Taiwania 64(3): 269-279, 2019 DOI: 10.6165/tai.2019.64.269 Development of pollinium and associated changes in anther of Calanthe tricarinata Lindl., an epidendroid orchid Ravi KANT Department of Botany, Sri Guru Teg Bahadur Khalsa College, Sri Anandpur Sahib- 140118, Punjab, India. *Corresponding author’s E mail: [email protected]; Phone number: +917986616044; Fax number: +9101887232037 (Manuscript received 30 June 2018; accepted 23 June 2019; online published 12 July 2019) ABSTRACT: In Calanthe tricarinata Lindl., anther primordium develops two thecae each with a mass of densely cytoplasmic archesporial cells surrounded by the protoderm. A six-layered anther wall develops from the archesporial cells of hypodermal layer. Cells in the tapetum are binucleate, only reported in a few primitive orchids. Formation of two septa in each theca results into eight microsporangia which is a primitive character. Pollen mother cells undergo simultaneous cytokinesis and form tetrahedral, rhomboidal, decussate, T-shaped, square and linear type of microspore tetrads. Pollen grains unite to form eight pollinia, which is a primitive feature in the family Orchidaceae. Pollinia unite to the viscidium through long caudicles to form a structure called the pollinarium unit. Pollen grains are monoaperturate with smooth exine. Present observations contribute significantly in the taxonomy and understanding character evolution in the family Orchidaceae. KEY WORDS: Anther wall, Binucleate tapetum, Calanthe tricarinata, Caudicle, Orchid, Pollinium, Septum. INTRODUCTION periclinal and anticlinal divisions and form primary parietal layer (Sood, 1986, 1989; Bhanwra and Vij, Orchids belong to the largest and highly evolved 2003). In contrast to this, in some other taxa, all the cells family of flowering plants called Orchidaceae. The present in an anther locule are differentiated into family Orchidaceae have been divided into five archesporial cells. In these taxa, the archesporial cells of subfamilies, i.e., Apostasioideae, Cypripedioideae, hypodermal layer form the anther wall whereas the Vanilloideae, Orchidoideae and Epidendroideae (Chase archesporial cells present inner to the hypodermal layer et al., 2003). Orchids constitute a peculiar group of form the sporogenous cells by repeated mitotic divisions plants among angiosperms for their floral diversity, (Bhanwra et al., 2006a; Kant and Hossain, 2010; Kant et presence of variously modified column and labellum, al., 2013). In most of the orchids, anther wall is four- different types of pollen dispersal units, numerous, layered (Sood, 1986). However, in some other orchids, smallest and non endospermic seeds, and requirement of anther wall is five-eight layered (Sood and Rao, 1988). a symbiotic association with species specific fungal Innermost middle layer develops fibrous thickenings partner for seed germination in nature (Hossain et al., similar to those of endothecium (Bhanwra et al., 2006a, 2013). In the family Orchidaceae, anther hosts several b; Kant et al., 2013). Tapetum is mostly uninucleate, important features which are frequently used in the however, binucleate tapetum has also been reported in a traditional system of classification, cladistic analysis and few primitive taxa (Swamy, 1949; Sood, 1992; Bhanwra to understand the character evolution at the various et al., 2006b). Number of pollinia varies from two, two- taxonomic levels. These features include variation in the bipartite, four, six and eight in the family (Dressler, 1993; number of anther wall layers (Arekal and Karanth, 1981; Stenzel, 2000). In the family Orchidaceae, the structure Sood and Rao, 1988; Bhanwra et al., 2006a; Kant and of exine is highly variable which is employed as an Hossain, 2010; Kant et al., 2013), pollen grains important taxonomic tool (Williams and Broome, 1976; aggregation into tetrads, massulae, soft or hard pollinia Stenzel, 2000; Kocyan and Endress, 2001; Barone (Dressler, 1993; Singer et al., 2008), types of massulae Lumaga et al., 2006; Singer et al., 2008). In case of (Dressler, 1993), variation in the number of pollinia primitive genera, exine surface of pollen grains exhibit (Freudenstein and Rasmussen, 1999), pollen wall more variations than the advanced one. These variations sculpturing etc. (Stenzel, 2000). are even prominent at the species level and have Literature reveals that orchids show variations taxonomic significance (Kapil and Arora, 1990). regarding the origin and number of anther wall layers. In Calanthe tricarinata Lindl., also called ‘Monkey majority of the taxa, only two-three archesporial cells are Orchid’ belongs to the subtribe Bletiinae, tribe differentiated in each anther locule. These cells are Arethuseae, subfamily Epidendroideae and family divided periclinally into primary parietal cell towards Orchidaceae (Dressler, 1993). In this taxon, plants are outer side and primary sporogenous cell towards inner 30- 40 cm long and develop in groups (Fig. 1A). Flowers side. The primary parietal cells undergo repeated are very beautiful and 2.2- 2.5 cm across (Fig. 1B). 269 Taiwania Vol. 64, No. 3 Flowers resemble a monkey and have a single fertile pollen grains were observed and photographed under a anther. This taxon is distributed in the temperate parts of JEOL JSM-600 Scanning Electron Microscope using Western Himalaya including India, Nepal and China at black and white photographic film on ‘Olympus’ the elevations between 2000-3000 m (Deva and Naithani, photomicroscope model BH-2. 1986). C. tricarinata is used in the traditional system of medicine to cure jaundice, sores, eczema and as an aphrodisiac (Joshi et al., 2009; Sharma and Samant, 2017). Despite the significance of anther characters in the traditional system of classification, cladistic analysis and understanding the character evolution, information regarding early anther development, origin and differentiation of anther wall layers, microsporogenesis, microgametogenesis, development of pollinium and surface features of pollen grains is lacking in this important taxon. Occurrence of eight pollinia has been reported in this taxon. However, mode of their formaion is not known. Therefore, C. tricarinata Lindl. was selected for the present study to investigate these features in detail. MATERIALS AND METHODS Periodic field trips were made in July- August to the forests of Kufri, at mean sea level of 2720 m (31.10°N’ 77.25°E’) and Narkanda, at mean sea level of 2710 m (31°16’ 77°27E’). Kufri and Narkanda are situated at a distance of 23 km and 60 km from Shimla, the capital city of Himachal Pradesh, India respectively. Flower buds and open flowers at different stages of development were collected and fixed in a solution of formalin-acetic acid-ethanol in 1:1:18 ratio (5 ml formalin: 5 ml acetic acid: 90 ml 50% ethanol) for 24–48 hours and subsequently transferred to 70% ethanol for the long term storage and further use. These floral buds and open flowers were dehydrated in ethanol-tertiary-butyl alcohol series followed by their infiltration with paraffin wax at 60°C. Subsequently blocks were made in the paraffin wax and serial sections were cut with a Spencer 820 Rotary Microtome (American Optical Company, U.S.A.) at a thickness of 5-7m. Serial sections were stained with safranin-fast-green series. Permanent slides were prepared through mounting the sections with DPX (a mixture of distyrene, a plastilizer and xylene; HiMedia Mumbai, India) (Johansen, 1940; Berlyn and Miksche, 1976). Slides were observed and micrographs were taken Fig.1. Plants growing in natural habitat and morphology of flower in Calanthe tricarinata. A. Plants growing in natural habitat; B. on ‘Olympus’ photomicroscope model BH-2. For A flower showing sepals (1, 2, 3) and petals (4, 5, 6) where la= Scanning Electron Microscope (SEM) observations, labellum, an= anther cap, co= column. anthers with anther wall removed and pollinaria units were fixed in 2% glutaraldehyde, 2.5% p-formaldehyde RESULTS in 0.05 M phosphate buffer (pH 7). This material was then fixed in 1% osmium tetroxide and dehydrated in a Early anther development and differentiation of anther graded series of acetone and finally subjected to critical wall point drying in a CPD 7501 critical point dryer. After Transverse sections of flower buds and open flowers drying, gold coating was done on the aluminium stub were observed at different stages of development. It was using double adhesive tape with JFC 1100 sputter observed that anther primordium was initiated as a (Postek et al., 1980). After gold coating, anthers and squarish mass of meristematic cells surrounded by the 270 Sep 2019 Kant, R. : Development of pollinium Calanthe tricarinata Lindl. protoderm (Fig. 2A). In the next stage of development, Microsporogenesis, microgametogenesis and associated anther primordium was developed into two lateral thecae changes in the anther wall oriented towards the labellum (Fig. 2B). Each theca The pollen mother cells were hexagonal or polygonal developed a mass of densely cytoplasmic archesporial in shape. The outermost pollen mother cells, i.e. cells cells of uniform size as compared to the larger close to the tapetum were arranged in a plane at right vacuolated cells of connective region. These angle to the tapetal cells. As meiotic divisions were archesporial cells were increased in number by the started in the pollen mother cells, the cells in the tapetum repeated mitotic divisions. Out of these cells, the were divided mitotically. But, nuclear divisions in the archesporial cells of hypodermal