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Mahendran 2012

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Acta Physiol Plant (2013) 35:829–840

DOI 10.1007/s11738-012-1127-3

ORIGINAL PAPER

Asymbiotic seed germination of Cymbidium bicolor Lindl.


(Orchidaceae) and the influence of mycorrhizal fungus
on seedling development
G. Mahendran • V. Muniappan • M. Ashwini •

T. Muthukumar • V. Narmatha Bai

Received: 21 February 2012 / Revised: 11 October 2012 / Accepted: 15 October 2012 / Published online: 4 November 2012
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2012

Abstract An in vitro plant regeneration protocol was Abbreviations


successfully established for Cymbidium bicolor an epi- BAP Benzyl amino purine
phytic orchid by culturing seeds from green pods. Imma- KIN Kinetin
ture seeds were germinated on four basal media viz., TDZ Thidazuron –N phenyl-N0 -1,2,3-thadiazol-5-ylurea
Murashige and Skoog (MS) medium, Knudson C (KC) LO Lindemann orchid medium
orchid medium, Knudson C modified Morel (KCM) med- KCM Knunson C modified Morel medium
ium and Lindemann orchid (LO) medium. Seed germina- NAA a-Napthalene acetic acid
tion and protocorm development was significantly higher MS Murashige and Skoog medium
in LO medium (96.6 %) followed by KCM (89 %), MS KC Knudson C orchid medium
(77.5 %) and KC (62.7 %) media after 56 days. For mul- a.s.l Above sea level
tiple shoot induction the protocorms were transferred to B5
medium supplemented with cytokinin. Among the various
cytokinins tested, BAP (4.42 lM) induced maximum
(27.59) number of multiple shoots per explant. IBA was Introduction
effective in inducing healthy roots. Tissue-cultured proto-
corms and seedlings of C. bicolor were inoculated with Orchidaceae is one of the largest and advanced plant
AC-01 fungal strain (Moniliopsis sp.) isolated from the families with 20,000–30,000 species (Chugh et al. 2009).
mycorrizal roots of an epiphytic orchid Aerides crispum. Orchids are fascinating groups of ornamental plants with
Mycorrhizal fungi significantly enhanced number of roots, numerous exotic novel cultivars possessing elegant flowers
root length and shoot number. produced by interspecific, as well as intergeneric hybrid-
ization. However, orchids in the wild are endangered as a
Keywords Cymbidium bicolor  Asymbiotic seed consequence of environmental disruption, succession of
germination  Protocorm  Multiple shoots  natural habitats and over exploitation for horticultural
Aerides crispum  Mycorrhiza  Fungal co-culture purposes. In situ conservation of dwindling populations of
endangered orchid species is very difficult because of their
Communicated by J. van Staden. slow growth and low seed germination. Thus, in recent
years, the maintenance of living collections of orchids has
G. Mahendran (&) been considered to be an important aspect of conservation
Department of Botany, Plant Tissue culture lab,
(Sharrock 2007; Somiya 2007).
Bharathiar University, Coimbatore 641046, India
e-mail: mahendran0007@gmail.com All orchids have an obligate relationship with mycor-
rhizal fungi during seed germination and development
V. Muniappan  M. Ashwini  T. Muthukumar  (Rasmussen 1995). The mycorrhizal fungi provide the
V. Narmatha Bai
carbon source necessary for seed germination and seedling
Department of Botany, School of Life Sciences,
Bharathiar University, establishment (Yam and Arditti 2009). In some green and
Coimbatore 641046, Tamilnadu, India achlorophyllous orchids, the dependency on mycorrhizal

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830 Acta Physiol Plant (2013) 35:829–840

fungi may extend into adulthood (Julou et al. 2005; Abadie Previous studies have shown that mycorrhization of
et al. 2006; Rasmussen and Rasmussen 2007). Moreover, tissue-cultured orchid plantlets reported to enhance, plant
the application of mycorrhizal fungi in horticulture and for height, stem diameter, root number, biomass, plant survival
conservation purposes has recently gained considerable and establishment percentages (Chang and Chou 2007;
attention (Rasmussen 2002; Rasmussen and Rasmussen Fang et al. 2008; Hou and Guo 2009). Mass propagation for
2007; Swarts and Dixon 2009; Zettler et al. 2007). commercial cultivation requires a simple, economical,
Cymbidium or ‘‘boat orchid’’ is a popular orchid grown rapidly multiplying and highly reproducible protocol.
commercially worldwide (Chugh et al. 2009). Today, Keeping these in mind the present study was carried out to
orchids such as Cymbidium, Dendrobium, Oncidium and (a) select the appropriate medium for seed germination and
Phalaenopsis are marketed globally and the orchid industry protocorm development (b) induce multiple shoot buds
contributes substantially to the economy of many the South from seed derived protocorm and (c) test the effectiveness
East Asian countries. Cymbidium bicolor Lindl. is an of the mycorrhizal fungus on the growth and development
important horticultural orchid known for its beautiful of protocorm and seedling development under in vitro
flowers (Chugh et al. 2009). In Cymbidium, plantlets were condition and consequently on the ex vitro acclimatization
regenerated in in vitro using shoot tips (Morel 1964), process.
mature and immature seeds (Chung et al. 1985; Shimasaki
and Uemoto 1990), green capsules (Hossain et al. 2010;
Deb and Pongener 2011), flower stalks (Wang 1988), Materials and method
pseudo bulbs (Shimasaki and Uemoto 1990), shoot seg-
ments (Nayak et al. 1997), flower buds (Shimasaki and Preparation of green capsules
Uemoto 1990), protocorm-like bodies (PLBs) (Begum
et al. 1994a; Huan and Tanaka 2004; Teixeira da Silva Green capsules of C. bicolor (naturally pollinated) were
et al. 2007), thin cell layers of PLBs (Malabadi et al. 2008), collected from National Orchidarium, Yercaud (11°480 N
artificial seeds (Nhut et al. 2005) and through somatic latitude 10°550 and 78°-130 E longitude, 1,500 m a.s.l),
embryogenesis (Begum et al. 1994b; Chang and Chang Salem, Tamilnadu, India. The freshly collected capsules
1998; Huan and Tanaka 2004; Mahendran and Narmatha were surface sterilized in 0.01 % (w/v) mercuric chloride
Bai 2012). Hoque et al. (1994) found that Phytomax solution for 2 min, rinsed thoroughly thrice with sterile
medium was the best among the five different media distilled water, dipped in 70 % (v/v) ethanol for 30 s and
(Phytomax, Modified Vacin and Went, KC, KCM and LO flamed. Seeds from the surface sterilized capsules were
medium) tested for large scale multiplication of C. bicolor. extracted by longitudinally splitting the capsule with a
Transplantation stage continues to be a major bottleneck sharp sterilized surgical blade. The seeds were then spread
in the micropropagation of orchids. A substantial number as thin film in the test tube containing 20 ml of culture
of micropropagated plantlets fail to survive when trans- media. The cultures were maintained at 25 ± 2 °C under
ferred from in vitro conditions to a greenhouse or field cool white fluorescent tubes at a light intensity of
environment. The greenhouse or field environment have 50 lmol m-2 s-1 with 16/8 h L/D photoperiod in culture
substantially lower relative humidity, higher and intense room conditions.
light levels that are stressful to micropropagated plants
compared to in vitro conditions (Chugh et al. 2009). The Seed viability test
manipulation of acclimatization conditions reduce the
plantlet losses, however, it incurs an additional production Seed viability was tested according to Vellupillai et al.
cost. Attempts have been made to reduce transplantation (1997). For enumerating the seed viability percentage,
losses by the use of CO2 enriched environment and high seeds from the fresh capsules were treated with 1 % (w/v)
light intensity for autotrophic miropropagation (Vyas and 2,3,5-triphenyl tetrazolium chloride (pH 7.0) in the dark
Purohit 2003; Dave and Purohit 2004). This technology is overnight. Treated seeds were observed with a light
yet to gain popularity (Nowak 1998). Alternatively the microscope and scored as either viable (red embryo) or
naturally occurring beneficial mycorrhizal association nonviable (white embryo).
between fungi and orchids could be exploited for better
nutrient acquisition (Zhu and Miller 2003), improved plant Asymbiotic seed culture
water relations and imparting tolerance to abiotic stresses
such as drought and salinity (Kapoor et al. 2008) in in vitro Four asymbiotic orchid seed germination media namely
derived plantlets. Such biologically hardened plants could MS (Murashige and Skoog 1962), KC (Knudson 1946),
perform better and consequently, reduce losses due to KCM (Morel 1965) and Lindemann orchid (LO) (Linde-
environmental stresses. mann et al. 1970) were tested to select a suitable medium

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Acta Physiol Plant (2013) 35:829–840 831

for seed germination. All the media were supplemented taxon was identified using morphological and growth
with 3 % (w/v) sucrose and solidified with 0.8 % (w/v) characteristics as described by Currah et al. (1997).
agar (Hi media-India).The pH of the media was adjusted to
5.6–5.8 with 1 N NaOH or HCl before autoclaving at Orchid mycorrhizal fungus inoculation
121 °C, 105 kPa for 20 min.
The protocorms (60-days old) and seedlings (120-days old)
Induction of multiple shoots of C. bicolor were implanted on B5 Basal medium con-
taining 2 % sucrose and 0.8 % agar for co-cultivation in
Lindemann orchid medium was supplemented with cyto- test tube (protocorm) and conical flasks (seedling). One
kinins such as BAP, KIN and TDZ for multiple shoot PDA agar (6 mm in diameter) disc containing the fungal
induction. The media turned brown and arrested the seed- mycelium was taken from the margin of the fungal colony
ling development. So, the protocorms (60-days old) and placed 2 cm away from the protocorm or seedling.
obtained from LO medium were subcultured in B5 medium Uninoculated protocorms (60-days old) or seedlings (120-
supplemented with cytokinins including BAP (1.10, 2.21, days old) served as control. All treatments were maintained
4.42, 8.84 or 13.26 lM), Kin (1.16, 2.32, 4.64, 9.28 or under fluorescent lights (50 lmol m-2 s-1) with a 16-h
13.92 lM) and TDZ (1.13, 2.26, 4.52, 9.24 or 13.76 lM) photoperiod at 25 ± 2 °C. The influence of the orchid
individually to asses effect on multiple shoot development. mycorrhizal fungus on plant growth parameters such as
plant height, number of new roots, root length, number of
Rooting new shoots per protocorm or seedlings were assessed
125 days after inoculation. Each treatment had ten repli-
For root development, individual shoots each with 2–3 cates and the experiment repeated thrice.
expanded leaves were detached from the shoot clumps and
transferred to B5 liquid medium containing either IBA Estimation of fungal colonization
(4.92–14.76 lM) or NAA (5.37–16.11 lM).
Free-hand transverse sections of the roots were made and
Fungal isolation and morphological characterization stained with trypan blue. Stained sections were mounted
and observed for presence of mycorrhizal structures
Fresh roots of the epiphytic orchid Aerides crispum Lindl. (pelotons). To determine the ratio of cortical cells con-
were collected from Vellingiri hill (longitude 6°400 and taining pelotons, the total number of cortical cells per
7°100 E and latitude 10°550 and 11°100 N, 1,400 m a.s.l) microscopic field (five microscopic fields were examined
Coimbatore, Tamilnadu, India. Mycorrhizal fungus (des- for each section) and the number of cortical cells con-
ignated as AC-01) was isolated using modification of taining intact and lysed pelotons were counted and recor-
Masuhara and Katsuya (1994) method. The roots were ded. Pelotons with distinguishable hyphae were considered
soaked in teepol detergent solution for about 5–6 min and intact and those in which the hyphae were indistinguishable
thoroughly washed with running water. The roots were were considered lysed.
surface sterilized with 0.05 % (w/v) mercuric chloride for
2 min and washed 3–7 times with sterile distilled water. Ex vitro plant establishment
The roots were then cut into longitudinal thin sections after
the removal of the velamen tissue and transferred on to For ex vitro establishment, well-rooted mycorrhizal and
potato dextrose agar (PDA) medium containing 100 mg/l non-mycorrhizal plantlets were rinsed thoroughly with tap
streptomycin. After 10 days of incubation at 30 °C, the water to remove residual nutrients from the plant body and
hyphal tips emerging from the root segments were trans- transplanted into plastic pot (10 9 8 cm; height 9 diam-
ferred onto fresh PDA medium. eter) containing sterilized vermiculite (50 g per pot). The
The fungal isolate was maintained on PDA at 30 °C in plastic pots were covered by polyethylene bags and
the dark. The diameter of five colonies were measured maintained under fluorescent lights at 50 lmol-l m-2 s-1
daily for 2 weeks. Growth rate was calculated by plotting with a 16-h photoperiod at 23 ± 2 °C.
colony diameters against time (Nontachiyapoom et al.
2010). For microscopic observation, the fungal hyphae Experimental design and data analysis
were stained in trypan blue (0.005 %) (w/v) lacto glycerol
solution and mounted in clean lacto glycerol and examined The germination percentage was recorded at 14, 28, 42 and
under an Olympus BX51 compound microscope attached 56 days of culture. Percent germination was calculated by
with a ProgRes C3 camera. The number of nuclei per fungal dividing the number of germinated seeds by the total
cell was enumerated as per Leonerd (1979). The fungal number of seeds observed. Number of shoots, height, root

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832 Acta Physiol Plant (2013) 35:829–840

number and root length was recorded after 56 days of Fig. 2 Asymbiotic seed germination, multiple shoot development c
culture. Each treatment was repeated thrice with ten rep- and effect of mycorrhiza (AC-01) on seedling development in
Cymbidium bicolor. a Formation of protocorms on Lindmann
licates. Data were subjected to analysis of variance medium after 14 days inoculation (bar 150 lm). b Embryo emerging
(ANOVA) and the mean values were separate using Dun- out of the seed by rupturing the testa on Lindemann orchid medium
can’s Multiple Range Test (DMRT). One and two-sample (bar 100 lm). c 56 days old protocorms developed on LM orchid
t test was used to compare the effects of the presence of the medium (bar 0.5 cm). d Formation of multiple shoots on B5
supplemented with 13.92 lM of KIN (bar 0.5 cm). e Multiple shoots
orchid mycorrhizal fungus in protocorms and seedlings. developed on B5 medium supplemented with 4.42 lM of BAP (bar
The statistical package SPSS (Version-17) was used for the 0.5 cm). f Rooting of micropropagated plant on B5 medium
analyses. supplemented with 14.76 lM of IBA (bar 0.5 cm). g Effect of
isolated orchid mycorrhiza (AC-01) on protocorm (bar 0.5 cm).
h Influence of fungus on new shoot formation after 4 months of
mycorrhizal inoculation (bar 1.0 cm). i Influence of fungus on root
Results growth (bar 1.0 cm). j Hardened plantlet after 2 months under ex
vitro condition (bar 2.0 cm). E embryo. S seed coat
Tetrazolium (TZ) viability test indicated a mean embryo
viability of 73 %. The embryos enlarged and occupied the inoculation, whereas protocorm development was observed
entire seed coat after 14 days of culture (Fig. 2a). Germi- only after 70 days of culture in KC medium.
nation as evidenced by enlargement of the embryo was first When LO medium was supplemented with cytokinins
observed in LO medium, followed by both KCM and MS such as BAP, KIN and TDZ for multiple shoot induction,
medium. The seed germination was delayed in KC medium the media turned brown with time possibly indicating the
by 42 days. phenolic exudation. Browning of the media coincided with
After 42 days, the embryos by repeated cell divisions the arrest of seedling development, so the seedlings were
emerged rupturing the testa in KCM medium (Fig. 2b). transferred to B5 medium supplemented with three differ-
The percentage seed germination was 73.5 % in KCM ent cytokinins such as BA or TDZ or Kin individually
basal medium, 55.6 % in MS medium and 43.5 % in KC (Table 1). Two shoots were formed in B5 medium devoid
medium. In LM the percentage seed germination was of growth regulators at 90 days. Among the three cytoki-
89.2 % (Fig. 1). The embryos in LO medium swelled and nins tested, multiple shoot induction was frequent in BAP
transformed into globular hairy protocorm (Fig. 2c), which (4.42 lM), followed by Kin (13.92 lM). Among the dif-
subsequently produced promeristem. The germination ferent levels BAP tested, the maximum number of shoots
process was initially erratic and inconsistent, but the per- was observed on the B5 medium containing 4.42 lM of
centage of germination increased gradually and reached the BAP (27.59 ± 0.88) (Table 1; Fig. 2d, e). The shoot buds
significantly highest value at 56 days after culture. Seed first appeared as small white protuberances over the surface
germination was 96.6 % in LO medium followed by 89 % of the protocorm which eventually developed into multiple
in KCM medium, 77.5 % in MS medium, and 62.7 % in shoots within 35–56 days. The number of shoot buds
KC medium. After 56 days, a pair of leaves emerged from increased with increasing concentration of BAP up to an
the surface of the protocorms. MS and KCM basal media optimal level of 4.42 lM. Among the various concentra-
supported protocorm development after 56 days of tions of Kin and TDZ tested, maximum number of the
multiple shoots were recorded in B5 medium supplemented
with 13.92 lM Kin (20.75 ± 0.35 shoots/explant) and
120
KC KCM MS LO 13.76 lM TDZ (5.25 ± 0.31 shoots/explant).
Seed germination (%)

100
Rooting
80
IBA significantly promoted all the rooting parameters,
60
including root length and number of root (Table 2; Fig. 2f).
40
Significantly higher root numbers were recorded in
14.76 lM of IBA (5.40 ± 0.24).
20
Characterization of the fungal morphology
0
14 28 42 56
Colonies of the isolate AC01 cultured on PDA and iden-
Number of days
tified as Moniliopsis sp. grew at the rate of 3.41 mm per
Fig. 1 Effects of different culture media on seed germination of day. The colonies were effuse, dirty white and turning
Cymbidium bicolor. Error bars indicates ±SE brown with age. The mycelium was partly superficial and

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Table 1 Effect of various


BA (lM/l) Kin (lM/l) TDZ (lM/l) No of multiple shoots Shoot length (cm)
cytokinins on multiple shoot
induction from seed derived – – – 2.20 ± 0.20i 3.58 ± 0.19d,e
protocorms of Cymbidium e
bicolor 1.10 – – 9.60 ± 0.40 2.44 ± 0.15h,i
c
2.21 – – 17.80 ± 0.20 2.56 ± 0.12g,h
a
4.42 – – 28.00 ± 0.31 2.88 ± 0.74f,g,h
b
8.84 – – 19.80 ± 0.37 2.98 ± 0.83f,g
13.26 – – 18.60 ± 0.50c 3.74 ± 0.78c,d
g,h
– 1.16 – 3.40 ± 0.24 4.14 ± 0.11b,c
f
– 2.32 – 5.20 ± 0.20 4.76 ± 0.12a
e
– 4.64 – 10.40 ± 0.50 3.34 ± 0.27d,e,f
d
– 9.28 – 15.60 ± 0.40 3.20 ± 0.27e,f
– 13.92 – 20.40 ± 0.24b 2.02 ± 0.14j
*** Significant at P \ 0.001 – – 1.13 2.00 ± 0.29 i
4.40 ± 0.24a,b
Values represent mean ± SE of – – 2.26 2.60 ± 0.24 h,i
4.60 ± 0.15a,b
three repeated experiments each h,i
– – 4.52 3.00 ± 0.18 3.76 ± 0.10c,d
with 10 replications. Means in a g
column with the different letter – – 9.24 4.20 ± 0.20 3.32 ± 0.21d,f
(superscript) are significantly – – 13.76 5.20 ± 0.20f 2.36 ± 0.16i,j
different according to DMRT F-value15, 761.68*** 24.56***
64
(P \ 0.05)

Table 2 Effect of auxins on root induction from regenerated shoots Mycorrhizal association significantly enhanced the number
of Cymbidium bicolor of roots, root length and shoot number (Table 3; Fig. 2i).
Growth hormone No of roots/plant Root length/
plant (cm) Ex vitro establishment of plantlets
IBA (lM/l) NAA (lM/l)

4.92 – 3.40 ± 0.24c,d 3.48 ± 0.34c After 60 days, the cover was gradually loosened, thus
b,c
9.84 – 4.00 ± 0.00 4.70 ± 0.15b dropping the humidity (65–70 %). The procedure adopted
14.76 – 5.40 ± 0.24a 5.82 ± 0.14a subsequently resulted in successful in vitro hardening of the
5.37 2.40 ± 0.24e 2.84 ± 0.15d plants (Fig. 2j). Within 2 weeks, the plants were completely
10.74 3.20 ± 0.20d 3.78 ± 0.21c acclimatized and survived in open containers. This resulted
b
16.11 4.20 ± 0.20 4.48 ± 0.37b in 90 % survival compared to 50 % in control.
F value5, 24 24.15*** 88.87***
Fungal colonization
*** Significant at P \ 0.001
Values represent mean ± SE of three repeated experiments each with
10 replications. Means in a column with the different letter (super- The root of C. biolor consisted of velamen, exodermis with
script) are significantly different according to DMRT (P \ 0.05) passage cells, cortex, endodermis and vascular region. The
fungal hyphae entered the roots through the root hairs
partly immersed in the medium (Fig. 3a, b). The hyphae (Fig. 4a) or well-developed multilayered velamen before
were regularly septate, brown, 4.37–(5.52)–8.05 lm wide entering into exodermal passage cells. Passage cells
and multinucleate (5–8 per cell). Rhizomorphs and clamp appeared as sunken or broken cells in the exodermis where
connections were absent. Monilioid cells when present the fungal hyphae aggregated (Fig. 4b). Pelotons were
were terminal short chains and ellipsoidal [6.9–(12.96)– numerous in the cortical cells. The pelotons were irregu-
23.0 9 6.9–(11.08)–16.1 lm] (Fig. 3c, d). Numerous larly distributed in the cortex and were more frequent in the
cluster of sclerotia formed a brown crust on the colony outer cortex than in the inner cortex (Fig. 4c). In colonized
surface (Fig. 3e, f). cortical cells, the nucleus was displaced to the periphery of
the cell (Fig. 4d, e). The hyphae penetrated the cortical cell
Fungal effects on plant growth wall and continuously spread inwards forming pelotons
(Fig. 4f). Individual hyphae constituting the peloton could
At the time of harvest (4 months after co-inoculation), be easily differentiated during initial stages but was grad-
mycorrhizal plantlets had better growth with new root and ually lost with time indicating its slow lysis and digestion
shoot production than non-mycorrhizal plantlets (Fig. 2g, h). (Fig. 4g, h). Intact and lysed pelotons occurred simulta-
However, the magnitude of response varied with treatments. neously in different cortical cells (Table 4).

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Acta Physiol Plant (2013) 35:829–840 835

Fig. 3 Morphological
characterization of Moniliopsis
sp. (AC-01). a, bMoniliopsis sp.
(AC-01) cultured on PDA
medium (bar 1.0 cm). c,
d Hyphae in short chains (bar
10 lm). e, f Monilioid cells (bar
20 lm)

Discussion to a great extent was influenced by the quality of basal


medium. Based on the previous reports on the effectiveness
In vitro seed germination has been suggested as a suitable of various basal media for asymbiotic seed culture of
propagation method for conservation of orchids (Kauth orchids, the four basal media were selected. Of the four
et al. 2006; Stewart and Kane 2006). According to Hartman media tested in the present study, LO medium showed
et al. (1997) specific endogenous growth promoting and significantly higher (96.6 %) percentage of seed germina-
inhibiting compounds are involved directly in control of tion after 56 days of culture initiation. Besides LO medium,
seed development, dormancy and germination. To initiate other three media also supported moderate germination
seed germination, three conditions must be fulfilled, which (89 % in KCM, 77. 5 % in MS medium and 62.7 % in KC);
includes seed viability, appropriate environmental condi- however, germination was delayed and the germinated
tions and overcoming primary dormancy. In the present seeds failed to differentiate into either PLBs or plantlets
study successful seed germination and protocorm formation properly. The nutrient regime for orchid culture is species

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836 Acta Physiol Plant (2013) 35:829–840

Table 3 Effect of Moniliopsis sp. on growth of Cymbidium bicolor tetrazolium test. This suggests that tetrazolium test is
Treatment
unreliable for assessing C. bicolor seed viability. This
contrasts the results reported for hard-seeded orchids such
stage Control Inoculated t value as Cypripedium (Lauzer et al. 1994; Vujanovic et al. 2000),
Protocorms Eulophia alta (Johnson et al. 2007) and Satyrium nepalense
Plant height (cm) 0.56 ± 0.05 2.20 ± 0.20 -9.698*** (Mahendran and Narmatha Bai 2009) where tetrazolium
No. shoots 2.00 ± 0.31 4.20 ± 0.37 -4.491** staining grossly overestimated germinability. Differences
Seedling between estimated viability and observed germinability in
Plant height (cm) 3.80 ± 0.37 5.40 ± 0.40 -5.715* C. bicolor may result from less optimal pretreatment or
No. shoots 3.40 ± 0.24 12.80 ± 0.37 -8.485*** staining methods. The observations of the present and other
No. new roots 4.00 ± 0.44 11.00 ± 0.44 -13.898 studies clearly exemplify the need for direct seed germi-
Length of root (cm) 5.80 ± 0.58 15.80 ± 0.37 -4.811**
nation tests in orchids rather than tetrazolium test for
asserting seed viability.
Values represent mean ± SE of three repeated experiments each with In C. bicolor, LO medium was supplemented with
10 replications based on Two Paired Samples test at P \ 0.05
*, **, *** cytokinins such as BAP, KIN and TDZ for multiple shoot
Significant at P \0.5, P \ 0.01, P \0.001 respectively
induction, the media turned brown with time possibly
indicating the phenolic exudation. Browning of the media
coincided with the arrest of seedling development, so the
specific and no single culture medium is universally appli- seedlings were transferred to B5 medium. Excessive phe-
cable for all the orchid species. For example MS medium nolic production is possibly due to increased polyphenol
for Malaxix khasiana (Deb and Temjensangba 2006), oxidase and catalase activity triggered by certain culture
Coelogyne suaveolens (Sungkumlong and Deb 2008) and conditions, such as improperly balanced nutrients or the
Cymbidium aloifolium (Deb and Pongener 2011), P723 lack of required growth stimulating substance as suggested
medium for Eulophia alta (Johnson et al. 2007) and New by Stoutamire (1974) and Harvais (1982).
Dogashima medium for Calanthe tricarinata (Godo et al. The success of rapid and direct shoot regeneration from
2010) were reportedly most suitable over other nutrient protocorm explant opens an efficient way to mass propa-
media. gate C. bicolor. The type and concentration of growth
In C. bicolor, the percentage of germination varied with regulators play an important role during in vitro propaga-
media. All the media used in the present study differed in tion of many orchid species (Arditti and Ernst 1993). In the
their chemical compositions. Mineral salts in the media present study protocorms developed multiple shoots
varied not only in their concentrations, but also in their directly on B5 medium supplemented with cytokinins.
available forms. Briefly, MS medium is highly enriched Protocorms can possess several centers of meristematic
with both macro- and micro-elements, whereas KC and activity, but usually develop only a single shoot as
KCM medium contained low amounts of both macro- and observed in nature (Tatarenko and Vakhrameeva 1998),
micro-elements and lacked vitamins and amino acids. whereas, in vitro, two pathways of protocorm cloning have
Conversely, LO medium is moderate in both macro- and been revealed: the formation of a great number of shoot
micro-elements but is enriched with amino acids and apices, with further endogenous production of adventitious
vitamins, thus conditions are suspected to be responsible roots and the formation of numerous secondary protocorms
for enhancing seed germination which is in agreement with from epidermal cells of a single protocorm (Andronova
many workers (Sharma et al. 1991; Kauth et al. 2006; et al. 2000). In C. bicolor, the protocorm produced multiple
Stewart and Kane 2006; Roy et al. 2011). Mariat (1949) shoots directly on the medium fortified with different
reported that vitamin B favoured germination and differ- cytokinins individually. Among the cytokinins tested, BAP
entiation of Cattleya seedlings; thiamine, nicotinic acid and was found more effective for inducing multiple shoots
biotin were most effective for Cattleya hybrids. Pyridoxine (28.00 ± 0.31/explant). BAP was also found suitable for
with nicotinic acid and biotin favoured better germination protocorm and shoot multiplication in Geodorum densi-
of Orchis laxiflora seeds (Mead and Bulard 1979). These florum (Sheelavantmath et al. 2000), Cymbidium pendulum
findings indicate the existence of species–medium speci- (Pathak et al. 2001), Geoderum purpureum (Mohapatra and
ficity. Such species-specific medium for seed germination Rout 2005), Dendrobium tranparens (Sunitibala and
have also been reported by many workers (Arditti and Kishor 2009) and Eulophia nuda (Panwar et al. 2012),
Ernst 1993; Bhadra et al. 2002; Kauth et al. 2008; Roy whereas TDZ was beneficial in Satyrium nepalense
et al. 2011). (Mahendran and Narmatha Bai 2009), Aerides vanda-
The percentage of seed germination recorded in the rum 9 Vanda stangeana (Kishor and Sunitibala Devi
germination test was higher compared to those recorded in 2009).

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Acta Physiol Plant (2013) 35:829–840 837

Fig. 4 Anatomical studies showing association of the fungus in the Pelotons—the nucleus is pushed aside in the cell protoplasm (arrow).
roots of Cymbidium bicolor. a The entry (arrow) of the fungal hyphae fArrow points to the hyphae passing through the cell wall. g Cortical
(H) through the root hair (RH). b Colonized exodermal passage cell— cells isolated and stained with tryphan blue, showing pelotons
Note the colonized cell with a fungal hyphae (arrow). c Formation of (arrow). h Lysing pelotons. Nucleus is pushed aside in the cell
Pelotons in the cortical cell. d Transverse section of myorrhizal root protoplasm (arrow). P pelotons. C cortical cell, LP lysing pelotons,
showing the cortical cells packed with pelotons. e Cortical cells with H hyphae, RH roots hairs

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838 Acta Physiol Plant (2013) 35:829–840

Table 4 Frequency of colonization in the root section of Cymbidium Conclusion


bicolor
Root and mycorrhizal characteristics An efficient method for the in vitro germination of seeds
and for the regeneration of a large number of plantlets from
Number of root hairs 7.00 ± 0.316 protocorms for C. bicolor has been described. We isolated
Number of passage cells 12.60 ± 0.510 Moniliopsis sp. from A. crispum. This fungal isolate was
Number of pelotons in cortical layer 84.80 ± 5.791 capable of enhancing the growth of C.bicolor protocorm
Number of digested Pelotons in cortical layer 17.40 ± 1.029 and seedling. As shown here, seeds of C. bicolor could
Total number of cortical cells 180.00 ± 26.840 germinate and seedlings could develop without fungal
Values represent mean ± SE of three repeated experiments each with symbionts. However, inoculation of C. bicolor seedling
10 replications based on one sample test at P \ 0.05 with Moniliopsis sp. significantly increased plant growth
and their survival rate. Further experiments are now being
The phenomena of mycotropism and mycorrhiza are carried out to assess the in vitro growth promoting ability
discussed by many workers in both terrestrial and epiphytic of this fungal strain in other orchid taxa.
orchids (Chang and Chou 2007; Chang et al. 2007;
Author’s contribution G. Mahendran carried out the
Fang et al. 2008). In the present study, the fungal isolate
entire research work on tissue culture and mycorrhiza.
Moniliopsis sp. played a very important role in the seedling
V. Muniappan helped in designing the experiment per-
development. The mycorrhizal seedlings developed several
taining to mycorrhiza. M. Ashwini carried out a part of the
new and long roots and shoots compared to their non-
tissue culture work. T. Muthukumar helped with experi-
mycorhizal conspecifics. The fungal hyphae formed tightly
mental design, co-ordination of mycorrhizal part and
interwoven pelotons which are considered to be the most
interpretation of data. V. Narmatha Bai helped with
distinctive characteristic future of orchid mycorrhiza
co-ordination of tissue culture and interpretation of data.
(Currah and Zelmer 1992). Thus, an appropriate symbiotic
relationship has been established between the inoculated Acknowledgments The financial support by University Grants
orchid mycorrhizal fungus and C. bicolor seedlings. Commission (UGC), 37-97/2009 (SR) Dated: 19.12.2009. New Delhi
The fungal entry into roots was through the root hair or is gratefully acknowledged.
the velamen cells and spread through the cortical cells as
observed in C. sinense and C. goeringii (Fan et al. 1999;
Wu et al. 2005). The results of the present study clearly References
showed that mycorrhizal fungi improved the development
Abadie JC, Puttsepp U, Bonfante P, Selosse MA (2006) Cephalan-
of C. bicolor protocorms and seedlings. In the present thera longifolia (Neottieae, Orchidaceae) is mixotrophic: acom-
study we just demonstrated the growth promoting ability of parative study between green and non photosynthetic
the endophyte, but the real mechanism remains unknown individuals. Can J Bot 84:1462–1477
and needs further investigation. Observation similar to the Andronova EV, Batygina TB, Vasilyeava VE (2000) Protocorm. In:
Batygina T (ed) Embryology of flowering plants. Terminology
present study was made in Cymbidium kanran cv. Hook. and concepts 3: 329–334
where mycorrhizal fungus inoculation stimulated plant Begum AA, Tamaki M, Kako S (1994a) Formation of protocorm-like
growth parameters like plant height, leaf and root devel- bodies (PLBs) and shoot development through in vitro cultures
opment and fresh weight (Lee et al. 1998). In addition, of outer tissue of Cymbidium PLB. Jpn Soc Hortic Sci
63:663–673
uptake of N by mycorrhizal C. kanran roots was more than Begum AA, Tamaki M, Tahara M, Kako S (1994b) Somatic
non-mycorrhizal roots. Some studies do indicate that embryogenesis in Cymbidium through in vitro culture of inner
orchid mycorrhizal fungi secrete plant hormones which tissue of protocorm-like bodies. J Jpn Soc Hortic Sci 63:419–427
have been shown to have strong effects in improving the Bhadra SK, Bhattacherjee B, Barua AK, Hossain MM (2002)
Micropropagation of Dendrobium aphyllum (Roxb.)
growth of orchids (Liu et al. 2010). Moniliopsis sp. isolated G.E.C.Fisher. Bang J Gent Biotech 3:47–50
from A. crispum was able to improve the growth of Chang C, Chang WC (1998) Plant regeneration from callus cultures
C. bicolor. This clearly shows the lack of host specificity of of Cymbidium ensifolium var misericors. Plant Cell Report
this fungal strain and its host benefit across species. This is 17:251–255
Chang DCN, Chou LC (2007) Growth responses, enzyme activities
in accordance with the observation where an Epulorhiza and component changes as influenced by Rhizoctonia orchid
repens strain (UAMH9824) isolated from Spiranthes mycorrhiza on Anoectochilus formosanus Hayata. Bot Stud
brevilabris was able to promote seedling development in 48:445–451
other taxa (see Massey and Zettler 2007). It can be con- Chang DCN, Chou LC, Lee GC (2007) New cultivation methods for
Anoectochilus formosanus Hayata. Orchid Sci Biotech 1:56–60
cluded that the fungal strain Moniliopsis sp. used in the Chugh S, Guha S, Usha Rao I (2009) Micropropagation of orchids:
present study clearly enhanced the growth of C. bicolor a review on the potential of different explants. Sci Hort
in vitro. 22:507–520

123
Acta Physiol Plant (2013) 35:829–840 839

Chung JD, Chun CK, Choi SO (1985) Asymbiotic germination of germination. In: Teixeira da Silva JA (ed) Floriculture,
Cymbidium ensifolium. II. Effects of several supplements to the Ornamental and Plant Biotechnology: Advances and Topical
medium, pH values and light and/or dark culture periods on Issues, vol V, 1st edn. Global Science Books Ltd., Isleworth,
growth of rhizome and organogenesis for rhizome. J Korean Soc pp 375–391
Hortic Sci 26:86–92 Kishor R, Sunitibala Devi H (2009) Induction of multiple shoots in a
Currah RS, Zelmer C (1992) A key and notes for the genera of fungi monopodial orchid hybrid (Aerides vandarum Reichb.f x Vanda
mycorrhizal with orchids and a new species of the genus stangeana Reichb.f) using thidiazuron and analysis of their
Epuhorhiza. Rep Tottori Mycol Inst 30:43–59 genetic stability. Plant Cell Tiss Organ Cult 97:121–129
Currah RS, Zelmer CD, Hambleton S, Richardson KA (1997) Fungi Knudson L (1946) A new nutrient solution for the germination of
from orchids mycorhizas. In: Arditti J, Pridycon AM (eds) orchid seed. Am Orchid Soc Bull 15:214–217
Orchid Biology: Reviews and Prespectives. VII. Kluwer Aca- Lauzer D, St- Arnaud M, Barabe D (1994) Teterazolium straining and
demic publishers, Great Britan, pp 117–170 in vitro germination of mature seeds of Cypripedium calceolus
Dave N, Purohit SD (2004) In vitro growth shoot multiplition of (orchidaceae). Lindeleyana 9:197–204
Achras sapota a controlled carbon dioxide environment. Biol Lee SS, Oh CH, Paek KY, Lee TS (1998) Isolations of the orchid
Plant 48:621–624 mycorrhizal fungi from the roots of the Korean native orchids
Deb CR, Pongener A (2011) Asymbiotic seed germination and and inoculations of the isolates to four different orchids. Korean
in vitro seedling development of Cymbidium aloifolium (L.) Sw.: J Plant Pathol 14:536–542
a multipurpose orchid. J Plant Biochem Biotechnol 20:90–95 Leonerd JH (1979) Practical nuclear staining procedures for Rhizoc-
Deb CR, Temjensangba (2006) In vitro propagation of threatened tonia like fungi. Phytopathol 69:958–961
terrestrial orchid. Malaxis khasiana Soland ex. Swartz through Lindemann EGP, Gunckel JE, Davidson OW (1970) Meristem culture
immature seed culture. Indian J Exp Biol 44:762–766 of Cattleya. Am Orchid Soc Bull 39:1002–1004
Arditti J, Ernst, R (1993) Micropropagation of orchids. Wiley, New Liu H, Luo Y, Liu H (2010) Studies of mycorrhizal fungi of Chinese
York orchids and their role in orchid conservation in China—a review.
Fan L, Guo SX, Xiao PG (1999) Study on the structure and Bot Rev 76:241–262
localization of acid phospatase of mycorrhiza root of Cymbidium Mahendran G, Narmatha Bai V (2009) Mass propagation of Satyrium
sinense (Orchidaceae). Acta Bot Yunnan 21:197–201 nepalense D.Don- A medicinal orchid via seed germination. Sci
Fang D, Hongxia LIU, Hui JIN, Yibo LUO (2008) Symbiosis between Hort 119:203–207
fungi and the hybrid Cymbidium and its mycorrhizal micro- Mahendran G, Narmatha Bai V (2012) Direct somatic embryogenesis
structures. For Stud China 10:41–44 and plant regeneration from seed derived protocorms of Cym-
Godo T, Komori M, Nakaoki E, Yukawa T, Miyoshi K (2010) bidium bicolor Lindl. Sci Hort 135:40–44
Germination of mature seeds of Calanthe tricarinata Lindl. an Malabadi RB, Teixeira da Silva JA, Nataraja K, Mulgund GS (2008)
endangered terrestrial orchid, by asymbiotic culture in vitro. Shoot tip transverse thin cell layers and 2, 4-epibrassinolide in
In Vitro Cell Dev Biol Plant 46:323–328 the micropropagation of Cymbidium bicolor Lindl. Floricult
Hartman H, Kester D, Davis F, Geneve R (1997) Plant propagation; Ornamental Biotechnol 2:44–48
Principles and Practices, 6th edn. Prentice-hall, New Jersey, Mariat F (1949) Action de l’acide nicotinique sur la germination et al.
pp 125–144 developmentdes embryous de Cattleya. Compt Rend Acad Sci
Harvais G (1982) An improved culture medium for growing the 229:1355–1357
orchid Cypripedium reginae axienically. Can J Bot 51:327–332 Massey EE, Zettler L (2007) An expanded role for in vitro symbiotic
Hoque MI, Roy AR, Sarker RH, Haque MM (1994) Micropropagation seed germination as a conservation tool: two case studies in
of Cymbidium bicolor through in vitro culture. Plant Tissue Cult North America (Platanthera leucophaea and Epidendrum no-
4:45–51 turnum). Lankesteriana 7:303–308
Hossain MM, Sharma M, Teixeira da Silva JA, Pathak P (2010) Seed Masuhara G, Katsuya K (1994) In situ and in vitro specificity between
germination and tissue culture of Cymbidium giganteum Wall. ex Rhizoctonia sp. and Spiranthes sinensis (Persson) Ames.
Lindl. Sci Hortic 123:479–487 var.amoena (M. Bieberstein) Hara (Orchidaceae). New Phytol
Hou XQ, Guo SX (2009) Interaction between a dark septate 127:711–718
entophytic isolate from Dendrobium sp. and roots of D. nobile Mead JW, Bulard C (1979) Vitamins and nitrogen requirements of
seedlings. J Integ Plant Biol 51:374–381 Orchis laxiflora Lamk. New Phytol 83:129–136
Huan LVT, Tanaka M (2004) Callus induction from protocorm-like Mohapatra A, Rout GR (2005) In vitro micropropagation of
body segments and plant regeneration in Cymbidium (Orchida- Geoderum purpureum R.Br. Indian J Biotech 4:568–570
ceae). J Hortic Sci Biotechnol 79:406–410 Morel G (1964) Tissue culture—a new means of clonal propagation
Johnson TR, Stewart SL, Daniela D, Kane ME, Richardson L (2007) of orchids. Am Orchid Soc Bull 33:437–478
A symbiotic and symbiotic seed germination of Eulophia alta Morel GM (1965) Clonal propagation of orchids by meristem culture.
(orchidaceae)—Preliminary evidence for the symbiotic culture Cymbidium Soc News 20:3–11
advantage. Plant Cell Tiss Org Cult 90:313–323 Murashige T, Skoog F (1962) A revised medium for rapid growth and
Julou T, Burghardt B, Gebauer G, Berveiller D, Damesin C, Selosse bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497
M (2005) Mixotrophy in orchids: insights from a comparative Nayak NR, Rath SP, Patnaik S (1997) In vitro propagation of three
study of green individuals and non-photosynthetic individuals of epiphytic orchids, Cymbidium aloifolium (L.) Sw., Dendrobium
Cephalanthera damasonium. New Phytol 166:639–653 aphyllum (Roxb.) Fisch. and Dendrobium moschatum (Buch-
Kapoor R, Sharma D, Bhatnagar AK (2008) Arbuscular mycorrhizae Ham) Sw. through thidiazuron-induced high frequency shoot
in micropropation system and their potential applications. Sci proliferation. Sci Hortic 71:243–250
Hortic 116:227–239 Nhut DT, Tien TNT, Huong MTN, Hien NTH, Huyen PX, Luan VQ,
Kauth PJ, Vendrame WA, Kane ME (2006) In vitro seed culture and Le BV, Teixeira da Silva JA (2005) Artificial seeds for
seedling development of Calopogon tuberosus. Plant Cell Tiss preservation and propagation of Cymbidium spp. Prop Orna-
Org Cult 85:91–102 mental Plants 5:67–73
Kauth PJ, Dutra D, Johnson TR, Stewart SL, Kane ME, Vendrame W Nontachiyapoom S, Sasirat S, Manoch L (2010) Isolation and
(2008) Techniques and applications of in vitro orchid seed identification of Rhizoctonia-like fungi from roots of three

123
840 Acta Physiol Plant (2013) 35:829–840

orchids genera, Paphiopedium, Dentrobium and Cymbidium Gardens, National Science Museum and Botanic Gardens
collected in Chiang Rai and Chiang Mai provinces of Thailand. Conservation International, Tokyo, pp 9–23
Mycorrhiza 20:459–471 Stewart SL, Kane ME (2006) Symbiotic germination of Habenaria
Nowak J (1998) Benefits of in vitro biotization of plant tissue cultures macroceratitis (Orchidaceae), a rare Florida terrestrial orchid.
with microbial inoculants. In Vitro Cell Dev Biol Plant Plant Cell Tiss Org Cult 86:159–167
34:122–130 Stoutamire WP (1974) Terrestrial orchid seedlings. In: Withner CL
Panwar D, Ram K, Harish, Shekhawat NS (2012) In vitro propagation (ed) The Orchids Scientific Studies. John Wily and Son, New
of Eulophia nuda Lindl., an endangered orchid. Sci Hort York, pp 101–128
139:46–52 Sungkumlong, Deb CR (2008) Effects of different factors on
Pathak P, Mahant KC, Gupta A (2001) In vitro propagation as an aid immature embryo culture, PLBs differentiation and rapid mass
to conservation and commercialization of Indian orchids: seed multiplication of Coelogyne suaveolens (Lindl.) Hook. Indian J
culture. In: Pathak P, Sehgal RN, Shekhar N, Sharma M, Sood A Exp Biol 46:243–248
(eds) Orchids: Science and Commerce. Bishen Singh Mahendra Sunitibala H, Kishor R (2009) Micropropagation of Dendrobium
Pal Singh, India, pp 319–362 transparens L. from axenic pseudobulb segments. Indian J
Rasmussen H (1995) Terrestrial orchids from seed to mycotrophic Biotech 8:448–452
plant. Cambridge University Press, Cambridge Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the
Rasmussen HN (2002) Recent developments in the study of orchid age of extinction. Ann Bot 104:543–556
mycorrhiza. Plant Soil 244:149–163 Tatarenko IV, Vakhrameeva MG (1998) On vegetative propagation in
Rasmussen HN, Rasmussen FN (2007) Trophic relationships in orchids. Byulleten’ Botanicheskogo Sada I S Kosenko
orchid mycorrhiza diversity and implications for conservation. 7:155–157 (in Russian)
Lankesteriana 7:334–341 Teixeira da Silva JA, Giang DTT, Chan MT, Sanjaya Norikane A,
Roy AR, Patel RS, Patel VV, Sajeev S, Deka BC (2011) Asymbiotic Chai ML, Chico-Ruı0 z J, Penna S, Granstrom T, Tanaka M
seed germination, mass propagation and seedling development (2007) The influence of different carbon sources, photohetero-,
of Vanda coerulea Griff ex. Lindl. (BlueVanda):an in vitro photoauto- and photo mixotrophic conditions on protocorm-like
protocol for an endangered orchid. Sci Hort 128:325–331 body organogenesis and callus formation in thin cell layer
Sharma SK, Tandon P, Mishra RR (1991) Vitamins as related to culture of hybrid Cymbidium (Orchidaceae). Orchid Sci Bio-
axenic seed germination and seedling growth of Cymbidium technol 1:15–23
elegans Lindl. and Coelogyne punctulata Lindl. J Orchid Soc Vellupillai M, Swaru PS, Goh CJ (1997) Histological and protein
India 5:25–28 changes during early stage of seed germination in the orchid
Sharrock S (2007) Botanic gardens and their role in national plant Dendrobium curmenatum. J Hortic Sci 76:941–948
conservation strategies. In: Ishida G, Iwashinam T, Konishi T, Vujanovic V, St-Arnaud M, Barabe D, Thibeault G (2000) Viability
Kurashige Y, Oikawa J, Yukawa T (eds) Conservation of plant testing of orchid seed and the promotion of coloration and
diversity in Japanese botanic gardens. Japan Association of germination. Ann Bot 86:79–86
Botanical Gardens, Tsukuba Botanical Gardens, National Sci- Vyas S, Purohit SD (2003) In vitro growth and shoot multiplication of
ence Museum and Botanic Gardens Conservation International, Wrightia tomentosa (Roxb.) Roem et Shult in controlled carbon
Tokyo, pp 2–8 dioxide environment. Plant Cell Tiss Org Cult 75:283–286
Sheelavantmath SS, Murthy HN, Pyati AN, Ashok Kumar HG, Wang X (1988) Tissue culture of Cymbidium: plant and flower
Ravishanker BV (2000) In vitro propagation of the endangered induction in vitro. Lindleyana 3:184–189
orchid Geodorum densiflorum (Lam.) Schltr. through rhizome Wu JR, Ham SF, Zhu YY, Lv M, Wang HP, Guo W (2005) Ultra-
section culture. Plant Cell Tiss Org Cult 60:151–154 structure of symbiosis mycorrhizal between Cymbidium goerin-
Shimasaki K, Uemoto S (1990) Micropropagation of a terrestrial gii and Rhizoctonia.sp. Nanjing for Univ Nat Sci Ed 29:105–108
Cymbidium species using rhizomes developed from seeds and Yam TW, Arditti J (2009) History of orchid propagation: a mirror of
pseudo bulbs. Plant Cell Tiss Org Cult 22:237–244 the history of biotechnology. Plant Biotechnology Reports 3:1–56
Somiya K (2007) Role of botanical gardens from the standpoint of the Zettler LW, Poulter SB, McDonald KI, Stewart SL (2007) Conser-
national biodiversity strategy of Japan. In: Ishida G, Iwashinam vation-driven propagation of an epiphytic orchid (Epidendrum
T, Konishi T, Kurashige Y, Oikawa J, Yukawa T (eds) nocturnum) with a mycorrhizal fungus. Hort Sci 42:135–136
Conservation of plant diversity in Japanese botanic gardens. Zhu YG, Miller RM (2003) Carbon cycling by arbuscular mycorrhizal
Japan Association of Botanical Gardens, Tsukuba Botanical fungi in soil-plant systems. Trends Plant Sci 8:407–409

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