Seed Propagation of Habeneria radiata. Morphological and

Article
Seed
Propagation
of Habenaria
radiata
Morphological and Physiological Characteristics of Plants
Derived from in vitro Cultured Seedlings
Teruto Nagayoshi* Tomoko
Hatanaka* and Takeshi Suzuki*
Abstract
The habitat and the number
of Habenaria radiata, one of the vulnerable and endangered wild orchids in Japan, decreases
year by year. It is necessary to develop an effectivemethod
to propagate thisspecies.By
applying in vitro germination and
culture techniques, it was found that the seeds of thisspecies germinated in a few weeks on artificial
media and acclimated
easily.Furthermore,
the term of growth from seedlings to full-grown was significantlyshorter than other Orchidaceae in-
vestigated. A large number of bulbs were obtained and the morphological and physiological characteristicsof plants grown
from them were examined in 1994 and 1995. Some characteristicsof individual clones, e.g.number of leaves,size of the
flowers, and arc of the lip remained stable over the two years. Positive comparative correlationswere found between many
characteristics,for example
leaf and the number
weight of the bulb and stem diameter, length of the stem and length of the leaf,length of the
of leaves, the length of the leaf and the number
of the leaf and stem diameter, the number
dwarfs, variegated leaves, unopened
of leaves and the number
of flowers,length of the leaf and stem diameter, width
of flowers.Several variant phenotypes such as albinos,
flowers and green-striped flowers that are similar to reported mutants, were produced.
The effectivenessof seed propagation and problems associated with variantsis discussed.
Key
Words:
Recent
Habenaria
radiata, vulnerable wild orchids, seed propagation, variants.
progress in genetic engineering
to manipulation
has allowed
of genes and their transfer even among
tantly related species. Since
all species may
may
prove
to be important. Thus,
of wild life reduces
the pool
only now
exploitable.
One
gene
becoming
of the main
bank
all
extinction
of genetic information
functions
that is
of our museum
is to collect
and rare wild plants mainly
growing
in Hyogo
In addition to categorizing
are surveying
in gathering
plants with in Hyogo
their habitats. We
prefec-
are also engaged
and stocking seeds and cultivating several wild
species, from
seedlings
and
cuttings, in the experimental
project was
initiated to increase Habenaria
radiata,
one of the rare wild orchids in the prefecture ( Hyogo
fecture, 1995
), which
concentrated
survey
WWFJ,
1989
is also designated
For a long time, some
have
been
develop
be bred, it might
picking
help pre-
the wild variety.
wild orchids, including
H. radiata,
cultivated by flower enthusiasts. However,
their
easy. It is therefore necessary
an effective methodology
Orchidaceae
the den-
year by year.
to propagate
requires an atypical germination
to development
to
this species.
process, such
as the help of specific orchid fungi. Thus, ordinary sowing
been
reported that if planted in vitro on artificialmedium
sterile germination
without
of
it has
for
and culture, seedlings could be obtained
the aid of fungi (Knudson,
reports on the method
1922,1924).
This study
of in vitro seed propagation
interim results on morphological
ological characteristics examined
in 1994
of H. raand physi-
and 1995.
pre-
as one of the seven
Materials
and
Method
species of vulnerable plants in Japan (
). H. radiata
is one of the most
orchids native to Japan. However,
and human
Seeds
popular wild
habitat destruction, due
exploitation has threatened
this
Konda
was
collected.
of Nature and Human
people
Activities,Hyogo,
pick it from
its
marshy
area. In August
tiful white
flying-bird shape, many
of Habenaria
lected in open
species. In addition, since this flower is favored for itsbeau-
* Museum
varieties could
vent the flower enthusiasts from
diata and some
field at the museum.
A
If consumer
a consequence,
of this species decreases
seeds in pots or seedbeds is not successful. However,
prefecture.
ture, we
sity and the number
cultivation has not been
data for conservation, and to increase the number
of endangered
natural habitat for personal use. As
ultimately be
able to share genetic material through such manipulation,
existing genes
us
dis-
radiata used
1992
were
and
colthe
and 1993, artificialpollination
carried out and one month
Yayoi-ga-oka 6, Sanda, 669-13 Japan
this study
fields to the north of Kobe
later, many
capsules were
Table 1. Composition of theHyponex medium.
Fig. 2. Frequency
% of flowered plants and their bulb weights in
1995.
In 1994
and 1995
the bulbs were
diameter
weighed
and 8 cm
and planted
individually
in 9 cm
deep
plastic pots.
Considering
the possibility of breeding prominent
in the future, plants susceptible to disease were
1994;
were
when
some
plants became
not used. The
vived
infected, germicidal
plants grown
the disease from
varieties
discarded in
from
agents
bulbs in 1995
the previous year. Many
sur-
plants were
again infected by disease in 1995, and this time germicidal
agents were
The
used.
following
ing plants: date
and width
characteristics were
of the leaf, the number
diameter, date of blooming,
and length of the spur. Only
in the data. Weight
Stem
of flowers per plant,
in the lip,arc of the lip,
blooming
plants were
of the bulb and blooming,
plants were
length was measured
the leaf were measured
enaria radiata.
From
early September
H. radiata
conditions
were
to late December
sown
on
1992,
artificial medium
and cultured using the following
the seeds were
hypochlorite
surface sterilized with
seeds
under
an intensity of 2000
green
lux. Within
and germinated.
leaves
and some
After
acclimation
greenhouse
at 25 °C under
had made
Seedlings
to obtain bulbs. More
were
than 650 bulbs were
bulbs
had propagated
bulbs
were
from
1400,
and about
seeds using the same
the first flower bloomed.
measured
between
Size
at the
of the flower was
the edges of the lip of the flower bloomwhen
In 1994, out of 650 bulbs planted, 470 plants bloomed.
Amongst
them
istics. In
1995,
378 plants were
about
2500
than 850 plants bloomed,
mother
plants bloomed
studied for these character-
bulbs were
amongst
and were
which
planted and more
358 plants whose
used in 1994
were
studied
again.
by March.
by the end of 1993. After one year the number
produced
several
cultivated in a
duced
to about
day when
first,
the seeds
of
the leaf sheaths at the
date was recorded
except for the sprouting date.
fluorescent light at
bulbs in the medium
in spring, seedlings
over
characteristics were measured
medium
produced
measured
basal part of the stem. Blooming
the firstflower bloomed
method:
one or two weeks
was
and width
using the longest leaf of each plant.
sterile
for ten minutes and then rinsed three times with
( Table 1) and incubated
diameter
and
from the top of the bulb to the
ing first(Fig. 1). These
one percent sodium
sterilized water; they were then placed on Hyponex
turned
of
Stem
included
blooming
included in the data.
basal part of the firstflower (Fig. 1 ). Length
Fig. 1. Schematic picture of measured characteristics
in latinHab-
for bloom-
of leaves per plant, stem
the number
size of the flower, length of the neck
non-blooming
measured
of germination, length of the stem, length
pro-
of these
1100
method.
new
In 1994,
Most
all plants were
of the plants produced
cultivated in the greenhouse.
more
than two bulbs ( mostly
three bulbs). In 1995, in such cases of more
being
available from
one mother
than two bulbs
bulb, they were simultane-
ously cultivated in and outside of the greenhouse.
Fig. 3. Distribution of date of the germination. A: In the green-
Fig. 4. Distribution of length of the stem (mm). A: In the green-
house 1994. B: In the greenhouse 1995. C: Outside 1995.
house 1994. B: In the greenhouse 1995. C: Outside 1995
cases, the first two leaves sprouted
Results
during
their early growing
longest
Bulb
Weight
The
vs. Blooming
mean
weight
Rate
of the bloomed
1995,
bulbs was
560.3
(42
mg-1549
mg), whereas
that of the non-blooming
was
501.7 mg
(33 mg-1900
mg)
in 1995
mg
bulbs
leaf was 97.8 mm
it was 85.1 mm
the group
(Fig. 2 ).
The
Date
From
early January
to February,
bulbs
pots, and kept either in the greenhouse
minated
from
germination
late March
were
planted in
or outside. They
mm-9.0
The
Number
238.9
mm
mm-403
(42 mm-404
mm)
132.5
mm
(Fig.
4).
mm).
in the group
(61 mm-249
mm)
in 1994
was
In 1995, it was 236.5 mm
(93
cultivated in the greenhouse
and
in the group
Length
Width
cultivated outside
of the Leaf
mean
Stem
mm)
(3.0
cultivated outside (Fig. 6).
number
firstone
very small. In some
per
Plant
of leaves per plant was 6.9 (4-11) in
cultivated in
and 7.3 (5-10) in the group cultivated outside
Diameter
When
covered
group
mm)
the stem
diameter
was measured,
tightly with the leaf sheaths. Thus,
the stems
diameter thus measured
was 3.7 mm
In 1995, it was 3.7 mm
cultivated in the greenhouse
in the group
were
the measured
included the thickness of the leaf sheaths. The
in 1994.
and width of leaves varied greatly. The
or two leaves and last two or three were
(4.0 mm-
and 6.0 mm
In 1995, it was 7.9 (5-12) in the group
ameter
and
in
(Fig. 7).
stem
Length
in the group
the greenhouse
stem length of plants that bloomed
mm)
(4.0 mm-9.0
cultivated in the greenhouse
of the Leaves
The
the Stem
mean
cul-
ger-
to April ( Fig. 3 ) and the date of
was recorded.
of
(23 mm-80
width of the longest leaf was 7.7 mm
mm)
1994.
Length
In
in the group
in 1994. In 1995, it was 6.8 mm
in the group
of Germination
in 1994.
mm)
and 52.2 mm
and died
length of the
cultivated outside (Fig. 5).
mean
12.0 mm)
mean
(39 mm-193mm)
(33 mm-141
tivated in the greenhouse
underground
stage. The
(1.1 mm-7.6
(1.7 mm-6.1
and 3.0 mm
cultivated outside (Fig. 8).
mm)
di-
mean
mm)
in the
(1.8 mm-8.7
Fig. 5. Distribution
1994.
Fig. 6. Distribution
1994.
of length of the leaf (mm). A: In the greenhouse
B: In the greenhouse
1995. C: Outside
1995.
of width of the leaf (mm). A: In the greenhouse
B: In the greenhouse
1995. C; Outside
1995.
Fig. 7. Distribution of number of the leaves. A: In the greenhouse
1994. B: In the greenhouse 1995. C: Outside 1995.
Fig. 8. Distribution of diameter of the stem(mm).
A: In the green-
house 1994. B: In the greenhouse 1995. C: Outside 1995.
Fig. 9. Distribution of date of blooming. A: In the greenhouse
Fig. 11. Distribution of size of the flower (mm). A: In the green-
1994. B: In the greenhouse 1995. C: Outside 1995.
house 1994. B: In the greenhouse 1995. C: Outside 1995.
Fig. 10. Distribution of number
Fig. 12. Distribution of length of the neck (mm). A: In the green-
house
of the flowers. A: In the green-
1994. B: In the greenhouse 1995. C: Outside 1995.
house 1994. B: In the greenhouse 1995. C: Outside 1995.
Fig. 13. Distribution of arc of the lip(°).A: In the greenhouse
Fig. 14. Distribution of length of the spur (mm). A: In the green-
1994. B: In the greenhouse 1995. C: Outside 1995.
house 1994. B: In the greenhouse 1995. C: Outside 1995.
Fig. 15. Percent distributionof date of the germination, A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 16. Percent distributionof length of the stem(mm),
A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 17. Percent distributionof length of the leaf(mm), A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 18. Percent distributionof width of the leaf(mm), A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 19. Percent distributionof number of the leaves, A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 20. Percent distributionof diameter of the stem (mm), A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 21. Percent distributionof date of the blooming, A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 22. Percent distributionof number
of the flowers, A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 23. Percent distributionof sizeof the flower (mm), A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Date
Fig. 15,
of Blooming
Flowers
vated
started to open
in the greenhouse
in early July in the group
there were
plant, they usually bloomed
more
combining
the percent
the three graphs
Figs. 16-26
also shows
than two buds per
in the same
manner.
1994,
( B ) represents greenhouse
either every other or every third
group
the combined
Group
environments
Number
of the Flowers
The
1994.
mean
number
per
), stem
Plant
of flowers per plant was 4.0 (1-10) in
In 1995, it was 2.9 (1-7) in the group
greenhouse
and 2.1 (1-5) in the group
cultivated in the
cultivated outside (
Fig. 10).
were
However,
when
three graphs of 4-13
plants from
1995. When
considered
length of was almost
the same
the same
), the stem
group
showed
shows
that the stem
remarkably
its environment
( A and B groups
over the two years.
were
different
length was different ( e.g. C
shorter stem
length ). The
of the Flower
The
mm)
mean
(Fig. 16 ). The
same
ers per plant (Fig. 22 ), however,
size of the flowers was
in 1994. In
1995,
it was
32.0 mm
30.0 mm
(12 mm-47
(19 mm-45
in the group
cultivated in the greenhouse
mm-41
in the group cultivated outside (Fig. 11). Sizes
mm)
of the firstflowers were
ond
or third one blooming
were
almost the same
in the same
and 29.1 mm
mm)
(13
tendency
as those of the sec-
between
of the neck
B and C groups
The
1995
in the
Lip
mm
mean
length of the neck in the lip (Fig. 1) was 12.2
(5.5 mm-19.0
(5.0 mm-15.0
and 9.5 mm
mm)
mm)
in 1994. In 1995,
it was 10.8 mm
in the group cultivated in the greenhouse
(2.0 mm-15.0
mm)
no differlength
(Fig. 24
the data in 1994
in Tables 2 and 3. Amongst
and stem
positive correlations between
diameter, stem
and
the characterbulb
length and the number
of
leaves, length of the leaf and stem diameter, length of the leaf
and
The
than in C group
correlation coefficients between
are shown
weight
of the Neck
B and C groups (Fig.
).
istics in 1994, there were
Length
of flow-
no significant
(Figs. 23, 26 ). The
was longer in B group
by
was shown
18 ). As to flower size and spur length, there were
plant, but later flowers
a littlesmaller.
there were
differences in the leaf width between
ences
result
length of this species is influenced
in the length of the leaf (Fig. 17 ) and the number
Size
1995,
the cultivating
the cultivating environments
( B and C groups
3C
( A ) represents all plants from
and ( C ) outside plants from
day.
3A, 3B, and
distribution of characteristics studied.
cultivated
and late July in the group
outside (Fig. 9 ). When
culti-
shows
in the group
cultivated out-
side (Fig. 12).
the number
of leaves, length of the leaf and the number
of flowers, leaf width and stem diameter, and the number
leaves
and number
of flowers. In 1995, there were
correlations between
stem length and the length of the leaf,
the length of the leaf and the number
tion there were
of
positive
some
of flowers. In
positive correlations between
addi-
the data
collected the previous year.
Arc
Germinating
of the Lip
The
mean
arc of the lip (Fig. 1 ) was 76.1 ° (35 °-150 °)
among
frequency
and frequency
of variants varied
capsules.
in 1994. In 1995, it was 67.7 ° (30 °-130 °)in the group cultivated
in the greenhouse
and 62.8 ° (10 °- 130 °) in the group
cultivated outside (Fig. 13 ).
Length
of the Spur
The
mm-53
mm)
Discussion
mean
mm)
in 1994. In 1995, it was 35.4 mm
in the group
(24 mm-47
The
length of the spurs (Fig. 1) was 38.2 mm
mm)
cultivated in the greenhouse
in the group
(20
(23 mm-47
and 35.3 mm
outside (Fig. 14).
seeds of Habenaria
artificialmedia. By
the media,
adding
radiata easily germinated
on the
different organic compounds
to
e.g. either yeast extract, malt extract, casanino
acid or peptone, some
differences resulted. Those
will be discussed in another report.
results
Fig. 25. Percent distributionof arc of the lip(°),A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Table
2. Correlation coefficientsbetween characteristics
measured in 1994.
Table 3. Correlation coefficientsbetween characteristics
measured in 1995.
In bulbous
enced
plants, generally, blooming
by bulb volume.
is strongly influ-
In the case of H. radiata, when
bulb was heavier than 201 mg, the blooming
high (68.7
%
). By
than 200 mg, the frequency
%).
of bloomed
nated
(1995) reported that seeds of H. radiata gathered
and grew
bulbs were
Tegarayama
by the end of June. They
stage in September.
Botanical
Garden
(1991)
using our method,
one year. Thus,
throughout
also reported similar
it took only one or two
sowing
of which
seeds in autumn,
bloomed
incubating
the winter, and acclimatizing them in spring
shortens the growing
Comparing
in September.
two years for the firstflower to bloom.
years to obtain adult phase bulbs, some
within
In the next spring
potted and one plant bloomed
it took almost
results. However,
in January the following year, germi-
seedlings of 3-5 cm
entered the dormant
Thus,
was
2-3 weeks
earlier than the latter (Fig. 21 ). Higuchi
al., (1987) reported that flowering of Habenaria
temperature
from
only. When
April to August
of 1.3°c and a minimum
It was warmer
comparing
in 1994
the blooming
dates between
of the plants cultivated in the greenhouse,
1994
and 1995
the former group
on
the daily temperatures
difference
difference of 0.9°c was observed.
than in 1995
(Fig. 27 ).
been
several mu-
tants reported in the leaf characteristics of this species (
Kimura,1988
Concerning
ported
). Most
of these were
discovered
flower characteristics, some
in the field.
mutants
during the past fifteen years (Kimura,
present investigation, several variants were
1995
were
re-
). In the
produced
from
seedlings, e.g. albinos, dwarfs, variegated leaves, unopened
flowers, and green-striped flowers, etc.. Some
ants appeared
the same
this species propagates
might
of these vari-
as the reported mutants. Usually
vegetatively in the field.Thus,
have been accumulated
there
recessive genes in the popula-
tion of thisspecies in the field.
Whether
period.
et
depends
of the two years, a maximum
For the past thirty years, there have
in October, then sowed
them
not grow to adult
(Fig. 2 ).
Takeda
150
was
was lighter
plants was low (35.0
It ispossible that the lighter bulbs may
phase
frequency
contrast, if the bulb weight
the
specific variants result from
conditions, or genetic components
developmental
is currently under
inves-
tigation.
Fig. 26. Percent distributionof length of the spur (mm), A in the greenhouse 1994, B in the greenhouse 1995, C outside 1995 respectively.
Fig. 27. Temperature curve from April to September
(°c), A maximum
1995, C maximum
temp, in the greenhouse 1994, E minimum
temp, outside 1995, D minimum
temp, in the greenhouse 1994, B maximum
temp, in the greenhouse
temp, in the greenhouse 1995, F minimum
temp, outside 1995 respectively.
Table 4. Mean
values and correlationcoefficientsof various morphological and physiological characteristics
between parent and offsprings.
*The
mean
value in 1995 was totaled only cultivatedin the greenhouse. R : Correlation coefficients
All bulbs investigated in 1995
gated. Thus,
the same
genotypes
were
were
vegetatively propaused in the investiga-
tions in these two years. The
characteristics measured
the two years were
( Table 4 ). There
compared
positive correlations between
were
over
some
the two years data in number
of leaves (r=0.59), size of the flower (r=0.57), and arc of the
lip (r=0.54). There
were
years data in stem
length (r=0.46), stem
and spur
bulbs
obtained from
correlations between
diameter
the two
(r=0.41)
results indicated that once
seed, various characteristics might
stable.
The
Albert
length (r=0.44). The
were
remain
weak
authors wish
to express their thanks to Mr. Daniel
for his review
of the manuscript.
References
Higuchi,
(1987)
H., Kimura
Flowering
N., Hiyama,
I., and
characteristics and
Habenaria
(Pecteris) radiata. Proc. 12th
conference,
Tokyo,
284.
Mizuno,
its regulation
world
N.
of
orchids
Hyogo prefecture (1995) The important nature in Hyogo,
Red data book of Hyogo prefecture.l-268.(in Japanese)
Kimura, N. (1988) The variant of the Habenaria radiata.
Published by Mitsuhashi Syunji. Curious wild orchids.
20-24, 115- 119.(in Japanese)
Kimura, N. (1995) The fancy flower of Habenaria radiata.
The Wild Orchid Journal, No. 109, 18-22.(in Japanese)
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seeds. Bot.Gaz., 73, 1-25.
Knudson, L. (1924) Further observations of non symbiotic
germination of orchid seeds. Bot. Gaz., 77, 212-219.
Takeda, M. (1995) Information of Sagisou group. The circle news of Kurashiki museum of naturalhistory.No.45,
12.(in Japanese)
Tegarayama
Botanical Garden (1991) Sagisou. 1-25.(in
Japanese)
World Wide Funds for Nature, Committee
of Japan
(1989) Red data book of Japanese plants. 1-320.
Japanese)
Received: October
Accepted: February
(in
5,1995.
15,1996.
Photo. 1. Variants derived from seedlings.A: Short neck B: Long
neck C: Dwarfs (center and left)D: Unopened
flower
Photo. 2. Variants derived from seedlings.E: Multi-bud F: Albinos G: Twin flowers H: Cross lip flower