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_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 CytologicalStudieson 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 1of 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. Dyad and triad
sporads constituted 8.4% and 0.8% of sample respectively. As there were about 10% of PMCs
showing complete desynapsis, apparently some of the desynaptic PMCs did not end up as dyad
sporads.

Microspore mitosis
The number of chromosomes in each microspore could be counted at prophase and meta
phase (Figs. 18, 19). It was observed that microspores within the same tetrad might not contain
the same chromosome numbers, apparently due to random distribution of univalents and lag
gards at anaphase I. Mitosis in dyad sporad (Fig. 20) was usually normal except for their
doubled chromosome numbers. At anaphase, the chromosomes in each microspore formed
an inner and outer groups (Fig. 21). The outer or peripheral groups of chromosomes later
differentiated into deeply stained generative nuclei, while the vegetative nuclei were only lightly
stained and less condensed (Figs. 22, 23).

Discussion

In the present study, Arundinagraminifolia was found to have 40 chromosomes in somatic

cells. These consist of 2 pairs of telocentric chromosomes and 18 pairs of metacentric or sub
metacentric chromosomes. However, Sharma and Chatterji (1966) reported that all 20 pairs
of chromosomes in their material were either metacentric or submetacentric.
Considerable meiotic irregularites were observed in PMCs from the present material.
Most of them had 2 or more unpaired univalents at metaphase I, thus accounting for the ap
pearance of microcytes in some tetrad sporads. Surprisingly, about 10% of PMCs showed
complete or near-complete failure of homologous pairing due to unknown causes. Apparent
ly, this group of PMCs gave rise to dyad sporads and later unreduced gametes through the
formation of restitution nuclei in the first meiotic division. This explained the comparable
percentages of such PMCs (10%) and that of dyad sporads (8.4%).
It is interesting to note that despite of considerable meiotc disturbances in the materi
al, fertility appeared to be very good. The plant produced many seed pods with open-pollina
tion. Controlled self-pollination was always successful in inducing pod formation. Apparently,
the plant could produce enough viable male and female gametes for successful fertilization.
However, it is not known whether all its selfed progeny are euploid with 40 chromosomes.
The occurrence of dyad sporad was also reported in other diploid orchids such as Calanthe
and Spathoglottis species (Teoh 1984). Den Njis and Peloquin (1977) found that unreduced
gametes were commonly formed in diploid species of Solanum. It is therefore probable that
many diploid plant species can produce a small percentages of unreduced gametes under natural
1987 CytologicalStudieson Arundinagraminifolia
(Orchidaceae) 273

conditions. Such mechanism might have played a role in the evolution of polyploid forms in
many sexually reproducing diploid populations. However, such polyploids might not be suc
cessful in their competition with diploid parents and were eventually eliminated from the
populations.

Summary

A clone of Arundina graminifolia from Malaysia was found to be diploid with 2n=40
chromosomes. This is in agreement with 3 earlier reports on materials collected elsewhere.
The present study revealed a considerable degree of meiotic irregularities in this clone. These
were in the forms of asynapsis, desynapsis, chromosome laggards at anaphase I, and formation
of restitution nuclei at both meiotic division I and II. The cause and significance of such ir
regularities in this species remain to be studied.

References
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Lenz,L. W.and Wimber,D. E. 1959. Hybridizationand inheritancein orchids. In: The Orchids,a Scientific
Survey. C. L. Withnered., pp. 261-314.
Mehra,P. N. and Vij,S. P. 1970. IOPBchromosomenumberreportsXXV. Taxon19: 102-113.
Pancho,J. B. 1965. IOPBchromosomenumberreportsIII. Taxon14(3): 86-87.
Sharma,A. K. and Chatterji,A. K. 1966. Cytologicalstudieson orchidswithrespectto their evolutionand
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Sheehan,T. and Sheehan,M. 1983. Orchidgenera,illustrated-Arundiana.Amer.OrchidSoc.Bull.52 (3):
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Tanaka,R. 1965. Chromosomenumbersof somespeciesof Orchidaceae fromJapanand its neighbouring area.
J. Jap. Bot.40 (3): 65-77.
Teoh,S. B. 1984. Polyploidspore formationin diploidorchidspecies. Genetica63: 53-59.