_??_1987 by Cytologia, TokyoCytologia 52: 267 -273 , 1987 Cytological Studies on Arundina graminifolia (Orchidaceae) Y. H. Lee Botany Department, National University of Singapore, Singapore Accepted March 17, 1986 Arundiana graminifolia (Don.) Hochr. is a terrestrial orchid whose native habitat extends from Sri Lanka and India through South East Asia to Tahiti (excluding the Philippines) . As there are considerable morphological variations among different populations , some taxonomists classified the genus into as many as 8 species (Sheehan and Sheehan 1983) . However, Holttum (1964) considered it as one highly variable species and presented good supporting arguments. His viewpoint is generally accepted at present. There were very few reports on cytological studies on this species. Pancho (1965) reported a somatic chromosome number of 32 for his material, while Tanaka (1965) as well as Sharma and Chatterji (1966) reported 40 chromosomes. The latter also noted some meiotic irregul arities as early separation of homologous chromosomes and laggards at anaphase I. More recently, Mehra and Vij (1970) observed a haploid set of 20 chromosomes in gametes of this species. Further observations on meiotic chromosome behaviour are described in this paper. Materials and methods Arundina graminifolia is commonly grown in Singapore and Malaysia. Each plant often consists of many stems growing close together. One such plant with a height of about 1 meter growing in the garden of the Botany Department of the National University of Singapore was used for the present study. Young flower buds at desirable stages were collected in the morning. These pollinia were dissected out and fixed in 45% acetic acid for 10 minutes at room temper ature. The tissue was then squashed and stained in 1% aceto-orcein for about 10 minutes. For chromosome counts, actively growing root tips were collected in the morning, pretreated in saturated solution of 8-hydroxyquinonine for 3 hours at 18°C, and then fixed in 1:1:2 mixture of 95% ethanol, chloroform, glacial acetic acid for 24 hours at room temperature. The root tips were then hydrolysed in 1N HCl at 60•Ž for 5 minutes and stained in 1% aceto-orcein for 1/2 -1 hour with intermittent warming over an alcohol lamp. Observations Mitosis The chromosome number in somatic cells was found to be 40 (Fig. 1). The chromosomes are rather small at mitotic metaphase, mostly range from 2 to 3ƒÊm. There are 2 pairs of exceptionally small chromosomes less than 2ƒÊm in length, and appeared to be telocentric. They tended to show some degree of somatic association, so that the members of each homo logous pair often laid a short distance from each other at metaphase or sometimes loosely associated. Meiosis The size of the flower buds in relation to the meiotic stages were given below as a general guide: 268 Y. H. Lee Cytologia 52 Less than 7mm Early prophase I 7-8mm Meiotic division I 8-8.5mm Meiotic division II 8.5-10mm Sporad stage 10-11mm Pollen mitosis 11-12mm Binucleate microspore Meiotic asynchrony was common in PMCs of a pollinium. Two or more stages of meiotic division were often encountered in PMCs from the same pollinium. For example, a pollinium taken from a flower bud of 11 mm in length was found to contain PMCs at binu cleate stage, and also some PMCs at various stages of meiotic division II. First meiotic division: Prophase I Pachynema chromosomes (Fig. 2) consist of long strands of 2 different thickness. The thicker strands represented paired homologues after successful synapsis, while the thinner strands were single unpaired chromosomes due to asynapsis. A number of deeply stained bodies along the chromosomes were the chromomeres. Diplonema (Fig. 3) was the first stage in which bivalents and univalents could be counted. A very pronounced repulsion between the paired homologues was apparent at this stage. In PMCs where pairing was regular, 20 bivalents colud be observed. By late diplonema, the chromosomes had undergone further contraction and condensation. At diakinesis, most chromosomes remained as bivalents after terminalisation of chiasmata, while a few might dissociate to become univalents, although these homologous pairs often laid close to each other (Fig. 4). In general, very few remained as ring bivalents indicating low frequency of chiasma formation. In extreme cases, almost all bivalents within a PMC dis sociated as a result of desynapsis. There were also PMCs where desynapsis did not occur, so that 20 bivalents were observed. Metaphase I-anaphase I The configuration of first meiotic metaphase can be grouped into 3 main patterns (Figs. 5-7) as follows: a) Normal alignment of bivalents: In 15% of PMCs sampled, all bivalents aligned normally along the equatorial plate (Fig. 5). No univalents were observed. These PMCs would give rise to haploid microspores if later meiotic stages were normal. b) Normal alignment of bivalents plus univalents: This class accounted for nearly 75 of PMCs sampled. The number of univalents ranged from 2 to 12, lying outside the equatorial plate (Fig. 6). As the univalents were not properly oriented, they would be distributed random ly to either poles. As a result, a high proportion of resulting microspores might therefore contain different aneuploid chromosome numbers. c) Desynapsis. About 10% of PMCs showed complete or near-complete desynapsis with univalents scattered all over the cytoplasm, without any clear polar orientation (Fig. 7). Figs. 1-10. Mitosis and fist meiotic division in Arundiana graminifolia. 2400•~, except Fig . 1. 1, mitotic metaphase (2n=40) of a root-tip cell. Arrows indicate 2 pairs of telocentric chromosomes . 5250•~. 2, pachynema. Both single (asynaptic) and double-stranded (synapitc) chromosomes can be recognised. 3, diplonema. Paired members of bivalents repel each other. 4, diakenesis . Bivalents and univalents are present. Pairs of univalents lying close to one antoher indicate de synapsis. Arrows indicate the 2 pairs of 'small' chromosomes. 5, metaphase I. Complete pairing of homologous chromosomes is shown. 6, metaphase I. Partial pairing failure, with 2 pairs of chromosomes remained as univalents. 7, metaphase I. Complete failure of homologous pairing . 8, anaphase I. Sequential separation of homologous chromosomes. 9, late anaphase I. Two loosely associated pairs of chromosomes lagged between 2 chromosome groups. 10, telophase I . Condensation of 2 groups of chromosomes at the end of meiotic division I. 1987 Cytological Studies on Arundina graminifolia (Orchidaceae) 269 270 Y. H. Lee Cytologia52 The failure of separation of the univalents into 2 groups at anaphase I laid the ground for the formation of unreduced gametes. Disjunction of homologous chromosomes at anaphase I appeared to be sequential (Fig. 8). Loosely associated bivalents often separate earlier than the others. Some bivalents remained associated even at anaphase I and appeared as paired laggards (Fig. 9). One or more paired or unpaired laggards were observed in nearly 50% of the PMCs at anaphase I. Anaphase I-telophase I Figs. 11-17. Second meiotic division in PMCs of Arundina graminifolia. 2400•~. 11, interphase. Two daughter nuclei formed without followed by cytokinesis. 12, metaphase II. Two chromo some groups prepared for equational divisions. 13, anaphase II. Regular equational division resulted in 4 haploid groups of chromosomes in the PMC, 2 of which appeared to be moving towards each other. 14, anaphase II. Failure of clear separation of 2 haploid chromosome groups in one of the PMC might lead to the formation of a restitution nucleus. 15, anaphase II. Two groups of unreduced chromosome numbers could lead to the formation of unreduced gametes. 16, telophase II. Two PMCs appeared to form unreduced restitution nuclei while a third formed four normally reduced haploid nuclei. 17, sporad formation. Four haploid nuclei were formed in each PMC prior to cytokinesis. 1987 CytologicalStudies on Arundinagraminifolia (Orchidaceae) 271 The 2 groups of chromosomes in PMCs condensed at telophase I and eventually formed 2 nuclei (Figs. 10, 11). This was not followed by cytokinesis, a common charactersistic of orchids (Lenz and Wimber 1959). Second meiotic division The second meiotic division appeared to be relatively normal in most PMCs. At meta phase II (Fig. 12), the 2 linearly arranged groups of chromosomes lined up parallel or at dif ferent angles to each other. When chromosome disjunction occurred following equational division, 2 of the 4 resulting chromosome groups might move towards one another (Fig. 13). If the 2 groups happened to meet, they might combine to form a single group with twice the Figs. 18-23. Microspore mitosis in Arundina graminifolia. 2400•~. 18, all 4 microspores were synchronised at the mitotic prophase. 19, mitotic metaphase in all 4 microspores of a sporad. 20, mitotic metaphase in 2 microspores of a dyad sporad, each having an unreduced chromosome number. 21, mitotic anaphase in a tetrad sporad. 22, mitotic telophase in a tetrad sporad. 23, binucleate stage in microspores of a tetrad sporad. 272 Y. H. Lee Cytologia52 normal chromosome number, thus giving rise to a triad sporad containing 1 unreduced micro spore plus 2 reduced haploid microspores. On the other hand, failure of segregation in 1 of the 2 chromosome groups at anaphase II (Fig. 14) might also result in a triad sporad. Apparently, the 2 events rarely occurred as the number of triads were relatively small among the sporads formed. A few PMCs were found to show. unreduced chromosome number at anaphase II (Fig. 15). Restitution of pairs of chromosome groups (Fig. 16) also appeared to occur at telophase II, another possible cause of dyad formation. Sporad formation In a sample of 345 sporads, 89.1% were normal tetrads (Fig. 17) and 1.7% were tetrads with microcytes. These were presumably derived from PMCs with patterns (a) and (b) at metaphase I. The relatively small number of tetrads with microcytes indicates that univa lents and laggards were often incorporated during the formation of nuclei.