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Intergeneric hybrid of two rare and endangered orchids, Renanthera

imschootiana Rolfe and Vanda coerulea Griff. ex L. (Orchidaceae): Synthesis
and characterization

Article  in  Euphytica · January 2008

DOI: 10.1007/s10681-008-9755-9

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Euphytica
DOI 10.1007/s10681-008-9755-9

Intergeneric hybrid of two rare and endangered orchids,

Renanthera imschootiana Rolfe and Vanda coerulea Griff.
ex L. (Orchidaceae): Synthesis and characterization
Rajkumar Kishor Æ Gurumayum Jitendra Sharma

Received: 1 November 2007 / Accepted: 16 June 2008

Ó Springer Science+Business Media B.V. 2008

Abstract Renanthera imschootiana and Vanda
coerulea are two rare and endangered orchid species
featuring in IUCN Red List of Threatened Plants as well
as in Red Data Book of Indian Plants, and are renowned
for their unique ornamental traits. Renanthera imscho-
otiana and V. coerulea have different flowering periods
and by taking opportunity of off-season flowering of
V. coerulea which happened during the flowering
season of R. imschootiana, the hybridization was
effected by hand-pollination. Viable pollinia were
present in the off-season flowers of V. coerulea and
one out of four cross was successful when R. imscho-
otiana was taken as female parent. Reciprocal cross was
unsuccessful. The resulting immature hybrid embryos
were germinated in vitro on Vacin and Went medium
supplemented with 15% v/v coconut water. Best
seedling growth was observed on half-strength Murash-
ige and Skoog medium devoid of any plant growth
regulators while the transplanted seedlings grew best
on brick chips:charcoal pieces (2:1) potting mix in
community earthen trays. First flowering of the hybrid
seedlings happened four years ten months after transfer
to ex vitro environment. RAPD markers generated by
the decamer primer OPA1 convincingly confirmed the
hybridity. Registration of the hybrid was made with the
Royal Horticultural Society with the grex Renantanda
Kebisana Shija.
Keywords Renanthera imschootiana
Vanda
coerulea
Hybridization
Off-season flowering

Orchidaceae
Polymerase chain reaction
Randomly
amplified polymorphic DNA
Rare and endangered
Abbreviations
CW Coconut water
MS Murashigeand Skoog medium
PCR Polymerase chain reaction
RAPD Randomly amplifiedpolymorphic DNA
VW Vacin and Went medium

R. Kishor (&)
G. J. Sharma

Department of Life Sciences, Manipur University,
Imphal 795 003, India
e-mail: rajkumarkishor@yahoo.com Introduction

R. Kishor Renanthera imschootiana Rolfe and Vanda coerulea

Medicinal Plants and Horticultural Resources Division, Griff. Ex L. are two beautiful rare and endangered
Institute of Bioresources and Sustainable Development
(IBSD) Takyelpat Institutional Area, orchids featuring in the IUCN Red List of Threatened
Imphal 795 001, India Plants (Walter and Gillett 1998) as well as the Red

123

Data Book of Indian Plants (Nayar and Sastry 1987–
1990). They belong to the tribe Vandae of the
angiospermic family Orchidaceae. They are natives
of the Indo-Burmese mega-biodiversity hotspot
occurring mostly in the North-eastern States of India
and the adjoining areas of Myanmar. Renanthera
imschootiana has a long and branching inflorescence
bearing more than 30 flowers and it is very popular
for its bright red flowers. It normally flowers during
April–May. Renanthera imschootiana is listed in
Appendix I of the Convention on International Trade
in Endangered Species of Wild Fauna and Flora
(CITES) since 1979. Vanda coerulea also has long
and erect inflorescences bearing more than 20 flowers
and it is popular for its large, blue, tessellated flowers
which generally bloom in September–October. It was
in CITES Appendix I since 1979 but downlisted to
Appendix II in 2004 at the 13th Meeting of the
Conferences of the Party in Bangkok.
Due to their rich floricultural traits these two
species have been over collected from their natural
habitat and thus become threatened rare and endan-
gered orchids. Both R. imschootiana and V. coerulea
have also been used as potential parents for production
of a number of internationally renowned orchid
hybrids. Their great popularity even at species level
has indicated that a hybrid involving both would
produce a novel one having a pool of dominant
characters derived from each of them. However,
synthesizing a hybrid involving both of them remained
unsuccessful for so long due to distant flowering
periods. Separation of their flowering period by about
four to five months posed as the sole barrier in the
breeding programme. Off-season flowering is an
important event that happens with almost all the
angiosperm. A perusal of literature, however, appar-
ently indicates that the event is poorly documented.
Off-season flowering happens sometimes in Orchid-
aceae and this event can be judiciously utilized for
synthesis of rare hybrids (Kishor et al. 2006).
Ever since Knudson (1922) demonstrated asymbi-
otic germination of seeds of the orchids Cattleya,
Laelia and Epidendrum, this technique has been used
for germination of seeds of both epiphytic and
terrestrial orchids. Asymbiotic germination of imma-
ture seeds of V. coerulea in vitro on Vacin and Went
medium was reported by Nath et al. (1991). So, far
there is no report on in vitro germination of seeds of
R. imschootiana.
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Euphytica
Orchids have a long juvenile period. For vanda-
ceous hybrid orchids it has been shown that the
maturation period ranges from two (Kishor et al.
2006) to ten years (Teoh 1986). Hence, a breeder has
to wait for a long time to see the first flowering, the
key marker of the success of his hybridization. Such a
delayed flowering has been the major problem to a
breeder as the final point of interest i.e. the charac-
teristics of the hybrid flower could not be determined
within a short period. Therefore, the development of
a molecular marker for rapid confirmation of hybrid-
ity using simple and reliable biotechnological tools is
required. Many breeders have employed polymerase
chain reaction (PCR) based randomly amplified
polymorphic DNA (RAPD) markers for determina-
tion of hybridity in a number of crops. In orchids this
has been reported in Vanilla (Besse et al. 2004;
Divakaran et al. 2006) and intergeneric slipper orchid
hybrids (Handa et al. 1998).
Here, we are presenting the technical report on the
synthesis of the F1 hybrid of R. imschootiana and
V. coerulea. Development of in vitro culture proto-
col, successful transplantation, molecular and
morphological characterization of the hybrid orchid
are also discussed.
Materials and methods
Plant material and artificial hybridization
Plants of R. imschootiana (Fig. 1) and V. coerulea
(Fig. 2) were collected from their natural habitats in
different parts of Manipur (23o470 –25o410 north
latitude and 93° 610 –94°480 east longitude) and
preserved under the ambient condition of day/night
temperature, humidity and sunlight. Some of the
plants were grown in teak baskets of 15
(length) 9 15 (width) 9 5 (height) inches dimension
in the Orchidarium of Manipur University while other
remained attached on live trees in the Experimental
Garden.
Hybridization was performed when a single plant
of V. coerulea plant exhibited off-season flowering
during June 1998, overlapping the closing end of
flowering period of R. imschootiana. After 3–4 days
of anthesis, pollinia of V. coerulea (%) were removed
using fine sterilized forceps and deposited on the
stigma of R. imschootiana (&). The pollinia from the
Euphytica
Fig. 1 Renanthera imschootiana
Fig. 2 Vanda coerulea
female parent were removed to prevent self-pollina-
tion. Reciprocal cross was also performed. The hand–
pollinated flowers were bagged for 7 days to prevent
unwanted pollination and labelled individually with
tags giving the date and the time of pollination. After
hybridization, the capsules were allowed to develop
by maintaining the plants for 150 days. Four flowers
could be pollinated with R. imschootiana as female
parent while only two could be pollinated with
V. coerulea as female.
In vitro germination of immature hybrid seeds
and seedling growth
The 150 day-old capsule containing the hybridized
seeds was collected from the plant. It was soaked in
an aqueous solution of the laboratory detergent
Labolene (Qualigens, India) for 30 min. Solid dirt
particles adhered to the surface of capsule were
removed using a brush followed by rinsing with
sterile distilled water. The capsule was then surface-
disinfected successively using 70% (v/v) ethanol for
30 s and 0.1% (w/v) aqueous mercuric chloride
solution for 15 min. Afterwards, it was rinsed four
times each of 5 min with sterile double-distilled
water before air-drying in a laminar airflow cabinet
for 5 min. The sterilized capsules were dissected
transversely with a sterile surgical blade. Immature
seeds were scooped out of the sterilized capsules
and small mass (5 mm 9 5 mm) of the aggregated
seeds was shown on culture medium in test tube
(32 9 200 mm) for in vitro asymbiotic seed
germination.
Vacin and Went (VW; Vacin and Went 1949)
medium containing 2% (w/v) sucrose supplemented
with 15% (v/v) coconut water (CW) with varying
concentrations of plant growth regulators, viz., a-
naphthalene acetic acid (NAA; 0.5 and 1.0 mg/l), N6-
benzylaminopurine (BAP; 0.5 and 1.0 mg/l) and
kinetin (KN; 0.5 and 1.0 mg/l) (Sigma, USA) was
used. Coconut water from green nut was drained in a
500 ml glass beaker and boiled for 5 min. Then, it
was filtered through a Whatman No. 4 and added to
the VW medium. After adjusting the pH to 5.2 with
1N NaOH, the medium was gelled with 1% (w/v)
agar (Hi-Media, India). Approximately, 15 ml of the
medium was dispensed into each culture tube (32
mm 9 200 mm), closed with cotton plugs and
sterilized by autoclaving at 15 psi and 121°C at
1.05 kg cm-2 for 20 min. The scooped aggregated
mass of seeds was placed on the medium in the
culture tubes and incubated at 25 ± 2°C under
28 lmol m-2 s-1 illumination from cool white fluo-
rescent tubes (Philips, India) for 16/8 h light/dark
photoperiod.
Two different sets of experiments were tried to
see the performance of in vitro growth of the
developing seedlings. In the first set of experiments,
two different basal media, viz., VW medium and
half-strength Murashige and Skoog medium (MS;
Murashige and Skoog 1962) were tested. Half-
strength MS medium was prepared by reducing the
mineral, vitamins and sucrose concentration of MS
to half-strength while the amount of agar remained
123

unchanged at 0.8% (w/v). pH of the medium was
adjusted to 5.9 for proper gelling of the medium. In
the second set of experiments, MS medium at half-
strength was supplemented with varying concentra-
tions of BAP at 0.5 mg/l, KN at 0.5 mg/l and NAA at
0.5 mg/l. Conical flask of 100 ml volume was used
as the culture vessel. Seventy five-day-old seedlings
with 1–2 leaves and 0–1 roots were taken for the
experiment. Observations were taken after 150 days.
Morphological changes in terms of number, length
and width of leaves and roots were recorded.
Transplantation and flowering
In vitro raised hybrid seedlings having an average of
6.5 leaves and 4.0 roots were removed from the flasks
and washed in tap water. Subsequently, they were
treated with 5% (w/v) fungicide (Carbendazim) for
15 min. Transplantation of the seedlings was tried on
three potting media, viz., (i) brick chips:charcoal
pieces (2:1), (ii) brick chips:charcoal pieces (2:1)
mulched with moss (Sphagnum sp.) and (iii) brick
chips only. The brick chips and charcoal pieces (ca 2
cu. cm.) were autoclave-sterilized prior to potting
while the moss was treated with 5% (w/v) fungicide.
They were put into earthen community trays (height,
4 cm 9 diameter, 15 cm). Five community trays (per
treatment) having thirty seedlings each were kept
directly in the poly-house condition with average
temperature of 28°C and 90% RH. The intermediate
exposure to growth chamber or greenhouse environ-
ment was skipped.
PCR-RAPD for hybridity confirmation
Young leaves from the hybrid seedlings and the
parent species were used as source for DNA extrac-
tion. Total genomic DNA was extracted following the
protocol of Doyle and Doyle (1987) with minor
modifications. The quality and quantity of the DNA
samples was determined by observing the ratio of
No: of enlarged seeds with developed chlorophyll
Germination percentage ¼
Total number of seedsper microscopic field
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absorbance at A260/A280. Intactness of genomic
DNA was further checked by subjecting it to 0.8%
agarose gel electrophoresis.
Amplification was done in 25 ll reaction volume
containing 19 Taq polymerase assay buffer (10 mM
Tris–HCl pH 8.0, 50 mM KCl, and 2.5 mM MgCl2),
0.2 mM of dNTPs, 1 lM primer, 1.0 unit of Taq
polymerase and 25 ng of template DNA. All reagents
used were from Bangalore Genie Pvt. Ltd., India.
Initially, fifteen arbitrarily chosen 10-mer random
primers, synthesized at Bangalore Genie Pvt. Ltd.,
India by providing the sequences (Operon Technol-
ogies, USA), were screened. Depending upon the
amplification of the desired banding pattern only
OPA1 (50 -CAGGCCCTTC-30 ) was selected for
hybridity confirmation. High annealing temperature
(HAT) amplification was performed using a thermal-
cycler (Model: iCycler, Biorad, USA) programmed
for an initial denaturation at 94°C for 4 min for 1
cycle, then 94°C for 1 min, 53°C for 1 min, 72°C for
2 min for 40 cycles with 5 min final extension at
72°C. The amplified DNA fragments were resolved
in 2% agarose gel electrophoretically at 70 V, using
19 TAE buffer. A 1 kb ladder (Promega, USA)
served as the standard molecular weight marker. The
ethidium bromide stained (0.5 lg/l) gels was visual-
ized and photographed using a Gel Documentation
System (Biorad, USA). The RAPD reaction was
carried out at least three times to ensure the
reproducibility of the amplified bands.
Experimental design and data analysis
In vitro culture experiments were set up in com-
pletely randomized design. For in vitro seed
germination 10 replicates (Single culture tube is one
replicate) were taken. One-tenth of the germinating
seeds were removed from each tube and spread on a
glass slide. The seeds were then counted using a
stereozoom microscope. Germination percentage is
calculated using the formula:
 100%
Euphytica

For the growth experiment five seedlings per culture Table 1 Influence of plant growth regulators on in vitro seed
flask was considered and twenty five flasks per germination of the hybrid orchid R. imschootiana 9 V. coerulea
treatment were taken. One seedling per flask was PGRs Germination Time taken for development (days)
selected randomly for morphological observation. For (mg/l) (%)
Green Leaf First First
the transplantation experiment only 10 plants were globular primordia leaf root
selected randomly from each community trays for stage
recording the morphological data. Significance of
Control 100 28.2d 35.5e 42.8e 60.0c
treatment effects was determined using analysis of
BAP
variance (ANOVA). Variation among treatments was
0.5 70 37.6b 54.2b 70.6b 90.0a
analyzed using Tukey’s test. a a a
1.0 65 45.0 60.4 77.2 90.0a
Kn
Results 0.5 100 31.7c 35.5e 49.2d 60.4c
b cd d
1.0 100 37.4 42.4 49.9 72.5b
The off-seasonally developed inflorescence of V. co- NAA
erulea was unhealthy and had only two flowers. From 0.5 100 37.3b 41.2d 49.0d 54.6d
b c c
those flowers four pollinia were obtained for hybrid- 1.0 100 39.5 45.6 53.3 57.4d
ization with R. imschootiana. One out of four crosses In each column the mean followed by the same letter are not
was successful with capsule development when significantly different as indicated by Tukey’s test (P = 0.05)
R. imschootiana was taken as female parent. How- Control—VW medium supplemented with 15% (v/v) coconut
ever, the reciprocal cross with V. coerulea as female water without plant growth regulators
parent was not successful as indicated by the abortion Data recorded every alternate day
of the two developing capsules. The resulting hybrid
embryos were allowed to develop for 150 days.
Visible morphological changes associated with
seed germination were observed after 20 days of
inoculation on culture medium. The germinating
seeds swelled and turned green. The germination
percentage ranged from 65–100. On average the best
germination response was observed on VW medium
supplemented with 15% (v/v) CW and devoid of
any plant growth regulators (Table 1, Fig. 3). On
this medium, the germinating seeds took minimum
time for reaching green globular stage (28.2 days),
development of the leaf primordia (35.5 days) and
first leaf (42.8 days). However, development of first
root was observed on the medium supplemented
with 0.5 mg/l NAA (54.6 days). Supplementation of
BAP in the medium retarded the germination
response. Fig. 3 Asymbiotic seed germination on VW medium supple-
Seedling growth response of R. imschooti- mented with 15% (v/v) coconut water (control)
ana 9 V. coerulea on half-strength MS medium
was found to be better than that on VW medium In the second experiment on seedling growth,
(Table 2, Fig. 4). Seedlings grown on half-strength seedlings cultured on half-strength MS medium
MS medium attained maximum leaf number (4.8 per supplemented with various plant growth regulators
seedling), leaf length (15.4 mm) leaf width (4.1 mm) individually showed a varied response (Table 3).
and root number (2.6 per seedling) after 150 days of Optimum response of growth was observed on half-
inoculation. However, root length remained more or strength MS medium devoid of any plant growth
less the same on both the media. regulators (control) after 150 days of inoculation.

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Table 2 Comparative effect of the media, VW and half- Table 3 Effect of plant growth regulators on growth response
strength MS, on growth of the seedlings of the hybrid orchid R. of the seedlings of hybrid R. imschootiana 9 V. coerulea on
imschootiana 9 V. coerulea in vitro half-strength MS medium supplemented with different PGRs
Media Growth responses of the seedlings PGRs Growth responses of the seedlings
(mg/l)
Leaf Root Leaf Root
Number/ Length Width Number/ Length Number/ Length Width Number/ Length
seedling (mm) (mm) seedling (mm) seedling (mm) (mm) seedling (mm)

VW 3.2b 13.0b 2.3b 1.5b 7.1a Control 4.8a 15.4a 4.1a 2.6ab 8.6b
a a a a a
‘ MS 4.8 15.4 4.1 2.6 8.6 BAP
In each column the mean followed by the same letter are not 0.5 3.4b 11.1b 3.3b 2.2b 5.3c
significantly different as indicated by Tukey’s test (P = 0.05) Kn
The data recorded after 150 days of inoculation 0.5 3.9b 11.6b 4.8a 2.1b 9.4b
NAA
0.5 3.7b 11.0b 3.8b 3.9a 11.6a
In each column means followed by the same letter are not
significantly different as indicated by Tukey’s test (P = 0.05)
Control—Half-strength MS medium supplemented without
plant growth regulators
Data recorded after 150 days of inoculation

flowered successfully. The inflorescence was 250–

Fig. 4 Seedling growth on half-strength Murashige and Skoog
medium
Responses on media supplemented with BAP
(0.5 mg/l), KN (0.5 mg/l) and NAA (0.5 mg/l) were
not better than that of the control. However, the
medium supplemented with NAA (0.5 mg/l) exerted
better response on root development. The response of
the seedling growth varied with the three different
substrates used for hardening. Of the three different
potting substrates used, survival percentage and the
growth performance of the seedlings were found to be
highest (80 %) in the community tray having brick
chips:charcoal pieces (2:1) as potting substrates
(Table 4, Fig. 5).
The hybrid seedlings of R. imschootiana 9 V. co-
erulea flowered four years and ten months after
transfer to ex vitro environment. Ten percent of
the hardened seedlings developed floral buds and
300 mm long with 8–12 flowers approximately
(Fig. 6). A single flower was 45–50 mm across,
bluish mauve in colour. The dorsal sepal was with or
without a purple mark at its tip. The lateral sepals had
darker tessellation, inherited from its male parent
(V. coerulea). The petals also had purple markings at
the tips, inherited from the female parent (R. imscho-
otiana). However, there was no sign of spots like the
scarlet spots scattered over the dorsal sepal and petals
of R. imschootiana. The lip was purple towards the
tip and light pink inside. The hybrid has a long
flowering period extending from February till July.
The flowers lasted for about 45 days. This hybrid has
been registered with the Royal Horticultural Society
with the grex Renantanda Kebisana Shija bearing
registration number T:128725. Comparative account
of the hybrid and the parent species are given in
Table 5.
Out of 15 RAPD primers assessed only one primer,
OPA1, showed convincingly the success of hybrid-
ization by generating reproducible polymorphic
banding patterns of the parents, viz., R. imschootiana
and V. coerulea and their inheritance in the hybrid
(Fig. 7). The primer OPA1 generated six markers for
the female parent, R. imschootiana, out of which an
amplicon of about 837 bp was monomorphic with that

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Table 4 Effect of different potting media on transplantation success and growth response of seedlings of hybrid R. imschooti-
ana 9 V. coerulea
Potting media Survival (%) Responses of the seedlings
Leaf Root
Number/seedling Length (mm) Width (mm) Number/seedling Length (mm)

PM1 80 6.9a 55.3a 12.3a 7.0a 50.9a

a b b b
PM2 73 5.7 46.9 8.1 4.7 37.5b
a b b b
PM3 60 5.3 45.3 7.8 4.2 36.3b
In each column means followed by the same letter are not significantly different as indicated by Tukey’s test (P = 0.05)
Potting mixture (PM1) contains brick chips: charcoal pieces (2:1); Potting mixture (PM2) contains brick chips: charcoal pieces (2:1)
mulched with moss (Sphagnum sp.); Potting mixture (PM3) contains brick chips only
Data recorded two years after transplantation

Fig. 5 Transplanted seedlings in brick chips : charcoal pieces

(2:1) potting substrate

of the male parent, V. coerulea and the hybrid while

three other amplicons of 541, 611 and 1998 bp were
also generated in the hybrid. For V. coerulea, OPA1
amplified 4 markers, one (837 bp) was monomorphic
while three polymorphic amplicons of 794, 941 and
1618 bp were also generated in the hybrid.

Discussion Fig. 6 Renanthera imschootiana 9 Vanda coerulea hybrid

flowers

Many vandaceous orchids are important in hybrid-

ization programmes for the cut flower industry (Goh
and Kavaljian 1989). Vandaceous orchids offer great
variations both at species and generic levels in the
flower colour, size, shape texture and number per
spike in addition to their vegetative features. Taking
all these into consideration, many breeders attempted
at artificial hybridization of different species resulting
in new combinations of forms, colours sizes, etc.
Renantanda represents a vandaceous hybrid genus
resulting from the cross involving the genera Renan-
thera and Vanda. The orchids of this genus have a
morphology intermediate between the original forms
and shapes resulting from introgression of genes of
Renanthera and Vanda.

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Table 5 Morphological characteristics of R. imschootiana, V. coerulea and their hybrid

Plant parts Morphological characteristics
R. imschootiana$ V. coerulea# Hybrid

Leaves 80–120 9 20–30 mm, dark green,
oblong-lanceolate, apex unequally
lobed
Inflorescence Many flowered, longer than
leaves, [300 mm in length,
horizontal
Flower 45 mm across, dorsal sepal
16 9 5.0 mm spathulate with red
blotch at tip; laterals 22 9 12 mm
crimson, orbicular-oblong; lateral
petals 12–03 mm yellowish with red
spots, linear spathulate; lip 06–
07 mm long, red with 5–7 creamish
calli
100–200 9 20–40 mm, narrowly 80–150 9 15–25 mm, green,
oblong, apex obliquely truncate, lanceolate, oblong, apex unequally
toothed lobed and toothed
Many flowered, loose, longer than 250–300 mm long, erect, 8–12
leaves, 250–400 mm long, erect flowered
55–65 mm across, pink, sepals and 45–50 mm across, dorsal sepal
petals subequal, darker tessellation 27 9 08 mm; laterals 32 9 25 mm
seen; lip 20-.2 mm long, deep bluish mauve with dark tessellation;
pink, apex emarginated lateral petals 27 9 07 mm with
purple marking at tips; lip 12–
13 mm long, pink inside and purple
at tip with five white ridges, obtuse

means if we have to cross two plants having different

Fig. 7 PCR-RAPD profiles of R. imschootiana, V. coerulea
and the hybrid ‘R. imschootiana x V. coerulea’ generated by
the primer OPA1 (50 -CAGGCCCTTC-30 ); .-bands specific of
R. imschootiana; ..-bands specific of V. coerulea; Lanes,
M-1 kb ladder, 2-R. imschootiana, 3-V. coerulea, 4-R.
imschootiana x V. coerulea
Synchronous flowering in different individuals is
necessary to perform hybridization through artificial
pollination. Otherwise pollen preservation is the only
and distant flowering periods. However, we did not
have to try for pollen preservation as we noticed a
single plant of V. coerulea to show off-season flower-
ing during June, 1998 towards the closing end of the
flowering period of R. imschootiana. Taking this
opportunity, its hybridization with R. imschootiana
was achieved. Some horticulturists have tried to induce
off-season flowering (forced flowering) in some crops
by manipulating growth regulator (Junthasri et al.
2000), nutrient regime (Manochai et al. 2005), photo-
period (Darnell et al. 2006) and temperature (Lewis
et al. 1999) and other factors. Manochai et al. (2005)
reported year round off-season flower induction in
Dimocarpus longan by KClO3 applications. Junthasri
et al. (2000) induced off-season flowering in mango by
application of paclobutrazol, a plant growth retardant,
in combination with thiourea.
Fruit setting provides a common measure of repro-
ductive success in orchid breeding (Proctor 1998). The
lower percentage of fruit setting in the successful
crosses may be due to intergeneric incompatibilities
(Shiau et al. 2002), experimental mishandling, or
difference in age of the flowers of the male and female
parents. Unsuccessful reciprocal cross, in our experi-
ment, might perhaps be due to unhealthy state of the
off-season flowers of V. coerulea.
It was observed that the mature capsules of R. im-
schootiana dehisced approximately 240 days after
pollination. Therefore, we used 150 day-old immature
seeds for in vitro germination. Seeds from immature

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Euphytica
capsules are suitable for in vitro germination as
embryos become viable and easy to surface-sterilize
them (Arditii 1967). Germination frequencies of many
orchids are found to be higher on culturing immature
seeds than mature seeds (Withner 1955). Yamazaki
and Miyoshi (2006) reported that mature seeds of the
orchid Cephalanthera falcata deposited lignin in the
inner integument of the mature seeds and this inhibited
embryo growth by mechanical restriction. Also, strin-
gent surface-sterilization of the ripe seeds affects the
viability and reduces the germination percentage in
orchids (Van Waes and Debergh 1986). The orchid
seeds are minute and exhibit poor level of differenti-
ation as indicated by the absence of endosperm and
arrested development of the embryo which remains at
globular stage (Arditii 1992). They have limited food
reserves especially lipid droplets and small amounts of
proteins. Despite this poor organization and limited
food reserve, they can be germinated in vitro by
providing specific nutritional and environmental con-
ditions (Knudson 1922).
Coconut water (CW) was supplemented in the
germination medium because its beneficial effect on
seed germination have been reported for orchids like
Bletia urbana (Rubluo et al. 1989), Cattleya (Kerbuay
and Handro 1981), Rhynchostylis retusa and V. coeru-
lea (Nath et al. 1991). Lo et al. (2004) reported that an
endogenous or exogenous supply of growth regulators
is essential for lipid mobilization during germination of
orchid seeds. Hence, the responses of plant growth
regulators, viz., BAP, KN and NAA on in vitro
asymbiotic germination of the hybrid seeds of R. imx-
chootiana 9 V. coerulea were investigated. Results
obtained from our study inferred better germination
response of the hybrid on VW medium devoid of plant
growth regulators suggesting that there might be
sufficient endogenous growth hormones required for
the initial stages of germination.
Further development of orchid seedlings from
germinated seeds involves steps like, protocorm
formation and organ differentiation. In vitro seedling
growth of orchids depends greatly on the types of
media used. To achieve further active growth of the
seedlings the performance of another medium, half-
strength MS medium, was compared with VW
medium. Seedlings of R. imschootiana 9 V. coeru-
lea attained maximum growth on half-strength MS
medium and this might, perhaps, be due to its
richness in macro and micro-element regime.
The delicate phenotype of the in vitro raised
plantlets cannot sustain direct exposure to harsher
ex vitro environment until or unless they are properly
acclimatized prior to transplantation. Acclimatization
of in vitro raised plantlets in the greenhouse under
controlled humidity and temperature is found to be
highly beneficial for successful transplantation. A
higher number of plants successfully acclimatized in
the community pot rather than individuals which may
be due to community effect (Potter 2000). Hence, we
used earthen community trays for transplantation.
Brick chips and charcoal pieces were used as potting
substrate as it had properties such as maximum water
holding capacity, porosity and good drainage which
were essential for proper growth and development of
in vitro raised seedlings of orchids. Mulching of the
medium with moss also increased the water retaining
capacity of the medium. However, for this particular
hybrid mulching was not very desirable as evidenced
by the lower survival percentage (Table 4).
Morphological observation of the hybrid flower
revealed that some of the dominant traits of the parent
species were inherited. The long erect inflorescence
with medium size flowers with intense colour, texture,
longevity and extended flowering period are some of
the desired characters being pooled into this hybrid.
The intermediate form of the vegetal and floral parts
also confirmed the success of the hybridization.
The random primer, OPA1, could generate the
desired banding patterns exclusive of the two parental
orchid species. At the same time the dominant
parental bands were also found to be present in the
hybrid proving that the synthesized hybrid carried the
parental characters. Lim et al. (1999) suggested that
RAPD could be used to indicate the genetic closeness
of orchid species and hybrids, and thus help to predict
the outcome of a cross, based on genotypic informa-
tion. In our experiment we used a high annealing
temperature (HAT) of 53°C for the PCR reaction and
it resulted in reproducible clear and distinct bands.
Yamagishi (1995) also used the same annealing
temperature and similar PCR conditions for RAPD
studies of Lilium.
Even though our experiment was based on a single
event of off-season flowering it has a significant role in
the development of a rare orchid hybrid using two rare
and endangered orchids having distant flowering
periods. This hybrid with a pool of the dominant
characters from both the parent represents a bluish
123

Renantanda orchid, which is a new addition to the
group. Renantanda Kebisana Shija can either be
commercially exploited at the F1 generation or form
the backbone for a whole new range of secondary
hybrids of vandaceous orchids especially for interna-
tional cut flower industry. Synthesis of hybrids using
rare and endangered orchids for commercial purposes
will certainly reduce the threatening pressure on their
wild parents. Such hybrids also carry the genes of the
parental species which may be retrieved, if needed.
Acknowledgement The authors are thankful to the
Department of Biotechnology, Govt. of India for the award
of Postdoctoral Fellowship to Rajkumar Kishor, which enabled
the study of molecular confirmation of hybridity.
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