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REVIEW ARTICLE
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.
novel cultivars with desirable traits are urgently needed in proembryos could be observed within the capsules, although
Paphiopedilum improvement programs, and to address this, the result was not quantified. By the early globular stage (90
tissue culture techniques are likely to play a vital role. DAP), additional cell divisions had occurred in the inner tiers
as well as in the surface layer, resulting in the growth of the
Paphiopedilum species/hybrids propagated in vitro embryo proper. A distinct protoderm layer was found at
To date, 75 studies have reported on the in vitro propagation approximately 105 DAP, when 105 DAP, plastids with starch
of Paphiopedilum species and cultivars, including nearly 32 granules, mitochondria, lipid bodies and small vacuoles were
native species and more than 30 hybrids. However, 58 studies readily seen in the cells of the embryo proper. As the embryo
reported for seed germination and PLB formation subse- reached the mature globular stage (150 DAP), mitotic activity
quently. Most explants of Paphiopedilum micropropagation ceased. The volume of large vacuoles within the cytoplasm
used derived from in vitro material of Paphiopedilum hybrids decreased and the cytoplasm became dense. The testa
and only three reports from ex vitro-derived explants originated from the inner and outer integuments of the
ovule. Cells of the inner layers of the testa took on a slender
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15
(Supplementary material).
shape, whereas those of the outer layers were broader. By the
Asymbiotic seed germination globular stage (150 DAP), the outer layer of the testa began to
Until fairly recently, due to the limited success in tissue culture compress and shrivel gradually, and cuticular substances
protocols, commercial Paphiopedilum propagation by growers enveloped the entire embryo and inner integument and
had still been entirely through asymbiotic seed germination embryo, creating a hydrophobic barrier to water and nutrient
(Hong et al., 2008). Asymbiotic seed germination of fully uptake in fully mature P. delenatii seeds. As in other orchid
mature Paphiopedilum seeds is often difficult and different species, no endosperm was observed since the testa of most
(Pierik et al., 1988; Stimart & Ascher, 1981). Paphiopedilum orchids is normally derived from the outer integument (Yeung
seed germination and development are significantly influenced et al., 1996) although the P. delenati testa is derived from both
by several factors, including capsual maturity and pre- the inner and outer integument. In addition, the morpho-
treatment of seeds, medium composition, culture conditions logical features of the transfer cell and the absence of
and culture method. Some experimental results, such as the cuticular material in the suspensor cell wall corroborated the
germination percentage of seeds of the same species on the hypothesis that the suspensor is the major site of nutrient
same medium, have been shown to be inconsistent, for uptake for the developing embryo in P. delenatii (Lee et al.,
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 may have become reduced within the seeds (Lee, 2007; Van
et al. (2012) reported that pre-treatment of mature P. wardii der Kinderen, 1987). The lower seed germination percentage
seeds (360 DAP) with NaOCl solution containing 0.5% or lack of germination might be due to seed damage caused by
available chlorine for 60 min or 1.0% available chlorine for a long duration of NaOCl treatments (Kaneko & Morohashi,
40 min significantly increased germination percentage (70.33 2003).
and 67.67%) than other pre-treatments and the control (0.1%
HgCl2 aqueous solution) and all pre-treatments significantly Effect of culture media on asymbiotic seed
shortened the period for initiating germination. Pretreatment germination
of NaOCl, NaOH or Ca(OCl)2 may have eroded the testa and
Effect of basal media and pH
increased the permeability of the seed to oxygen and nutrients
(Major & Wright, 1974; Rasmussen, 1995; Van Waes & The impact of many mineral-basal media has been tested on
Debergh, 1986a,b) while the level of inhibitors such as ABA in vitro germination of Paphiopedilum seed. Both seed
4 S. Zeng et al. Crit Rev Biotechnol, Early Online: 1–14
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
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 different minerals also have a significant effect on P. wardii
strongly affected by the mineral composition of the basal seed germination and seedling development of P. wardii. For
medium (Table 2). Most Paphiopedilum species prefer a low example, highest seed germination was on Vacin and Went
mineral medium for seed germination and the inhibition of medium (VW; Vacin & Went, 1949), but only 14% of
Paphiopedilum germination on Murashige and Skoog medium protocorms developed to Stage 5 seedlings, significantly
(MS, Murashige & Skoog, 1962) may be due to its high total lower than on 1/2 MS and Hyponex N026 media in which
mineral content (Chen et al., 2004a; Ding et al., 2004; Long 52% of protocorms in Stage 2 died (Zeng et al., 2012).
et al., 2010; Pierik et al., 1988), such that 1/2, 1/4, 1/6, 1/5 or Pierik et al. (1988) reported highest seed germination of
1/8 MS (i.e. a half, quarter, fifth, sixth or eighth of the content P. ciliolare on KC and 1/4 MS medium and much lower levels
of micro- and macro-nutrients) have been shown to be more on MS medium; KC resulted in plant death. Modified Thomale
suitable (Ding et al., 2004; Lee & Lee, 1999; Zhou et al., GD1 medium (Thomale, 1954) was superior to 1/4 MS
2013). medium for protocorm development and seedling growth.
The ideal medium for germination of the same Germination of P. ciliolare seed was significantly lower on 1/4
Paphiopedilum species differs. For example, Nhut et al. MS medium, which contained 3/4th- or full-strength micro-
(2005) reported 9-month-old seeds of P. delenatii cultured on salts (except iron) than medium without micronutrients, but
Knudson C (KC) medium (Knudson, 1946), to be optimal for seed germination of P. ciliolare was not significantly different
in vitro germination than on MS or 1/2 MS medium. Similarly, on 1/4 MS medium containing 1/4-, 1/2-, 3/4- or full-strength
P. wardii similarly showed significantly lower seed germin- micronutrients. Seed germination of P. ciliolare was not
ation on MS than on 1/2 MS medium (Zeng et al., 2012). significantly affected by the concentration of NaFeEDTA,
However, minerals were not the only factor affecting P. wardii 25 mg/L being optimal. Plant development was strongly
seed germination because seed germination percentage was promoted by iron, and all protocorms and plantlets died
significantly lower on 1/4 MS medium – which contained a during light treatment in iron-free medium.
lower total mineral concentration – than on 1/2 MS media. Seed germination of P. villosum var. densissimum was
However, the exact medium composition and the proportion of 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
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 (1.84 mM), although high phosphate concentration inhibited
relatively low mineral salt and inorganic N concentrations subsequent protocorm development.
in VW medium (mineral concentrations: KC, 13.42 mM, VW, In most Paphiopedilum studies, seed germination was
16.3 mM; inorganic N concentrations: KC, 12.25 mM, VW, possible at a pH of 5.0–6.0 (Ernst, 1975, 1980; Fast, 1971;
3.78 mM) and high phosphate concentration (4.74 mM) of Flamee, 1978; Thomale, 1957; Zeng et al., 2012). Pierik et al.
VW compared to 1/4 MS (0.32 mM), RE (2.21 mM) and KC (1988) reported that the pH (5.5, 6.0, 6.5 and 7.0) of the
DOI: 10.3109/07388551.2014.993585 In vitro propagation of Paphiopedilum orchids 7
medium had hardly any effect on seed germination of coconut water (CW), banana homogenate (BH), potato
P. ciliolare, reacting slightly better at pH 5.5 and 6.0, homogenate (PH), and organomineral complexes such as
although not significantly, than at pH 6.5 and 7.0. hydrolysed casein (HC), yeast extract, tryptone and peptone
Furthermore, development in the light was clearly better at (Curtis, 1947; Fast, 1971; Lee & Lee, 1999; Long et al., 2010;
a low pH, and pH 7.0 strongly inhibited the growth of Pierik et al., 1988).
seedlings (Pierik et al., 1988). In experiments with media supplemented with organomin-
eral complexes, Chen et al. (2004b) reported that when KC
Carbon source medium was supplemented with 1.0 g/L yeast extract, the seed
germination percentage of P. armeniacum and P. micranthun
Carbohydrates as an energy source in medium and osmotica decreased significantly. Curtis (1947) reported seed germin-
have significant effects on Paphiopedilum seed germination ation percentages of P. insigne and P. hirsutissimum to be
and protocorm development. Germination of Paphiopedilum higher with 1.0–2.0 g/L peptone than without peptone, while
seed did not occur without sugar (Fast, 1971; Pierik et al., Zeng et al. (2012) reported that seed germination percentage
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 of P. wardii increased significantly when the seeds were
germination of Paphiopedilum orchids (Chen et al., 2004a; cultured on 1/2 MS medium supplemented with 0.5, 1.0 or
Ding et al., 2004; Hossain et al., 2013; Zeng et al., 2012). 1.5 g/L peptone than without peptone (Figure 1G). Tryptone
However, other carbon sources including glucose, fructose, has also had a dominant influence on the germination of
maltose and mannitol, were also used in some studies (Long Paphiopedilum seeds and subsequent development (Fast,
et al., 2010; Pierik et al., 1988). Pierik et al. (1988) reported the 1971; Flamee, 1978; George et al., 2008; Lee & Lee, 1999;
highest germination (48%) on modified Thomale medium with Pierik et al., 1988; Stimart & Ascher, 1981; Thomale, 1957;
the lowest sugar concentration (0.5% glucose + 0.5% fructose), Zeng et al., 2012). Pierik et al. (1988) claimed that tryptone
which was significantly higher than on medium with 1.0% (1.5, 2.0 or 2.5 g/L) significantly promoted P. ciliolare seed
glucose + 1.0% fructose (37%) or on medium with 1.25% germination and the further development of seedlings. Lee &
glucose + 1.25% fructose (29%), but not significantly higher Lee (1999) reported that 1/4 MS medium containing 2 g/L
than on medium with 0.75% glucose + 0.75% fructose (41%). tryptone promoted the germination of P. delenatii,
Long et al. (2010) reported a significantly higher (19%) P. bellatulum and P. primulinum seeds. Zeng et al. (2012)
germination percentage of P. villosum var. densissimum on also reported that the germination percentage of P. wardii
For personal use only.
2 g/L glucose-amended medium than on 2 g/L sucrose- seeds increased significantly when they were cultured on 1/2
amended medium, or medium amended with 2 g/L maltose or MS medium supplemented with 1.0 g/L tryptone than without
mannitol (0.08%). Maltose is an effective sugar source for tryptone. Organic compounds (HC, peptone and tryptone)
Oncidium ‘‘Gower Ramsey’’ plantlet growth (Jheng et al., generally consist of low molecular weight proteins, amino
2006) although Long et al. (2010) found that maltose was less acids, vitamins and plant growth substances, which are able to
effective than glucose or sucrose in promoting germination of enhance plant growth by providing plant cells with a readily
Paphiopedilum. available source of nitrogen (George et al., 2008). In addition,
the vitamins biotin and pyridoxine, and in particular nicotinic
Plant growth regulators acid, have positive effects on seed germination and subse-
Different plant growth regulators (PGRs) have different quent seedling growth (Arditti, 1967; Lucke, 1971; Thomale,
effects on seed germination of different orchid species 1957; Withner, 1959). Lucke (1971) reported that 0.0001%
(Teixeira da Silva, 2013a). Most Paphiopedilum seed could biotin resulted in increased synthesis of chlorophyll and
germinate normally in the absence of exogenous PGRs. promoted the development of Paphiopedilum seedlings.
Hegarty (1955) observed a stimulatory effect of indole-3- In experiments of media supplemented with fruit juice,
butyric acid (IBA) on the germination of Paphiopedilum Long et al. (2010) reported highest germination percentage of
hybrids, Pierik et al. (1988) also found auxin [0.001–1.0 mg/L P. villosum var. densissimum seeds with 10% coconut milk,
indole-3-acetic acid (IAA) and 0.001–1.0 mg/L IBA] had a while germination percentage using apple and potato hom-
slightly promotive effect, but neither cytokinin [0.001– ogenates never exceeded 3%, and 0% with chayote and
1.0 mg/L N6-benzyladenine (BA) and 0.001–1.0 mg/L lactalbumin hydrolysate. Zeng et al. (2012) also reported a
Kn[nor gibberellic acid (0.001–1.0 mg/L GA3) affected ger- significant increase in germination percentage when P. wardii
mination. However, seedling growth and development in the seeds were cultured on 1/2 MS medium supplemented with
light were increasingly inhibited and distorted by an 7.5, 10 and 15% CW (but not 5%) than without CW. CW
increasing auxin, cytokinin and GA3 concentration. IBA was contains many different types of biochemicals, including
more toxic (although why it was considered to be toxic was amino acids, vitamins, sugar, minerals and phytohormones
not defined) than IAA, and inhibition started at 0.5 mg/L of (Yong et al., 2009) and their natural inhibitors and regulators
cytokinin and at 0.01 mg/L of GA3 (Pierik et al., 1988). which include ethylene, ABA, phenols and flavonols
(Mamaril et al., 1988), all of which may influence seed
germination or seedling development. CW also includes
Organic supplements
various inorganic ions such as phosphorus, magnesium,
Orchid seed germination and protocorm development are potassium and sodium (Raghavan, 1977), which benefit
stimulated or inhibited by organic amendments (Chen et al, orchid seed germination (Hossain et al., 2013; Teixeira da
2004b; Harvais, 1973; Zeng et al., 2012). Organic amend- Silva, 2013a). The chemical composition of CW is affected by
ments used include a number of fruit juicesuch such as several factors including the stage of maturity of the coconut,
8 S. Zeng et al. Crit Rev Biotechnol, Early Online: 1–14
soil and environmental conditions (Jackson et al., 2004; Yong 35.7%, respectively). Ding et al. (2004) reported that the seed
et al., 2009). The addition of BH to the medium promoted the germination percentage was not significantly different in liquid
seed germination of Paphiopedilum species and hybrids (Ernst, or solid culture, although the period of germination was
1980), but an opposite effect was also observed (Fast, 1971; shortened and the degree of synchronization was enhanced in
Pierik et al., 1988). The addition of BH to the medium also liquid medium, although none of these effects was quantified.
promote shoot and root development of Paphiopedilum Vasudevan & Van Staden (2010) suggested that enhanced
seedlings in vitro (Ernst, 1974, 1975, 1980; Fast, 1971; germination in liquid suspension cultures might be due to
Pierik et al., 1988; Zeng et al., 2012; Figure 1H–J). Lee & Lee leaching of the seed testa and the dilution of phyto-inhibitors.
(1999) reported that 20 g/L PH could promote the germination
of P. bellatulum, P. delenatii and P. primulinum seed, while
Culture environment influencing germination of
20 g/L BH decreased the germination of P. bellatulum,
Paphiopedilum seeds
P. delenatii and P. primulinum seed. Zeng et al. (2012)
reported no differences in seed germination percentage on 1/2 Culture temperature
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by South China Institute of Botany,CAS on 01/13/15
formation in other orchids (Arditti, 1967; Harvais, 1982). effectively limited bacterial contamination in initial cultures.
Photoinhibition on Norstog medium might take place due to Liao et al. (2011) reported that scape transverse slices of
the presence of amino acids, whose utilization may be affected Paphiopedilum hybrids (P. Deperle and P. Armeni White)
by light (Arditti, 1967; Spoerl, 1948). could induce adventitious buds and regenerate.
Zeng et al. (2012) reported that P. wardii seeds could
germinate under a 16-h photoperiod without dark pre-culture Modes of in vitro development and factors influencing
or in complete darkness and that germination percentage was modes of development
not significantly different on 1/2 MS medium containing
Callus and PLB induction
0.5 mg/L NAA, 10% CW and 1.0 g/L AC, although seed
germination percentage following pre-culture for 45 days Plant growth regulators (PGRs), including auxins (2,4-D),
in the dark then transferred to a 16-h photoperiod was cytokinins [BA, thidiazuron (TDZ) and Kn] and other com-
significantly higher than a 16-h photoperiod without dark pre- pounds such as PBOA (phenylboronic acid), were used to
culture, or complete darkness. The exact function of dark pre- induce callus and PLB formation (Lin et al., 2000; Long et al.,
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 2010; Luan et al., 2012; Stewart & Button, 1975). The most
protocorms cultured continuously in the dark did not develop suitable PGRs were 2,4-D, TDZ or their combination. Stewart
to Stage 5. These results suggest that light is an important & Button (1975) reported that terminal buds could produce
physical factor controlling seedling growth, but not necessar- callus on Heller medium (Heller, 1965) containing 1.0 mg/L
ily germination. 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
Tissue culture of Paphiopedilum orchids ‘‘Oakhi’’ P. lawrenceanum ‘‘Tradition’’) could be induced
from seed-derived protocorms on 1/2 MS medium supple-
The birth of orchid micropropagation was in 1960 when Morel mented with 1–10 mg/L 2,4-D and 0.1–1 mg/L TDZ in the
first cultured Cymbidium shoot meristems (Morel, 1960) but dark. Luan et al. (2012) also reported 1/2 MS medium
the pioneering work to micropropagate Paphiopedilum was supplemented with 10 mg/L 2,4-D and 0.1 mg/L TDZ to be
carried out by Bubeck (1973), while Morel (1974) recorded most suitable for callus induction of P. delenatii and
the first successful tissue culture propagation protocol for P. callosum. In Lin et al.’s (2000) study, on PGR-free
Paphiopedilum shoot tips. However, Paphiopedilum are con- medium, subcultured callus showed little growth and eventu-
sidered to be difficult to regenerate from tissue culture
For personal use only.
on 1/2 MS supplemented with 4 mM (0.86 mg/L) Kn and 2 g/L Hong et al. (2008) reported a suitable procedure for PLB
peptone. The induced callus could proliferate and form PLBs induction of P. Alma Gavaert from seed-derived callus on 1/2
from the surface of the proliferating callus when subcultured MS medium with 2.69 mM (0.5 mg/L) NAA combined with
onto the same medium. The highest number of secondary 0.45 mM (0.1 mg/L) TDZ. When transferred to 1/2 MS
PLBs formed on 1/2 MS medium supplemented with 4.0 mM medium supplemented with 26.85 mM (5.0 mg/L) NAA, an
(0.86 mg/L) Kn with an average of 4.1 PLBs/explant after 8 average of 4.7 PLBs/shoot bud formed from each explant after
weeks of culture. Conversely, the addition of BA inhibited the 120 days of culture. The suitable PGR concentration for shoot
induction of secondary PLB formation. The secondary PLBs multiplication was 4.65 mM (1.0 mg/L) Kn, which could
continued to proliferate and formed 9.5–12.1 new PLBs/ induce an average of 3.0 shoot from a single young shoot
secondary PLB after subculturing on 1/2 PGR-free MS after 60 days of culture.
medium supplemented with 60 g/L BH. Long et al. (2010) reported that the length and number of
Ng & Saleh (2011) tested 1/2 MS medium with different shoots of four Paphiopedilum spp. were influenced by the
concentrations of BH, PH and TH (15, 30, 45 or 60 g/L, concentration and combination of cytokinins and auxins
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. added to the medium. There was no advantage by replacing
A 20% (v/v) CW was the most effective organic amendment BA with TDZ in terms of inducing shoot organogenesis in all
to facilitate PLB formation of P. rothschildianum and four species. A relatively high BA concentration increased the
subsequent differentiation into plantlets. 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
Shoot multiplication
highest organogenesis of different Paphiopedilum spp.
PGRs, including auxins (2,4-D and NAA), cytokinins (BA, occurred with different combinations of cytokinins and
TDZ, zeatin and Kn) and other compounds such as 2iP were auxins in the medium. Shoot number of P. armeniacum was
used to shoot multiplication. Huang (1988) reported that highest with 4.0 mg/L BA and 0.1 mg/L NAA, with 1.0 mg/L
adventitious buds and plantlets could develop from the shoot BA and 0.5 mg/L NAA for P. insigne, with 3.0 mg/L BA and
tips of Paphiopedilum hybrid (P. philippinense P. Susan 1.0 mg/L NAA or with 6.0 mg/L BA and 0.1 mg/L NAA for
Booth) seedlings in vitro and MS medium supplemented with P. villosum var. densissimum and with 5.5 mg/L BA and
3.0 mg/L 2iP, 0.1 mg/L NAA, 30 mg/L adenine sulfate and 0.5 mg/L NAA for P. bellatulum.
For personal use only.
15% CW was effective for the establishment and aseptic Nhut et al. (2007) reported that TDZ induced shoots in
growth of explants, while MS medium supplemented with P. delenatii more effectively than BA or zeatin, with 75% of
100 mg/L BA (this level of BA is toxic to most plants) could nodal segments forming shoots when cultured on modified
be used to multiply shoots by enhancing axillary branching MS medium supplemented with 1.5 mg/L TDZ after 30 days
(Huang, 1988; Wu et al., 2012). in culture. When explants were cultured on modified MS
Huang et al. (2001) found that TDZ was not more medium supplemented with 2.0 mg/L BA or 1.5 mg/L zeatin,
effective than BA for shoot proliferation and rooting of 54.5 and 58% of explants, respectively formed shoots.
three Paphiopedilum hybrids (P. philippinense P. Susan Liao et al. (2011) reported that scape transverse slices of
Booth; P. bellatulum ‘‘Big spot’’ P. Jo Ann’s Wine; Paphiopedilum hybrids (P. Deperle and P. Armeni White)
P. micranthum P. glaucophyllum). The number of could induce adventitious buds and regenerate into whole
Paphiopedilum hybrid shoots doubled every 12 weeks when plants on modified MS medium supplemented with 4.43 mM
treated with 13 mM (2.93 mg/L) BA and 1.6 mM (0.3 mg/L) (1.0 mg/L) BA and 4.52 mM (1.0 mg/L) 2,4-D, or on modified
NAA while TDZ inhibited shoot proliferation of the same MS medium supplemented with 44.39 mM (10.0 mg/L) BA
hybrids. NAA did not benefit shoot formation, but slightly and 26.85 mM (5.0 mg/L) NAA, respectively. Liao et al.
inhibited it at the highest concentration (1.0 mg/L). (2011) found that sections of flower buds 1.5–3.0 cm long
Chen et al. (2002) reported that modified 1/2 MS medium from P. Deperle could produce shoots, but only sections of
supplemented with 0.45 or 4.45 mM (0.1 or 1.0 mg/L) TDZ, or flower buds42.5 cm from P. Armeni White were regenerable.
4.52 or 45.25 mM (1.0 or 10.0 mg/L) 2,4-D, alone or in Microscopic observations revealed that the small bract at the
combination, could control the development of multiple base of flower buds harbored a new miniature flower bud,
shoots from stem nodal explants of P. philippinense hybrids which further harbored primitive flower buds with dome-
(PH59, PH60). Modified 1/2 MS medium supplemented with shaped meristem-like tissues that presumably led to the
a combination of 4.52 mM (1.0 mg/L) 2,4-D and 0.45 mM induction of plants. The reiteration of this pattern resulted in a
(0.1 mg/L) TDZ induced a higher percentage of explants with scorpioid cyme inflorescence architecture in the multifloral
shoots and increased shoot number/explant more than the Paphiopedilum species, and its failure to reiterate resulted in a
PGR-free treatment in hybrid PH59. In hybrid PH60, although single flower.
the percentage of explants forming shoots was significantly Nhut et al. (2005) reported that the physical state of the
promoted on modified 1/2 MS medium supplemented with medium (liquid or semi-solid) substantially affected shoot
4.52 mM 2,4-D (1.0 mg/L) and 0.45 mM (0.1 mg/L) TDZ, formation in wounded in vitro seedlings of P. delenatii. Not
highest shoot number/explant was possible with 4.52 mM being completely submerged in the culture medium, wounded
(1.0 mg/L) 2,4-D, but without TDZ. However, Luan et al. seedlings could take up nutrient components and PGRs more
(2012) found that 1/2 MS supplemented with 0.5 mg/L TDZ easily than liquid medium. This stimulated the differentiation
and 0.1 mg/L NAA was most suitable for proliferation of of the affected tissue and resulted in a higher number
P. delenatii and P. callosum shoot buds. 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
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