The micromorphology of the leaves, pollen, and fruits of different taxa of Camptotheca was studied under light microscope and scanning electron microscope. The key diagnostic characters can be used for the identification of taxa within Camptotheca include leaf shape, venation type, vein number, stoma size and frequency, shape of outer stomatal rim, subsidiary cell number, and glandular trichome size; cotyledon shape, venation type, and vein number; and fruit surface texture, disc, length, and color. Other characters could not dependably be used for species level discrimination

Li, Shiyou

Taylor, Josephine

Shiyou Li

Josephine Taylor

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Pharmaceutical Crops

Micromorphological Variations of Camptotheca Decaisne

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Stephen F. Austin State UniversitySFA ScholarWorksNCPC Publications and Patents National Center for Pharmaceutical Crops2014Micromorphological Variations of CamptothecaDecaisneShiyou LiStephen F Austin State University, Arthur Temple College of Forestry and Agriculture, lis@sfasu.eduJosephine TaylorStephen F Austin State University, Department of Biology, jtaylor@sfasu.eduFollow this and additional works at: http://scholarworks.sfasu.edu/ncpc_articlesTell us how this article helped you.This Article is brought to you for free and open access by the National Center for Pharmaceutical Crops at SFA ScholarWorks. It has been accepted forinclusion in NCPC Publications and Patents by an authorized administrator of SFA ScholarWorks. For more information, please contactcdsscholarworks@sfasu.edu.Recommended CitationLi, Shiyou and Taylor, Josephine, "Micromorphological Variations of Camptotheca Decaisne" (2014). NCPC Publications and Patents.Paper 36.http://scholarworks.sfasu.edu/ncpc_articles/36Send Orders for Reprints to reprints@benthamscience.ae 116 Pharmaceutical Crops, 2014, 5, (Suppl 2: M4) 116-119    2210-2906/14 2014 Bentham Open Open Access Micromorphological Variations of Camptotheca Decaisne Shiyou Li1,* and Josephine Taylor2 1National Center for Pharmaceutical Crops, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University, Nacogdoches, TX 75962, USA; 2Department of Biology, College of Sciences and Mathematics, Stephen F. Austin State University, Nacogdoches, TX 75962, USA Abstract: The micromorphology of the leaves, pollen, and fruits of different taxa of Camptotheca was studied under light microscope and scanning electron microscope. The key diagnostic characters can be used for the identification of taxa within Camptotheca include leaf shape, venation type, vein number, stoma size and frequency, shape of outer stomatal rim, subsidiary cell number, and glandular trichome size; cotyledon shape, venation type, and vein number; and fruit sur-face texture, disc, length, and color. Other characters could not dependably be used for species level discrimination. Keywords: Camptotheca, glandular trichome length, lower leaf surface, pollen, stomatal length, upper leaf surface. INTRODUCTION Camptotheca was formerly a monotypic genus, and, prior to this investigation, anatomic and micromorphological data were limited to C. acuminata [1-3]. In the present study, we describe the micromorphological variations of leaf, pollen, and fruit surfaces of three different species. Some characters (e.g., size, frequency, outer rim, and subsidiary cell number of stomata, and glandular trichome shape and size) can be used to distinguish the species within Camptototheca. For other characters, however, there were no significant varia-tions observed.  MATERIALS AND METHODS The micromorphology of the lower leaf surface was stud-ied for several populations of each of three different species by stripping fresh leaf epidermis in the morning and observ-ing the samples under a light microscope. Three trees were selected and 20 cells were examined from one leaf of each tree. The sample size (n) of C. acuminata was 600 cells, C. lowreyana was 120 cells, and C. yunnanensis was 60 cells. Stomatal density and size (length and width), subsidiary cell number, and glandular trichome length were recorded for each leaf. Fresh leaf tissue was also prepared for scanning electron microscopy (SEM). Pollen samples of C. acuminata and C. lowreyana were collected by Dige Chen in Huaiji, Guangdong, China, in 1996. The pollen sample of C. yunnanensis was collected from a dried specimen. At least 30 pollen grains of each spe-cies were examined and photographed with SEM.  Three fruits from each of the species were used for SEM examination. Three 2  2 mm samples were selected, respec-tively, from the top, center, and bottom of each fruit.  *Address correspondence to this author at the National Center for Pharma-ceutical Crops, Arthur Temple College of Forestry and Agriculture, Stephen F. Austin State University, Nacogdoches, TX 75962, USA; Tel: 936-468-2071; Fax: 936-468-7058; E-mail: lis@sfasu.edu All fresh samples were prepared for SEM by overnight fixation at 4 °C in a 1:1 mixture of 5% glutaraldehyde and 0.1M potassium phosphate buffer, pH 6.8. The samples were rinsed three times with potassium phosphate buffer 0.05 M at 10 to 15 minute intervals and kept refrigerated. A 1:1 mix-ture of OsO4 (2%) and Potassium phosphate buffer 0.1 M was added and the samples were refrigerated for two hours. Following osmication, specimens were rinsed twice with distilled water at 10 to 15 minutes each time, then dehy-drated in a graded ethanol series at 10 to 15 minutes each step (10%, 40%, 60%, 80%, 95%, 100%, and 100% ETOH). Specimens were then critical point dried using carbon diox-ide as the transitional fluid, mounted on SEM stubs, sputter coated with gold palladium, and examined using a Hitachi S405-A scanning electron microscope operating at 15 KeV. Air-dried pollen grains were dusted onto adhesive-coated SEM stubs, then sputter coated as examined as described.  Variation among populations was compared using ANOVA and Tukey's HSD Test for each of the quantitative measures. Means, standard deviations, and ranges of all characters were calculated and are presented for all samples. Statistical significance levels employed are 0.05, 0.01, and 0.001. Frequency distributions of all characters in each sam-ple are also presented to reveal overlap that are often ob-scured with means-based methods of comparison.  RESULTS AND DISCUSSION Our phenotypic analysis showed that key diagnostic characters for the identification of taxa within Camptotheca include leaf shape, venation type, vein number, stoma size and frequency, shape of outer stomatal rim, subsidiary cell number, and glandular trichome size; cotyledon shape, vena-tion type, and vein number; and fruit surface texture, disc, length, and color. Other characters could not dependably be used for species level discrimination. The informative char-acters for which microscopic analysis was needed are de-scribed below, including stomatal density and size (length and width), subsidiary cell number, and glandular trichome Micromorphological Variations of Camptotheca Decaisne Pharmaceutical Crops, 2014, Volume 5    117 length. Like Nyssa [2], Camptotheca has simple unbranched non-glandular hairs, external unicellular glands, and stomata on the lower surface of the leaf (hypostomatous).  Upper Leaf Surfaces The adaxial surfaces of mature leaves of various Camp-totheca species are glassy with a thick cuticle and occasional non-glandular trichomes occasionally (Fig. 1). There is no diagnostic difference among the species.    Fig. (1). Upper leaf suface of Camptotheca acuminata ( 500).  Lower Leaf Surfaces Stomata provide an essential connection between the in-ternal air spaces of plants and the external atmosphere [4]. Most authors agree that stomatal size is usually sufficiently stable to be used as a diagnostic character [2]. Related spe-cies often have stomata of similar size [2]. Species with smaller stomata usually have a greater number than those species with larger stomata [2, 4]. Smaller stomata (<15 mm long) are often at higher density, while larger stoma (>38 mm) are observed to have lower density [2, 4]. This is not the case in Camptotheca, however.  The lower leaf suface has stomata and both glandular and non-glandular trichomes (Fig. 2). Stomatal size in Camp-totheca ranges from 20 to 35 μm in length and 10 to 25 μm in width, considered medium-sized in the vascular plants. C. acuminata has smaller stomata (average 22.85 15.30 mm) than both C. lowreyana and C. yunnanensis (29.33  20.08 mm and 31.05  20.23 mm, respectively).    Fig. (2). Lower leaf suface of Camptotheca lowreyana ‘Katie’  ( 300). The species with larger stomata (C. lowreyana and C. yunnanensis) have greater stomatal frequency (average 263.20 and 221.22 stomata per mm of leaf area) than the species with smaller stomata (C. acuminata: 180.39) (Fig. 3). In addition, we found that the thickness and form of the outer stomatal rim are of diagnostic value at the species level. Similar to Nyssa sylvatica Marsh [2], C. lowreyana has the outer rings of striae of stomata and not the concentric rings of striae, but differs from the other species of Camptotheca. Subsidiary cell number per stoma is higher in C. yun-nanensis (average 5.83 cells) than the other two species (C. acuminata: 4.86, C. lowreyana: 5.23) (Fig. 3).   Fig. (3). Leaf stomatal density, stomatal length, and subsidiary cell number of C. acuminata (n = 600), C. lowreyana (n = 120), and  C. yunnanensis (n = 60). 118    Pharmaceutical Crops, 2014, Volume 5 Li and Taylor  Fig. (4). Leaf glandular trichome length of C. acuminata (n = 600), C. lowreyana (n = 120), and C. yunnanensis (n = 60).   Fig. (5). Scanning electron micrographs of stomata and glandular trichomes on lower leaf surface (stomata  1,000; glandular trichomes  1,400): C. acuminata (a and b), C. lowreyana (c and d), and C. yunnanensis (e and f).   Fig (6). Scanning electron micrographs of pollens of Camptotheca species (upper:  750; Lower:  3,750) C. acuminata (a and b), C. lowrey-ana (c and d), and C. yunnanensis (e and f).  The size and shape of glandular trichomes on the lower leaf surface vary in Camptotheca and are diagnostic charac-ters for species identification. C. lowreyana has pandurate glandular trichomes that are larger (average 46.49 μm in length) than those in the other two species (C. acuminata: 34.87 μm and C. yunnanensis: 35.20 μm in length and both are obelliptic in shape) (Figs. 4 and 5).  Leaf micromorphology provides important quantitative and qualitative characters for species identification in Camp-totheca. Stomatal size and frequency, shape of outer stomatal rim, subsidiary cell number, and glandular trichome shape and size are of diagnostic value.  Pollen and Fruit Surfaces Pollen grains of Camptotheca are 3-colporate, suboblate, obtuse-triangular in polar view, 29-38 m (polar axis)  33-54 m (equatorial axis), and sexine punctitegillate (Fig. 6). Colpi are not very distinct; colpi margins have nexinous 0 5 10 15 20 25 30 Frequency (%) 26 28 30 3C. acuminata2 34 36 38Gl 40 42 44 4and Length C. yuananensi 6 48 50 52(um)  sC. lowr54 56 58eyana Micromorphological Variations of Camptotheca Decaisne Pharmaceutical Crops, 2014, Volume 5    119 thickenings [5]. SEM revealed no significant differences in pollen surface texture among the three species. In terms of pollen size, grains of C. acuminata are 34.4 (28.6-40.2) m  39.8 (34.5-42.5) m. Pollen of C. lowreyana are very simi-lar in size to those of C. acuminata, measuring 34.4 (28.6-40.2) m  39.8 (34.5-42.5) m in size. The only difference is that the pollen of C. lowreyana is more oblate. Pollen grains of C. yunnanensis are very similar to the other two species, but of smaller size, measuring 31.2 X 35.5um.  The fruit surface of C. acuminata is rugose, while both C. lowreyana and C. yunnanensis have smooth and lustrous fruit surfaces.  CONFLICT OF INTEREST The authors confirm that this article content has no con-flict of interest.   ACKNOWLEDGEMENTS Declared none. PATIENT’S CONSENT Declared none. REFERENCES [1] Li, S. Y.; Adair, K. T. Camptotheca acuminata Decaisne, Xi Shu, A Promising Anti-tumor and Anti-viral Tree for the 21st Century. Henry M. Rockwell Monograph, Stephen F. Austin State Univer-sity: Nacogdoches, 1994. [2] Metcalfe, C. R.; Chalk, L. Anatomy of the Dicotyledons. 2nd ed.; Clarendon Press: Oxford, 1979. [3] Liu, W. Z. Secretory structures and their relationship to accumula-tion of camptothecin in Camptotheca acuminata (Nyssaceae). Acta Bot. Sin., 2004, 46(10), 1242-1248. [4] Kramer, P. J.; Boyer, J. S. Water Relations of Plants and Soils. Academic Press: San Diego, 1995. [5] Erdtman, G. Pollen Morphology and Plant Taxonomy, Angio-sperms. 2nd ed.; Hafner Publishing Company: New York, London, 1966.   Received: December 30, 2013 Revised: September 02, 2014 Accepted: September 24, 2014 © Li and Taylor; Licensee Bentham Open.  This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/-licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. 

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