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ORIGINAL PAPER
Effect of sucrose concentration and seed maturity on in vitro
germination of Dendrobium nobile hybrids
Waraporn Udomdee
•
Pei-Jung Wen
•
Chen-Yu Lee
•
Shih-Wen Chin
•
Fure-Chyi Chen
Received: 28 February 2013 / Accepted: 5 October 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Orchid is a major floral crop around the world
and Dendrobium hybrids are considered to be one of the
most popular orchids. In vitro germination of hybrid seeds
is a common practice among orchid growers, however, in
many cross pollinations the embryos may not develop to
maturity, leading to poor seed germination. The effect of
seed maturity and sucrose concentration were investigated
via asymbiotic germination of nobile Dendrobium hybrids.
Capsules were harvested from two hybrids (Den. Lucky
Girl 9 Den. Second Love ‘Kirameki’ and Den. Lucky
Girl 9 Den. Hamana Lake ‘Kumi’) and one selfing of Den.
Second Love ‘Kirameki’ at 2, 3, 4, and 5 months after
pollination and immature seeds were taken. Immature
seeds from 3- to 5-month old capsules could be success-
fully germinated on Hyponex based medium. Immature
seeds from 4-month old capsules showed greatest germi-
nation rate of tested treatments, whereas 3-month old
immature seeds showed the least germination. After
6 weeks of in vitro culture, protocorms derived from
embryos developed on every concentration of sucrose, but
germination was greater at lower concentrations. Greater
concentration of sucrose decreased normal-developed
protocorms.
Keywords Nobile Dendrobium Asymbiotic seed
germination Capsule maturity Embryo rescue
Abbreviations
AC Activated charcoal
CW Coconut water
MS Murashige and Skoog medium
PPF Photosynthetic photon flux
Introduction
Dendrobium hybrids hold a prominent position in the
ornamental orchid industry because of its high number of
flowers per inflorescence, longlasting and recurrent flow-
ering (Martin and Madassery 2006). Furthermore, the
variety of flower colors and color patterns, and the rela-
tively short production cycle further increase their com-
mercial value (Vendrame et al. 2007). Dendrobiums are
usually propagated asexually by division of offshoots, but
the multiplication rate is extremely low (Martin and Ma-
dassery 2006).
Hybrid seeds produced from cross pollinations are
commonly germinated by in vitro asymbiotic technique. It
has been shown that in asymbiotic in vitro cultures the
medium provides all the necessary nutrients, vitamins and
hormones to support germination and protocorm/seedling
development (Kananont et al. 2010). The technique of
asymbiotic seed germination, first introduced by Knudson
(1946), has revolutionized the concept of orchid cultiva-
tion. Since then, in vitro seed germination protocols have
been established for many orchid species and advocated as
a suitable propagation technique for mass production
(Arditti and Ernst 1993; Roy et al. 2011). Besides, the
W. Udomdee
Department of Tropical Agriculture and International
Cooperation, National Pingtung University of Science and
Technology, Pingtung, Taiwan
P.-J. Wen C.-Y. Lee S.-W. Chin F.-C. Chen (&)
Department of Plat Industry, National Pingtung University of
Science and Technology, Pingtung, Taiwan
e-mail: fchen@mail.npust.edu.tw
S.-W. Chin
e-mail: swchin@mail.npust.edu.tw
123
Plant Growth Regul
DOI 10.1007/s10725-013-9856-x
growth and development of orchid seeds under in vitro is
affected by culture period and culture conditions (Ichihashi
1978;A
´
vila-Dı
´
az et al. 2009).
Optimization of in vitro seed germination techniques
specific for different Dendrobium hybrids is highly desir-
able as there is a wide genetic diversity among the hybrids.
In addition immature embryos are often produced in distant
hybridization, where the in vitro germination technique
alone can regenerate the seedlings from such immature
embryos. This would further enhance the efficiency of
in vitro protocols of any specific Dendrobium hybrid for
better growth and development of protocorms and thus
seedlings (Kamemoto et al. 1999; Kananont et al. 2010).
The immature seeds of various developmental stages ger-
minate variably depending on their maturity and the tech-
nique is variously referred as ovule/embryo/green pod/
green fruit culture (Deb and Pongener 2011). Lo et al.
(2004) reported that the maximum number of Dendrobium
tosaense seedlings was obtained from the immature seeds
of 12-week-old capsules on half-strength MS basal med-
ium. The use of immature orchid seeds for in vitro culture
involves the identification of a proper time for the harvest
of capsules when they reach the optimum stage of devel-
opment (Vasudevan and Staden 2010a, b).
In vitro germination of hybrid seeds is a common prac-
tice among orchid growers and most orchid seeds can either
be readily germinated after harvest from the mother plant or
stored for later germination (Vendrame et al. 2007). How-
ever, lower percentage or null of seed germination in distant
hybridizations may occur quite often between interspecific
hybrids (Lee et al. 2010), especially when abnormal meiotic
behaviors encountered in many amphidiploid parents which
may lead to breeding barriers (Kamemoto et al. 1999).
Therefore, optimized in vitro germination protocols and
culture media are required for different cross combinations
to obtain maximum germination.
The aims of this study were to determine the optimal
seed maturity and sucrose concentration in germination
medium for improving germination rate of nobile type
Dendrobium hybrids which usually are derived from
interspecific hybridization.
Materials and methods
Plant materials
Three nobile type Dendrobium hybrids; Den. Lucky Girl,
Den. Second Love ‘Kirameki’, and Den. Hamana Lake
‘Kumi’ were used as female parents and hand-pollinated
under greenhouse condition at National Pingtung Univer-
sity of Science and Technology. Immature (green) capsules
from self- and cross-pollination were harvested between 2
and 5 months after pollination. Capsule growth was taken
visually and measured by a ruler. Seeds with part of the
ovary tissue were taken from capsules at different times (2,
3, 4, and 5 months) after pollination and observed under a
dissecting microscope (Stemi SV6 Zeiss, Germany). The
appearance of capsules and seeds on placenta were pho-
tographed by a digital camera (Nikon, Coolpix 950).
Capsule sterilization, seed germination and culture
conditions
The capsules were surface-sterilized by dipping into 70 %
ethanol for 3 min and 0.6 % sodium hypochlorite with a
drop of Tween-20 for 10 min. Subsequently the capsules
were washed three times with sterile water and dipped into
95 % ethanol for 15 s followed by flaming for 2–3 s in a
lamina flow hood. The surface sterilized capsules were slit
longitudinally and the seeds were scooped out then sown
by spreading thinly as possible over the surface of medium
in 90 9 15 mm petri dishes (Alpha Plus Scientific Corp.,
Taiwan). Each petri dish contained 25 ml of medium and
sealed by clear plastic tape. The medium consisted of
2gL
-1
Hyponex
Ò
(N:P:K = 7:6:19) (Hyponex Corpora-
tion, Marysville, OH 43041, USA), vitamins and glycine as
described in Murashige and Skoog (1962) supplemented
with 2 g L
-1
activated charcoal, 1 g L
-1
Bacto-tryptone
(HiMedia Laboratories, India), 50 g L
-1
potato extract,
25 g L
-1
ripen banana pulp and solidified with 5 g L
-1
Sigma agar (Sigma-Aldrich Co. LLC., St Louis, USA)
combined with 1 g L
-1
Gelrite (Cheng Hsin Tang Chem-
ical Co. Ltd., Taiwan) with different concentration of
sucrose (0, 10, 20 and 40 g L
-1
). The pH of the media was
adjusted to 5.6–5.8 prior to autoclaving at 121 °C and
1.21 kg cm
-2
for 30 min. All cultures were maintained at
25 ± 2 °C under a 12/12-h (day/night) photoperiod pro-
vided by cool white fluorescent lamps (Starcoat
TM
F28
W/T5/840,170 MA, Hungary) at photosynthetic photon
flux (PPF) of 40 ± 10 lmol m
-2
s
-1
.
Experimental design and data analysis
Seed germination was observed weekly under stereomi-
croscope (Stemi SV6 Zeiss, Germany) and seed germina-
tion percentage was calculated 6 weeks after sowing by the
following procedure:
Germination percentage
¼
No: of seeds showing swelling of the embryo 100
Total no: of seeds sowed
Experiments were performed in a completely randomized
design. For seed maturity experiment, two capsules form
each pollination were used and the experiment of sucrose
Plant Growth Regul
123
concentrations consisted of four replicates of approxi-
mately 500 seeds per replicate. Analysis of variance was
performed by using SAS version 8.2 and mean separation
with the Fisher’s protected LSD test. The significance level
was set at p \0.05.
Results
Capsule development
Capsules were successfully harvested only from the cross
of Den. Lucky Girl 9 Den. Second Love ‘Kirameki’ at
5 months after pollination since other combinations failed
to develop beyond this stage. Capsule length increased
after pollination until 2-months (2.47 cm) and then slightly
increased 3 months post-pollination (2.53 cm) (Fig. 1a, b)
and after 4 and 5 months no more further growth for the
capsule and its surface withered (Fig. 1c, d). Seed size
increased and seed color changed from white to light green
to creamy yellow during the development of pollinated
capsules from 2 to 5 months (Fig. 1e–h).
Seed germination and seedling development
Viable immature seeds from capsules of 3–5 months post
pollination were swollen, color changed to green, embryo
enlarged and testa ruptured at the first week of germination,
and designated as Stage 1 (Fig. 2a). Later, appearance of
pro-meristem/trichomes on germinated protocorms within
2 weeks of culture, designated as Stage 2 (Fig. 2b). Sub-
sequently, at stage 3 leaves emerged from protocorm and
elongation occurred at the third week of culture (Fig. 2c).
At the fourth week or Stage 4, protocorms with developing
leaves and rhizoids were observed (Fig. 2d). At Stage 5
two leaves and one or more roots emerged after 5 weeks of
culture (Fig. 2e). At final stage, or Stage 6, two or more
leaves and roots were presented in seedlings at 6 weeks
after sowing (Fig. 2f).
Immature seeds from 2-month-old capsules from all
crosses tested did not germinate and subsequently died
after sowing because the embryos were still under-devel-
oped and the ovules remained in white color (Fig. 1e). The
highest germination percentage was observed from
4 month-old capsules of all cross combinations as well as
self pollination (Table 1) irrespective of sucrose concen-
tration in the culture medium. The results indicated that
certain periods of capsule growth after pollination is nec-
essary for immature seeds to attain better germination
ability.
Effect of sucrose concentration on seed germination
Different sucrose concentrations did not show significant
difference on seed germination percentage in all stages of
capsules (Fig. 3). However, sucrose concentration affected
protocorm development. The number of germinated seed-
lings at stage 6 is decreased when the concentration of
sucrose increased (Fig. 4a, d). All germinated seedlings
developed completely to stage 6 on media without or with
10 g L
-1
sucrose (Fig. 4a, b) in a shorter time. In contrast,
in medium supplemented with 40 g L
-1
sucrose the pro-
tocorms showed yellowish color, rough surface, and
developed seedlings were shorter.
Discussion
The germination capability of immature seeds of hybrids of
nobile Dendrobium varies with the harvesting time of
capsules. Therefore this study was carried out to identify
the appropriate harvesting time of capsules of Dendrobium
nobile hybrids following self and cross pollination during
breeding program. In addition, asymbiotic culture of orchid
Fig. 1 Development of capsules and immature seeds of Den. Lucky Girl 9 Den. Hamana Lake ‘Kumi’ at 2 (a, e), 3 (b, f), 4 (c, g) and 5 (d,
h) months after cross pollination. Scale bars a–d 10 mm e–h 1mm
Plant Growth Regul
123
seeds (undifferentiated embryos) is similar to embryo res-
cue of other crops (Bridgen 1994; Deng et al. 2011) and has
become a practical tool for most orchid propagation sys-
tems (Lee 2011). The rescue of hybrid embryos resulted
from intra- and inter-generic crosses is commonly applied
in Phalaenopsis breeding programs (Tsai et al. 2009). The
effects of seed maturity and sucrose concentrations on seed
germination and seedling growth of Denbrobium nobile
hybrids were studied in this experiment. The results
showed that seed maturity did affect germination percent-
age that germination was better with mature hybrid seeds
than with the immature seeds. Two month old capsules did
not produce germinable immature seeds. Probably at this
period the fertilization process has yet to be completed, as
has been reported in embryogenesis of Dendrobium and
Phalaenopsis amabilis var. formosa (Niimoto and Sagawa
1961; Lee et al. 2008), Cymbidium aloifolium (Deb and
Pongener 2011), Calanthe (Lee et al. 2010), Paphiopedi-
lum wardii Sumerh (Zeng et al. 2012), Dendrobium tosa-
ense Makino (Lo et al. 2004), and Phalaenopsis Silky
Moon (Thongpukdee et al. 2010). Green pod age/devel-
opmental stage of immature embryos were found to be a
key factor for successful asymbiotic germination (Lee et al.
2010; Deb and Pongener 2011). Mweetwa et al. (2008)
showed that immature seeds (90 DAP) of Phalaenopsis did
not germinate well probably due to their underdevelopment
in such short period. Lee et al. (2007) reported that the
optimum time for asymbiotic seed germination of Calanthe
tricarinata was obtained from immature seeds at 150 days
after pollination. At that time the embryos reached the
maximum size and, seed coat became dehydrated and
gradually shrunk into a thin layer, and with relatively low
level of endogenous abscisic acid. The results of this study
showed that the germination of 5 month-old seeds of Den.
Lucky Girl 9 Second Love ‘Kirameki’ was significantly
Fig. 2 Different stages of protocorms and young seedlings from
immature seeds of Den. Lucky Girl 9 Den. Hamana Lake ‘Kumi’.
Stage 1: a swollen seed with yellow protocorm (a), stage 2:
appearance of rhizoids (b), stage 3: emergence and elongation of
cotyledon (c), stage 4: protocorm with developing leaves and rhizoids
(d), stage 5: presence of two or more leaves (e) and stage 6: presence
of two or more leaves, with roots emerged (seedling) (f). RZ rhizoids,
CT cotyledon, TL true leaf, RT root. Scale bars a–e 1mm(f)5mm
Table 1 Effect of seed maturity on germination percentage of nobile
Dendrobium hybrids after 6 weeks of culture
Hybrid combination Maturity
(months)
Germination
rate (%)
Lucky Girl 9 Second Love ‘Kirameki’ 2 0 c
3 34.23b
4 80.03a
5 65.75a
Lucky Girl 9 Hamana Lake ‘Kumi’ 2 0 c
3 40.32b
4 83.41a
5ND
Second Love ‘Kirameki’ (selfing) 2 0 c
3 28.13b
4 90.06a
5ND
Treatments followed by the same letter are not statistically different at
p \ 0.05 by Fisher’s protected LSD test
ND not determined (the capsules failed to develop beyond this stage)
Plant Growth Regul
123
lower than that of 4 month-old (Table 1). It has been
reported that immature seeds of Cypripedium formosanum
germinated faster and better than mature seeds (Lee et al.
2005). Also, Lee et al. (2005) described that the reason for
the low germination of mature seeds may be due to the
physical constraint of the testa and the existence of the
germination inhibitors such as abscisic acid (Lee 2003).
Some orchids require lower concentration of cytokinins for
optimized germination, such as Habenaria macroceratitis
(Stewart and Kane 2006).
In addition to capsule maturity, the successful seed ger-
mination was affected by quality of medium composition,
sucrose concentration and quality and quantity of PGRs
(Deb and Pongener 2011). Lee (2011) reported that media
of different compositions can be used for culturing imma-
ture embryos and is species dependent. Sucrose is usually
added to a culture medium as energy source so that tissues
not dependent on photosynthetic activity for their primary
growth (Islam and Ichihashi 1999). Also, sucrose could
supply a balanced carbon source for cell growth with the
released hexoses directly participating in glycolytic and
pentose phosphate pathways (Zha et al. 2007). In develop-
ing seeds, it has been shown that the main function of
transfer cells is the uptake of the solutes such as sugars and
Fig. 3 Effect of different
sucrose concentration (0, 10, 20,
40 g L
-1
) on 4 month-old seed
germination of immature nobile
Dendrobium hybrids; Lucky
Girl 9 Second Love
‘Kirameki’, Lucky
Girl 9 Hamana Lake ‘Kumi’,
and Second Love ‘Kirameki’.
Vertical bars represent
mean ± SE
Fig. 4 Seed germination of
Den. Lucky Girl 9 Den.
Hamana Lake ‘Kumi’ on
different sucrose concentration;
0(a), 10 (b), 20 (c) and 40
(d)gL
-1
after 6 weeks of
culture. Scale bars 5mm
Plant Growth Regul
123
amino acids for storage product production (reviewed by
Lee and Yeung 2010). Deb and Pongener (2011) found that
different concentration of sucrose affected on immature
seed germination of Cymbidium aloifolium. Moreover,
Johnson et al. (2011) reported that increasing sucrose con-
centration from 10 to 50 mM resulted in decreased germi-
nation and development of Bletia purpurea. In contrast, our
study of different sucrose concentration did not affect seed
germination percentage of immature nobile Dendrobium
hybrids seeds except protocorm development (Fig. 3). The
formation and proliferation of PLB of Phalaenopsis aph-
rodite Rchb. F. were enhanced at low sucrose concentration
(0.3 %), while high concentration was inhibitory (Hsu and
Chen 2003). The number of protocorms at advanced stage
of development was significantly greater in media con-
taining lower concentration of sucrose than higher con-
centration of sucrose (Fig. 4). This result is consistent with
Wotavova
´
-Novotna
´
et al. (2007) which indicated that a
higher concentration of sucrose inhibits orchid seedling
growth, expressed by the growth rate of shoots and roots of
Dactylorhiza species. Deb and Sungkumlong (2008)
showed that at high sucrose concentration (4 %) germina-
tion decreased and stunted growth observed in immature
embryo culture of Coelogyne suaveolens (Lindl.) Hook, and
on media devoid of sucrose no germination was registered.
According to Johnson et al. (2011), the decreasing of ger-
mination and development at high level of sucrose suggests
either that seeds experienced osmotic stress at the higher
molarity or that growth and development was inhibited by
products of sucrose hydrolysis during autoclaving. Imma-
ture seeds of Dendrobium aggregatum had higher germi-
nation percentage, higher number of protocorms,
production of maximal number of shoots, shoot elongation,
as well as root formation on MS medium containing 3 %
sucrose and coconut water (Vijayakumar et al. 2012). In
addition, seedlings that were grown in the absence of
sucrose will be provided an opportunity for further devel-
opment under ex vitro conditions (Tan et al. 1998).
In this report, the immature seeds could germinate on
medium without added sucrose. This could be explained
that other organic additives such as potato extract and
banana paste in the medium contributed to the basal level
of carbon source. Sharrock and Lusty (2000) reported that
ripe banana contains high sugar content which is 23.43/
100 g raw edible portion, and during ripening; the sugars
are in the approximate ratio of glucose 20: fructose 15:
sucrose 65. The amount of sucrose in the banana added to
the medium is probably enough for germination without
extra sucrose (Fig. 3), although subsequent seedling
growth required certain levels of sucrose. Vyas et al.
(2009) reported that apart from the presence of minerals in
banana, the presence of IAA, GA
7
and GA
x
, zeatin, zeatin
riboside and 2iP have also been reported. In many cases,
banana homogenate promotes growth of protocorms and
seedlings (Lee 2011). Pierik (1988) found that banana
homogenate was slightly inhibitory to the germination of
Paphiopedilum ciliolare but promoted the growth of
seedlings once germination had taken place. Also, Lo et al.
(2004) reported that using banana and potato as medium
additives supported vigorous growth. Peptone and yeast
extract are also good organic sources for orchid seedling
development (Lee 2011). Also, coconut water and banana
homogenate can be used as organic additives to stimulate
seed germination and development of Dendrobium orchid
seeds (Ngampanya and Homla-aor 2010).
Conclusion
An efficient and simple method of embryo rescue for nobile
Dendrobium hybrids has been developed through asymbiotic
seed germination. Immature seeds harvested from 3 to
5 months old capsules after pollination were able to ger-
minate well on nutrient medium. Immature seeds harvested
4 months after pollination showed better asymbiotic ger-
mination than seeds harvested at other time intervals. Fur-
thermore this would probably shorten the time course for the
breeding program of nobile Dendrobium hybrids. Though
seed germination at different sucrose concentration was not
different, the number of embryo that developed into seedling
stage decreased when concentration of sucrose increased.
Seed germination and seedling development seemed to be
faster in the banana supplemented medium without extra
sucrose that may avoid the toxicity due to high sucrose
concentration and also reducing the production cost. This
technique will be valuable for improving breeding efficiency
of commercial nobile Dendrobium hybrids and also for large
scale production of uniform seedlings.
Acknowledgments The first author thanks National Pingtung Uni-
versity of Science and Technology for providing a scholarship during
her PhD study. We also thank members of the laboratory of plant
biotechnology, Department of plant industry, National Pingtung
University of Science and Technology for assistance. This work was
supported by grants supported by the Council of Agriculture, Taiwan
with grant numbers 100AS-5.3.1-ST-aB and 101AS-5.3.1-ST-aB.
References
Arditti J, Ernst R (1993) Micropropagation of orchids. Wiley, New
York, p 682
A
´
vila-Dı
´
az I, Oyama K, Go
´
mez-Alonso C, Salgado-Garciglia R
(2009) In vitro propagation of the endangered orchid Laelia
speciosa. Plant Cell Tissue Organ Cult 99:335–343
Bridgen MP (1994) A review of plant embryo culture. HortScience
29:1243–1246
Deb CR, Pongener A (2011) Asymbiotic seed germination and
in vitro seedling development of Cymbidium aloifolium (L.) Sw.:
a multipurpose orchid. J Plant Biochem Biotechnol 20:90–95
Plant Growth Regul
123
Deb CR, Sungkumlong (2008) Effects of different factors on
immature embryo culture, PLBs differentiation and rapid mass
multiplication of Coelogyne suaveolens (Lindl.) Hook. Indian J
Exp Biol 46:243–248
Deng Y, Chen S, Chen F, Cheng X, Zhang F (2011) The embryo
rescue derived intergeneric hybrid between chrysanthemum and
Ajania przewalskii shows enhanced cold tolerance. Plant Cell
Rep 30:2177–2186
Hsu CC, Chen FC (2003) Plant regeneration from protocorm-like
bodies induced in etiolated leave of Phalaenopsis aphrodite
Rchb. f. J Chinese Soc Hortic Sci 49(4):335–342 (In Chinese
with English abstract.)
Ichihashi S (1978) Studies on the media for orchid seed germination
II. The effects of anionic and cationic combinations relevant to
seeding populations and culture periods on the growth of Bletilla
striata seedlings. J Jpn Soc Hortic Sci 46:521–529
Islam MO, Ichihashi S (1999) Effects of sucrose, maltose and sorbitol
on callus growth and plantlet regeneration in Phalaenopsis,
Coritaenopsis and Neofinetia. J Japan Soc Hortic Sci 68:
1124–1131
Johnson TR, Kane ME, Perez HE (2011) Examining the interaction of
light, nutrients and carbohydrates on seed germination and early
seedling development of Bletia purpurea (Orchidaceae). Plant
Growth Regul 63:89–99
Kamemoto H, Amore TD, Kuehnle AR (1999) Breeding Dendrobium
Orchids in Hawaii. University of Hawaii Press, Hononolu,
pp 55–61
Kananont N, Pichyangkura R, Chanprame S, Chadchawan S,
Limpanavech P (2010) Chitosan specificity for the in vitro seed
germination of two Dendrobium orchids (Asparagales: Orchid-
aceae). Sci Hortic 124:239–247
Knudson L (1946) A new nutrient solution for germination of orchid
seed. Am Orchid Soc Bull 15:214–217
Lee YI (2003) Growth periodicity, changes of endogenous abscisic
acid during embryogenesis, and in vitro propagation of Cypri-
pedium formosanum Hay. PhD Dissertation. National Taiwan
University, Taipei, Taiwan
Lee YI (2011) In vitro culture and germination of terrestrial Asian orchid
seeds. In: Thorpe TA, Yeung EC (eds) Plant embryo culture:
methods and protocols. Humana Press, New York, pp 53–62
Lee YI, Yeung EC (2010) Embryo development and in vitro seed
germination of Bulbophyllum fascinator. Acta Hortic 878:243–250
Lee YI, Yeung EC, Cheng MC (2005) Embryo development of
Cypripedium formosanum in relation to seed germination
in vitro. J Am Soc Hortic Sci 130:747–753
Lee YI, Lu EF, Chung MC, Yeung EC, Lee N (2007) Developmental
changes in endogenous abscisic acid concentrations and asym-
biotic seed germination of a terrestrial orchid, Calanthe tricar-
inata Lindl. J Am Soc Hortic Sci 132:246–252
Lee YI, Yeung EC, Lee N, Chung MC (2008) Embryology of
Phalaenopsis amabilis var. formosa: embryo development. Bot
Stud 49:139–146
Lee SJ, Liao FS, Cheng SH (2010) Effects of culture media and capsule
maturity on seed germination of intraspecific and interspecific
crosses in the genus Calanthe. Acta Hortic 878:231–235
Lo SF, Nalawade SM, Kuo CL, Chen CL, Tsay HS (2004)
Asymbiotic germination of immature seeds, plantlet develop-
ment and ex vitro establishment of plants of Dendrobium
tosaense Makino—a medicinally important orchid. In Vitro Cell.
Dev Biol Plant 40:528–535
Martin KP, Madassery J (2006) Rapid in vitro propagation of
Dendrobium hybrids through direct shoot formation from foliar
explants, and protocorm-like bodies. Sci Hortic 108:95–99
Mweetwa AM, Welbaum GE, Tay D (2008) Effects of development,
temperature, and calcium hypochlorite treatment on in vitro
germinability of Phalaenopsis seeds. Sci Hortic 117:257–262
Ngampanya B, Homla-aor W (2010) Simple media for Dendrobium
orchid seed germination and protocorm development. Acta
Hortic 878:219–223
Niimoto DH, Sagawa NY (1961) Ovule development in Dendrobium.
Am Orchid Soc Bull 30:813–819
Pierik RLM (1988) In vitro culture of higher plants as a tool in the
propagation of horticultural crops. Acta Hortic 226:25–40
Roy AR, Patel PS, Patel VV, Sajeev S, Deka BC (2011) Asymbiotic
seed germination, mass propagation and seedling development
of Vanda coerulea Griff ex. Lindl. (Blue Vanda): an in vitro
protocol for an endangered orchid. Sci Hortic 128:325–331
Sharrock S, Lusty C (2000) Nutritive value of banana. In: INIBAP
annual report 1999. INIBAP: Montpellier (FRA), pp 28–31
Stewart SL, Kane ME (2006) Asymbiotic seed germination and in vitro
seedling development of Habenaria macroceratitis (Orchidaceae), a
rare Florida terrestrial orchid. Plant Cell Tissue Organ Cult 86:147–158
Tan TK, Loon WS, Khor E, Loh CS (1998) Infection of Spathoglottis
plicata (Orchidaceae) seeds by mycorrhizal fungus. Plant Cell
Rep 18:14–19
Thongpukdee A, Thepsithar C, Rojanawong T (2010) Optimum
conditions for seed germination of Phalaenopsis Silky Moon.
Acta Hortic 878:237–242
Tsai CC, Chiang YC, Huang SC, Liu WL, Chou CH (2009)
Intergeneric hybridization, embryo rescue and molecular detec-
tion for intergeneric hybrids between Ascocenda and Phalaen-
opsis. Acta Hortic 829:413–416
Vasudevan R, Staden JV (2010a) In vitro asymbiotic seed germina-
tion and seedling growth of Ansellia africana Lindl. Sci Hortic
123:496–504
Vasudevan R, Staden JV (2010b) Fruit harvesting time and
corresponding morphological changes of seed integuments
influence in vitro seed germination of Dendrobium nobile Lindl.
Plant Growth Regul 60:237–246
Vendrame WA, Carvalho VS, Dias JMM (2007) In vitro germination
and seedling development of cryopreserved Dendrobium hybrid
mature seeds. Sci Hortic 114:188–193
Vijayakumar S, Rajalkshmi G, Kalimuthu K (2012) Propagation of
Dendrobium aggregatum through the culture of immature seeds
from green capsules. Lankesteriana 12:131–1135
Vyas S, Guha S, Bhattacharya M (2009) Rapid regeneration of plants
of Dendrobium lituiflorum Lindl. (Orchidaceae) by using banana
extract. Sci Hortic 121:32–37
Wotavova
´
-Novotna
´
K, Vejsadova
´
H, Kindlmann P (2007) Effects of
sugars and growth regulators on in vitro growth of Dactylorhiza
species. Biol Plant 51:198–200
Zeng S, Wu K, Teixeira da Silva JA, Zhang J, Chen Z, Xia N, Duan J
(2012) Asymbiotic seed germination, seedling development and
reintroduction of Paphiopedilum wardii Sumerh., an endangered
terrestrial orchid. Sci Hortic 138:198–209
Zha XQ, Luo JP, Jiang ST, Wang JH (2007) Enhancement of
polysaccharide production in suspension cultures of protocorm-
like bodies from Dendrobium huoshanense by optimization of
medium compositions and feeding of sucrose. Process Biochem
42:344–351
Plant Growth Regul
123
