Cultivo de Paphiopedilum
-
Upload
pablo-gomez -
Category
Documents
-
view
43 -
download
2
Transcript of Cultivo de Paphiopedilum
ORIGINAL PAPER
In vitro propagation of four threatened Paphiopedilum species(Orchidaceae)
Bo Long • Alex X. Niemiera • Zhi-ying Cheng •
Chun-lin Long
Received: 27 March 2009 / Accepted: 11 January 2010 / Published online: 2 February 2010
� Springer Science+Business Media B.V. 2010
Abstract The effects of seed maturity, media type, carbon
source, and organic nutrient additives on seed germination,
protocorm development, and plant growth of Paphiopedi-
lum villosum var. densissimum Z. J. Liu et S. C. Chen were
investigated. Micropropagation frequency was enhanced
through the use of 200-day-old seed, Knudson C (KC)
medium, and the presence of both glucose and coconut milk
in the medium. The effects of various plant growth regula-
tors on the frequency of shoot organogenesis in four
Paphiopedilum species were also investigated. Explants of
P. villosum var. densissimum and P. insigne (Lindl.) Stein
incubated in the presence of 5 mg l-1 6-benzyladenine (BA)
with 0.5 mg l-1 a-naphthalene acetic acid (NAA) and
0.2 mg l-1 BA with 0.1 mg l-1 NAA, respectively, showed
a twofold increase in the frequency of shoot organogenesis.
For explants of P. bellatulum (Rchb. f.) Stein and P. arme-
niacum S. C. Chen et F. Y. Liu, the combination of
5.5 mg l-1 BA with 0.5 mg l-1 NAA and 4 mg l-1 BA with
0.1 mg l-1 NAA, respectively, resulted in the highest fre-
quencies of shoot organogenesis.
Keywords Micropropagation � Orchid �Seed germination � Shoot proliferation
Abbreviations
2,4-D 2,4-Dichlorophenoxyacetic acid
AC Activated charcoal
BA 6-Benzyladenine
IAA Indole-3-acetic acid
IBA Indole-3-butyric acid
KT Kinetin
LH Lactalbumin hydrolysate
NAA a-Naphthalene acetic acid
TDZ 1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl)-urea
Introduction
Paphiopedilum spp. (Orchidaceae), commonly known as
Lady’s slipper orchids, are often marketed as attractive and
desirable container-grown plants. A few species are
regarded as endangered or even extinct in the wild due to
over-collection from natural areas and large-scale illegal
trade. Paphiopedilum spp. have been designated as
endangered by the Convention on International Trade in
Endangered Species of Wild Fauna and Flora (CITES;
CITES Appendices 2008). In a natural setting, Paphio-
pedilum seed germinates relatively slowly due to the
absence of an endosperm. In P. armeniacum, the interval
between seed germination and tiller production is about 4
years (Liu et al. 2006), although some orchid hybrids and
tropical orchids initiate tiller production within 6 months
B. Long � Z. Cheng � C. Long (&)
Kunming Institute of Botany, Chinese Academy of Sciences,
650204 Kunming, China
e-mail: [email protected]; [email protected]
B. Long
e-mail: [email protected]
B. Long
Graduate School, Chinese Academy of Sciences, 100039
Beijing, China
A. X. Niemiera
Department of Horticulture, Virginia Polytechnic
and State University, Blacksburg, VA 24061, USA
e-mail: [email protected]
C. Long
College of Life and Environmental Sciences,
Minzu University of China, 100081 Beijing, China
123
Plant Cell Tiss Organ Cult (2010) 101:151–162
DOI 10.1007/s11240-010-9672-1
of germination and tiller yearly (Chen and Tsi 2003).
P. villosum (Lindl. ex Hook.) Stein and P. barbigerum
T. Tang et F. T. Wang both show low frequencies of fruit
set, with average fruit set of 8 and 26%, respectively
(Banziger 1996; Shi et al. 2008). Although Paphiopedilum
spp. produce abundant seeds, germination levels in nature
are low and, consequently, Paphiopedilum spp. are rela-
tively rare in the wild (Arditti and Ernst 1993).
In general, the growth and development of in vitro-
grown plants depend on factors such as macro- and micro-
element composition, carbon source, and plant growth
regulator (PGR; Murashige and Skoog 1962; Xiong and
Wu 2003). Although sucrose is the most utilized carbon
source for plant tissue culture, other sugars, such as glu-
cose, fructose, maltose, and mannitol, are also effective (Li
2002). The frequencies of in vitro seed germination of
P. insigne var. sanderae (Rchb. f.) Stein and P. armenia-
cum are also influenced by seed maturity and organic
nutrient additives, such as banana, potato, and coconut
juice (Nagashima 1982; Pierik et al. 1988; Ding et al. 2004;
Lee 1998, 2007). Asymbiotic seed germination of fully
mature orchid seeds is often difficult (Lee et al. 2006), with
the frequency ranging from 35 to 68% in 12 Paphiopedi-
lum spp. (Lee 1998; Tay et al. 1988; Chen et al. 2004b;
Zeng et al. 2006; Lee 2007). In contrast, the germination
frequency is 97 and 100% for P. ciliolare (Rchb. f.) Stein
and P. callosum (Rchb. f.) Stein, respectively (Stimart and
Ascher 1981; Pierik et al. 1988).
There are relatively few published reports on the
micropropagation of Paphiopedilum spp. using tissue cul-
ture technology, primarily due to the difficulty in main-
taining explants in culture. P. delenatii has been induced to
form shoot buds by wounding and liquid culture of nodal
segments (Nhut et al. 2005) with a regeneration frequency
of 75% (Nhut et al. 2007). Efforts to establish long-term
Paphiopedilum callus cultures from stem segments have
been relatively unsuccessful. However, a few Paphioped-
ilum hybrids have been successfully propagated from seed-
derived protocorms, callus, shoot, leaf, and nodal tissues
(Stewart and Button 1975; Lin et al. 2000; Chen et al.
2004a; Huang et al. 2001). It is likely that Paphiopedilum
hybrids are easier to micropropagate than native species.
Consequently, the propagation of Paphiopedilum spp. is
mainly achieved by asymbiotic seed germination due to the
limited success of tissue culture protocols. Given the
environmental status of Paphiopedilum spp., its high
desirability in commercial horticultural markets, and the
need to save several Paphiopedilum species from extinc-
tion, it is essential that an efficient tissue culture micro-
propagation method for Paphiopedilum be established. The
existence of a rapid and large-scale in vitro propagation
protocol would also assist in restoring Paphiopedilum spp.
to native environments. Tissue culture-produced plantlets
can also be used in ex situ preservation, which is an
important approach to plant germplasm preservation, par-
ticularly in terms of pest-free cultures, high propagation
frequency, and gene stability.
The in vitro propagation of P. villosum var. densissimum
and shoot multiplication of P. bellatulum, P. insigne, and
P. armeniacum have not been documented in the literature.
In the study reported here, we carried out a series of
experiments aimed at establishing an efficient microprop-
agation system for native Chinese Paphiopedilum spp.,
including P. villosum var. densissimum, P. bellatulum,
P. insigne, and P. armeniacum. In particular, the influence
of seed maturity, medium composition, sugar type, and
organic nutrient additives on in vitro seed germination and
plant growth has been investigated. The effects of combi-
nations of cytokinins and auxins on in vitro callus induc-
tion and shoot proliferation were also investigated.
Materials and methods
Plant material
Paphiopedilum var. densissimum, P. insigne, P. bellatulum,
and P. armeniacum were collected from wild populations
in Wenshan County of Yunnan Province (southwestern
China). The plants were transplanted into containers and
grown in the greenhouse at the Kunming Institute of Bot-
any in Kunming, China.
Basal culture media and general culture conditions
The basal culture medium consisted of quarter-strength MS
(Murashige and Skoog 1962) micro- and macro-elements,
100 mg l-1 myoinositol, 1 g l-1 niacin, 1 g l-1 pyridoxine
HCl, 1 g l-1 thiamine HCl, and 6 g l-1 agar (plant tissue
culture tested; Sanland Int, Tokyo, Japan). The pH of the
medium was adjusted to 5.8 with 1 N KOH or HCl. Prior to
use, the medium was autoclaved for 20 min at 121�C. All
other experimental media treatments are described
separately.
For each treatment in different media, either 100 seeds or
three explants were used per vessel, and the treatments were
tested in triplicate. Seeds or explants were placed in the dark
for a period of either 2 or 4 weeks and then transferred to
light conditions under a 16/8-h (light/dark) photoperiod with
light provided by 36-W cool-white fluorescent bulbs
(36 lmol m-2 s-1) at 22 ± 2�C for a period of 3 months.
Seed germination and protocorm growth experiments
Flowers of plants from all four species were hand-polli-
nated. The time from hand pollination to inoculation
152 Plant Cell Tiss Organ Cult (2010) 101:151–162
123
treatment (placement on medium) varied in the different
experiments. Seed capsules were collected and sterilized
with 75% (v/v) ethanol for 30 s and then dipped into 0.1%
HgCl2 for 15–20 min followed by a thorough rinsing in
sterile distilled water. Approximately 300–400 extracted
seeds were placed on the basal medium amended with
20 g l-1 sucrose, 1 g l-1 activated charcoal (AC), and
100 ml l-1 coconut milk (liquid endosperm of freshly
harvested Cocos sp.) in 200-ml glass vessels (height
8 cm 9 width 7 cm) sealed with a plastic cap.
P. villosum var. densissimum seed maturity experiment
To determine the influence of seed maturity on seed ger-
mination, P. villosum var. densissimum seed capsules were
collected at 120, 130, 140, 150, 160, 170, 180, 190, 200, or
300 days after pollination (DAP). At each collection date,
seeds were placed on the basal medium supplemented with
100 ml l-1 coconut milk, 20 g l-1 sucrose, and 1 g l-1AC.
The germination frequency of about 100 seeds at 20, 40,
60, or 80 days after inoculation (placement on amended
medium) was determined by microscopic observation
(Axioskop 40; Zeiss, Gottingen, Germany). Germination
was verified by the appearance of a white protocorm and
was expressed as the frequency of all seeds sown. Proto-
corms turned green after approximately 100 days in cul-
ture, were re-measured, and then used in the plant growth
experiments. Plantlet leaf size was measured after 200
days.
P. villosum var. densissimum media composition
experiments
To determine the effect of media composition on germi-
nation and growth, 300-DAP seeds were placed on one of
the following media: Knudson (KC; 1946), Vacin and
Went (VW; 1949), quarter-strength MS (basal culture
medium) or half-strength MS. Each of these media were
supplemented with 100 ml l-1 coconut milk, 20 g l-1
sucrose, and 1 g l-1 AC. Germination frequency and
growth were measured as described above.
P. villosum var. densissimum media supplement
experiments: carbon source
To determine the effect of carbon source on seed germi-
nation, 300-DAP seeds were placed on MS basal culture
medium amended with 100 ml l-1 coconut milk and
1 g l-1 AC. This medium was then amended with 20 g l-1
sucrose, glucose, maltose, or mannitol. Germination fre-
quency and growth were measured as described above.
P. villosum var. densissimum media supplement
experiments: organic nutrient additives
To determine the effect of organic nutrient additives on
seed germination, 300-DAP seeds were placed on MS basal
culture medium amended with 20 g l-1 sucrose and
1 g l-1 AC and then amended with 100 g l-1 potato mash,
100 g l-1 apple mash, 100 g l-1 chayote mash, 4 g l-1
lactalbumin hydrolysate (LH), or 100 ml l-1 coconut milk.
The source of these additives were fresh potato, apple,
chayote, Solanum tuberosum L., Malus domestica Borkh.
‘Red Fuji’, Sechium edule (Jacq.) Swartz, respectively, that
were skinned and homogenized in a blender. Germination
frequency and growth measurements were measured as
described above.
Experiments testing the effect of PGRs on callus
formation
To determine the influence of PGRs on callus formation of
the four species, protocorms and leaf fragments (from the
seed germination and protocorm growth experiments) were
cultured in media amended with various PGR combina-
tions (described below). There were at least 30 protocorms
of each of the four species per container. Leaf fragments
from aseptic plantlets of all species were cut into 0.5-, 1.0-,
1.5-, or 2.0-cm2 pieces. Each of the 200-ml containers
contained four fragments (0.5, 1.0, 1.5, and 2.0 cm2,
respectively). The media treatments consisted of various
combinations of three cytokinins and two auxins: (1)
0.1 mg l-1 kinetin (KT) in combination with 1.0, 2.0, 4.0,
or 8.0 mg l-1 2, 4-dichlorophenoxyacetic acid (2,4-D); (2)
0.02, 0.05, 0.1, or 0.5 mg l-1 1-phenyl-3-(1, 2, 3-thia-
diazol-5-yl)-urea (TDZ) in combination with 1.0, 2.0, 4.0,
or 8.0 mg l-1 2,4-D; (3) 1.0, 2.0, 4.0, or 8.0 mg l-1 indole-
3-butyric acid (IBA) in combination with 0.1, 0.5, or
1.0 mg l-1 a-naphthalene acetic acid (NAA). The MS
basal culture medium was amended with 100 ml l-1
coconut milk, 20 g l-1 sucrose, and 1 g l-1 AC. The fre-
quency of callus induction and browning were recorded
after 3 months.
Experiments testing the effect of PGRs on shoot
multiplication
Protocorms (all four species; from seed germination and
protocorm growth experiments) were transferred to new
200-ml containers (three plantlets per vessel) and cultured
for 1 year to become 3- to 5-cm plantlets; these plantlets
were used as a source of shoot multiplication material. In
addition, 3-cm P. armeniacum rhizome pieces, each with a
node, were taken from the aseptic plantlets derived in the
seed germination experiment and also used as shoot
Plant Cell Tiss Organ Cult (2010) 101:151–162 153
123
multiplication material. The PGR treatments (Table 3)
were: (1) the cytokinin TDZ (0.01, 0.02, 0.05, 0.1 and
0.2 mg l-1) in factorial combination with the auxin NAA
(0.1 and 0.5 mg l-1); (2) various combinations of the
cytokinin BA (range: 0.2–6.0 mg l-1) with the auxin NAA
( 0.1 and 1.0 mg l-1). The basal medium was amended with
100 g l-1 potato mash, and 20 g l-1 glucose. The number of
new shoots and shoot length were recorded after 3 months.
After 2 months, seedlings in uncapped containers were
maintained under culture room conditions at 70% relative
humidity. After a 3-day acclimation period (gradual tran-
sition from culture room conditions to a high-humidity
greenhouse environment), the plantlets were removed from
the containers and the roots washed in tap water to remove
residual agar. The plantlets were then transplanted into
10-cm-diameter pots with a peat moss substrate and grown
in the greenhouse. The survival rate of the plantlets after
1 month in the greenhouse was recorded.
Statistical analyses
Analysis of variance (ANOVA) was performed with the
SPSS ver. 16.0 statistical package (SPSS, Chicago, IL) and
mean separation with the LSD test. A significance level of
P \ 0.05 was applied. Figures were constructed using
Sigmaplot 10.0 (Systat Software Richmond, CA).
Results
Seed germination and protocorm growth experiments
P. villosum var. densissimum seed: maturity experiment
A minimum of 150 DAP was required for the seeds to
germinate (Table 1; Fig. 4a). The highest germination
frequencies, 31 and 13%, were obtained for 200-day-old
seeds inoculated (placed on medium) for 80 and 40 days,
respectively. Germination frequencies for 300-day-old
seeds were lower than those for 200-day-old seeds, but in
both the germination rate 80 days following inoculation
(13 vs. 31%, respectively) was higher than that 40 days
following inoculation (0 vs. 13%, respectively; Table 1).
P. villosum var. densissimum media composition
experiment
The highest germination frequency of P. villosum var.
densissimum, 56%, was obtained on KC medium 80 days
after inoculation (Fig. 1). The germination frequency rate
in the VW treatment (37%) was lower than that in the KC
treatment but higher than that in the quarter- and half-
strength MS treatments (21 and 14%, respectively). The
protocorms were larger on the VW medium (2.6 mm2) than
on the one-half-strength MS medium after 100 days of
culture, while protocorm sizes in the quarter-strength MS
and KC treatments were the same as those in the VW
treatment (Figs. 1, 4b). Rhizoids were observed on VW
medium after 100 days (data not shown).
P. villosum var. densissimum media supplements
experiments: carbon source
The frequency of P. villosum var. densissimum seed ger-
mination on the glucose-amended medium (19%) was
higher than that on media containing other carbon sources
after 100 days of culture (Fig. 2). The germination fre-
quency was lowest for the mannitol treatment (0.08%).
Leaf length and width were greater in the glucose treatment
than in the mannitol treatment, but they were the same in
the sucrose, glucose, and maltose treatments.
P. villosum var. densissimum media supplements
experiments: organic nutrient additives
Seed germination frequency was highest in the coconut
milk treatment (10%; Fig. 3). Germination frequencies in
the apple and potato treatments were less than 3%, while
those in the chayote and LH treatments were 0%. Leaf
length was lowest in the LH treatment, and lengths and
widths were the same for all other treatments, with the
exception of leaf width in the LH treatment.
Table 1 Effect of seed maturity on in vitro seed germination of Paphiopedilum villosum var. dentissimum
Inoculation timea (days) Frequency of in vitro germination (%)
120 DAP 130 DAP 140 DAP 150 DAP 160 DAP 170 DAP 180 DAP 190 DAP 200 DAP 300 DAP
40 0 b 0 b 0 b 0 b 0 b 0 b 0 b 0.43 b 13 a 0 b
80 0 c 0 c 0 c 0.12 c 0.14 c 0.74 c 0.9 c 0.71 c 31.33 a 13.33 b
Data are given as the mean in vitro germination frequency of seed of P. villosum var. densissimum collected from fruit harvested between 120
and 300 days after pollination (DAP). Each mean is based on microscopic observation. Means followed by different lower-case letters are
significantly different at P \ 0.05 level by the LSD testa Duration of time between placement of medium (plating) and germination
154 Plant Cell Tiss Organ Cult (2010) 101:151–162
123
The effect of PGRs on callus formation
Few seed-derived protocorms induced callus (data not
shown; Fig. 4c, d). All leaf pieces taken from 1.5-year-old
plantlets (cultured in containers; Fig. 4e) as callus induction
material turned brown and ultimately died. The combination
of 0.5 mg l-1 NAA with 2.0 mg l-1 BA resulted in leaves
that remained green (no browning, no callus) during the
Medium
Fre
quen
cy o
f ger
min
atio
n %
¡r/ p
roto
corm
siz
e m
m2
0
10
20
30
40
50
60
70Frequency of germination after 80days following inoculationPromcorm size after 100days following inoculation
C
A
B
C
ab ab ab
1/4MS KC VW 1/2MS
Fig. 1 Effect of medium composition on seed germination and
protocorm size of in vitro Paphiopedilum villosum var. dentissimum.Mean in vitro seed germination and protocorm size of P. villosum var.
densissimum in relation to composition of the medium. Data were
recorded 3 months following culture initiation. Each mean is based on
a visual observation. a Data were square-root transformed prior to
analysis of variance (ANOVA) and converted to the original scale for
demonstration in the figure. b Means with different letters within a
column are significantly different at P \ 0.05 level by the LSD test.
Bars: Standard errors. KC Knudson medium, VW Vacin and Went
medium, MS Murashige and Skoog medium (1/4, 1/2 quarter- and
half-strength, respectively). Details on the various media are given in
the text
Carbon sourceSucrose Glucose Maltose Mannitol
Freq
uenc
y of
ger
mia
ntio
n (
%)
/ Lea
f len
gth
(mm
)
0
5
10
15
20
25
Frequency of germinationLeaf lengthLeaf width
A
B
C
D
aba
ab
b
a a
ab
b
/ Lea
f wid
th (
mm
)
Fig. 2 Effect of carbon source
on mean in vitro seed
germination and leaf size of in
vitro Paphiopedilum villosumvar. dentissimum. Data were
recorded 3 months following
culture initiation. Each mean is
based on a visual observation. aData were expressed as
germination frequency; leaf
length was square-root and
reciprocal square-root
transformed prior to ANOVA
and converted to the original
scale for demonstration in the
figure. b Means with different
letters within a column are
significantly different at the
P \ 0.05 level by the LSD test.
Bars: Standard errors
Plant Cell Tiss Organ Cult (2010) 101:151–162 155
123
0
5
10
15
20
25
Frequency of germinationLeaf lengthLeaf width
Potato Apple Chayote LH Coconut
Organic nutrient additives
Fre
quen
cy o
f ger
min
atio
n (
%)
/ Le
af le
ngth
(m
m)
B
B
C C
A
a
a
a
b
a
a aab
bab
/ Le
af w
idth
(m
m)
Fig. 3 Effect of organic
nutrient additives on mean seed
germination and leaf size of
P. villosum var. dentissimum in
vitro. Data were recorded
3 months following culture
initiation. each mean is based on
a visual observation. Means
with different letters within a
column are significantly
different at the P \ 0.05 level
by the LSD test. LHLactalbumin hydrolysate. Bars:
Standard errors
Fig. 4 In vitro propagation process of Paphiopedilum spp. aGermination of 300-DAP (day after pollination) seed of P. villosumvar. densissimum: swollen embryo and ruptured testa (Bar: 0.1 mm).
b Protocorm growth (Bar: 1 mm), c Callus mass developed from a
protocorm of P. bellatulum. d Shoot cluster formed from callus of P.bellatulum. e 1.5-year-old P. insigne in vitro seedling with roots. f–I
Shoot multiplication from seedlings of P. villosum var. densissimum(f), P. insigne (g), P. bellatulum (h), and P. armeniacum (I).
j P. insigne root tubercles. k–m Transplanted seedlings of P. villosumvar. densissimum (k), P. insigne (l), and P. bellatulum (m) in a pot
(Bar: 1 cm)
156 Plant Cell Tiss Organ Cult (2010) 101:151–162
123
3-month culture period, but after subculture, the leaves
gradually turned brown (Table 2). A number of PGR com-
binations reduced the amount of browning, such as in the
case of P. insigne treated with 2,4-D (at 2.0, 4.0, and
8.0 mg l-1) in combination with KT (0.1 mg l-1) and TDZ
(0.02, 0.05 mg l-1; Table 2). Explant size also affected the
browning phenomenon; 1.5-cm2 leaf pieces had less
browning than all other sizes, and 2-cm2 pieces showed the
most browning (data not shown).
The effect of PGRs on shoot multiplication
The length and number of shoots of the four Paphiopedi-
lum spp. was influenced by the concentrations and com-
binations of cytokinins and auxins contained in the medium
(Table 3; Fig. 4f-I). In a 3-month period, three new
plantlets were produced (per three plants) by: (1) P.
villosum var. densissimum for the combinations of
3.0 mg l-1 BA with 1.0 mg l-1 NAA, 5.0 mg l-1 BA with
0.5 mg l-1 NAA, and 6.0 mg l -1 BA with 0.1 mg l-1
NAA; (2) P. insigne for the combinations of 0.2 mg l-1
BA with 0.1 mg l-1 NAA and 1.0 mg l-1 BA with
0.5 mg l-1 NAA. For P. bellatulum, the combination
5.5 mg l-1BA with 0.5 mg l-1 NAA induced maximum
shoot organogenesis (0.33; one new plantlet per three
plants) and produced the longest shoots (0.6 cm); this same
result was obtained with the 1.0 mg l-1 BA and 0.5 mg l-1
NAA treatment. Organogenesis occurred im all PGR
combinations for P. armeniacum; for this species, shoot
number was highest in the 4.0 mg l-1 BA and 0.1 mg l-1
NAA treatment (Table 3).
After 2 months, plantlets with three or more 3- to 6-cm-
long roots and four to five leaves were transferred to pots
with a peat moss substrate (Fig. 4k–m). Although the
survival frequency of these greenhouse-grown rooted
plants was about 60%, the seedlings grew very slowly.
Discussion
Seed germination and protocorm growth in vitro
Seed maturity significantly affected the germination fre-
quency of P. villosum var. densissimum (Table 1). The
germination frequency of 200-DAP seeds was highest on
the basal medium (inoculation); seed coat formation started
about 200 DAP. Seeds\200-DAP turned brown soon after
inoculation, indicating that the embryo was seemingly too
underdeveloped to absorb nutrients from the medium. In
contrast, the frequency of germination for 300-DAP seed
was lower than that of 200-DAP seed, likely because the
seed coat had already formed (microscopic observation),
possibly attenuating nutrient uptake. A similar effect of
seed maturity on the germination of Paphiopedilum seed
has been shown by Nagashima (1982) and Ding et al.
(2004). Ren and Wang (1987) found that if a P. godefroyae
(Godef.) Stein embryo had 10–40 cells at 100 ± 5 DAP,
then suspensor and endosperm nuclei degenerated at
120 ± 5 DAP; however, at 200 ± 10 DAP, the globular
embryo of mature seed had 120–140 cells, and there was a
single cell layer in the seed coat. Lee (1998) reported
optimal seed germination of P. delenatii Guillanmin at 150
DAP (68%), with the germination frequency decreasing
Table 2 Effect of cytokinins in combination with auxins on brown-
ing of leaf fragments
Cytokinins (mg l-1) Auxin (mg l-1) Browninga (%)
2,4-D NAA
KT
0.1 1 0 100
0.1 2 0 66.7
0.1 4 0 57.1
0.1 8 0 42.9
TDZ
0.02 1 0 28.6
0.02 2 0 71.4
0.05 4 0 33.3
0.05 8 0 71.4
0.1 1 0 85.7
0.1 2 0 100
0.5 4 0 100
0.5 8 0 100
BA
1 0 0.1 100
2 0 0.1 100
4 0 0.1 100
8 0 0.1 100
1 0 0.5 100
2 0 0.5 0
4 0 0.5 100
8 0 0.5 100
1 0 1 100
2 0 1 100
4 0 1 100
8 0 1 100
KT Kinetin, TDZ 1-phenyl-3-(1, 2, 3-thiadiazol-5-yl)-urea, BA 6-ben-
zyladenine, 2,4-D 2,4-dichlorophenoxyacetic acid, NAA a-naphthalene
acetic acida Mean browning of Paphiopedilum insigne leaf fragments following
culture in medium containing various combinations and concentra-
tions of cytokinins and auxins. Browning was recorded after 3 months
of culture. Each mean is based on a visual observation
Plant Cell Tiss Organ Cult (2010) 101:151–162 157
123
Ta
ble
3E
ffec
to
fcy
tok
inin
sin
com
bin
atio
nw
ith
aux
ins
on
mo
rph
og
enes
isd
uri
ng
the
cult
ure
of
exp
lan
ts
Cy
tok
inin
sA
ux
inP
.vi
llo
sum
var
.d
ensi
ssim
um
P.
insi
gn
eP
.b
ella
tulu
mP
.a
rmen
iacu
m
TD
Z
(mg
l-1)
BA
(mg
l-1)
NA
A
(mg
l-1)
Sh
oo
t/ex
pla
nt
(no
.)
Sh
oo
tle
ng
th
(cm
)
Sh
oo
t/ex
pla
nt
(no
.)
Sh
oo
tle
ng
th
(cm
)
Ro
ot
tub
ercl
e
(cm
3)
Sh
oo
t/ex
pla
nt
(%)
Sh
oo
tle
ng
th
(cm
)
Sh
oo
t/n
od
e
(no
.)
Sh
oo
tle
ng
th
(cm
)
0.0
10
0.1
00
00
00
00
0.6
71
.05
a
0.0
20
0.1
00
00
00
00
10
.66
b
0.0
50
0.1
00
00
00
00
1.0
81
.18
a
0.1
00
0.1
00
00
00
00
1.1
30
.93
a
0.2
00
0.1
00
00
00
00
1.0
70
.73
b
0.0
10
0.5
00
00
00
00
0.7
20
.75
a
0.0
20
0.5
00
.33
0.8
0a
00
0.4
00
.33
0.5
0b
0.9
60
.53
b
0.0
50
0.5
00
.33
1.1
0a
00
00
01
0.7
3b
0.1
00
0.5
00
.33
0.3
3c
00
00
00
.98
0.6
4b
0.2
00
0.5
00
00
00
00
1.1
00
.90
b
00
.20
0.0
20
00
00
00
10
.76
b
00
.50
0.0
20
.33
0.3
0c
0.3
30
.15
c0
00
0.8
00
.68
b
01
.00
0.1
00
00
00
00
0.8
30
.82
a
01
.50
0.1
00
00
.67
0.5
8b
00
01
.67
0.9
7a
02
.00
0.2
00
00
.33
0.2
6c
00
.33
0.3
0b
1.0
01
.36
a
02
.50
0.2
00
00
.33
1.0
0b
00
00
.88
1.1
4a
03
.00
0.3
00
.33
1.6
0a
00
00
01
0.2
3c
03
.50
0.3
00
00
01
.00
00
10
.70
b
00
.20
0.1
00
01
1.3
7a
00
00
.78
0.2
7c
00
.50
0.1
00
00
00
00
0.8
00
.42
c
01
.00
0.5
00
01
1.2
3a
00
.33
0.6
2a
1.1
40
.27
c
01
.50
0.5
00
.67
2.1
5a
00
00
00
.71
0.8
8a
02
.00
1.0
00
00
00
00
1.1
70
.75
a
02
.50
1.0
00
00
01
.00
00
1.1
70
.30
c
03
.00
1.0
01
00
1.8
5a
00
2.0
00
01
.20
0.8
0a
03
.50
1.0
00
.67
0.4
5c
00
1.5
00
01
.11
0.6
1b
04
.00
0.1
00
0B
row
n0
00
02
1.2
0a
04
.50
0.1
00
.33
0.5
0c
Bro
wn
00
00
1.1
40
.82
a
05
.00
0.1
00
0B
row
n0
00
01
1.0
3a
05
.50
0.1
00
.33
0B
row
n0
00
0–
–
06
.00
0.1
01
00
.00
0.6
8b
Bro
wn
00
00
11
.10
a
04
.00
0.5
00
0B
row
n0
00
01
.38
1.7
5a
04
.50
0.5
00
.33
1.0
0a
Bro
wn
00
00
––
158 Plant Cell Tiss Organ Cult (2010) 101:151–162
123
drastically at 210 DAP when the seeds were fully mature.
At 150 DAP, the cuticular layer was not fully formed and
suspensor cells were vacuolated, thereby enabling func-
tional nutrition uptake (Lee 1998). Our results as well as
those of Ren and Wang (1987), Lee (1998, 2007), and Ding
et al. (2004) show that the influence of seed maturity on
germination is species-specific. Orchid seed germination is
relatively low due to the fact that an endosperm fails to
develop during seed development (Lee et al. 2006); in
addition, a thin seed coat may not sufficiently protect the
embryo from desiccation. The formation of a prominent
cuticle on the embryo surface may ensure moisture reten-
tion by embryo cells as well as offer physical protection.
However, the cuticle lowers the success rate of in vitro
orchid seed germination (Lee et al. 2006).
We found that the relatively low mineral salt and
inorganic N concentrations in KC and VW medium pro-
moted seed germination of P. villosum var. densissimum
(mineral salt concentrations: KC, 13.42 mM, WV,
16.3 mM; inorganic N concentrations: KC, 12.25 mM,
VW, 3.78 mM; Fig. 1). Germination frequency was high-
est on the medium amended with KC; this result is similar
to that reported by Johnson and Kane (2007) working with
Vanda hybrids (orchid). In contrast, Bhaskar and Rajeevan
(1996) reported a higher frequency of germination for
Vanda ‘John Club’ on half-strength MS than on KC, while
Roy and Banerjee (2002), working with Vanda tessellata
(Roxb.) Hook. ex Don, obtained similar germination rates
on half-strength MS and KC. The stimulative effect of the
KC medium on germination frequency may be related to
the fact that KC has a relatively high calcium concentra-
tion (4.23 mM) compared to VW (1.94 mM), and quarter-
and half-strength MS (0.75 and 1.5 mM, respectively). The
supply of calcium significantly influences plant growth and
cell karyokinesis. Larger protocorms were obtained on VW
than on half-strength MS (Fig. 1), which may be related to
the relatively high phosphate concentration (4.74 mM) of
VW compared to quarter- and half-strength MS (0.32 and
0.62 mM, respectively) and KC (1.84 mM). Phosphate
uptake by rhizoids (which emerged after embryo swelling
and testa breakage) may have stimulated protocorm
growth. This response is similar to other observations on
the orchid Bletia purpurea (Lam.) DC (Dutra et al. 2008).
The germination frequency of P. villosum var. densiss-
imum was highest on the glucose-amended medium and
was almost twofold higher than that on the sucrose-amen-
ded medium (Fig. 2), which is consistent with the obser-
vations of Cao and Jia (2003), Xie (2003), Cuenca and
Vieitez (2000), and Abdoulaye and Mark (2006). Maltose
has been reported as being an effective sugar source for
Oncidium ‘Gower Ramsey’ plantlet growth (Jheng et al.
2006). However, our findings showed that maltose was less
effective in promoting germination in Paphiopedilum,Ta
ble
3co
nti
nu
ed
Cy
tok
inin
sA
ux
inP
.vi
llo
sum
var
.d
ensi
ssim
um
P.
insi
gn
eP
.b
ella
tulu
mP
.a
rmen
iacu
m
TD
Z
(mg
l-1)
BA
(mg
l-1)
NA
A
(mg
l-1)
Sh
oo
t/ex
pla
nt
(no
.)
Sh
oo
tle
ng
th
(cm
)
Sh
oo
t/ex
pla
nt
(no
.)
Sh
oo
tle
ng
th
(cm
)
Ro
ot
tub
ercl
e
(cm
3)
Sh
oo
t/ex
pla
nt
(%)
Sh
oo
tle
ng
th
(cm
)
Sh
oo
t/n
od
e
(no
.)
Sh
oo
tle
ng
th
(cm
)
05
.00
0.5
01
1.9
3a
Bro
wn
00
00
0.9
00
.92
a
05
.50
0.5
00
0B
row
n0
00
.33
0.6
4a
––
06
.00
0.5
00
0B
row
n0
00
00
.80
0.7
0b
Mea
nin
vit
ro-i
ncu
bat
edex
pla
nts
of
fou
rP
ap
hio
ped
ilu
msp
ecie
sfr
om
cyto
kin
ins
and
aux
inco
mb
inat
ion
s.D
ata
wer
ere
cord
ed3
mo
nth
sfo
llo
win
gth
ein
itia
tio
no
ftr
eatm
ent.
Eac
hm
ean
isb
ased
on
av
isu
alo
bse
rvat
ion
Dat
aw
ere
log
arit
hm
ical
lytr
ansf
orm
edp
rio
rto
AN
OV
Aan
dco
nv
erte
dto
the
ori
gin
alsc
ale
for
dem
on
stra
tio
nin
this
tab
le
Mea
ns
foll
ow
edb
yd
iffe
ren
tlo
wer
-cas
ele
tter
sw
ith
ina
colu
mn
are
sig
nifi
can
tly
dif
fere
nt
atth
eP
\0
.05
lev
elb
yth
eL
SD
test
–,
Lac
ko
fp
lan
tm
ater
ial
Plant Cell Tiss Organ Cult (2010) 101:151–162 159
123
which is consistent with the observations of Huang et al.
(2001), Shen and Xu (1997), Oldach et al. (2001), Cao and
Jia (2003), and Abdoulaye and Mark (2006).
Potato is generally used more frequently than apple as a
natural substance in tissue culture work. Our results show
that the germination frequency and leaf size of P. villosum
var. densissimum in the apple and chayote treatments were
the same as those found in the potato treatment (Fig. 3).
Potato is most likely used for its easy storage and inex-
pensiveness. Because components of natural addenda differ
depending on variety, production area and methods, and
fruit maturity, our results serve mainly as an indication of
the relative influence of different natural sources, espe-
cially in terms of Paphiopedilum germination.
Callus induction
Our results demonstrate that, irregardless of the callus
induction media treatment and size of the in vitro leaf
fragment, the inducement of callus formation was generally
unsuccessful. Only a few calli were induced from proto-
corms (Table 2), indicating that either the composition of
the medium was not suitable for Paphiopedilum spp. or the
methodology was not suitable, since brown exudates cov-
ered most of the solid medium during explant culture and
may have affected the capacity of explants to absorb var-
ious nutrients. Lin et al. (2000) also found the inducement
of callus from protocorms of a Paphiopedilum hybrid
[P. callosum (Rchb.f.) Stein ‘Oakhil’ 9 P. lawrenceanum
(Rchb.f.) Pfitzer ‘Tradition’] to be extremely slow, but
about 42–65% of the protocorms in their experiment pro-
duced yellowish-white calli; however, stem, root tip, and
green leaf explants of another Paphiopedilum hybrid
[P. henryanum Braem ‘#1’ 9 P. philippinense (Rchb.f.)
Stein ‘#10’] failed to produce viable callus. The frequency
of explants forming callus of a Cymbidium hybrid was 50%
(Huan et al. 2004). These results imply that callus induc-
tion for interspecific hybrids is challenging and show that
the induction success for hybrids was greater than that for
the natural species since the PRG treatment combinations
in our investigation were relatively ineffective in inducing
Paphiopedilum callus (Table 2). Thus, methods and
materials other than those used in our study need to be
developed for the mass propagation of these orchid species.
Shoot multiplication in vitro
Nhut et al. (2007), working with Paphiopedilum delenatii,
showed that TDZ was more effective than BA in inducing
shoot organogenesis, even though the regeneration rate for
TDZ was only 75%. Our investigation revealed no advan-
tages of replacing BA with TDZ in terms of inducing shoot
organogenesis in our four Paphiopedilum species (Table 3).
Huang et al. (2001) found that the shoot number of Pa-
phiopedilum hybrids doubled every 12 weeks when treated
with BA and NAA, and that TDZ inhibited shoot prolifer-
ation and rooting of Paphiopedilum hybrids. Yan et al.
(2006) and Lu et al. (2001) reported that as many as two to
seven shoots per plant were produced on Cypripedium fla-
vum P. H. Hunt. et Summerh and Cymbidium ensifolium cv.
Yuh Hwa rhizomes when the latter were treated with BA.
Twelve shoots per node were induced for Vanda spathulata
(L.) Spreng. when treated with 44.4 lM BA with 17.1 lM
or 28.5 lM indole-3-acetic acid (IAA; William et al. 2003);
Dendrobium candidum Wall. ex Lindl. produced five shoots
per explant when treated with half-strength MS supple-
mented with 2 mg l-1 BA, 0.2 mg l-1 NAA, and
0.1 mg l-1 IBA (Luo et al. 2006). The maximum number of
adventitious buds per explants that has been reported to
date—an average of 24.5—were generated from Dendro-
bium candidum explants treated with 1.2 mg l-1 BA and
1.2 mg l-1 NAA (Zhao et al. 2007). Results from Martin
(2003), Chen et al. (2004a), Thomas and Michael (2007),
and Hong et al. (2008) show that, in most cases, Paphio-
pedilum spp. induce fewer shoots than other orchids.
The concentration of exogenous PGRs required for the
micropropagation of Paphiopedilum was found to be spe-
cies-specific (Table 3). A relatively high BA concentration
increased the shoot number in P. villosum var. densissimum
and P. armeniacum but decreased it in P. insigne. This
finding is similar to those of Molina et al. (2007) working
with Troyer citrange (Citrus sinensis [L.] Osbeck 9 Pon-
cirus trifoliata [L.] Raf.). In our study, a BA concentration
[3.5 mg l-1 caused extensive necrosis of P. insigne
explants. For P. bellatulum, organogenesis occurred with
only four PGR combinations. Shoot numbers in all four
orchid species were the same at TDZ concentrations
ranging from 0.01 to 0.1 mg l-1.
Organogenesis occurred on P. armeniacum rhizomes for
all PGR combinations (Table 3). Underground rhizomes of
temperate terrestrial Cymbidium species are useful organs
for micropropagation (Shimasaki and Uemoto 1990), but
this type of morphogenetic development is not a common
phenomenon in the Orchidaceae. There are 18 Chinese
species of Paphiopedilum, and only P. armeniacum and
P. micranthum have underground rhizomes, although a few
short rhizomes are occasionally observed on P. malipoense
S. C. Chen et Z. H. Tsi (Chen and Tsi 2003). Thus, prop-
agation of rhizomatous species, such as P. armeniacum,
facilitates clonal reproduction and enables mass propaga-
tion, whereas this is not true for the other three species used
in this study.
We found that when the BA concentration C2 mg l-1 or
higher, root tubercles were formed for P. insigne (Table 3,
Fig. 4j) and that the size of the tubercle and the fate of
plantlets depended on BA concentration. Root tubercles are
160 Plant Cell Tiss Organ Cult (2010) 101:151–162
123
atypical of Paphiopedilum spp. roots. P. insigne plantlets
turned brown when the concentration of BA was
[3.5 mg l-1. Thus, the concentration of the PGR is an
important factor to be taken into consideration in Paphio-
pedilum in vitro culture.
In most cases, a high ratio of cytokinin to auxin enhanced
shoot formation (PGR experiment, Table 3). This effect
was species-specific. Maximum shoot production for
P. villosum var. densissimum, P. insigne, P. bellatulum, and
P. armeniacum occurred at approximate ratios of 10:1, 2:1,
11:1, and 40:1 or 8:1, respectively. In some cases, such as
P. armeniacum, BA was unnecessary for shoot production;
in other cases, there was a wide range of ratios at which
maximum shoot formation occurred (e.g., P. armeniacum).
Shoot proliferation from leaf tissue is common in ferns
and dicotyledons and less common in monocotyledons
(Xiong and Wu 2003). The results from our investigation
indicate that the frequency of in vitro seed germination and
shoot organogenesis in Paphiopedilum species is lower
than that of most other orchid species, even when in vitro
techniques are used. However, our work sheds light on
techniques to maximize propagation success by employing
in vitro seed germination and shoot organogenesis meth-
odologies. As such, our results are relevant to the perpet-
uation of endangered Paphiopedilum spp.
Conclusion
The results of this study are promising in terms of devel-
oping a propagation protocol for the mass production of
P. villosum var. densissimum, P. bellatulum, P. insigne,
and P. armeniacum. Conventional seed propagation tech-
niques for Paphiopedilum spp. are relatively simple, but
the approximately 4-year period from seedling to seed
production renders seeds as an unlikely propagation start-
ing point, especially in terms of meeting horticultural
market demands. Thus, the use of plantlets or rhizomes
derived from in vitro seed germination for shoot multipli-
cation will significantly shorten the production cycle. Our
methods may prove to be of valuable for both horticultural
and conservation purposes.
Acknowledgments The authors thank Chunyan Han, Guangwan
Hu, and Yuan Liu for their kind help. This work was supported by the
Natural Science Foundation of Yunnan (2005C0053 M), the Ministry
of Science and Technology of China (2005DK21006) and the Min-
istry of Education of China (B08044 & MUC-985-3-3).
References
Abdoulaye T, Mark J (2006) Effects of carbon source and explant
type on somatic embryogenesis of four Cacao genotypes.
HortScience 41(3):753–758
Arditti J, Ernst R (1993) Micropropagation of orchids. John Wiley
and Sons, New York
Banziger H (1996) The mesmerizing wart: the pollination strategy of
epiphytic lady slipper orchid Paphiopedilum villosum (Lindl.)
Stein (Orchidaceae). Bot J Linn Soc 121:59–90
Bhaskar J, Rajeevan PK (1996) Embryo culture of Vanda ‘John
Club’. S Indian Hortic 44:36–38
Cao SD, Jia HT (2003) Effects of different carbon source on tissue
culture of Strawberry. Shandong For Sci Technol 126(3):6–7
Chen SC, Tsi ZH (2003) The orchids of China, 2nd edn. Chinese
Forest Press, Beijing, pp 116–126
Chen TY, Chen JT, Chang WC (2004a) Plant regeneration through
direct shoot bud formation from leaf cultures of Paphiopediulumorchids. Plant Cell Tiss Organ Cult 76:11–15
Chen ZL, Ye XL, Liang CY, Duan J (2004b) Seed germination in
vitro of Paphiopedilum armeniacum and P. micranthum. Acta
Hortic Sinica 31(4):540–542
Convention on International Trade in Endangered Species of Wild Fauna
and Flora Appendices I, II and III (2008) Geneva, Switzerland.
Available at: http://www.cites.org/eng/app/E-Jul01.pdf
Cuenca B, Vieitez M (2000) Influence of carbon source on shoot
multiplication and adventitious bud regeneration in vitro beech
cultures. Plant Growth Regul 32:1–12
Ding CC, Wu H, Liu FY (2004) Factors affecting the germination of
Paphiopedilum armeniacum. Acta Bot Yunnanica 26(6):673–677
Dutra D, Johnson TR, Kauth PJ, Stewart SL, Kane ME, Richardson L
(2008) Asymbiotic seed germination, in vitro seedling develop-
ment, and greenhouse acclimatization of the threatened terrestrial
orchid Bletia purpurea. Plant Cell Tiss Organ Cult 94:11–21. doi:
10.1007/s1124000893820
Hong PI, Chen JT, Chang WC (2008) Plant regeneration via
protocorm like body formation and shoot multiplication from
seed derived callus of a maudiae type slipper orchid. Acta
Physiol Plant 30:755–759. doi:10.1007/s1173800801582
Huan LVT, Takamura T, Tanaka M (2004) Callus formation and
plant regeneration from callus through somatic embryo struc-
tures in Cymbidium orchid. Plant Sci 166:1443–1449
Huang LC, Lin CJ, Kuo CI, Huang BL, Murashige T (2001)
Paphiopedilum cloning in vitro. Sci Hortic 91:111–121
Jheng FY, Do YY, Liauh YW, Chung JP, Huang PL (2006)
Enhancement of growth and regeneration efficiency from
embryogenic callus cultures of Oncidium ‘Gower Ramsey’ by
adjusting carbohydrate sources. Plant Sci 170:1133–1140
Johnson TR, Kane ME (2007) Asymbiotic germination of ornamental
Vanda: in vitro germination and development of three hybrids.
Plant Cell Tiss Organ Cult 91:251–261
Knudson C (1946) A nutrient for germination of orchids. Am Orchid
Soc Bull 15:214–217
Lee YI (1998) The study of embryo development and seed
germination in vitro in slipper orchids. MSc thesis. National
Taiwan University, Taiwan
Lee YI (2007) The asymbiotic seed germination of six Paphiopedilumspecies in relation to the time of seed collection and seed
pretreatment. Acta Hortic 755:381–385
Lee YI, Yeung EC, Lee N, Chung MC (2006) Embryo development
in the Lady’s slipper orchid, Paphiopedilum delenatii, with
emphasis on the ultrastructure of the suspensor. Ann Bot
98:1311–1319
Li JM (2002) Textbook of plant tissue culture, 2nd edn. Agriculture
University of China, Beijing, p 25
Lin YH, Chang C, Chang WC (2000) Plant regeneration from callus
culture of a Paphiopedilum hybrid. Plant Cell Tiss Organ Cult
62:21–25
Liu ZJ, Liu KW, Chen LJ (2006) Conservation ecology of endangered
species Paphiopedilum armeniacum (Orchidaceae). Acta Ecol
Sinica 26(9):2791–2800
Plant Cell Tiss Organ Cult (2010) 101:151–162 161
123
Lu I, Sutter E, Burger D (2001) Relationships between benzyladenine
uptake, endogenous free IAA levels and peroxidase activities
during upright shoot induction of Cymbidium ensifoilum cv. Yuh
Hwa rhizomes in vitro. Plant Growth Regul 35:161–170
Luo JF, Cheng ZY, Long CL (2006) Studies on the rapid propagation
and in vitro storage of Dendrobium candidum. Guihaia 26(1):
69–73
Martin KP (2003) Clonal propagation, encapsulation and reintroduc-
tion of Ipsea malabarica (Reichb.f.) J. D. Hook., an endangered
orchid. In Vitro Cell Dev Biol Plant 39:322–326
Molina RV, Castello S, GarcıaLuis A, Guardiola JL (2007) Light
cytokinin interactions in shoot formation in epicotyl cuttings of
Troyer citrange cultured in vitro. Plant Cell, Tissue Organ Cult
89:131–140
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bio assays with tobacco tissue cultures. Physiol Plant 15:473–
497
Nagashima T (1982) Studies in the seed germination and embryo-
genesis in goeringii Rchb. f. and Paphiopedilum insigne var.
sanderae Rchb. J Jpn Soc Hortic Sci 51:94–105
Nhut DT, Trang PTT, Vu NH, Thuy DTT, Van Khiem D, Van Binh
N, Van KTT (2005) A wounding method and liquid culture in
Paphiopedilum delenatii propagation. Propag Ornam Plants
5:158–163
Nhut DT, Thuy DTT, Don NT, Luan VQ, Hai NT, Van KTT,
Chinnappa CC (2007) In vitro stem elongation of Paphiopedilumdelenatii Guillaumin and shoot regeneration via stem node
culture. Propag Ornam Plants 7:29–36
Oldach KH, Morgnestern A, Rother S, Girgi M et al (2001) Efficient
in vitro plant regeneration from immature zygotic embryos of
pearl millet (Pennisetum glaucum L. R. Br.) and Sorghumbicolor L. Moench. Plant Cell Rep 20:416–421
Pierik RLM, Sprenkels PA, Vanderharst B, Vandermeys QG (1988)
Seed germination and further development of plantlets of
Paphiopedilum ciliolare Pfitz in vitro. Sci Hortic 34:139–153
Ren L, Wang FX (1987) Embryological studies of Paphiopedilumgodefroyae stein. Acta Bot Sinica 29(1):14–21
Roy J, Banerjee N (2002) Optimization of in vitro seed germination,
protocorm growth and seedling proliferation of Vanda tessellata(Roxb.) Hook ex G Don. Phytomorph 52:167–178
Shen XL, Xu Z (1997) Studies on the conditions of tissue culture in
Stevia Rebaudiana BertoniII. Effect of different elementary
culture medium and carbon resource for callus formation and
germination. Sugar Crops China 4:9–10
Shi J, Luo YB, Bernhardt P, Ran JC, Liu ZJ, Zhou Q (2008)
Pollination by deceit in Paphiopedilum barbigerum (Orchida-
ceae): a staminode exploits the innate colour preferences of
hoverflies (Syrphidae). Plant Biol 11:17–28
Shimasaki K, Uemoto S (1990) Micropropagation of a terrestrial
Cymbidium species using rhizomes developed from seeds and
pseudobulbs. Plant Cell Tiss Organ Cult 22:237–244
Stewart J, Button J (1975) Tissue culture studies in Paphiopedilum.
Am Orchid Soc Bull 44:591–599
Stimart DP, Ascher PD (1981) In vitro germination of Paphiopedilumseed on a completely defined medium. Sci Hortic 14:165–170
Tay LJ, Takeno K, Hori Y (1988) Culture conditions suitable for in
vitro seed germination and development of seedling in Paphio-pedilum. J Jpn Soc Hortic Sci 57:243–249
Thomas TD, Michael A (2007) High frequency plantlet regeneration
and multiple shoot induction from cultured immature seeds of
Rhynchostylis retusa Blume., an exquisite orchid. Plant Bio-
technol Rep 1:243–249
Vacin EF, Went FW (1949) Some pH changes in nutrient solutions.
Bot Gaz 110:605–613
William DS, Gangaprasad A, Seeni S, Sarojini MV (2003) Micro-
propagation and ecorestoration of Vanda spathulata, an exquisite
orchid. Plant Cell Tiss Organ Cult 72:199–202
Xie ZB (2003) Effect of casein hydrolyzed and different carbon
source on tissue culture of kiwifruit. Agric Technol 23(4):56–59
Xiong L, Wu LF (2003) Ornamental tissue culture and large scale
production. Chemical Industry Press, Beijing
Yan N, Hu H, Huang JL, Xu K, Wang H, Zhou ZK (2006)
Micropropagation of Cypripedium flavum through multiple
shoots of seedlings derived from mature seeds. Plant Cell,
Tissue and Organ Cult 84:114–118
Zeng SJ, Chen ZL, Duan J (2006) Asepsis sowing and in vitro
propagation of Paphiopedilum hirsutissimum Pfitz. Plant Physiol
Commun 42(2):247
Zhao P, Wang W, Feng FS, Wu F, Yang ZQ, Wang WJ (2007) High
frequency shoot regeneration through transverse thin cell layer
culture in Dendrobium candidum Wall ex Lindl. Plant Cell Tiss
Organ Cult 90:131–139
162 Plant Cell Tiss Organ Cult (2010) 101:151–162
123