A cytogenetical study of the fertility of three local orchid hybrids

Chin Chee Wei Winston1, Aung Thame 2, Yam Tim Wing2

ABSTRACT

Renanthera Singapore Botanic Gardens, an intrageneric orchid hybrid, Arachnoglottis, a bigeneric orchid
hybrid and Mokara Singa Gold, a trigeneric orchid hybrid were subjected to a cytogenetical analysis of their
pollen in order to predict their fertility. In flowers pollen mother cells divide via meiosis to form gametes. It
was found that in the Renanthera Singapore Botanic Gardens the chromosome pairing during Metaphase I of
meiosis was very regular while in the Mokara Singa Gold and Arachnoglottis it was not. The sporad analysis
showed that mature Renanthera Singapore Botanic Gardens pollen consisted of a very high percentage of
tetrads (a group of 4 gametes formed from normal meiosis) while the Mokara Singa Gold and Arachnoglottis
pollen had a much lower percentage of tetrads. Based on these results, it was hypothesized that the
Renanthera Singapore Botanic Gardens is a fertile hybrid while the Mokara Singa Gold and the
Arachnoglottis are highly infertile. This hypothesis was confirmed by breeding experiments.

INTRODUCTION

The Orchidaceae family is the largest family of flowering plants in the world, having 20,000- 25,000 species
(Yam, 1995). An unusual aspect of the orchid family is the ability of different orchid genera to cross-breed to
form hybrids. For example, a complicated artificial genus Brilliandeara involves up to 6 genera. This is
possible because hybrids breed with other species or hybrids to form more advanced generations of hybrids
(Arditti, 1992). The Singapore Botanic Gardens orchid breeding programme’s first hybrid, the Spathoglottis
Primson, flowered in 1931 since then the Gardens has produced more than 300 hybrids (Yam, 1995). The
Renanthera Singapore Botanic Gardens, Arachnoglottis and Mokara Singa Gold are relatively new hybrids
so little is known about their cytogenetics. The Renanthera Singapore Botanic Gardens orchid is a
intrageneric cross between two Renanthera orchids. The Arachnoglottis is a bigeneric cross between an
Arachnis hybrid and a Trichoglottis species. The Mokara Singa Gold is a trigeneric cross between the three
genera Arachnis, Ascocentrum and Vanda.

The Renanthera Singapore Botanic Gardens and Arachnoglottis are diploid orchids, which means they
contain two sets of chromosomes. In diploid orchids, pollen mother cells, which contain the diploid number
(2n) of chromosomes, divide by the process of meiosis to form the male gamete pollen cells, which contain
the haploid number (n) of chromosomes. Meiosis consists of two successive divisions, meiosis I and
meiosis II (refer to diagram).

At the end of meiosis I, two daughter cells are formed, each with the haploid number of chromosomes. Each
chromosome is made up of two identical sister chromatids joined together. During meiosis II, the second
division, the two daughter cells both divide and the sister chromatids of the chromosomes are separated, so
that four daughter cells each with the haploid number of chromosomes are formed as the end product of
meiosis. In orchid pollen the daughter cells resulting from Meiosis II are grouped together to form a sporad,
and when there are four it is called a tetrad. Each daughter cell is called a microspore.

It is crucial that at Metaphase I, the homologous chromosomes pair up correctly at the equator so that they
can be separated. This separation process is called disjunction. A pair of homologous chromosomes is called
a bivalent. The extent of the pairing of chromosomes is dependent on the level of homology between parental
sets of chromosomes. In orchids often there is a lack of homology between parental chromosomes and hence
incomplete pairing or in extreme cases no pairing occurs. An unpaired chromosome called a univalent. When
this happens there is an unequal division of chromosomes between daughter cells during meiosis, thus the
microspores produced will be aneuploid in nature (having irregular numbers of chromosomes) and are non-
functional. Hence tetrads may prove to be non-functional despite normal disjunction in both meiosis I and II
due to unequal division of chromosomes (Chan, 1996).
__________________________________________________________________________________________________________
1
SRP Student, Anglo-Chinese Junior College, 25 Dover Close East, Singapore 139748
2
Orchid Research Laboratory, Singapore Botanic Gardens, Cluny Road, Singapore 259569
 Prophase I Metaphase I Anaphase I Telophase I
The chromosomes The homologous The homologous The cell divides into two
condense and become chromosomes pair up at chromosomes are forming two daughter
visible. the equator of the cell. separated and pulled to cells.
opposite poles of the cell

Prophase II Metaphase II Anaphase II Telophase II

Chromosomes prepare to Chromosomes line up at Sister chromatids are 4 daughter cells (tetrads)
migrate to equator the equator of the cells separated and pulled to are formed.
opposite poles of the cell

Due to incomplete pairing of chromosomes, when cytokinesis (the division of the cell) occurs, some
chromosomes do not lie within the vicinity of the microspores. These chromosomes may have been laggards,
univalents that do not migrate to either pole, or univalents that have migrated but failed to incorporate
themselves with the rest of the chromosomes. These disorientated chromosomes may eventually form
microcytes, additional microspores apart from the normal four or micronuclei, additional nuclei. Presence of
microcytes or micronuclei is an indication of infertility.

Also, when meiosis is irregular sporads with numbers of other than 4 microspores may be observed- monads,
dyads, triads and polyads. Polyads are formed either because of numerous laggards or scattered univalents,
or by supernumerary divisions caused by certain genes which usually results in sterility (Tanaka and
Kamemoto, 1984). Polyads are very unlikely to be functional gametes. Monads are formed when non-
disjunction occurs in both meiosis I and II. Monads contain the full set of chromosomes and can become
tetraploid gametes as they contain 4 sets of chromosomes. Dyads can be formed in two ways- either non-
disjunction during meiosis I followed by a normal second meiotic division or a normal first meiotic division
followed by non-disjunction in both daughter cells. Non-disjunction occurs when the presence of numerous
laggards during Anaphase causes restitution of the two segregated groups of chromosomes and the laggards
into a single nucleus. Triads are formed when either non-disjunction occurs in only one of two daughter cells
in meiosis II, or when two groups of chromosomes are united in Anaphase II due to proximity of meiotic
spindles. Both triads and dyads have the potential to be functional gametes if they contain full sets of
chromosomes. (Chan, 1996)

The Mokara Singa Gold is a triploid hybrid, which means it contains the triploid number (3n) of
chromosomes or three sets of chromosomes. This is because the Mokara Singa Gold was a result of a cross
between a tetraploid (4n) orchid and a diploid (2n) orchid. This presents complications that will be discussed
later in the Results and Discussion section.

This investigation aimed to observe the extent of chromosome pairing during Metaphase I of meiosis in the
three hybrids as well as the nature of the sporads of their mature pollen and from this information
hypothesize whether they were fertile. The hypotheses would then be compared with breeding experiments.
This study also aimed to develop a feasible, suitable and effective method to “catch” the pollen cells at the
correct stage (Metaphase I), study the chromosome pairing and hence deduce whether the hybrid under study
was fertile. The advantages of such a method is that it takes comparatively much less time than breeding
experiments, which could take up to months.
MATERIALS AND METHODS

Fresh stalks with a good number of buds were cut from plants in the National Orchid Gardens Nursery and
the Pasir Panjang Nursery and kept in the laboratory in beakers of water. The buds were measured using
vernier calipers from the pedicel to the end of the bud (for the Renanthera Singapore Botanic Gardens it was
from the spur to the end of the bud), dissected under a dissecting microscope and the pollen removed. A
small portion of the pollen was removed for study while the rest was stored in 45% acetic acid at 4°C.

The pollen to be studied was fixed in 45% acetic acid at 4°C for about 10-20 min. A drop of aceto-orcein
stain was added to the pollen and the slide left for 30-45 min inside a glass chamber saturated with 45%
acetic acid. A cover slip was placed on top of the pollen and pressed down hard. A pen was then used to tap
the cover slip repeatedly in order to further spread the pollen cells as well as the chromosomes out. The slide
was heated in a direct alcohol lamp flame for about 5-6 seconds, pressed once again to further spread the
chromosomes and finally washed with a drop of 45% acetic acid.

The slides were observed under the microscope and the stage of division was recorded along with the bud
length. Cells in Metaphase I and Anaphase I in which the chromosomes were clearly separated were
photographed and screened for the number of bivalents and univalents and observed for other features such
as laggards. For the sporad analysis, mature pollen cells taken from the largest buds on the stalks were scored
for the percentages of monads, dyads, triads, tetrads and polyads among 200 sporads. This was done three
times and the averages and standard deviations calculated.

If a bud had already passed Metaphase I, then the next smallest bud on the same stalk was dissected and the
pollen studied. This process was continued until the bud with pollen at the right stage was found. Also, if it
were discovered that buds of a certain length had pollen at too early a stage, all other buds of that length were
marked, left on the stalks and studied the next day.

RESULTS AND DISCUSSION

Mapping of Stages of Development of Pollen

Renanthera Singapore Botanic Gardens

Bud Length (mm) Stage

<4.0 Pollen Mother Cells
4.0-4.5 Meiosis I
4.5-5.5 Meiosis II
>5.5 Sporads

For the Renanthera Singapore Botanic Gardens, Meiosis I was very active from 4.0- 4.5 mm, while meiosis
II was predominant from 4.5- 5.5 mm. An excellent slide showing virtually all the stages of meiosis was
obtained at 4.5 mm. Buds larger than 5.5mm showed all the cells in sporad state.

Mokara Singa Gold

Bud Length (mm) Stage

<4.5 Pollen Mother Cells
4.5-6.0 Meiosis I
6.0-7.0 Meiosis II
>7.0 Sporads
For the Mokara Singa Gold, from 4.5mm onwards traces of Prophase, Metaphase and Anaphase I were seen.
At 6.0mm, an excellent slide showing all the stages of meiosis I was obtained. At 7 mm, all the cells were
clearly in the sporad stage. Above 7mm would hence be the ideal length to carry out sporad analysis.

Arachnoglottis

Bud Length (mm) Stage

<4.5 Pollen Mother Cells
4.5- 6.5 Meiosis
>6.5 Sporads

For the Arachnoglottis, from 4.5 onwards cells in Prophase, Metaphase and Anaphase I could be seen with
their chromosomes very clear. For this hybrid separate ranges for meiosis I and meiosis II were not able to be
determined because most of the sporads formed were dyads and it could not be determined whether meiosis I
or II took place. However at 6.5 mm, most of the cells were clumped together and appeared to have
completed meiosis and just reached the Telophase I stage. At 7.0 mm all the cells were clearly in the sporad
stage.

The wider range for meiosis for the Arachnoglottis and Mokara Singa Gold as compared to the Renanthera
Singapore Botanic Gardens is probably due to the lack of homology between parental chromosomes, hence
the cells do not divide as readily.

In conclusion, the study has been able to determine the range of bud lengths for each hybrid over which the
entire process of meiosis has been observed to take place. In searching for buds at Metaphase I, relying on
bud lengths is not accurate because rate of bud growth is dependent on many external factors such as
presence of water and nutrients and the surrounding temperature. Furthermore, measuring buds with vernier
callipers is inaccurate and unprecise. Hence it is ideal to start with buds of a length near the middle of the
given range for meiosis and then used the methods outlined earlier to find a bud at the right stage.

This method has proven to be very effective with all three hybrids and is easily reproducible, hence is a
suitable method to be used in the future with other new hybrids.

Chromosome Pairing and Sporad Analysis

No. of Cells No. of Range of no. of Mean no. of

Studied chromosomes bivalents bivalents

Renanthera S.B.G. 5 38 18 – 19 18.2 ± 0.7

Mokara Singa Gold 7 57 8 – 14 10 ± 1.9

Arachnoglottis 2 38 16 16

% Renanthera S.B.G. Mokara Singa Gold Arachnoglottis

Polyads 0% 45.8 ± 3.6% 0.3 ± 0.4%
Tetrads 89.2 ± 1.7% 37.2 ± 3.7% 15.4 ± 5.3%
Triads 10.5± 2.0% 6.0 ± 0.9% 16.4 ± 4.6%
Dyads 0.3 ± 0.4% 9.3 ± 10.0% 56.0 ± 8.4%
Monads 0% 1.7 ± 1.0% 11.9 ± 2.5%

In the Renanthera Singapore Botanic Gardens, the chromosome pairing was regular (refer to fig.1A).
Numerous cells in Anaphase showed 2 clear groups of 19 segregating chromosomes that could be counted.
(refer to fig. 1B-J). Cells at Telophase II showed 4 clear groups of chromosomes (refer to fig. 1K) indicating
that meiosis had been regular. Mature sporads had a very high percentage of tetrads (nearly 90%) refer to fig.
1L ). There were very few tetrads with microcytes or micronuclei observed.

From these results it was hypothesized that the hybrid is fertile, which is confirmed by breeding experiments.
When crossed with other fertile hybrids, the Renanthera Singapore Botanic Gardens produces regular and
healthy seed pods.

A B C

D E F

G H I

J K
 L M

Fig. 1 (A) Cells showing regular chromosome pairing (x400). (B-J) Cells at Anaphase showing two clear
groups of segregating chromosomes (x400). In (B), (D) and (E) 19 chromosomes can be counted in each
group. (K) Cells at Telophase II showing 4 clear groups of chromosomes (x400). (L and M) Sporads showing
a very high percentage of tetrads (x400)

In the Mokara Singa Gold, the chromosome pairing was irregular, with many trivalents and univalents. (refer
to fig. 2A-E). Many cells in Metaphase and Anaphase showed massive univalents and laggards respectively
(refer to fig. 2F-N). Sporads showed a very relatively high percentage of polyads as well as tetrads with
microcytes (refer to fig. 2O and P), indicating that meiosis had not been normal. Although there were a
moderate percentage of tetrads, the tetrads formed were unlike regular tetrads and not well shaped. Numerous
dyads and triads with microcytes and micronuclei were also observed.

Irregular chromosome pairing came about because there were three sets of chromosomes and thus an odd
number of chromosomes, hence the presence of numerous univalents, trivalents and laggards. These
chromosomes grouped together to form micronuclei and microcytes, resulting in polyad formation later as
observed. Pairing most likely occurred between related pairs of parental chromosomes, for example Vanda-
Vanda or Vanda- Arachnis (Lee Y.H., 1994). Division of chromosomes between the two daughter cells in
meiosis I was unequal, thus even if normal disjunction occurred at meiosis II the microspores in the tetrads
formed would most likely have aneuploid chromosome numbers. From these results it was hypothesized that
the hybrid is infertile, which is confirmed by breeding experiments, where when crossed with another fertile
hybrid, the Mokara Singa Gold hybrid produced seed pods which aborted at one month old.

Similar results have been observed in studies done on other Mokara hybrids in Singapore. Pollen from
Mokara Chark Kuan, Mak Chin On, Princess Mikasa and Cheah Loong Fun have all displayed numerous
univalents and laggards with a maximum of 19 bivalents formed. Princess Mikasa has displayed a relatively
high percentage of dyads with microcytes as a result of frequent fusion of segregating chromosomes at
Telophase due to numerous laggards (Lee Y.H., 1994).
 A B

U
U
U

C D E
U

U
T
L
F G H
L

L
L

I J K
U

L
 L M
U

N P O P
P TR

TM

Fig. 2 (A-E) Cells showing poor chromosome pairing with numerous univalents (U) and trivalents (T)
(x400). (F-N) Cells in Metaphase and Anaphase showing massive univalents (U)and laggards (L)
respectively (x400). (O and P) Sporads showing polyads (P), tetrads with microcytes (TM) and triads (TR)
(x400).

In the Arachnoglottis, the chromosome pairing was fairly regular, displaying univalents similar to that in the
Mokara Singa Gold (refer to fig. 3A-G ) but not as massive. The chromosomes in some cells also showed the
syndrome of stickiness (refer to fig. 3H ) Stickiness is caused by certain abnormal genes and is another
indication of sterility (Tanaka and Kamemoto, 1984). Some cells in Anaphase displayed laggards (refer to
fig. 3I). The sporads showed a very high percentage of dyads (approx. 50%) and there were very few sporads
with microcytes or micronuclei observed. Monads were also observed (refer to fig. 3J-K).

From this information it was hypothesized that the Arachnoglottis is fertile to a very small extent due to the
dyads and monads, which was confirmed by breeding experiments. When the Arachnoglottis was crossed
with a fertile hybrid, seed pods with very few seeds were produced. The tetrads were unlikely to be
functional because of the incomplete chromosome pairing and hence unequal division of chromosomes.

The results for all three hybrids can be easily correlated to the nature of the hybrids. In intergeneric hybrids
like the Mokara Singa Gold and Arachnoglottis there is a lack of homology between parental chromosomes,
thus partial pairing or a complete lack of pairing occurs and the hybrid is usually infertile (Tanaka and
Kamemoto, 1984). The opposite happens in intrageneric hybrids like the Renanthera Singapore Botanic
Gardens, where the parental chromosomes show a high degree of homology and perfect pairing occurs.
 U
A U
B C
U

U
U
U

D E F

G H I

L L

D
J D
K

M M

D T

T
Fig. 3 (A-G) Cells showing fairly regular chromosome pairing with univalents (U) (x400). (H) Syndrome of
stickiness (x400). (I) Cell in Anaphase showing laggards (L) (x400). (J and K) Sporads showing tetrads (T),
dyads (D) and monads (M) (x400).
However for intrageneric hybrids this is not always the case. Studies showed that hybrids created
by crossing different species within the genera Dendrobium displayed different levels of irregular
meiotic behaviour, with univalents and trivalents observed (Kamemoto, 1994). Thus the level of
fertility of intrageneric hybrids is once again dependent on the level of homology between the
parental chromosomes.

Triploid orchids are known to be poor breeders and notoriously sterile (Dodson and Gillespie,
1967) because of the unequal distribution of three sets of chromosomes (Tanaka and Kamemoto,
1984). However, occasionally triploids produce offspring, for example when the triploid Vanda
Nellie Morley is crossed with diploid strap-leaved Vanda individuals with 38, 39, 52, 57, 70, 71,
73, 75, 76 chromosomes are produced. (Tanaka and Kamemoto, 1984)

FURTHER IMPROVEMENTS AND STUDY

Instead of removing whole buds from the stalk, the buds could be dissected while on the stalk and only half
the pollen removed from them for study. If the pollen is at too early a stage, the other half could be left to
develop and studied at a later date.

Study of the Arachnoglottis has proven to be interesting and thought provoking because if it is the dyads or
monads that have caused the few seeds, then these seeds would be polyploid in nature. Another experiment
could be done where the Arachnoglottis is crossed with a fertile diploid hybrid such as the Renanthera, and a
ploidy analysis done on the resultant hybrids. If they are pentaploid, the monads are responsible for
fertilisation; if they are triploid, the dyads are responsible for fertilisation.

REFERENCES

1. Calaway and Gillespie (1967). The Biology of the Orchids. Mid-America Orchid Congress Inc., America
2. Chan Leong Teck (1996). Interspecific and Intergeneric Hybrids of Arachnis. BSc. Hon. Dissertation,
National University of Singapore.
3. Kamemoto, Amore and Kuehnle (1994). Breeding Dendrobium Orchids in Hawaii. University of
Hawai'i Press, Canada
4. Klug, W. and Cummings, M. (1997). Concepts of Genetics 5th Edition. Prentice-Hall, Inc. Upper Saddle
River, New Jersey 07458
5. Lee Y.H. (1994) Genomic and Meiotic Analysis of Mokara Orchids. In Journal of Heredity v. 85, Oxford
University Press 200 Madison Avenue, New York, NY 10016, pp. 39-43
6. Loke Wai Kit. (1996). Studies of Dendrobium Orchids in Singapore. BSc. Hon. Dissertation, National
University of Singapore.
7. Rost, Barbour, Thornton, Weier and Stocking (1984). Botany: A Brief Introduction to Plant Biology.
John Wiley and Sons, Canada
8. Tanaka, R. and Kamemoto, H. (1984). Chromosomes in Orchids: Counting and Numbers. In J. Arditti
(ed.), Orchid Biology: Review and Perspectives, III Cornell Univ. Press Ithaca, N.Y., pp. 323-410.
9. Yam Tim Wing (1995). Orchids of the Singapore Botanic Gardens. National Parks Board, Singapore
Botanic Gardens, Cluny Road, Singapore 259569.

ACKNOWLEDGEMENTS

I would like to thank Dr. Yam Tim Wing, my mentor for his invaluable guidance and assistance as well as his
assistant, Mr. Thame Aung, for his valuable assistance at the laboratory. I would also like to thank Mrs.
Fabiola Soong, my teacher-advisor for her help and advice. Finally, I would like to thank the National Parks
Board and the Singapore Botanic Gardens for allowing me to use their orchids without which this project
would not have been possible.