kumlungbai, personal communication). strong scent (Pooma, 2002), they have been used as Consequently, the taxonomic category is described in a ingredient for various perfumeries. Secondly, V. critically endangered, A1cd C2a version 2.3; species of diospyroides is one of medicinal plants that is being used the International Union for Conservation of Nature (IUCN) to treatment as blood tonics and assists in maintaining Red list (Ashton, 1998). In modern drug discovery program, stem of V. diospyroides in Peninsular Thailand consists of two main chemical constituents namely vaticaphenol A and *Corresponding author. E-mail: [email protected]. Tel: vatdiospyroidol. The vatdiospyroidol exhibits biological +66-0-7735-5040 ext. 8874 Fax: +66-0-7735-5453. activity including anticancer in oral epidermoid 3682 J. Med. Plants Res.
carcinoma (KB), colon cancer (Col2) and breast cancer Unfortunately, FCM and taxonomic study has never been (BC1) cell lines (Seo et al., 1999; Kinghorn, 2000). utilized for identifying plant variation. From this study, our However, the V. diospyroides trees show variable forms findings are the first report on FCM analysis of the genus in morphological traits especially in size, shape and color Vatica compared successfully to morphometric analysis. of leaves, flowers and fruits. The drug discovery research The variability problem in V. diospyroides is compre- on this species might be still unable to gain proper hensive, thus this study aims at clarifying the state of knowledge because of the separable chemical existing of one or more types of tree, particularly the constituents extracting among individuals, thus might question: how many type forms of V. diospyroides exist in have differences in curative properties. Unfortunately, the the region of Peninsular Thailand? Here, an attempt is specific individual form of V. diospyroides tree having the made to recognize tree types differing morphologically valuable chemical constituents has not been described to and molecularly within the species through estimation of date. Investigating the separated forms within the species leaf morphology and FCM analysis. The present study is therefore important to determine the tree-type was carried out to: (a) compare the morphological representation. Moreover, the specific form is influential variation in the species by using leaf morphometric study; to identifying unique curative properties that would help in and (b) assess the 2C-DNA content and genome size of developing of drug discovery program (Kinghorn et al., the species by using FCM. 2011). Two major approaches to the relative analysis together of plant variability for genus or species delimitation are MATERIALS AND METHODS morphological and molecular studies (Chiapella et al., 2011). The morphological comparison which is known as Study site and sample collection numerical taxonomy has been widely performed in V. diospyroides Symington leaves were randomly sampled from several doubtful plant genera such as genus Senna adult trees (about 5 m high) in the Nong Thung Thong non-hunting (Soladoye et al., 2010) and Cassia (Petchsri and area, Kiansa, Suratthani province, where the land are completely Bookerd, 2003; Saheed and Illoh, 2011). These confirm flooded with water for three to four months of the year (Pooma, that the most confusing genus differing in morphological 2002) and constituted with tropical-evergreen plant community and complex grassland. At least twenty mature leaves (about 3 m high) traits are able to delimit meaningfully by estimating the per tree were picked and estimated for morphology. potential organs such as leaf and flower. On the other Fresh leaf specimens in the area and herbarium specimens at hand, using morphological comparison lacks obvious limit the Forest Herbarium (BKF), Bangkok, were assessed by between asserted forms and stretched geographic range, measuring five leaf quantitative characters which are listed often bring about incomplete identification keys subsequently. Young intact leaf tissues of individual form of V. (Chiapella et al., 2011). The molecular investigation is diospyroides seedlings were prepared for 2C-DNA content analysis. Seeds of maize (Zea mays cv. CE-777), external reference plant, therefore used to carry out the limitation of the were kindly provided by Dr. Jaroslav Dolezel (Laboratory of conventional technique. There are many potential Molecular Cytogenetics and Cytometry, Institute of Experimental molecular markers to investigate relative plant genera or Botany, Sokolovska 6, Olomouc, CZ-77200 Czech Republic). species by applying together with morphological study such as Amplified Fragment Length Polymorphisms (AFLP), Restriction Fragment Length Polymorphisms Leaf morphometric study
(RFLP), Simple Sequence Repeats (SSR) and Random Leaf morphometric study was carried out on mature living Amplified Polymorphic DNA (RAPD) techniques (Young, specimens in the area and on herbarium specimens at the BKF 2000). Although the RAPD technique has been usually resulting in twenty-eight individual trees. Table 1 shows listed performed to preliminary study genetic polymorphism in samples, locations, voucher numbers and collector numbers of V. various plant species (Qiao-Ming et al., 2002; diospyroides specimens in the herbarium. Five quantitative morphological characters of leaves were lamina length, leaflet Kaewmuangmoon et al., 2010; Sheidai, et al., 2010), length, leaflet width, leaflet length/width and petiole length. difficulties have been encountered when using this Measurements were carried out using a metric ruler and tape procedure on a large scale (Rival et al., 1997) which measure; only correlated characters were subsequently analyzed cannot be replaced by the others. Flow cytometry (FCM) by cluster analysis. usually offer a simple, rapid and accurate analysis; thus is convenient analysis for determining cytogenetic Flow cytometric study variation. The effectiveness of FCM with plant has been mostly addressed in areas of 2C-DNA content, ploidy The flow cytometer was a FACSCalibur (Becton Dickinson level and genome size parameters (Suda et al., 2010). It is Biosciences, San Jose, CA) (Scientific Equipment Center, Prince of generally assumed that 2C-DNA and genome size Songkla University, Hat Yai campus) working with CellQuest parameters have been used as a marker for identifying software (Becton Dickinson) equipped with a 488 nm argon iron the variation of plant genera. For example, 166 samples laser. Propidium iodide (PI) was measured at 585 nm to read 2C nuclei histograms of 5,000 nuclei per sample. Young leaves of of Fallopia (Suda et al., 2010) and 3 cultivars of Elaeis maize were used as external reference plants (2C = 5.43 pg) (Srisawat et al., 2012) have been distinguished (Lysak and Dolezel, 1998). Approximately, 50 mg of V. successfully by using 2C-DNA and genome size values. diospyroides young leaves were finely chopped using a razor blade Srisawat et al. 3683
Table 1. Sample location, collection date, voucher number and collector number of representative specimens of Vatica diospyroides examined at the BKF.
Sample Location Collection date Voucher number Collector 1 Kiansa, Suratthani 5th March, 1976 BKF 62041, SN 018543 T. Smitinand 12143 2 nd 5th April, 1998 BKF 98178, SN 018544 Mitre 1 3 nd 9th March, 1957 BKF 15823, SN 018542 T. Smitinand 3823 4 nd nd BKF 109551, SN 100241 Bandit 5 Nong Thung Thong non-hunting area, Kiansa, Suratthani February, 1990 BKF 109551, SN 100240 Bandit 6 Klong Naka wildlife sanctuary, Suksamran, Ranong 28th June, 2004 BKF 154356, SN 163275 S. Gardner and P. Sidisunthorn ST 0883 7 nd nd BKF 109551, SN - Bandit
nd: no data recorded.
in 1.0 ml LB01 extraction buffer containing 15 mM Tris, 2 length/ width (P < 0.01). Table 4 also reveals that character analysis divided these three characters mM Na2 EDTA, 0.5 mM spermine. 4HCl, 80 mM KCl, 20 the statistical differences between V. diospyroides including lamina length, leaflet length/width and mM NaCl, 0.1% (v/v) Triton X-100 and pH 8.0 (Dolezel et trees were significant (P < 0.01). The Squared petiole length of V. diospyroides into two major al., 1989). After extraction, 50 µl of RNase and PI were added immediately prior to filtering through a 42 µm nylon Euclidean Distance value indicates the differences groups; 1) the tree number 4, 9, 26 and 27 were mesh (Pfosser et al., 1995). All samples were carried out of leaf morphological traits. For example, the more closely related which was defined as SS with 3 replicates per form. During analysis, after every cluster that exists between tree number 1 and 26, (small size) form having narrow leaf, and 2) the three replicates, the reference plant was used as a 1 and 10 were found to be 23.424 and 10.734, other tree samples (tree number 1 to 3, 5 to 8 and controller to check the calibration of the flow cytometer. respectively, which were distantly related leaf 10 to 25) were more closely related which was
morphological features and represented a highly defined as LS (large size) form having wide leaf RESULTS AND DISCUSSION different degree of variation. In contrast, the (Figure 1). closely related of leaf morphological features were In the sampling of V. diospyroides trees in the Leaf morphometric study found, for example, in the tree number 7 and 24, study site, we found that the most dominant form 11 and 18 (coefficients = 0.018 and 0.218, was wide-leaf tree followed by narrow-leaf tree In the present study, twenty-eight samples of V. respectively) showing great degree of similarity in which resembled the result of cluster analysis, diospyroides Symington including living and their features. However, some highly distant resulting in twenty-four LS samples and four SS herbarium specimens were compared morphologi- relationships represented by a high coefficient samples. With the previous report/herbarium cally. Leaf morphological comparison of the values were found to be remarkable similarities collections, some of V. diospyroides specimens quantitative features of studied V. diospyroides among leaf morphological traits, for example, of were collected from the Klong Naka wildlife leaves are shown in Table 2 and Figure 1. The tree number 1 and 2 (coefficient = 9.004). In sanctuary (Pooma, BKF, Table 1), Suksamran average means of lamina length, leaflet length, Figure 2, the degree of relationship between V. district, Ranong province, and some were leaflet width, leaflet length/width and petiole length diospyroides trees was represented by the collected from Takuapa district, Phang Nga ranged between 15.0 to 31.9, 16.4 to 33.4, 4.3 to dendrogram. It is clear that tree number 1 is province (Srinual and Thammathaworn, 2008). 8.7, 3.1 to 5.4, and 1.1 to 2.2 cm, respectively. distantly related to tree number 2, though they Interestingly, the alignments of the Suksamran The correlation coefficients of the three high appear to have some similarities with leaf and Takuapa borders are connectedly related by correlated quantitative parameters of V. morphology. It is interesting to note that three out along coast of the Andaman Sea while distance to diospyroides leaf are shown in Table 3. It was of the five leaf-quantitative characters have been the Nong Thung Thong non-hunting area is found that there is high positive significant shown by cluster analysis to have about 99% level related by rivers. Artorn Kumlungbai (Director of correlation between petiole length and leaflet of significance in delimiting the taxon. The Nong Thung Thong non-hunting area, personal 3684 J. Med. Plants Res.
Table 2. Mean and standard deviation of the quantitative characters of Vatica diospyroides leaves (cm).
Tree no. Lam. length Lflt. length Lflt. width Lflt. length/width Pet. length 1 25.07 ± 2.87 27.04 ± 3.06 6.20 ± 0.57 4.37 ± 0.30 2.05 ± 0.21 2 25.07 ± 1.74 27.02 ± 1.77 8.41 ± 0.75 3.23 ± 0.18 2.01 ± 0.10 3 24.10 ± 2.31 25.49 ± 2.20 6.10 ± 0.34 4.18 ± 0.18 1.42 ± 0.24 4 26.34 ± 0.87 27.41 ± 0.89 5.09 ± 0.36 5.42 ± 0.35 1.09 ± 0.05 5 24.64 ± 1.52 26.36 ± 1.59 6.72 ± 0.38 3.94 ± 0.29 1.74 ± 0.19 6 28.38 ± 4.39 30.39 ± 4.77 8.30 ± 1.25 3.67 ± 0.10 2.10 ± 0.38 7 23.24 ± 1.29 25.01 ± 1.43 6.50 ± 0.56 3.87 ± 0.27 2.06 ± 0.20 8 24.79 ± 3.44 26.72 ± 3.45 8.01 ± 0.78 3.34 ± 0.20 1.93 ± 0.10 9 26.03 ± 2.25 27.32 ± 2.49 5.62 ± 0.41 4.86 ± 0.11 1.39 ± 0.32 10 31.94 ± 3.23 33.42 ± 3.38 7.63 ± 0.72 4.39 ± 0.30 1.66 ± 0.18 11 25.65 ± 4.40 27.34 ± 4.66 7.18 ± 1.53 3.87 ± 0.38 1.61 ± 0.14 12 28.26 ± 4.93 30.05 ± 5.20 8.24 ± 2.35 3.73 ± 0.39 1.84 ± 0.41 13 30.10 ± 1.56 31.63 ± 1.80 8.43 ± 0.39 3.76 ± 0.05 1.50 ± 0.21 14 30.48 ± 1.67 31.68 ± 1.88 6.85 ± 0.52 4.66 ± 0.43 1.20 ± 0.23 15 27.10 ± 3.00 28.30 ± 3.22 7.47 ± 0.74 3.81 ± 0.27 1.37 ± 0.15 16 24.45 ± 2.40 25.93 ± 2.23 7.28 ± 0.81 3.58 ± 0.09 1.53 ± 0.25 17 27.32 ± 2.78 28.52 ± 3.31 6.52 ± 1.00 4.41 ± 0.20 1.72 ± 0.17 18 25.63 ± 1.10 27.00 ± 1.13 7.05 ± 0.28 3.83 ± 0.01 1.48 ± 0.25 19 23.56 ± 2.46 24.95 ± 2.53 5.71 ± 0.52 4.38 ± 0.20 1.28 ± 0.06 20 30.39 ± 1.71 32.45 ± 1.94 8.74 ± 0.81 3.73 ± 0.22 2.18 ± 0.40 21 25.79 ± 2.96 27.32 ± 2.75 6.68 ± 1.04 4.16 ± 0.36 1.50 ± 0.14 22 21.39 ± 1.39 23.52 ± 1.40 5.77 ± 0.66 4.11 ± 0.31 2.10 ± 0.26 23 21.95 ± 1.33 23.80 ± 1.88 6.48 ± 0.62 3.70 ± 0.35 1.78 ± 0.80 24 23.22 ± 1.51 25.16 ± 1.75 6.60 ± 0.49 3.83 ± 0.20 2.05 ± 0.33 25 23.01 ± 1.30 24.59 ± 1.39 5.98 ± 0.65 4.16 ± 0.35 1.58 ± 0.09 26 14.96 ± 2.16 16.42 ± 2.47 4.29 ± 0.94 3.88 ± 0.37 1.46 ± 0.31 27 17.76 ± 0.97 19.37 ± 1.05 6.38 ± 0.64 3.06 ± 0.26 1.67 ± 0.04 28 25.01 ± 1.77 26.56 ± 1.82 6.70 ± 0.78 3.84 ± 0.37 1.57 ± 0.20
Lam. length = Lamina length, Lflt. length = Leaflet length, Lflt. width = Leaflet width, Lflt. length/width = Leaflet length/width and Pet. Length = Petiole length.
SS and LS
Figure 1. Morphological difference among leaf blade narrowly (SS) and widely (LS) form of Vatica diospyroides leaves. Srisawat et al. 3685
Table 3. Correlation matrix of SS and LS forms of Vatica diospyroides using three correlated quantitative characters.
Correlation Lflt. length/width Pet. length Lam. length 0.28 -0.03 Lflt. length/width -0.52**
Lam. length = Lamina length, Lflt. length/width = Leaflet length/width and Pet. Length = Petiole length. **Significant difference at 99% (P < 0.01).
Table 4. Analysis of variance (ANOVA) of five characters.
Cluster Error Correlation df df F Sig. Mean square Mean square Lam. length 185.08 1 6.63 26 27.92 0.000* Lflt. length 178.41 1 6.70 26 26.64 0.000* Lflt. width 9.36 1 0.84 26 11.10 0.003* Lflt. length/width 0.09 1 0.25 26 0.38 0.545 Pet. Length 0.02 1 0.09 26 0.20 0.660
Lam. length = Lamina length, Lflt. Length = Leaflet length, Lflt. Width = Leaflet width, Lflt. length/width = Leaflet length/width and Pet. length = Petiole length.*Sig. = Significant (P < 0.01).
Figure 2. Cluster analysis showing the relationship of twenty-eight Vatica diospyroides specimens based on five characters (dendrogram using average linkage). 3686 J. Med. Plants Res.
communication) explained that the most wild populations flower parts of V. diospyroides should be estimated in of V. diospyroides in peninsular Thailand were generally further experiments. found in the Nong Thung Thong non-hunting area with a hundred number of population size. Although V. diospyroides trees were distributed in more locations than Flow cytometric study that of the study area, some were distributed by cultivation (Pooma and Newman, 2001). It is therefore The C-DNA values of V. diospyroides were taken from possibly to confirm that the samples of V. diospyroides young leaves of seedlings by using Z. may cv. CE-777 estimated in this study are reliable specimens (2C = 5.43 pg) as external reference plant. The DNA representing the most plant that are distributed in the contents (2C-values) were estimated to be 2.21 and 1.83 peninsular Thailand and might be the best genetic source pg for LS and SS form, respectively. Their genome sizes of the species. (1-C value) were therefore 1080 and 895 Mbp (Figure 3; V. diospyroides trees reveal difference in leaf size of an Table 5). These DNA content and genome size were individual specific form. This variation within the species firstly reported on Vatica and can be used as database in may be due to sunlight and genetic factor. An effect of plant systematic taxonomy and may be helpful in further environment on leaf shape and size variations on some drug discovery programs. plants have been reported by Wilson et al. (1998) and Our previous work reported that the C-DNA value was Campey et al. (2000), who studied the light intensity one of the informative parameters which proved to be of effected on shape, length and width of leaves. With the diagnostic cultivar classification of genus Elaeis (Srisawat same environment and age of plant effects, we et al., 2012). Knowledge of V. diospyroides 2C-DNA and suggested that the minimum and maximum distant values genome size values would thus certainly help with of V. diospyroides leaf characters are about two-folds clarifying systematic relationship between closely or different which might be due to the genetic factor. Several distantly related species, and improving species authors have emphasized the usefulness of leaf identification. Moreover, this could help in designing more morphological study, a part of numerical taxonomy, in specific marker in any further drug discovery programs, characterizing plant classification (Petchsri and Bookerd, especially this species have curative properties for 2003; Soladoye et al., 2010; Saheed and Illoh, 2011). cancer treatments (Seo et al., 1999). In this study, the Thus, we may disagree at present with the key to species grouping based on DNA content values (1.83 and 2.21 identification of V. diospyroides stated by Pooma and pg) was in agreement with the group based on the leaf Newman (2001) and Srinual and Thammathaworn morphological traits (SS and LS form). Therefore, not (2008). Although, both authors have used conventional only that the leaf morphometric study might be and Electron Microscopic techniques to clarify the key to recommended as useful method for species delimitation species in the genus Vatica, they provided insufficient but the FCM useful also might be the taxonomic utility information on variation within the species. when using both methods in combination. We Our results confirmed that V. diospyroides trees could recommend an application of these methods in a be divided into LS and SS patterns. Interestingly, not only complicated taxonomic review of various plant species in the lamina length, leaflet length/width and petiole length the future. However, the specific variety of V. of both V. diospyroides forms are comparatively diospyroides should be verified more extensively by using distinguished but their petal colors are also different, types of chemical constituent and genetic polymorphism revealing in yellowish and pinkish-white petal. It is particularly. Moreover, a study of their chromosome indicated that varied morphological traits of V. numbers should be recognized for estimating the diospyroides should be investigated more extensively. relationship among genome size from FCM and number Unfortunately, Thailand had encountered an effect of El of haploid chromosome, since the chromosome number Nino which resulted in severe and long drought and its relation to another parameters of plants in the phenomena between November 2009 and August 2010 Dipterocarpaceae have been reported by several authors in all parts of the country (www.hiso.or.th). On the other (Brandis, 1895; Dayanandan et al., 1999; Gamage et al., hand, the extreme climate change might have influenced 2006), resulting in mystifying genetic relationship of the the manner of living of V. diospyroides thus terminate puzzling genus in this family. their flowering. As mentioned earlier, we did not receive In conclusion, V. diospyroides trees differed by means the sufficient specimens of flower in blossoming period of leaf morphological characters, 2C-DNA contents and (February to April, 2011) and studied only leaf characters genome sizes. We believed that the systematic arranging then. In particular, morphometric study had provided of V. diospyroides by using DNA content and genome valuable taxonomic information at the species levels for size allowed taxonomist to distinguish more taxa than the different groups of plants (Stuessy and Crawford, 1990). conventional taxonomic based on morphology. However, This is therefore the first recommendation of leaf information for delimiting within the taxon is still morphological study that may be used as marker for completely lacking until the phytochemical and DNA revision of the taxon. To confirm the result of this study, markers will be performed. Srisawat et al. 3687
a
b
Figure 3. Histogram of relative fluorescence intensities (PI fluorescence) of Vatica diospyroides nuclei. (a) SS form; (b) LS form.
Table 5. Estimation of 2C-DNA content and genome size (C-DNA values) in two Vatica diospyroides forms using maize as external reference plant.
C-DNA values (Mean ± SD) V. diospyroides forms 2C (pg) Genome size (Mbp) Z. mays (external reference) 5.43 2655 LS form 2.21 ± 0.31 1080 SS form 1.83 ± 0.27 895
Values are given as mean and standard deviation of the mean genome in mass values (2C, pg) and base pair (1C, Mbp), 1 pg = 978 Mbp (Dolezel et al., 2003).
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