Embryology of Spathoglottis Plicata Blume: a Reinvestigation and Additional Data
Total Page：16
File Type：pdf, Size：1020Kb
Tropical Natural History 15(2): 97–115, October 2015 2015 by Chulalongkorn University Embryology of Spathoglottis plicata Blume: A Reinvestigation and Additional Data NARASAK SRIYOT1, ACHRA THAMMATHAWORN2 AND PIYADA THEERAKULPISUT1* 1 Applied Taxonomic Research Center, and Salt-tolerant Rice Research Group, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, THAILAND 2 Office of General Education, Khon Kaen University, Khon Kaen 40002, THAILAND * Corresponding author. Piyada Theerakulpisut ([email protected]) Received: 16 May 2015; Accepted: 6 September 2015 ABSTRACT.– Spathoglottis plicata Blume is very popular among terrestrial orchids because it is attractive, easy to grow and can flower all year round. The aim of this study was to reinvestigate the embryological characters using classical paraffin embedding and sectioning, along with ovule clearing and scanning electron microscopy analyses. The simultaneous cytokinesis results in decussate, isobilateral, linear, T-shaped and tetrahedral microspore tetrads. Mature pollen grains are two-celled with perforate sculpturing. The anther wall is comprised of an epidermis, two to three layers of endothecium, one layer each of middle layer and one layer of binucleate glandular tapetal cells. At anthesis, the mature anther wall is composed of epidermis and fibrous endothecium. At 9 d after pollination (DAP), the mature ovules are anatropous, bitegmic and tenuinucellate. The megaspore tetrad is linear. Development of the embryo sac conformed to the Polygonum type. The pollen tube enters the embryo sac through the micropyle (porogamy), and double fertilization is observed at 15 DAP. The zygote divides transversely to form a two-celled proembryo. The basal cell becomes the suspensor initial cell, whereas the terminal cell divides transversely to form a linear three-celled proembryo. The proembryonal tetrad is T-shaped. Embryogenesis is of the Onagrad type. The suspensor is single-celled, hypertrophied and cap-, globular- or sac- like. Fruit opens at 30 DAP. Seeds are fusiform in shape with longitudinally oriented testa cells with a smooth periclinal seed surface and straight anticlinal walls. The mature globular embryo is acotyledonous and lacks differentiation. Furthermore, cleavage polyembryony gives rise to twin embryos. KEY WORDS: Spathoglottis plicata, ground orchid, embryology, Orchidaceae INTRODUCTION & Wood, 1992). In Thailand, five species have been recorded (Seidenfaden, 1986). The genus Spathoglottis Blume is a Orchidaceae is of interest for embryo- member of the subfamily Epidendroideae. logical studies because of the diversity in All members of this genus are terrestrial and the development and organization of the most grow at low to moderate altitudes, female gametophyte, characteristics of although a few taxa occur at high altitudes suspensor and different patterns of in grasslands and open forests in moist areas polyembryony (Sood, 1985). (Dressler, 1993; Beltrame, 2006). This The ground orchid (Spathoglottis genus is composed of 10 species distributed plicata) is one of the most commonly from Sri Lanka, India and the Andaman cultivated orchids in Southeast Asia and Islands through Indochina to southern could be found in gardens throughout the China, Malaysia and Indonesia east to the region. It is suitable as a potted orchid Philippines, New Guinea, Australia and the because it is very attractive, fast growing, Pacific islands as far as Samoa (Seidenfaden easy to grow and with rapid fruit 98 TROPICAL NATURAL HISTORY 15(2), OCTOBER 2015 maturation. At maturity, plants flower MATERIALS AND METHODS almost throughout the year (Kheawwongjun & Thammasiri, 2008). These attributes have Plant Materials also led to it being used as a model for The samples of S. plicata (Ground orchid studies. This taxon has already been orchid) were grown in the greenhouse at the studied in many aspects, including artificial Department of Biology, Faculty of Science, propagation (Teng et al., 1997; Minea et al., Khon Kaen University. The ground orchid 2004; Sinha et al., 2009; Thakur & (Fig. 1A) is a perennial plant with ovoid Dongarwar, 2012; Aewsakul et al., 2013; pseudobulbs covered with fibrous leaf Sebastinraj & Muhirkuzhali, 2014), bases. There are 4–8 linear-lanceolate leaves cytology (Ramesh & Ranganathan, 2008), per pseudobulb, each up to 35 cm long and physiology (Wang et al., 2002, 2003; Novak 2–6 cm wide, yellow-green to dark-green & Whitehouse, 2013), pollination biology and with prominent veins abaxially. The (Soguillon & Rosario, 2007; Thakur & inflorescences are lateral racemes, up to 25– Dongarwar, 2012) and molecular biology 130 cm long, bearing many successive and (Ranghavan & Goh, 1994; Yang & Loh, loosely spaced flowers. Flowering can occur 2004). from August to March, the peak period The embryology of S. plicata has been being November to February. examined by a few authors, where the work of Swamy (1943a, 1949a,b), Prakash & Lee- Microsporogenesis, Microgametogenesis Lee (1973), Veyret (1974) and Ansari and Anther Wall Anatomy (1977) have detailed aspects of the The floral buds and blooming flowers sporogenesis, gametogenesis and embryo- (Fig. 1B) were harvested at different stages genesis, but the information is rather of development, during the flowering period fragmentary. In addition, these researches (November 2013–March 2014). The length were documented only by line drawings and of each floral buds was measured from the have not been reinvestigated so far. The aim tip to the base of the perianth, and they were of this study was to reinvestigate the divided into different stages based on their microsporogenesis and microgametogenesis length and fixed in 70% (v/v) FAA (1:1:18 of S. plicata in relation to the floral (v/v/v) ratio of formalin: acetic acid: 70% developmental stages using the classical (v/v) ethanol). Dehydration, infiltration and paraffin embedded serial section analysis, as embedding were performed in the well as the post-pollination development of conventional way. Sections were cut at 5 megasporogenesis, megagametogenesis and µm thick with a steel knife on a sliding embryogenesis using ovule clearing and microtome. They were stained with safranin scanning electron microscopy (SEM) and fast-green combination, then mounted analyses. The data presented here will in DePeX mountant (Thammathaworn, provide valuable background knowledge for 1995) and observed by light microscopy. both conservation and molecular biology The anther squash was also prepared and studies of this species. stained with 1% (w/v) aceto-orcein to clarify the mitotic division of microspore nuclei. SRIYOT ET AL. — EMBRYOLOGY OF SPATHOGLOTTIS PLICATA 99 FIGURE 1. Flower morphology of S. plicata. A. The front view of a blooming flower. B. Flowers at different stages of development were prepared for anther transverse sections with paraffin method. Scale bar = 1 cm. To study the pollinium morphology and gently removed by a toothpick. The pollinia the pollen grain surface features, the pollen were displaced from the column by applying grains were fixed in 70% (v/v) FAA and a slight upward pressure to the bottom of the then transferred to 70% (v/v) alcohol. To anther cap and the pollinia adhered to the preserve the exine, no acetolysis was toothpick on contact. After removal, the performed. Dehydration was performed in a pollinia were transferred to the same flower series of increasing % (v/v) acetone. by placing them onto the stigmatic surface Specimens were then critical point dried (Kauth et al., 2008). The efficiency of using CO2, placed on specimen stubs with pollination was checked by flower double sided silver tape and sputter coated senescence and fruit development. The with gold. The samples were examined resulting fruits at different stages of under SEM (Philips, XL-20) at the Institute development were chosen at intervals of 3 d. of Botany, Faculty of Biology, University of Approximately 30 developing fruits were Innsbruck, Austria. Pollen terminology chosen and fixed in 50% (v/v) FAA. The followed that defined by Punt et al. (2007). ovules were carefully removed to a slide with a few drops of Herr’s 4 ½ clearing Megasporogenesis, Megagametogenesis solution (Herr, 1971), no dehydration was and Embryogenesis necessary, and a cover glass was then added. One of the unique characters of ovule Herr’s clearing solution made the samples development in Orchidaceae is that ovules transparent in a couple of minutes. normally do not appear at the time of Photographs were captured under Nomarski anthesis. The development of ovules is illumination using an Olympus BX51 activated by pollination and normally takes microscope coupled to an Olympus DP11 several weeks to complete (Swamy, 1949a, digital camera. 1949b). In order to ensure a good fruit set, In order to study the morphological the stigma receptivity of the flower at characters of the seed coat, seeds at the fruit anthesis was tested using hydrogen dehiscence stage were prepared for SEM peroxide, where the appearance of bubbles analysis as described above. Measurements could be observed on receptive stigma were performed on the width and length of (Dafni & Maués, 1998). Flowers at similar the embryos and seeds using a minimum of stage were then artificially self-pollinated. 15 seeds. To this end the anther cap and pollinia were 100 TROPICAL NATURAL HISTORY 15(2), OCTOBER 2015 FIGURE 2. Anther anatomy, microsporogenesis and microgametogenesis. A. Transverse section of a young floral bud showing two rows