PLANT REGENERATION FROM PROTOCORM-DERIVED CALLUS OF CYPRIPEDIUM FORMOSANUM
YUNG-I LEE* AND NEAN LEE
Department of Horticulture, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei, Taiwan
(Received 27 September 2002; accepted 6 January 2003; editor S. Guha-Mukherjee)
Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived
protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52mM 2,4-dichlorophenoxyacetic
acid and 4.54mM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained
by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4mm callus
after being transferred to the medium with 4.44mM N6-benzyladenine after 8wk of culture. The regenerated protocorm-
like bodies formed shoots and roots on medium containing 1gl21activated charcoal and 20gl21potato homogenate. After
24wk of culture on this medium, well-developed plantlets ready for potting were established.
Key words: Cypripedium formosanum; callus induction; plant regeneration; protocorm-like body.
Cypripedium formosanum, commonly known as slipper orchid, is
a popular terrestrial orchid with attractive pinkish flowers which has
the potential for increased use in perennial gardening and as a pot
flower crop. The wild populations are now considered to be extinct
as a result of over-collection and habitat destruction. For
commercial requirements and the conservation of endangered
species, it is desirable to establish protocols for rapid and large-
scale clonal propagation.
Methods of micropropagation have been documented in many
orchids, but the slipper orchids are still difficult to propagate
in vitro (Arditti and Ernst, 1993). In Paphiopedilum (a relative of
Cypripedium), Stewart and Button (1975) reported that a few
plantlets could be regenerated from shoot apex-derived callus, but
that the callus eventually failed to survive during subcultures.
Recently, a procedure with reproducible results to obtain plantlets
from callus culture of a Paphiopedilum hybrid was reported
(Lin et al., 2000). However, there has been no such report for
Cypripedium. We report here a reliable in vitro regeneration
procedure of C. formosanum from subcultured protocorm-derived
callus. In the investigation reported here, seed-derived protocorm
segments were used for callus induction and subsequent plant
Materials and Methods
greenhouse at Mei-Fung farm (2000m above sea level) in Taiwan.
A number of flowers were self-pollinated manually by transferring pollinia
onto the stigma of the same flower in April (mean temperature 108C). Green
Plants of C. formosanum were cultivated in a
surface-sterilized with a 1% sodium hypochlorite solution for 20min and
rinsed three times with sterile distilled water. Capsules were cut open and
the seeds were scooped out with forceps onto the quarter-strength macro-
elements and full-strength micro-elements of MS medium (Murashige and
Skoog, 1962) supplemented with (mgl21): myo-inositol (100), niacin (0.5),
pyridoxine HCl (0.5), thiamine HCl (0.1), glycine (2.0), tryptone (1000),
sucrose (20 000), and Gelritee (2200).
The seed cultures were incubated in darkness at 25 ^ 28C for 12wk to
develop into small round protocorms, then transferred to illuminated
conditions at 25–30mmolm22s21(daylight fluorescent tube FL-20D/18, 20
W, China Electric Co., Taipei). Twenty weeks after sowing, cone-shaped
protocorms (approximately 5–6mm in diameter) were bisected transversally.
The segments were then used as explants for callus induction.
Callus induction from protocorm segments.
1/2-strength and 1/4-strength macro-elements of MS basal salts (1/2-MS
and 1/4-MS) with full-strength micro-elements supplemented with (mgl21):
myo-inositol (100), niacin (0.5), pyridoxine HCl (0.5), thiamine HCl (0.1),
glycine (2.0), sucrose (20 000), and Gelritee (2200) were selected for this
trial. Plant growth regulators were added prior to autoclaving as optional
additives according to the experimental objectives. 2,4-Dichlorophenoxy-
acetic acid (2,4-D; 0, 0.45, and 4.52mM), N6-benzyladenine (BA; 0, 4.44,
and 22.19mM), and thidiazuron (TDZ; 0, 0.45, and 4.54mM) were added to
the basal media in this experiment. The pH of the media was adjusted to 5.5
with 1N KOH or HCl prior to autoclaving at 1218C for 15min. Six
protocorm segments were inoculated onto 10ml of Gelrite-solidified medium
in 25 £ 100mm culture tubes and incubated in darkness.
Protocorm-like body (PLB) formation.
in diameter) was placed on 1/4-MS basal medium supplemented with
(mgl21): myo-inositol (100), niacin (0.5), pyridoxine HCl (0.5), thiamine
HCl (0.1), glycine (2.0), sucrose (20 000), and Gelritee (2200). The
combinations of plant growth regulators were the same as callus induction
except the removal of 2,4-D. The explants were cultured in 10ml of medium
in 25 £ 100mm culture tubes and incubated under illumination.
Plantlet regeneration. About 6mm-long PLBs were placed on 1/4-MS
basal medium supplemented with (mgl21): myo-inositol (100), niacin (0.5),
pyridoxine HCl (0.5), thiamine HCl (0.1), glycine (2.0), sucrose (20 000),
activated charcoal (1000), and Gelritee (2200). Peptone (0.5 and 1gl21),
potato homogenate (20 and 50gl21), and banana homogenate (20 and
50gl21) were added to the basal media in this experiment. Potato and ripe
banana were purchased from a local supplier, then peeled and cut into about
1cm3sections. These fresh materials were boiled for 10min with 100ml of
distilled water and homogenized with a kitchen blender. The homogenate
For callus induction,
One piece of callus mass (4mm
*Author to whom correspondence should be addressed: Email leeyungi@
In Vitro Cell. Dev. Biol.—Plant 39:475–479, September–October 2003
q 2003 Society for In Vitro Biology
was added to the basal medium as required before the pH was adjusted.
Explants were incubated in 25 £ 100mm culture tubes under illumination.
Cultural conditions.All cultures were incubated at 25 ^ 28C, and
under a 12/12-h photoperiod at 25–30mmolm22s21(daylight fluorescent
tubes FL-20D/18, 20W, China Electric Co.) or in the dark.
Histological observation.Some callus masses after 2 and 4wk of culture
were fixed in a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in
0.1M sodium phosphate buffer (pH 7.2) at 48C, overnight. After three
15-min buffer rinses, the materials were postfixed in 1% OsO4in the same
buffer for 4h at room temperature and then rinsed in three 15-min changes
of buffer. Following fixation, the materials were dehydrated in an acetone
series and embedded in Spurr’s resin (Spurr, 1969). Serial sections, 1mm
thick, were cut using a glass knife on a Richert-Jung Ultracut E
ultramicrotome. These sections were stained with periodic acid Schiff (PAS)
reagent and counter-stained with 1% toluidine blue O (Yeung, 1984).
Statistical analysis. Experiments were performed in a randomized
design and repeated twice. Eighteen replicates were taken for each
treatment in the experiment of callus induction, and six explants were
planted in each culture tube. Observations were made after 24wk of culture.
For PLB formation, 27 replicates were used for each treatment, and one
explant was planted in each culture tube. Observations were made after 8wk
of culture. For plantlet regeneration, 20 replicates were used for each
treatment, and two explants were planted in each culture tube. Observations
were made after 8wk of culture. The data were statistically analyzed and
means were compared using Duncan’s multiple range test (Duncan, 1955).
Induction and subculture of callus.
was extremely slow, and browning of protocorm segments was
common in most cases. The cultures on 1/4-MS medium showed
better induction of callus compared to those on 1/2-MS medium
(Table 1). More phenolic compounds were released in the 1/2-MS
medium that not only retarded the formation of callus but also
caused lethal effects on the explants. About 11.6–55.8% of
explants produced yellowish friable callus from the incision areas of
protocorm segments (Fig. 1A) in the dark on 1/4-MS basal media
Initially, callus formation
containing both 2,4-D (0.45–4.52mM) and TDZ (0.45–4.54mM)
after 16–24wk of culture. In contrast, 10.6–19.7% of callus
formation was obtained on 1/2-MS basal media. Small amounts of
yellowish and non-friable callus formed on 1/4-MS basal medium
containing 4.52mM 2,4-D alone, but they failed to proliferate. The
results showed that BA was neither able to induce callus formation
alone, nor in combination with 2,4-D. TDZ alone also failed to
induce callus formation. In most cases, explants formed multiple
shoots on media containing BA. The percentage of callus induction
was greater (55.8%) on medium containing 4.52mM 2,4-D and
4.54mM TDZ as compared to the other combinations of 2,4-D and
Protocorm-derived callus was subcultured at 8wk intervals on
1/4-MS medium in combination with 2,4-D (0.45–4.52mM) and
TDZ (0.45–4.54mM) (data not shown). After two subcultures, some
yellowish and friable callus differentiated into globular structures
on medium containing 0.45mM 2,4-D and 0.45mM TDZ. On the
other hand, most of the proliferated callus still remained yellowish
with a friable surface on the medium containing 4.52mM 2,4-D and
4.54mM TDZ. Hence, the medium supplemented with 4.52mM 2,4-
D and 4.54mM TDZ was selected as the standard maintenance
medium for proliferation of totipotent callus.
PLB formation. Within 24wk of subculture (subcultured
bimonthly), each callus mass (4mm diameter) was transferred
onto the media under illumination for PLB formation studies
(Table 2). About 11.1–96.2% of the callus produced globular
granuleswithin 4wkof culture.
transformed into PLBs after 8wk of culture (Fig. 1B, C). The
optimum growth regulator concentration for regeneration from
totipotent callus was found to be 4.44mM BA, where the highest
percentage of PLB formation (96.2%) and the maximum number of
PLBs per explant (13 PLBs) were observed. The medium with no
growth regulators or containing 2,4-D alone resulted in PLB
formation, but with a lower number of PLBs. On the media with
combinations of 2,4-D and TDZ, most callus turned yellowish green
and became compact in appearance under illumination that gave
rise to a lower percentage of PLB formation (11.1–12.9%).
Histological examination revealed that globular-shaped granule
formation occurred at the periphery of the callus mass. Small
clusters of cytoplasmic cells subtended by a few vacuolated cells
were discernible after 2wk of culture on the medium containing
4.44mM BA (Fig. 2A). These clusters of cells continued to grow,
and the globular-shaped granules started to emerge (Fig. 2B). They
were characterized by globular-shaped embryo proper, formed by
small cytoplasmic cells. Small starch grains began to appear within
the cells at the center of the embryo proper, and a differentiated
protoderm proper was observed at this time.
Regeneration of plantlets from PLBs.
peptone, potato homogenate, and banana homogenate were tested in
the experiments (Table 3). Survival percentage of PLBs was high on
media with 1% peptone and 20gl21potato homogenate. In contrast,
survival of PLBs was poor on media with 20 and 50gl21banana
homogenate. Shoot buds and root formation occurred after 3wk of
culture (Fig. 1D). The optimum rooting response was obtained on
the medium with 20gl21potato homogenate. In this medium,
plantlets produced an average of 7.2 thick, healthy roots, each
4.7cm in length within 10wk of culture (Fig. 1E). In a period of
24wk of culture, PLBs grew into plantlets about 6–7cm tall which
were ready for transplanting to pots (Fig. 1F).
Different concentrations of
EFFECT OF COMBINATIONS OF 2,4-D WITH TDZ OR BA ON
CALLUSING OF SEED-DERIVED PROTOCORM SEGMENTS OF
CYPRIPEDIUM FORMOSANUM WITH 1/2-STRENGTH AND 1/4-
STRENGTH MACRO-ELEMENTS OF MS BASAL SALTS (AFTER 8WK
OF CULTURE IN THE DARK)
Plant growth regulators (mM) % Callusing
2,4-D BATDZ 1/2-MS1/4-MS
Means of 18 replicates with the same letters are not significantly different
at P , 0.05 (Duncan, 1955).
LEE AND LEE
seed-derived protocorm segment after 24wk on basal medium with 4.52mM 2,4-D and 4.54mM TDZ following induction (bar ¼ 12mm).
B, PLBs formed after 8wk of culture from protocorm-derived callus on basal medium with 4.44mM BA (bar ¼ 1.2mm). C, An elongated
PLB (bar ¼ 1.2mm). D, A PLB developed into a shoot bud and a root (bar ¼ 1.5mm). E, Lateral buds proliferating from a sprouting shoot
(bar ¼ 4mm). F, Twelve-month-old PLB-derived plantlets ready for potting (bar ¼ 12mm).
In vitro plantlet regeneration from protocorm-derived callus of Cypripedium formosanum. A, Totipotent callus obtained from a
MICROPROPAGATION OF CYPRIPEDIUM
The four genera (Cypripedium, Paphiopedilum, Phragmipedium,
and Selenipedium) of the subfamily Cypripedioideae are considered
to be difficult to culture in vitro (Arditti and Ernst, 1993). Efforts to
develop tissue culture methods have not had much success (Stewart
and Button, 1975). Recently, plant regeneration from callus culture
of a Paphiopedilum hybrid was demonstrated by Lin et al. (2000).
In the present report, we have described a procedure for callus
induction and plant regeneration from seed-derived protocorm
segments of C. formosanum.
In the present study, of the two cytokinins, BA and TDZ, in
combination with 2,4-D tested for callusing, BA did not show any
response. The superiority of TDZ in promoting callus induction and
in vitro morphogenesis was observed in several orchid species
(Ernst, 1994; Chang and Chang, 1998; Chen et al., 1999). TDZ
alone was efficient at inducing direct somatic embryogenesis from
the leaf explants of Oncidium (Chen et al., 1999), but, as shown
here, was not effective for protocorm segments of C. formosanum.
Thus, the exogenous hormonal requirements for inducing in vitro
morphogenesis of orchid species are quite different. Previously, Lin
et al. (2000) used the medium containing 4.52–45.2mM 2,4-D and
0.45–4.54mM TDZ for induction of totipotent calluses of
Paphiopedilum hybrids. In the present study, both 2,4-D and TDZ
were essential for inducing totipotent callus of C. formosanum, even
at a low concentration (0.45mM). This suggests that the
combination of TDZ with auxins played a crucial role in callus
induction of slipper orchids. Such an effect of the combination of
TDZ with auxins in maintaining long-term totipotent callus culture
of Cymbidium has also been observed (Chang and Chang, 1998).
A small amount of callus was observed on the medium containing
4.52mM 2,4-D alone; but these calluses failed to survive during
subcultures. This result appeared to be similar to that of shoot apex-
derived callus of Paphiopedilum (Stewart and Button, 1975).
Although the effect of organic supplements is complex, many
organic forms of nitrogen and natural organic compounds have been
used in media in the micropropagation of orchids, such as peptone,
tryptone, coconut water, and banana homogenate (Arditti and Ernst,
1993). Of the three organic supplements used, peptone and potato
homogenate significantly promoted rooting of the plantlets. The
highest survival rate (80%) of PLBs and the optimum rooting were
homogenate showed inhibitory effects for PLB survival, as Ichihashi
and Islam (1999) found on callus growth in Phalaenopsis,
Doritaenopsis, and Neofinetia. Effects of organic supplements on
various orchid species may indicate different requirements for
organic compounds. Which factors are particularly responsible for
PLB growth were not made clear by our study and could be the
subject for further investigation.
In the presented regeneration protocol, one small protocorm-
derived callus mass (4mm diameter) yielded 13 PLBs. A large
cytoplasmic cells at the periphery of calluses were discernible after 2wk of culture on the regeneration medium with 4.44mM BA
(bar ¼ 20mm). B, Globular-shaped granules were present within 4wk of culture. They were characterized by a differentiated protoderm
(arrow), and starch grains (arrowhead) begin to appear within the cells at the center of the embryo proper (bar ¼ 50mm).
Formation of globular-shaped granules from protocorm-derived callus of Cypripedium formosanum. A, Small clusters of
EFFECT OF COMBINATIONS OF 2,4-D WITH TDZ OR BA ON
PROTOCORM-LIKE BODY FORMATION FROM PROTOCORM-
DERIVED CALLUS OF CYPRIPEDIUM FORMOSANUM (AFTER 8WK
Percent of explants
Number of PLBs
Means of 27 replicates with the same letters are not significantly different
at P , 0.05 (Duncan, 1955).
LEE AND LEE
number of plantlets can be obtained within 1yr if the totipotent
callus was induced from protocorm segments. This appeared to be a
more reliable method for mass propagation as compared to seed
propagation with low germination frequency. Application of this
protocol to other Cypripedium species or hybrids also needs to be
assessed. In addition, this regeneration system may provide a
suitable procedure for attempting transformation of Cypripedium
In summary, totipotent calluses of C. formosanum were initiated
from excised protocorm segments on basal medium containing
4.52mM 2,4-D and 4.54mM TDZ. For plant regeneration, the
subcultured callus was transferred to medium containing 4.44mM
BA. This callus demonstrated its totipotency by plant regeneration
via PLBs as an intermediate step. The PLBs developed into
plantlets after further incubation on basal medium plus 1gl21
activated charcoal and 20gl21potato homogenate. Following the
10 plantlets could be formed from one piece of protocorm-derived
callus (4mm diameter) after 16wk of culture.
We thank Dr. Edward C. Yeung of the University of Calgary and
Dr. Wei-Chin Chang of the Institute of Botany, Academia Sinica for reading
and commenting on the manucript. This paper represents a portion of the
first author’s dissertation presented to the Faculty of the Research Institute
of Horticulture of the National Taiwan University in partial fulfillment of the
requirements for the degree of Doctor in Agricultural Sciences. The financial
support of the Council of Agriculture of the Republic of China is gratefully
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EFFECT OF PEPTONE, POTATO, AND BANANA HOMOGENATE ON PROTOCORM-LIKE BODY SURVIVAL AND ROOTING OF PLANTLETS OF
CYPRIPEDIUM FORMOSANUM (AFTER 8WK OF CULTURE)
Organic supplement(gl21)Survival rate of PLBs (%) Mean root number per plantlet Mean root length (cm)
zQuarter-strength macro-elements and full-strength micro-elements of MS medium lacking organic supplements served as control.
Means of 20 replicates with the same letters are not significantly different at P , 0.05 (Duncan, 1955).
MICROPROPAGATION OF CYPRIPEDIUM