.dot. Mag.

Tokyo 84: 118-122 _(March 25, 1971)

Types of Resting Nuclei in Orchidaceae

by Ryuso TANAKA*

Received November 18, 1970

Abstract

Morphological characteristics of the resting nuclei and chromosomes observed

in 115 species of 52 genera of Orchidaceae were summarized, and the following
five types of nuclei were found in Orchidaceae. Diffuse type: Characterized by
the dark staining chromatins without conspicuous aggregation , and associated
with the large chromosomes without any particular heterochromatic segment.
Simple chromocenter type : Characterized by the light staining chromatins and
some dark staining chromocenters with rough surface, and has no connection
with chromosome size; some of the chromosomes of the complement have a
gradual heterochromatic segment at the proximal region. Complex chromocenter
type: Characterized by many dark staining chromocenters which vary in shape
and aggregate into several large chromatin blocks, and associated with either
medium or small chromosomes ; most of the chromosomes of the complement
have several gradual heterochromatic segments at proximal as well as interstitial
and distal regions. Rod prochromosome type: Characterized by the smooth-faced
rod prochromosomes, and associated with small chromosomes; most of the chromo-
somes of the complement have a clearly distinguishable heterochromatic rod seg-
ment at the proximal region. Round prochromosome type : Characterized by the
smooth-faced round prochromosomes, and associated with small chromosomes;
most of the chromosomes of the complement have a clearly distinguishable hetero-
chromatic round segment at the proximal region.

Interphase nuclei** in the cells of meristematic tissues show a large variation

in the morphological characteristics of chromatinsl'. In a previous paper2' the inter-
phase nuclei of Spiranthes sinensis, Orchidaceae, were found to be classified into
micro-condensed nuclei (G1 phase), extended nuclei (early S phase), partly condensed
nuclei (middle S phase, major condensed nuclei (late S phase), and macro-condensed
nuclei (G2 phase).
Resting nuclei** in the cells of permanent tissues also vary between plant
species1'3'4', as reported by Heitz5' and surveyed by Stebbinss' and Tschermak-
Woess7'. The present author has reported in a preliminary papers' that the resting
nuclei of the species of Orchidaceae showed variation in the morphological charac-
teristics of chromatins and that they can be classified into three categories : diffuse
nucleus, chromocenter nucleus and prochromosomes nucleus. In the present paper
a summary of other additional observations on the resting nuclei and the morphology
of chromosomes in the species of Orchidaceae will be dealt with.

* Botanical institute , Faculty of Science, Hiroshima University, Hiroshima, Japan.

** In the present paper the term ' interphase nucleus' is adopted only for the nucleus of
meristematic cells, while the nucleus in the cells of permanent tissues is referred to as

resting nucleus'.
March, 1971 Types of Resting Nuclei in Orchidaceae 119

Materials and Methods

Observations on the resting nuclei and chromosomes have been made in 115
species of 52 genera in Orchidaceae. The names of the species studied have been
reported in previous papers8'9'. For the observations of the resting nuclei the root
tip epidermal cells were used, since a preliminary test in several species showed
that the chromatins displayed their morphological characteristics most distinctly
and invariably in these cells. Mitotic chromosomes were observed in the root tip
meristematic cells.
Root tips were fixed in 45°o acetic acid at 1O-15 ° ° for about 10 minutes and
macerated in a mixture of two parts of 1 N HC1+one part of 45% acetic acid at
60° for 30 seconds. They were, then, stained with 1% aceto-orcein and squashed
on temporary preparates. For the observation of chromosomes at mitotic metaphase
root tips were pretreated with 0.002 mol. 8-hydroxyquinoline solution at 15° for 3
hours.

Results

Resting nuclei showed a large variation in the morphological characteristics of

chromatins between the species studied. In some species the chromatins were
stained darkly and homogeneously, while in others they were stained faintly and
formed some aggregations. The chromatins were fibrous in some species, while
they were chromomeric in others. It was, however, found that the resting nuclei
of each species showed respective morphological characteristics in the condensation
of chromatins, in size, shape and number of chromocenters or prochromosomes, and
in the distribution of chromomeres and chromonemata.
The resting nuclei of the species studied were found to be categorized into five
types: diffuse type, simple chromocenter type, complex chromocenter type, rod
prochromosome type, and round prochromosome type (Fig. 1). Three of these five
types, i. e., diffuse, complex chromocenter and round prochromosome types, were
found to be comparable with the categories proposed by Heitz5', Stebbins6' and
Tschermak-Woess7'. In Orchidaceae, however, the three types were found among
species under one family, while those of Heitz5', Stebbins6' and Tschermak-Woess7'
were found among the species belonging to different families of Angiosperms.
Each of the 115 species was found to show one of the five types, while occasion-
ally the species with the nuclei intermediate between some of these types were
observed. The characteristics of the respective types are as follows.
Diffuse type (Fig. 1 A) : This is stained darkly and has no chromatin bodies
remarkably aggregated. Chromatins show almost homogeneous distribution in the
whole regions of the nucleus forming a fibrous network of the chromonemata.
Nucleoli are large and are stained very slightly with aceto-orcein. This type is
composed of large chromosomes (Fig. 1 A-m). The chromosomes at mitotic prophase
are stained darkly and homogeneously showing several elastic small constrictions.
There are no segments which are conspicuously heterochromatic or euchromatic
(Fig. 1 A-p). This type was observed in Pogonia minor (2n=18), P. japonica (2n=
20), Cephalanthera falcata (2n=34), Epipactis thunbergii (2n=40), Listera makinoana
(2n=38), and Ephippianthes schmidtii (2n=36).
Simple chromocenter type (Fig. 1 B) : This is stained lightly and possesses
some darkly stained heteropycnotic bodies termed chromocenters. The chromo-
120 TANAKA, R. Vol. 84

centers show irregularly protruded rough surface which gradually transforms into
a diffused chromatin. The number of chromocenters is lower than the number of
chromosomes of the complement. The chromocenters form heterochromatic segments
which are located in the proximal regions of some chromosomes of the complement.
The heterochromatic segments transform gradually into partially aggregated distal
segments showing a gradual transition from heterochromatin to euchromatin (Fig.
1 B-p). The chromosomes at mitotic metaphase vary in size differing widely among
species. Most of the species with this type of nucleus were found to have medium
chromosomes (Fig. 1 B-m), while some had large chromosomes (Cypripedium and
Paphiopedilum) or small chromosomes (Dendrobium, Bulbophyllum, Cattleya and
Taeniophyllum). Nucleoli are relatively large and are stained slightly with aceto-
orcein. This type was observed in Cypripedium japonicum (2n=20), Paphiopedilum
insigne (2n=26), P. callosum (2n=32), Bletilla striata (2n=32), Blet. formosana (2n=
36), Malaxis paludosa (2n=28), Oberonia japonica (2n=30), Coelogyne cristata (2n=40),

Fig. 1. Types of nucleus and the morphology of the chromosomes in Orchidaceae. r,

resting nuclei, x 11.00. p and m, the longest chromosomes at mitotic prophase (p) and
metaphase (m) in respective nuclei, x3300. A, diffuse type in Listera makinoana (2n=38).
B, simple chromocenter type in B!eti.l!a striata (2n=32). C, complex chromocenter type in
Cymbidium kanran (2n=40). D, rod prochromosome type in Spiranthes sinensis (2n=30). E,
round prochromosome type in Goodyera maximowicziana (2n=28).
March, 1971 Types of Resting Nuclei in Orchidaceae 121

Pholidota chinensis (2n=40), Thunia alba (2n=42), Dendrobium moniliforme (2n=38),.

D. nobile (2n=38), D. phalaenopsis (2n=38), D. tosaense (2n=38), Cattleya labiata (2n=
40), Bulbophyllum inconspicuum (2n=38), B. japonicunz (2n=40), Tainia laxiflora (2n=
36+1-4 B), and Vanda and its allied genera including Taeniophyllum aphyllun2 (2n=38).
Complex chromocenter type (Fig. 1 C) : It has many darkly stained chromo-
centers occasionally show aggregations forming several large heteropycnotic blocks
which vary in size and number. In addition to the chromocenters there occur many
chromomeres localized in several regions of the nucleus. The chromomeres usually
show an irregular distribution, while sometimes they show a fibrous arrangement.
The chromocenters form the heterochromatic segments situated in the proximal
region as well as in the interstitial and distal regions of most chromosomes of the
complement (Fig. 1 C-p), thus showing a higher number than the chromosome
number. The chromosomes at mitotic metaphase are of either medium or small
size. The nucleoli are relatively large and are stained slightly with aceto-orcein.
This type was in many genera of Orchidaceae, e. g., Tropidia, Liparis, Pleione, Eria,
Oreorchis, Cymbidium, Ansellia, Grammatophyllum, Stanhopea, Calanthe, Phaius,
Cremastra, Bletia, Eulophia, Spathoglottis, Acanthephi~pium, Sobralia, Oncidiunz and
its allied general°,il', Masdevallia, Zygopetallum and its allied genera, and Maxillaria
and its allied genera. The resting nuclei of Microtis parviflora (2n=44), Orchis
graminifolia (2n=42), Amitostigma keiskei (2n=42), Platanthera hologlottis (2n=42),
Habenaria sagittifera (2n=28), Flab. radiata (2n=32) Eleorchis japonica (2n=40), and
Arundina sinensis (2n=40) are observed to be intermediate between the simple
chromocenter type and the complex chromocenter type.
Rod prochromosome type (Fig. 1 D) : It possesses many rod-shaped hetero-
pycnotic bodies which vary in size and shape. The number of heteropycnotic
bodies corresponds to the number of chromosomes with heterochromatic segments.
Thus, the heteropycnotic bodies are termed prochromosomes. The surface of the
prochromosomes are observed to be smooth and clear transforming abruptly into
diffused chromatins. The diffused chromatins are fibrous and are stained very
lightly. Each of the prochromosomes forms a heterochromatic segment located in
the proximal region of the chromosome (Fig. 1 D-p). The heterochromatic segments
transform abruptly into euchromatic segments located in the distal regions. There
occurs only one proximal heterochromatic segment per chromosome. The chromo-
somes at mitotic metaphase are of relatively small size. Nucleoli are of medium
size and are stained slightly with aceto-orcein. This type was observed in Spiranthes
sinensis (2n=30) and Pristiglottis tashiroi (2n=26).
Round prochromosome type (Fig. 1 E) : It possesses the round-shaped prochromo-
somes of almost equal size. Very clear distinctions can be seen between the pro-
chromosomes and the diffused chromatins. The diffused chromatins are either
fibrous or chromomerec. Each of the prochromosomes forms, respectively, the
proximal heterochromatic round segment which transforms abruptly into the distal
euchromatic segment (Fig. 1 E-p). Chromosomes at mitotic metaphase are the
smallest among those of the five types. Nucleoli are small and are stained faintly
with aceto-orcein. This type was observed in Goodyera maximowicziana (2n=28, 56),
C. repens (2n=32), G. macrantha (2n=30) G. procera (2n=42), and Anoectochilus
letsuoi (2n=40).
122 TANAKA, R. Vol. 84

Discussion

According to the observation of Heitz5' in Hordeum vulgare, Gramineae, another

type of nucleus known by the name of cup nucleus has been reported7'. In the
cup nucleus the heterochromatic materials are concentrated in the polar regions of
the mitotic spindle showing one-sided localization of chromocenters. In Orchidaceae
species having the cup nucleus was not found.
It can be noted that the taxonomic groups which show large variation in ex-
ternal morphology and rapid speciation, e. g., Orchis and its allied genera, Dendrobium
and its allied genera, Vanda and its allied genera, and Oncidium and its allied
genera, show either the simple chromocenter type or the complex chromocenter
type, while most of the taxonomic groups, which show small variation and seem
to be stable and slowly speciating, have connection with either one of the other
three types described in the present paper.

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