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In vitro propagation of Paphiopedilum orchids

Article  in  Critical Reviews in Biotechnology · April 2016

DOI: 10.3109/07388551.2014.993585 · Source: PubMed

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Crit Rev Biotechnol, Early Online: 1–14

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REVIEW ARTICLE

In vitro propagation of Paphiopedilum orchids

Songjun Zeng1, Weichang Huang2, Kunlin Wu1, Jianxia Zhang1, Jaime A. Teixeira da Silva3, and Jun Duan1,4
1
Key Laboratory of South China Agricultural Plant Molecular Analysis and Gene Improvement, South China Botanical Garden, Chinese Academy
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15

of Sciences, Guangzhou, China, 2Shanghai Chen Shan Botanical Garden, Shanghai, China, 3P. O. Box 7, Miki-cho post office, Ikenobe 3011-2,
Kagawa-ken, 761-0799, Japan, and 4Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden,
Chinese Academy of Sciences, Guangzhou, China

Abstract Keywords
Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are In vitro, micropropagation, Paphiopedilum,
sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under regeneration, seed germination,
the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their tissue culture
commercial value through large-scale propagation in vitro is an option to reduce pressure from
illegal collection, to attempt to meet commercial needs and to re-establish threatened species History
back into the wild. Although they are commercially propagated via asymbiotic seed
germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by Received 6 November 2013
plant regeneration from tissue culture. This review aims to cover the most important aspects Revised 26 May 2014
and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to Accepted 26 August 2014
emphasize the importance of further improving tissue culture protocols for ex vitro-derived Published online 13 January 2015
explants.
For personal use only.

Introduction Convention on International Trade in Endangered Species of

Paphiopedilum Pfitzer (Orchidaceae) orchids are commonly
known as the slipper orchids because of their resemblance to
the pouch-shaped lip of a lady’s slipper. Members of this
genus are undoubtedly one of the most popular and
commercialized orchids as pot plants and cut flowers due to
a wide variety of flower shapes, sizes and colors, which have
captured the interest of many orchid growers and hobbyists
and are one of the most popular and rare orchid genera being
sold and exhibited today (Cribb, 1998; Liu et al., 2009;
Sheehan & Sheehan, 1994). The genus Paphiopedilum is
comprised of about 96–100 species and its range extends from
India eastwards across southern China to the Philippines and
throughout South-east Asia and the Malay Archipelago to
New Guinea and the Solomon Islands (Cribb, 1998; Liu et al.,
2009; World Checklist of Selected Plant Families, 2014; Wu
et al., 2009). Wild populations are under the threat of
extinction as a result of over-collection and loss of suitable
habitats, and all Paphiopedilum species are listed in the
Address for correspondence: Songjun Zeng, Key Laboratory of South
China Agricultural Plant Molecular Analysis and Gene Improvement,
South China Botanical Garden, Chinese Academy of Sciences,
Guangzhou 510650, China. E-mail address: zengsongjun@scib.ac.cn
Weichang Huang, Shanghai Chen Shan Botanical Garden, Shanghai
201602, China. E-mail: hwc_zx@126.com
Jaime A. Teixeira da Silva, P. O. Box 7, Miki-cho post office, Ikenobe
3011-2, Kagawa-ken, 761-0799, Japan. E-mail: jaimetex@yahoo.com
Duan Jun, Key Laboratory of South China Agricultural Plant Molecular
Analysis and Gene Improvement, South China Botanical Garden,
Chinese Academy of Sciences, Guangzhou 510650, China. E-mail:
duanj@scib.ac.cn
Wild Fauna and Flora (CITES) Appendix I. According to
CITES, trade of these species is prohibited (Zeng et al., 2012).
Paphiopedilum orchids are generally propagated through
the division of axillary buds from the mother plant. This
conventional method of propagation is very inefficient and
time consuming. In a natural setting, Paphiopedilum seed
germinates relatively slowly due to seed morphological and
physiological characteristics, which is similar to other terres-
trial orchids (Lee et al., 2006; Rasmussen, 1995; Zeng et al.,
2012). The discovery of asymbiotic germination of orchid
seeds by Knudson (1921) was the first practical procedure for
the in vitro propagation of any plant under axenic conditions.
In vitro Paphiopedilum seed germination, through biotic
associations or abiotic culture, is a popular method for in vitro
culture (Zeng et al., 2013). In Paphiopedilum, 58 protocols for
in vitro seed germination have been described, or 80.6% of all
in vitro studies (Supplementary Material). However, seed
setting and germination percentage of many Paphiopedilum
species/cultivars are extremely low and are often affected by
many unknown factors (Arditti, 2008; Pierik et al., 1988). The
success of Paphiopedilum micropropagation from ex vitro-
derived explants has been relatively limited due to the rarity
of materials, difficulties related to bacterial and fungal
contamination of such explants and the poor development of
explants that survive under in vitro conditions (Huang, 1988;
Stewart & Button, 1975). Paphiopedilum species and hybrids
are the only commercially grown orchids that are not
cloned (Huang, 1988; Liao et al., 2011; Ng & Saleh, 2011).
However, the rapid multiplication of elite clones, production
of healthy and disease-free plants and faster introduction of
 2 S. Zeng et al.
novel cultivars with desirable traits are urgently needed in
Paphiopedilum improvement programs, and to address this,
tissue culture techniques are likely to play a vital role.
Paphiopedilum species/hybrids propagated in vitro
To date, 75 studies have reported on the in vitro propagation
of Paphiopedilum species and cultivars, including nearly 32
native species and more than 30 hybrids. However, 58 studies
reported for seed germination and PLB formation subse-
quently. Most explants of Paphiopedilum micropropagation
used derived from in vitro material of Paphiopedilum hybrids
and only three reports from ex vitro-derived explants
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15
(Supplementary material).
Asymbiotic seed germination
Until fairly recently, due to the limited success in tissue culture
protocols, commercial Paphiopedilum propagation by growers
had still been entirely through asymbiotic seed germination
(Hong et al., 2008). Asymbiotic seed germination of fully
mature Paphiopedilum seeds is often difficult and different
(Pierik et al., 1988; Stimart & Ascher, 1981). Paphiopedilum
seed germination and development are significantly influenced
by several factors, including capsual maturity and pre-
treatment of seeds, medium composition, culture conditions
and culture method. Some experimental results, such as the
germination percentage of seeds of the same species on the
same medium, have been shown to be inconsistent, for
For personal use only.
example, the germination percentage of 120-days after pollin-
ation (DAP) P. armeniacum seed was 25.2% by Chen et al.
(2004b) and 18.4% by Ding et al. (2004) on Robert Ernst
medium (RE; Arditti et al., 1982).
Paphiopedilum seed germination and protocorm
development
Seed germination and seedling growth and in vitro develop-
ment of Paphiopedilum are usually divided into five stages
(Figure 1A–F; Zeng et al., 2012): (1) rupture of the testa by an
enlarging embryo (¼germination); (2) appearance of the
shoot (¼protomeristem) and/or rhizoids; (3) emergence and
elongation of the first leaf; (4) the presence of one leaf and
one or more roots; (5) the presence of two or more leaves and
roots, i.e. a seedling. The speed of seed germination can be
simply calculated in each developmental stage (i.e. per unit
time) by dividing the number of seed by the number of
protocorms.
Effect of seed maturity and pre-treatment
on asymbiotic seed germination
In orchids, the zygotic embryo is poorly differentiated, and
meristems and cotyledons are usually not present at the time
of seed dispersal (Yeung et al., 1996). At present, information
on embryo development in Paphiopedilum species is limited,
and there are only a few reports about P. insigne (Nagashima,
1982; Zinger & Poddubnaya-Arnoldi, 1966), P. godefroyae
(Ren & Wang, 1987), P. delenatii (Lee et al., 2006) and
P. hirsutissinum, P. appletonianum and P. armeniacum
(Zhang et al., 2013). Important and detailed observations
were made by Lee et al. (2006), who found that fertilization
occurred at approximately 60 and 75 DAP, zygotes and
Crit Rev Biotechnol, Early Online: 1–14
proembryos could be observed within the capsules, although
the result was not quantified. By the early globular stage (90
DAP), additional cell divisions had occurred in the inner tiers
as well as in the surface layer, resulting in the growth of the
embryo proper. A distinct protoderm layer was found at
approximately 105 DAP, when 105 DAP, plastids with starch
granules, mitochondria, lipid bodies and small vacuoles were
readily seen in the cells of the embryo proper. As the embryo
reached the mature globular stage (150 DAP), mitotic activity
ceased. The volume of large vacuoles within the cytoplasm
decreased and the cytoplasm became dense. The testa
originated from the inner and outer integuments of the
ovule. Cells of the inner layers of the testa took on a slender
shape, whereas those of the outer layers were broader. By the
globular stage (150 DAP), the outer layer of the testa began to
compress and shrivel gradually, and cuticular substances
enveloped the entire embryo and inner integument and
embryo, creating a hydrophobic barrier to water and nutrient
uptake in fully mature P. delenatii seeds. As in other orchid
species, no endosperm was observed since the testa of most
orchids is normally derived from the outer integument (Yeung
et al., 1996) although the P. delenati testa is derived from both
the inner and outer integument. In addition, the morpho-
logical features of the transfer cell and the absence of
cuticular material in the suspensor cell wall corroborated the
hypothesis that the suspensor is the major site of nutrient
uptake for the developing embryo in P. delenatii (Lee et al.,
2006).
Under suitable germination conditions, the embryo fully
forms and the testa might not be lignified, allowing it to be
permeable to water and nutrients. Immature 90-DAP seeds of
Paphiopedilum wardii did not germinate and turned brown
soon after inoculation and 120-DAP seeds needed a period
longer than 150-DAP to germinate or more mature seeds,
indicating that very young orchid ovules are not appropriate
for germination and may need time for organogenesis or
synthesis of nutrients to occur or to help embryos recognize
stimulating agents present in the medium, thus allowing them
to germinate (Nhut et al., 2005; Zeng et al., 2012).
Alternatively, the embryo may be too underdeveloped to
absorb nutrients from the medium (Long et al., 2010). One-
hundred and eighty DAP seeds of P. wardii may have
germinated well due to efficient protein mobilization during
rehydration and an undeveloped embryonic envelope
(Table 1; Rasmussen, 1995; Zeng et al., 2012). Several
factors affect the low germination percentage of mature
Paphiopedilum seeds: an impermeable testa (Van Waes &
Debergh, 1986a), the presence of chemical inhibitors such as
abscisic acid (ABA), or the lack of certain germination-
promoting hormones (Van der Kinderen, 1987). Under
suitable germination conditions, the zygotic embryo of
almost fully mature seed forms fully and the testa might not
be lignified, allowing it to be permeable to water and nutrients
while mature seeds may have a greater potential for propa-
gation and storage because of a fuller testa and lower water
content (Miyoshi & Mii, 1998; Zhang et al., 2013).
Paphiopedilum seed germination and protocorm develop-
ment are stimulated by suitable pre-treatment including with
sodium hypochlorite (NaOCl) or NaOH (Lee, 2007; Liao &
Chen, 2006; Zeng et al., 2012). The kind, concentration of
 DOI: 10.3109/07388551.2014.993585 In vitro propagation of Paphiopedilum orchids 3
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For personal use only.

Figure 1. In vitro seed culture, seedling development and re-introduction of Paphiopedilum wardii Sumerh. (A) Stage 0, seed, ungerminated. (B) Stage
1, testa ruptured. (C) Stage 2, appearance of rhizoids. (D) Stage 3, emergence and elongation of first leaf. (E) Stage 4, one leaf and root present. (F)
Stage 5, presence of two or more leaves. (G) Asymbiotic seed germination on 1/2 MS medium supplemented with 0.5 mg/L NAA, 10% CW (v/v) and
1.0 g/L AC. (H) and (I) Development of seedlings on Hyponex N026 medium supplemented with 1.0 mg/L NAA, 10% CW and 1.5 g/L AC. (J) Seedling
growth on Hyponex N016 medium containing 1.0 mg/L NAA, 50 g/L BH and 1.5 g/L AC. (K) Flowering plants from seedlings in vitro to the
greenhouse. (L) Establishment of in vitro seedlings in the field 12 months after re-introduction. T: testa, FL: first emerged leaf, RZ: rhizoids, RT: root,
SL: second emerged leaf. Scale bars: (A) 50 mm, (B) 100 mm, (C) 0.1 mm, (D) 0.07 mm, (E) 0.2 mm, (F) 0.4 mm, (G–I) 3.0 cm, (J) 4 cm, (K) 20 cm, (L)
10 cm. [Reprinted from Zeng et al. (2012) with kind permission from Elsevier].

pre-treatment solution and its duration were key factors. Zeng
et al. (2012) reported that pre-treatment of mature P. wardii
seeds (360 DAP) with NaOCl solution containing 0.5%
available chlorine for 60 min or 1.0% available chlorine for
40 min significantly increased germination percentage (70.33
and 67.67%) than other pre-treatments and the control (0.1%
HgCl2 aqueous solution) and all pre-treatments significantly
shortened the period for initiating germination. Pretreatment
of NaOCl, NaOH or Ca(OCl)2 may have eroded the testa and
increased the permeability of the seed to oxygen and nutrients
(Major & Wright, 1974; Rasmussen, 1995; Van Waes &
Debergh, 1986a,b) while the level of inhibitors such as ABA
may have become reduced within the seeds (Lee, 2007; Van
der Kinderen, 1987). The lower seed germination percentage
or lack of germination might be due to seed damage caused by
a long duration of NaOCl treatments (Kaneko & Morohashi,
2003).
Effect of culture media on asymbiotic seed
germination
Effect of basal media and pH
The impact of many mineral-basal media has been tested on
in vitro germination of Paphiopedilum seed. Both seed
 4 S. Zeng et al. Crit Rev Biotechnol, Early Online: 1–14

Table 1. Effect of degree of seed maturity of Paphiopedilum on germination in vitro.

Days after Seed

pollination germination
Species/cultivars (DAP) Medium percentage (%) References
P. insigne var sanderae (Rchb.f.; O. Gruss & Roeth) 160 Hyponex 0.5 Nagashima (1982)
170 85
180 85
190 75
200 65
210 65
220 65
P. primulinum (M. W. Wood & P. Taylor) 70 1/4 MS 6.1 Lee & Lee (1999)
90 28.6
110 57.6
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130 41.1
150 43.6
P. philipinense (Rchb.f.) Stein 70 1/4 MS 9.0 Lee & Lee (1999)
90 32.4
110 23.1
130 32.0
150 0
P. bellatulum (Rchb.f.) Stein 70 1/4 MS 15.8 Lee & Lee (1999)
90 60.7
110 48.1
130 69.7
150 56.8
P. delenatii Guillaumin 70 1/4 MS 15.7 Lee & Lee (1999)
90 20.0
11 37.5
130 55.6
150 68.0
210 31.0
For personal use only.

P. armeniacum (S. C. Chen & F. Y. Liu) 60 1/5 MS 3.5 Ding et al. (2004)
80 10.3
100 22.3
120 40.6
140 32.8
160 24.7
180 22.9
P. armeniacum (S. C. Chen & F. Y. Liu) 120 1/4 MS 11.0 Chen et al. (2004a)
180 0
P. armeniacum (S. C. Chen & F. Y. Liu) 120 RE 32.4 Chen et al. (2004a)
180 0
P. micranthum (Tang & F. T. Wang) 120 RE 25.2 Chen et al. (2004a)
180 0
P. micranthum (Tang & F. T. Wang) 120 1/4 MS 11 Chen et al. (2004a)
180 0
P. delenatii Guillaumin 90 KC 0 Nhut et al. (2005)
180 8.8
270 90.1
P. armeniacum (S. C. Chen & F. Y. Liu) 86 1/4 MS 15.13 Liao & Chen (2006)
106 30.12
127 64.62
156 0
180 2.0
200 3.2
238 2.5
P. villosum var. densissimum (Z. J. Liu & S. C. Chen) 140 1/4 MS 0 Long et al. (2010)
150 0.12
160 0.17
170 0.74
180 0.9
190 0.71
200 31.33
300 13.33
P. callosum (Rchb.f.) Stein 180 1/2 RE 0
210 0
230 42
270 95
300 20
P. micranthum (Tang & F. T. Wang) 110 1/5 MS 35

(continued )
 DOI: 10.3109/07388551.2014.993585 In vitro propagation of Paphiopedilum orchids 5
Table 1. Continued

Days after Seed

pollination germination
Species/cultivars (DAP) Medium percentage (%) References
120 37
130 29
140 34
150 37
160 31
170 26
180 27
190 22
200 25
210 18
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P. wardii Summerh. 60 1/2 MS (half- strength 0f Zeng et al. (2012)

macro- and
micro-nutrients,
full-strength
other ingredient)
90 0f
120 10.00e
150 31.33c
180 65.33a
210 53.33b
240 30.33c
270 20.00d
300 6.33e
330 5.67e
360 6.00e
P. hangianum (Perner & Gruss) 60 H026 medium 0f Zeng et al. (2013a)
90 12.33e
120 45.67c
61.00b
For personal use only.

150
180 72.67a
210 59.33b
240 30.00d
270 17.67e
300 17.00e

KC, Knudson C medium; MS, Murashige and Skoog medium; RE, Robert Ernst medium. Values followed by different lower-case
superscript letters within a column for a species are significantly different at p50.05.

germination and seedling development of Paphiopedilum are
strongly affected by the mineral composition of the basal
medium (Table 2). Most Paphiopedilum species prefer a low
mineral medium for seed germination and the inhibition of
Paphiopedilum germination on Murashige and Skoog medium
(MS, Murashige & Skoog, 1962) may be due to its high total
mineral content (Chen et al., 2004a; Ding et al., 2004; Long
et al., 2010; Pierik et al., 1988), such that 1/2, 1/4, 1/6, 1/5 or
1/8 MS (i.e. a half, quarter, fifth, sixth or eighth of the content
of micro- and macro-nutrients) have been shown to be more
suitable (Ding et al., 2004; Lee & Lee, 1999; Zhou et al.,
2013).
The ideal medium for germination of the same
Paphiopedilum species differs. For example, Nhut et al.
(2005) reported 9-month-old seeds of P. delenatii cultured on
Knudson C (KC) medium (Knudson, 1946), to be optimal for
in vitro germination than on MS or 1/2 MS medium. Similarly,
P. wardii similarly showed significantly lower seed germin-
ation on MS than on 1/2 MS medium (Zeng et al., 2012).
However, minerals were not the only factor affecting P. wardii
seed germination because seed germination percentage was
significantly lower on 1/4 MS medium – which contained a
lower total mineral concentration – than on 1/2 MS media.
However, the exact medium composition and the proportion of
different minerals also have a significant effect on P. wardii
seed germination and seedling development of P. wardii. For
example, highest seed germination was on Vacin and Went
medium (VW; Vacin & Went, 1949), but only 14% of
protocorms developed to Stage 5 seedlings, significantly
lower than on 1/2 MS and Hyponex N026 media in which
52% of protocorms in Stage 2 died (Zeng et al., 2012).
Pierik et al. (1988) reported highest seed germination of
P. ciliolare on KC and 1/4 MS medium and much lower levels
on MS medium; KC resulted in plant death. Modified Thomale
GD1 medium (Thomale, 1954) was superior to 1/4 MS
medium for protocorm development and seedling growth.
Germination of P. ciliolare seed was significantly lower on 1/4
MS medium, which contained 3/4th- or full-strength micro-
salts (except iron) than medium without micronutrients, but
seed germination of P. ciliolare was not significantly different
on 1/4 MS medium containing 1/4-, 1/2-, 3/4- or full-strength
micronutrients. Seed germination of P. ciliolare was not
significantly affected by the concentration of NaFeEDTA,
25 mg/L being optimal. Plant development was strongly
promoted by iron, and all protocorms and plantlets died
during light treatment in iron-free medium.
Seed germination of P. villosum var. densissimum was
higher on VW than on 1/4 MS, RE or KC media, pH were
 6 S. Zeng et al. Crit Rev Biotechnol, Early Online: 1–14

Table 2. Effect of basal media on propagation in vitro of Paphiopedilum.

Seed germination frequency (%) or

Appropriate basal media
Burgeff EG-1; KC;
Norstog; Thomale GD
Hypnonex; MS
Norstog; Burgeff EG-1
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Species/cultivars description References
P. callosum (Rchb.f.) Stein Burgeff EG-1 was most suitable Stimart & Ascher (1981)a
P. hybrids (P. McLaren Park  P. White for germination and survival in
Fringe, P. Jack Tonkin the light.
Jason  P. Hellas Westonbint, Thomale GD was most suitable
P. parishii  P. curtisii var. Sandarae) for germination and survival in
the dark.
P. insigne var sanderae (Rchb.f.) O. Gruss Hypnonex was better than MS for Nagashima (1982)a
& Roeth seed germination.
P. sukhakulii Schoser & Senghas and its Norstog was suitable for seed Tay et al. (1988)a
four hybrids (P. sukhakulii  germination
P. nevium, P. sukhakulii  P. acmo- Burgeff EG-1 was suitable for

dontum, P. sukhakulii  P. bellatum, protocorm differentiation and

P. Clair de Lune  P. sukhakulii) subsequent growth of the
seedlings.
Modified Thomale GD1 P. ciliolare (Rchb.f.) Stein 75% Pierik et al. (1988)
KC 78%
1/4 MS 78%
1/2 MS 68%
3/4 MS 58%
MS 50%
MS P. delenatii Guillaumin, P. bellatulum 1/8 MS medium was most suitable Lee & Lee (1999)a
(Rchb.f.) Stein, P. primulinum for P. delenatii, 1/4 MS medium
(M. W. Wood & P. Taylor) was most suitable for P. bella-
tulum, P. primulinum.
KC; VW; Thomale GD; P. delenatii Guillaumin, P. bellatulum, Knudson C was most suitable for Lee & Lee (1999)a
Hyponex No. 1 3 g/L (Rchb.f.) Stein, P. primulinum P. bellatulum, Thomale GD was
(M. W. Wood & P. Taylor) most suitable for P. delenatii
and P. primulinum.
1/2 MS Paphiopedilum villosum var. densissimum 21 Long et al. (2010)
For personal use only.

(Z. J. Liu & S. C. Chen)

1/4 MS 14
VW 37
KC 56
MS P. armeniacum (S. C. Chen & F. Y. Liu) 2.9 Chen et al. (2004a)
1/4 MS 11.0
VW 17.3
RE 32.4
KC 16.1
Hyponex 1 9.5
1/2 MS P. armeniacum (S. C. Chen & F. Y. Liu) 15.8 Ding et al. (2004)
1/5 MS 42.5
1/10 MS 31.4
RE 18.4
Hyponex 1 23.1
MS P. micranthum (Tang & F. T. Wang) 0 Chen et al. (2004a)
1/4 MS 8.1
VW 7.2
RE 25.2
KC 11.6
Hyponex 1 5.9
KC P. delenatii Guillaumin 90.1 Nhut et al. (2005)
MS 9.0
1/2 MS 73.0
1/2 MS Paphiopedilum villosum var. densissimum 21% Long et al. (2010)
(Z. J. Liu & S. C. Chen)
1/4 MS 14%
VW 37%
KC 56%

KC, Knudson C medium; MS, Murashige and Skoog medium; RE, Robert Ernst medium; VW, Vacin and Went medium.
a
Percentage values not quantified in these studies.

all 5.8 (Long et al., 2010). This may be related to the
relatively low mineral salt and inorganic N concentrations
in VW medium (mineral concentrations: KC, 13.42 mM, VW,
16.3 mM; inorganic N concentrations: KC, 12.25 mM, VW,
3.78 mM) and high phosphate concentration (4.74 mM) of
VW compared to 1/4 MS (0.32 mM), RE (2.21 mM) and KC
(1.84 mM), although high phosphate concentration inhibited
subsequent protocorm development.
In most Paphiopedilum studies, seed germination was
possible at a pH of 5.0–6.0 (Ernst, 1975, 1980; Fast, 1971;
Flamee, 1978; Thomale, 1957; Zeng et al., 2012). Pierik et al.
(1988) reported that the pH (5.5, 6.0, 6.5 and 7.0) of the
 DOI: 10.3109/07388551.2014.993585
medium had hardly any effect on seed germination of
P. ciliolare, reacting slightly better at pH 5.5 and 6.0,
although not significantly, than at pH 6.5 and 7.0.
Furthermore, development in the light was clearly better at
a low pH, and pH 7.0 strongly inhibited the growth of
seedlings (Pierik et al., 1988).
Carbon source
Carbohydrates as an energy source in medium and osmotica
have significant effects on Paphiopedilum seed germination
and protocorm development. Germination of Paphiopedilum
seed did not occur without sugar (Fast, 1971; Pierik et al.,
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15
1988). Sucrose is the most utilized carbon source for seed
germination of Paphiopedilum orchids (Chen et al., 2004a;
Ding et al., 2004; Hossain et al., 2013; Zeng et al., 2012).
However, other carbon sources including glucose, fructose,
maltose and mannitol, were also used in some studies (Long
et al., 2010; Pierik et al., 1988). Pierik et al. (1988) reported the
highest germination (48%) on modified Thomale medium with
the lowest sugar concentration (0.5% glucose + 0.5% fructose),
which was significantly higher than on medium with 1.0%
glucose + 1.0% fructose (37%) or on medium with 1.25%
glucose + 1.25% fructose (29%), but not significantly higher
than on medium with 0.75% glucose + 0.75% fructose (41%).
Long et al. (2010) reported a significantly higher (19%)
germination percentage of P. villosum var. densissimum on
For personal use only.
2 g/L glucose-amended medium than on 2 g/L sucrose-
amended medium, or medium amended with 2 g/L maltose or
mannitol (0.08%). Maltose is an effective sugar source for
Oncidium ‘‘Gower Ramsey’’ plantlet growth (Jheng et al.,
2006) although Long et al. (2010) found that maltose was less
effective than glucose or sucrose in promoting germination of
Paphiopedilum.
Plant growth regulators
Different plant growth regulators (PGRs) have different
effects on seed germination of different orchid species
(Teixeira da Silva, 2013a). Most Paphiopedilum seed could
germinate normally in the absence of exogenous PGRs.
Hegarty (1955) observed a stimulatory effect of indole-3-
butyric acid (IBA) on the germination of Paphiopedilum
hybrids, Pierik et al. (1988) also found auxin [0.001–1.0 mg/L
indole-3-acetic acid (IAA) and 0.001–1.0 mg/L IBA] had a
slightly promotive effect, but neither cytokinin [0.001–
1.0 mg/L N6-benzyladenine (BA) and 0.001–1.0 mg/L
Kn[nor gibberellic acid (0.001–1.0 mg/L GA3) affected ger-
mination. However, seedling growth and development in the
light were increasingly inhibited and distorted by an
increasing auxin, cytokinin and GA3 concentration. IBA was
more toxic (although why it was considered to be toxic was
not defined) than IAA, and inhibition started at 0.5 mg/L of
cytokinin and at 0.01 mg/L of GA3 (Pierik et al., 1988).
Organic supplements
Orchid seed germination and protocorm development are
stimulated or inhibited by organic amendments (Chen et al,
2004b; Harvais, 1973; Zeng et al., 2012). Organic amend-
ments used include a number of fruit juicesuch such as
In vitro propagation of Paphiopedilum orchids 7
coconut water (CW), banana homogenate (BH), potato
homogenate (PH), and organomineral complexes such as
hydrolysed casein (HC), yeast extract, tryptone and peptone
(Curtis, 1947; Fast, 1971; Lee & Lee, 1999; Long et al., 2010;
Pierik et al., 1988).
In experiments with media supplemented with organomin-
eral complexes, Chen et al. (2004b) reported that when KC
medium was supplemented with 1.0 g/L yeast extract, the seed
germination percentage of P. armeniacum and P. micranthun
decreased significantly. Curtis (1947) reported seed germin-
ation percentages of P. insigne and P. hirsutissimum to be
higher with 1.0–2.0 g/L peptone than without peptone, while
Zeng et al. (2012) reported that seed germination percentage
of P. wardii increased significantly when the seeds were
cultured on 1/2 MS medium supplemented with 0.5, 1.0 or
1.5 g/L peptone than without peptone (Figure 1G). Tryptone
has also had a dominant influence on the germination of
Paphiopedilum seeds and subsequent development (Fast,
1971; Flamee, 1978; George et al., 2008; Lee & Lee, 1999;
Pierik et al., 1988; Stimart & Ascher, 1981; Thomale, 1957;
Zeng et al., 2012). Pierik et al. (1988) claimed that tryptone
(1.5, 2.0 or 2.5 g/L) significantly promoted P. ciliolare seed
germination and the further development of seedlings. Lee &
Lee (1999) reported that 1/4 MS medium containing 2 g/L
tryptone promoted the germination of P. delenatii,
P. bellatulum and P. primulinum seeds. Zeng et al. (2012)
also reported that the germination percentage of P. wardii
seeds increased significantly when they were cultured on 1/2
MS medium supplemented with 1.0 g/L tryptone than without
tryptone. Organic compounds (HC, peptone and tryptone)
generally consist of low molecular weight proteins, amino
acids, vitamins and plant growth substances, which are able to
enhance plant growth by providing plant cells with a readily
available source of nitrogen (George et al., 2008). In addition,
the vitamins biotin and pyridoxine, and in particular nicotinic
acid, have positive effects on seed germination and subse-
quent seedling growth (Arditti, 1967; Lucke, 1971; Thomale,
1957; Withner, 1959). Lucke (1971) reported that 0.0001%
biotin resulted in increased synthesis of chlorophyll and
promoted the development of Paphiopedilum seedlings.
In experiments of media supplemented with fruit juice,
Long et al. (2010) reported highest germination percentage of
P. villosum var. densissimum seeds with 10% coconut milk,
while germination percentage using apple and potato hom-
ogenates never exceeded 3%, and 0% with chayote and
lactalbumin hydrolysate. Zeng et al. (2012) also reported a
significant increase in germination percentage when P. wardii
seeds were cultured on 1/2 MS medium supplemented with
7.5, 10 and 15% CW (but not 5%) than without CW. CW
contains many different types of biochemicals, including
amino acids, vitamins, sugar, minerals and phytohormones
(Yong et al., 2009) and their natural inhibitors and regulators
which include ethylene, ABA, phenols and flavonols
(Mamaril et al., 1988), all of which may influence seed
germination or seedling development. CW also includes
various inorganic ions such as phosphorus, magnesium,
potassium and sodium (Raghavan, 1977), which benefit
orchid seed germination (Hossain et al., 2013; Teixeira da
Silva, 2013a). The chemical composition of CW is affected by
several factors including the stage of maturity of the coconut,
 8 S. Zeng et al.
soil and environmental conditions (Jackson et al., 2004; Yong
et al., 2009). The addition of BH to the medium promoted the
seed germination of Paphiopedilum species and hybrids (Ernst,
1980), but an opposite effect was also observed (Fast, 1971;
Pierik et al., 1988). The addition of BH to the medium also
promote shoot and root development of Paphiopedilum
seedlings in vitro (Ernst, 1974, 1975, 1980; Fast, 1971;
Pierik et al., 1988; Zeng et al., 2012; Figure 1H–J). Lee & Lee
(1999) reported that 20 g/L PH could promote the germination
of P. bellatulum, P. delenatii and P. primulinum seed, while
20 g/L BH decreased the germination of P. bellatulum,
P. delenatii and P. primulinum seed. Zeng et al. (2012)
reported no differences in seed germination percentage on 1/2
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15
MS containing PH, and significantly lower values on 1/2 MS
containing 75 or 100 g/L BH than without any organic
amendment. BH includes minerals, IAA, GA7 and GAx,
zeatin, zeatin riboside and N6-2(isopentenyl)adenine (2iP)
(Khalifah, 1966a,b; Van Staden & Stewart, 1975), which are
generally known to promote the germination of orchid seeds. In
contrast, the inhibitive effect of a high BH concentration on
germination may be that such medium contains too much sugar
or other substances to allow for the effective germination of
Paphiopedilum seed, even though it is suitable for plantlet
growth in vitro.
Activated charcoal
There are no across-the-board patterns as to whether adding
For personal use only.
activated charcoal (AC) to medium can improve the germin-
ation of orchid seed (Thomas, 2008). The first attempt to
darken culture medium used for orchid seed germination seems
to have been made in an effort to germinate native American
Cypripedium (Curtis, 1943). Darkening with charcoal (not AC)
had a clearly positive effect on seed germination and the
growth of Paphiopedilum seedlings (Ernst, 1974, 1975). Ding
et al. (2004) also reported that the seed germination percentage
of P. armeniacum was promoted on 1/5 MS medium containing
2 g/L AC, 36.1% higher than on 1/5 MS medium without AC
(9.6%). Lee & Lee (1999), however, reported that AC did not
promote seed germination of P. delenatii and P. primulinum,
but could promote seed germination of P. bellatulum. Pierik
et al. (1988) reported that 0.1 or 0.2% AC hardly affected
germination of P. ciliolare, but in the light, both concentrations
inhibited the further development of plants.
A tenable explanation for the positive effect of AC on seed
germination of orchids is that AC improves aeration and, at the
same time, should microelements be added, establish polarity,
affect substrate temperature or absorb toxic substances
including phenolics, but inversely absorbing certain com-
pounds, including certain vitamins and hormones, resulting in
a passive effect (Ernst, 1974; Thomas, 2008; Weatherhead
et al., 1978; Yam et al., 1990).
Liquid culture
There are no uniform or standardized results on liquid culture
to improve the germination of Paphiopedilum seed. Lee & Lee
(1999) reported that the seed germination of P. bellatulum,
P. delenatii and P. primulinum could be promoted to higher
percentages in liquid nutrient medium (58.2, 68 and 46.7%,
respectively) than in solid nutrient medium (36.5, 50.7 and
Crit Rev Biotechnol, Early Online: 1–14
35.7%, respectively). Ding et al. (2004) reported that the seed
germination percentage was not significantly different in liquid
or solid culture, although the period of germination was
shortened and the degree of synchronization was enhanced in
liquid medium, although none of these effects was quantified.
Vasudevan & Van Staden (2010) suggested that enhanced
germination in liquid suspension cultures might be due to
leaching of the seed testa and the dilution of phyto-inhibitors.
Culture environment influencing germination of
Paphiopedilum seeds
Culture temperature
In most studies on Paphiopedilum, seed germination was
possible at a temperature ranging from 22 to 28  C (Ernst,
1974). Pierik et al. (1988) reported a higher level of seed
germination of P. ciliolare at 25  C than at 21 or 29  C.
Light
Orchid seed germination has varying levels of response to light
or darkness (Arditti, 1967). The notion that epiphytic orchids
require light and terrestrial species require darkness for
germination is a widely accepted fact related to the require-
ments of natural conditions although germination responses to
photoperiod are often species specific, regardless of growth
habit (Kauth et al., 2008). Light also plays an important role in
the germination response of some Paphiopedilum species
which, when cultured in light, usually display inhibited
germination and induced dormancy. Pierik et al. (1988)
reported maximum germination of P. ciliolare in the dark
with germination becoming increasingly inhibited as irradi-
ance increased from 0 to 3.0 Wm2 during the first 12 weeks,
although only significant inhibition occurred at 3.0 Wm2.
However, light was necessary for plant development.
Surprisingly, not all seeds of different Paphiopedilum species
require a dark period to germinate. Zeng et al. (2012) reported
that the seed germination percentage of P. wardii was not
significantly different in continual darkness (68.33%) or under
a 16-h photoperiod without dark pre-treatment (65.33%).
However, the seed germination percentage of P. wardii was
highest (75.67%) when pre-treated with 45 days of darkness
followed by a 16-h photoperiod and was significantly higher
than in the two above-mentioned light conditions.
Stimart & Ascher (1981) reported that among four media
(Burgeff EG-1, Thomale GD, KC, Norstog), Burgeff EG-1 was
most suitable for seed germination and survival of three
Paphiopedilum hybrids (P. McLaren Park  P. White Fringe,
P. Jack Tonkin Jason  P. Hellas Westonbint and
P. parishii  P. curtisii var. Sandara) in the light while
Thomale GD was most suitable for germination and survival
in the dark. Tay et al. (1988) reported the best germination and
protocorm development of P. sukhakulii and its hybrids when
pre-treated on Norstog medium for 6 weeks or longer in the
dark. In that study, most protocorms in continuous light turned
brown and died off at an early stage of development while
seedlings that developed best were those obtained from
protocorms placed on Burgeff EG-1 medium in the light.
Amino acids, as used in Norstog medium, support seed
germination and protocorm growth but can also inhibit shoot
 DOI: 10.3109/07388551.2014.993585
formation in other orchids (Arditti, 1967; Harvais, 1982).
Photoinhibition on Norstog medium might take place due to
the presence of amino acids, whose utilization may be affected
by light (Arditti, 1967; Spoerl, 1948).
Zeng et al. (2012) reported that P. wardii seeds could
germinate under a 16-h photoperiod without dark pre-culture
or in complete darkness and that germination percentage was
not significantly different on 1/2 MS medium containing
0.5 mg/L NAA, 10% CW and 1.0 g/L AC, although seed
germination percentage following pre-culture for 45 days
in the dark then transferred to a 16-h photoperiod was
significantly higher than a 16-h photoperiod without dark pre-
culture, or complete darkness. The exact function of dark pre-
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15
culture is not understood at present. In addition, all
protocorms cultured continuously in the dark did not develop
to Stage 5. These results suggest that light is an important
physical factor controlling seedling growth, but not necessar-
ily germination.
Tissue culture of Paphiopedilum orchids
The birth of orchid micropropagation was in 1960 when Morel
first cultured Cymbidium shoot meristems (Morel, 1960) but
the pioneering work to micropropagate Paphiopedilum was
carried out by Bubeck (1973), while Morel (1974) recorded
the first successful tissue culture propagation protocol for
Paphiopedilum shoot tips. However, Paphiopedilum are con-
sidered to be difficult to regenerate from tissue culture
For personal use only.
due to the rarity of materials, difficulty caused by bacterial
and fungal contamination of ex vitro-derived explants and the
poor development of explants that survive under in vitro
conditions.
Explants, surface sterilization and disinfection
The morphogenetic response in orchids varies, depending on
the explant (reviewed by Chugh et al., 2009; Hossain et al.,
2013). The explants of Paphiopedilum hybrids have usually
been used in micropropagation, which indicates that
Paphiopedilum hybrids may be easier to micropropagate than
native species (Chen et al., 2002, 2004a,b; Huang, 1988; Liao
et al., 2011; Stewart & Button, 1975). Protocorms, PLBs and
seedlings in vitro, or parts thereof, are customarily used as
initial explants to study Paphiopedilum micropropagation,
including the shoot apex (Huang et al., 2001; Ng et al., 2010),
internodes (Ng et al., 2010; Ng & Saleh, 2011; Nhut et al.,
2007), PLBs (Lin et al., 2000; Zeng et al., 2013) or leaves
(Chen et al., 2004a; Lin et al., 2000), which did not need to be
sterilized. To date, only three reports of Paphiopedilum
micropropagation from ex vitro-derived explants exist
(Huang, 1988; Liao et al., 2011; Stewart & Button, 1975).
Stewart & Button (1975) used young and mature flower stems,
tips of leaves and roots, stamens, ovaries and terminal buds of
three outdoor-grown Paphiopedilum species (P. villosum,
P. fairrieanum, P. insigne). Huang (1988) used shoot apices
of Paphiopedilum hybrids as explants, disinfecting them by
immersion for 15 min under a mild vacuum in a solution
containing 0.5% NaClO, then, under a dissecting microscope,
shoot tips about 2–3 mm in size were excised, thus decreasing
infection. In addition, MS medium, containing 100–500 mg/L
of filter-sterilized carbenicillin and/or cefotaxime (antibiotics),
In vitro propagation of Paphiopedilum orchids 9
effectively limited bacterial contamination in initial cultures.
Liao et al. (2011) reported that scape transverse slices of
Paphiopedilum hybrids (P. Deperle and P. Armeni White)
could induce adventitious buds and regenerate.
Modes of in vitro development and factors influencing
modes of development
Callus and PLB induction
Plant growth regulators (PGRs), including auxins (2,4-D),
cytokinins [BA, thidiazuron (TDZ) and Kn] and other com-
pounds such as PBOA (phenylboronic acid), were used to
induce callus and PLB formation (Lin et al., 2000; Long et al.,
2010; Luan et al., 2012; Stewart & Button, 1975). The most
suitable PGRs were 2,4-D, TDZ or their combination. Stewart
& Button (1975) reported that terminal buds could produce
callus on Heller medium (Heller, 1965) containing 1.0 mg/L
2,4-D with or without 0.5 mg/L BA. Lin et al. (2000) reported
that totipotent callus of a Paphiopedilum hybrid (P. callosum
‘‘Oakhi’’  P. lawrenceanum ‘‘Tradition’’) could be induced
from seed-derived protocorms on 1/2 MS medium supple-
mented with 1–10 mg/L 2,4-D and 0.1–1 mg/L TDZ in the
dark. Luan et al. (2012) also reported 1/2 MS medium
supplemented with 10 mg/L 2,4-D and 0.1 mg/L TDZ to be
most suitable for callus induction of P. delenatii and
P. callosum. In Lin et al.’s (2000) study, on PGR-free
medium, subcultured callus showed little growth and eventu-
ally became brown and necrotic. The same events were
observed in callus maintained on medium containing TDZ or
2,4-D alone; the subcultured callus initially proliferated,
increased weight and then turned brown and necrotic. Callus
cultured on media containing both 2,4-D and TDZ proliferated
well and later regenerated on media designed for plantlet
regeneration. Media, supplemented with 5 mg/L 2,4-D and
1 mg/L TDZ, was selected as the standard maintenance
medium for proliferation of totipotent callus as this had the
best proliferation rate. Totipotent callus could grow on this
maintenance medium for 3 years without a loss of shoot
regeneration capacity. Callus developed further, forming
PLBs and eventually plantlets that could be transplanted
to pots and grew well. Little callus formed on medium
containing 1 mg/L 2,4-D or 100 mg/L PBOA alone, and
failed to proliferate and eventually died during subsequent
subculture. In all other combinations of PBOA and TDZ,
no callus formed. TDZ alone failed to induce callus.
However, stem, root tip and green leaf explants of another
Paphiopedilum hybrid (P. henryanum ‘‘#19’’  P. philippi-
nense ‘‘#10’’) failed to produce viable callus when cultured on
the media mentioned above as well as on various other media
(Lin et al., 2000).
Hong et al. (2008) reported that seeds from 5-month-old
green capsules of a Maudiae-type slipper orchid, P. Alma
Gavaert, could form totipotent callus on 1/2 MS medium
supplemented with 22.60 mM 2,4-D (5.0 mg/L) and 4.54 mM
(1.0 mg/L) TDZ in the dark. Callus was further proliferated
and maintained without any morphogenesis on the same
medium with a 2-month subculture interval for more than
2 years.
Ng & Saleh (2011) reported callus induction from nodal
stem explants of in vitro P. rothschildianum seedlings cultured
 10 S. Zeng et al.
on 1/2 MS supplemented with 4 mM (0.86 mg/L) Kn and 2 g/L
peptone. The induced callus could proliferate and form PLBs
from the surface of the proliferating callus when subcultured
onto the same medium. The highest number of secondary
PLBs formed on 1/2 MS medium supplemented with 4.0 mM
(0.86 mg/L) Kn with an average of 4.1 PLBs/explant after 8
weeks of culture. Conversely, the addition of BA inhibited the
induction of secondary PLB formation. The secondary PLBs
continued to proliferate and formed 9.5–12.1 new PLBs/
secondary PLB after subculturing on 1/2 PGR-free MS
medium supplemented with 60 g/L BH.
Ng & Saleh (2011) tested 1/2 MS medium with different
concentrations of BH, PH and TH (15, 30, 45 or 60 g/L,
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15
respectively) or 5, 10, 15 and 20% CW on PLB formation.
A 20% (v/v) CW was the most effective organic amendment
to facilitate PLB formation of P. rothschildianum and
subsequent differentiation into plantlets.
Shoot multiplication
PGRs, including auxins (2,4-D and NAA), cytokinins (BA,
TDZ, zeatin and Kn) and other compounds such as 2iP were
used to shoot multiplication. Huang (1988) reported that
adventitious buds and plantlets could develop from the shoot
tips of Paphiopedilum hybrid (P. philippinense  P. Susan
Booth) seedlings in vitro and MS medium supplemented with
3.0 mg/L 2iP, 0.1 mg/L NAA, 30 mg/L adenine sulfate and
For personal use only.
15% CW was effective for the establishment and aseptic
growth of explants, while MS medium supplemented with
100 mg/L BA (this level of BA is toxic to most plants) could
be used to multiply shoots by enhancing axillary branching
(Huang, 1988; Wu et al., 2012).
Huang et al. (2001) found that TDZ was not more
effective than BA for shoot proliferation and rooting of
three Paphiopedilum hybrids (P. philippinense  P. Susan
Booth; P. bellatulum ‘‘Big spot’’  P. Jo Ann’s Wine;
P. micranthum  P. glaucophyllum). The number of
Paphiopedilum hybrid shoots doubled every 12 weeks when
treated with 13 mM (2.93 mg/L) BA and 1.6 mM (0.3 mg/L)
NAA while TDZ inhibited shoot proliferation of the same
hybrids. NAA did not benefit shoot formation, but slightly
inhibited it at the highest concentration (1.0 mg/L).
Chen et al. (2002) reported that modified 1/2 MS medium
supplemented with 0.45 or 4.45 mM (0.1 or 1.0 mg/L) TDZ, or
4.52 or 45.25 mM (1.0 or 10.0 mg/L) 2,4-D, alone or in
combination, could control the development of multiple
shoots from stem nodal explants of P. philippinense hybrids
(PH59, PH60). Modified 1/2 MS medium supplemented with
a combination of 4.52 mM (1.0 mg/L) 2,4-D and 0.45 mM
(0.1 mg/L) TDZ induced a higher percentage of explants with
shoots and increased shoot number/explant more than the
PGR-free treatment in hybrid PH59. In hybrid PH60, although
the percentage of explants forming shoots was significantly
promoted on modified 1/2 MS medium supplemented with
4.52 mM 2,4-D (1.0 mg/L) and 0.45 mM (0.1 mg/L) TDZ,
highest shoot number/explant was possible with 4.52 mM
(1.0 mg/L) 2,4-D, but without TDZ. However, Luan et al.
(2012) found that 1/2 MS supplemented with 0.5 mg/L TDZ
and 0.1 mg/L NAA was most suitable for proliferation of
P. delenatii and P. callosum shoot buds.
Crit Rev Biotechnol, Early Online: 1–14
Hong et al. (2008) reported a suitable procedure for PLB
induction of P. Alma Gavaert from seed-derived callus on 1/2
MS medium with 2.69 mM (0.5 mg/L) NAA combined with
0.45 mM (0.1 mg/L) TDZ. When transferred to 1/2 MS
medium supplemented with 26.85 mM (5.0 mg/L) NAA, an
average of 4.7 PLBs/shoot bud formed from each explant after
120 days of culture. The suitable PGR concentration for shoot
multiplication was 4.65 mM (1.0 mg/L) Kn, which could
induce an average of 3.0 shoot from a single young shoot
after 60 days of culture.
Long et al. (2010) reported that the length and number of
shoots of four Paphiopedilum spp. were influenced by the
concentration and combination of cytokinins and auxins
added to the medium. There was no advantage by replacing
BA with TDZ in terms of inducing shoot organogenesis in all
four species. A relatively high BA concentration increased the
number of shoots in P. villosum var. densissimum and
P. armeniacum but decreased it in P. insigne. BA at 3.5 mg/
L caused extensive necrosis of P. insigne explants. The
highest organogenesis of different Paphiopedilum spp.
occurred with different combinations of cytokinins and
auxins in the medium. Shoot number of P. armeniacum was
highest with 4.0 mg/L BA and 0.1 mg/L NAA, with 1.0 mg/L
BA and 0.5 mg/L NAA for P. insigne, with 3.0 mg/L BA and
1.0 mg/L NAA or with 6.0 mg/L BA and 0.1 mg/L NAA for
P. villosum var. densissimum and with 5.5 mg/L BA and
0.5 mg/L NAA for P. bellatulum.
Nhut et al. (2007) reported that TDZ induced shoots in
P. delenatii more effectively than BA or zeatin, with 75% of
nodal segments forming shoots when cultured on modified
MS medium supplemented with 1.5 mg/L TDZ after 30 days
in culture. When explants were cultured on modified MS
medium supplemented with 2.0 mg/L BA or 1.5 mg/L zeatin,
54.5 and 58% of explants, respectively formed shoots.
Liao et al. (2011) reported that scape transverse slices of
Paphiopedilum hybrids (P. Deperle and P. Armeni White)
could induce adventitious buds and regenerate into whole
plants on modified MS medium supplemented with 4.43 mM
(1.0 mg/L) BA and 4.52 mM (1.0 mg/L) 2,4-D, or on modified
MS medium supplemented with 44.39 mM (10.0 mg/L) BA
and 26.85 mM (5.0 mg/L) NAA, respectively. Liao et al.
(2011) found that sections of flower buds 1.5–3.0 cm long
from P. Deperle could produce shoots, but only sections of
flower buds42.5 cm from P. Armeni White were regenerable.
Microscopic observations revealed that the small bract at the
base of flower buds harbored a new miniature flower bud,
which further harbored primitive flower buds with dome-
shaped meristem-like tissues that presumably led to the
induction of plants. The reiteration of this pattern resulted in a
scorpioid cyme inflorescence architecture in the multifloral
Paphiopedilum species, and its failure to reiterate resulted in a
single flower.
Nhut et al. (2005) reported that the physical state of the
medium (liquid or semi-solid) substantially affected shoot
formation in wounded in vitro seedlings of P. delenatii. Not
being completely submerged in the culture medium, wounded
seedlings could take up nutrient components and PGRs more
easily than liquid medium. This stimulated the differentiation
of the affected tissue and resulted in a higher number
of shoots/explant from wounded in vitro seedlings of
 DOI: 10.3109/07388551.2014.993585 In vitro propagation of Paphiopedilum orchids 11

P. delenatii. In semi-solid media containing 0.5 mg/L NAA, Ex vitro acclimatization and field establishment
the number of newly formed shoots tended to decrease (from
Culture flasks containing seedlings or micropropagated
2.3 to 0 shoots/explant) as the concentration of TDZ increased
in vitro Paphiopedilum plantlets are usually transferred to
(0.25–2.5 mg/L). In contrast, the number of shoots formed in
natural conditions from the culture chamber for 1–2 weeks
liquid medium increased (from 1.2 to 3.0 shoots/explant)
before they are transplanted to the field, which could
considerably when TDZ levels increased. The results suggest
strengthen their adaptability to external environments,
that the effects of TDZ on explants depended significantly on
useful for re-introduction projects into the wild (Chen et al.,
the physical properties of the medium. On liquid or semi-solid
2004b; Zeng et al., 2012). Supporting media often include
media containing 0.5, 1.0 or 3.0 mg/L TDZ, without the auxin
shattered bricks, volcanic rock, sphagnum moss, sieved peat,
NAA, the effect of liquid medium on shoot regeneration was
Zhijing stone for orchids (the water-retaining property is
better than semi-solid media. When TDZ was used at
superior to general stone for orchids), bark and other mixed
1.0 mg/L, the number of shoots/explant formed (5.2 shoots/
media. In a greenhouse environment with high air humidity,
explant) in liquid media was almost five-fold greater than on
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15

about 60% seedlings of P. villosum var. densissimum survived

semi-solid media (1.1 shoots/explant) and over two-fold
in a peat moss substrate (Long et al., 2010). Zeng et al. (2012)
greater (2.2 shoots/explant) than in media that contained
reported a mixed medium [2:1:1 (v/v) Zhijing stone for
auxin, suggesting that auxin may play an inhibitory role in
orchids: sieved peat: shattered fir bark] to be more suitable for
liquid media.
transplanting P. wardii seedlings than a substrate containing
Nhut et al. (2005) found that no shoots formed in control
sphagnum moss, sieved peat or Zhijing stone for orchids
treatments with non-wounded in vitro shoots of P. delenatii
alone, which had the ability to maintain moisture and air
seedlings in all types of media tested. An average of 2.3 green
simultaneously (Figure 1K).
and vigorous shoots was obtained when wounded seedlings
The best place to conserve plant germplasm and to
were cultured on solid-MS medium containing 0.25 mg/L
promote biodiversity is in the wild (McNaughton, 1989;
TDZ and 0.5 mg/L NAA, the wounding step being a pre-
Zeng et al., 2015). Re-introduction of native species has
requisite for shoot formation in seedlings. Wounded cells at
become increasingly important in conservation worldwide for
the damaged site may have responded more readily to
recovery of rare species and restoration purposes (Rout et al.,
stimulating agents such as certain PGRs present in media,
2009). Paphiopedilum wardii seedlings were successfully
allowing for differentiating into adventitious shoots.
established in the field and re-introduced into habitat and
For personal use only.

Ng et al. (2010) reported that multiple shoots could be

successfully induced from stem nodal and single shoot
explants of P. rothschildianum cultured on 1/2 MS medium
in the absence of PGRs and organic nitrogen additives
(control). The number of multiple shoots was further
increased by supplementing the culture medium with organic
nitrogen (peptone and tryptone–peptone). The highest number
of multiple shoots formed was scored on stem nodal explants
cultured on 1/2 MS medium supplemented with 1.0 g/L
peptone with an average of 2.9 shoots/explant after 16 weeks
of culture; however, the highest number of shoots formed on
medium supplemented with 2.0 g/L tryptone–peptone with an
average of 2.8 shoots forming per single shoot explant. In
contrast, peptone could not effectively induce the formation of
multiple shoots when single shoot explants were used, except
at a low concentration (0.5 g/L). The addition of a higher
amount of peptone (1.0 and 2.0 g/L) inhibited multiple shoot
formation.
Root induction
Root induction is easily achieved during the in vitro culture of
Paphiopedilum, and can be promoted by organic supplements
and/or PGRs (Huang et al., 2001; Zeng et al., 2013). Hong
et al. (2008) reported the suitable PGR for rooting was
26.85 mM (5.0 mg/L) NAA. Huang et al. (2001) found that
rooting was enhanced by 1.6 mM (0.3 mg/L) and 5.4 mM
(1.0 mg/L) NAA. Huang et al. (2001) also reported that CW
and HC enhanced root formation, 15% (v/v) CW and 1.0 g/L
HC being most suitable. Sucrose was better for rooting than
maltose, less rooting occurred at all maltose concentrations
while sucrose enhanced rooting at 0.18 M (62 g/L), but
suppressed it at a higher concentration (0.26 M or 89 g/L).
alien forest habitats (Zeng et al., 2012). The highest survival
rate (65%) occurred in the alien Ehuangzhang forest habitat
than in the Gaoligong Mountain habitat, possibly due to more
suitable irradiation, soil, water, nutrients, environmental
humidity and other factors in the former. Two years after
eco-reintroduction, 32% of flowering plants produced fruits
and seeds in loco, but no plants produced fruits and seeds in
the two alien forest habitats, possibly due to the lack of
suitable pollinators (Figure 1L; Zeng et al., 2012).
Conclusion and future prospects
Paphiopedilum available on the market come almost exclu-
sively from in vitro-germinated seeds of hybrid species. There
is abundant research of in vitro seed germination of
Paphiopedilum. However, biotic factors such as seed maturity,
age or developmental stage of the mother plant, and abiotic
factors related to basal medium, organic amendments, light,
temperature, carbon source, choice of PGRs, pH and culture
method have a strong impact on the outcome of in vitro
propagation of Paphiopedilum. An understanding of the
physiogy and ecology of seeds and whole plant ecology of
Paphiopedilum is necessary for commercial propagation.
The phenotype of plants must be predictable and uniform
to be commercially viable on a large scale, but the traits of
in vitro Paphiopedilum hybrid protocorms or seedlings are
variable and unpredictable, which is unacceptable in com-
mercial production (Liao et al., 2011). At present, there are
few protocols for the tissue culture of Paphiopedilum from
explants of mature plants, while Paphiopedilum species and
hybrids remain the only commercially grown orchids that are
not cloned because explants from mature plants of
Paphiopedilum species are recalcitrant to shoot induction
 12 S. Zeng et al.
and plant regeneration (Arditti, 2008) or as it is difficult to
obtain aseptic explants from mature plants through normal
surface sterilization steps for in vitro culture because of
endogenous bacteria (Chugh et al., 2009). Therefore, in the
future, it is very important to bring about further improve-
ments in in vitro tissue culture protocols of Paphiopedilum,
including the use of aseptic explants from mature plants and
increasing the speed of propagation and growth of
Paphiopedilum (Liao et al., 2011).
The explant sterilization protocol can affect the success of
asepsis and the subsequent quality and organogenic vitality of
the explant. Selecting a suitable explant source, using and
preparing an optimal explant, choosing a suitable disinfectant
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15
and applying an appropriate procedure can successfully reduce
contamination in the in vitro culture of orchids. Pre-in vitro
strategies for donor plants in the field or greenhouse, including
fertilizers, watering, plant maintenance and disease and pest
control can induce or create highly regenerative explant
sources (Winarto et al., 2013). In Paphiopedilum, a systematic
study of the sterilization protocol of explants is necessary.
Bioreactors, due to their high efficiency (Ziv, 2005), have
been applied to propagate a few orchids, including
Phalaenopsis (Park et al., 2000) and Oncidium ‘‘Sugar
Sweet’’ (Yang et al., 2010). However, there is no report yet
for Paphiopedilum. The liquid medium in a bioreactor allows
for close contact with the tissue which stimulates and facilitates
the uptake of nutrients and PGRs, leading to better shoot and
For personal use only.
root growth. Continuous shaking promotes less expression of
apical dominance and aeration which generally leads to
induction and proliferation of numerous axillary buds and
faster growth (Mehrotra et al., 2007), which may increase the
propagation and growth efficiency. In addition, a stricter
control of organogenesis might be possible if thin cell layers
derived from seed-derived protocorms are used (Teixeira da
Silva, 2013b). The use of permanent magnetic fields might also
be an innovative way to induce organogenesis in
Paphiopedilum, and has been successful in the tissue culture
of Cymbidium and Phalaenopsis (Van et al., 2011, 2012).
Some new biotechnological methods for in vitro propaga-
tion and growth of orchids, for example, CO2-enrichment
(Norikane et al., 2010) or alternative lighting systems [light-
emitting diodes (LEDs)/cold cathode fluorescent lamps
(CCFLs); Cybularz-Urban et al., 2007; Godo et al., 2011;
Tanaka et al., 1998] or a combination of both (Norikane et al.,
2013), could facilitate seed germination and stimulate
biomass production of orchids, which could also be very
useful for the in vitro propagation of Paphiopedilum due to its
slow speed of in vitro propagation and growth. However, there
are still no reports of LEDs and CO2-enrichment used on
in vitro culture of Paphiopedilum.
Acknowledgements
This study was supported by the Shanghai Administration of
Greenery and Cityscape Program (G142426), and the
Guangdong Key Technology Research and Development
Program (20131081030), and the Dongguan Social
Development Program (20131081030), and Guangzhou Key
Technology Research and Development Program
(Y233051001).
Crit Rev Biotechnol, Early Online: 1–14
Declaration of interest
The authors declare no conflicts of interest.
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