Plant Biotechnol Rep (2013) 7:71–82
DOI 10.1007/s11816-012-0238-z
ORIGINAL ARTICLE
The effects of soil sterilization, mycorrhizal inoculation, and rates
of phosphorus on growth and survival of Kalopanax septemlobus
microplants during the acclimatization period
Nelly S. Aggangan • Heung-Kyu Moon
Received: 24 August 2011 / Accepted: 5 June 2012 / Published online: 1 July 2012
Ó Korean Society for Plant Biotechnology and Springer 2012
Abstract In order to establish some cultural practices that
can improve growth and survival of somatic embryo (SE)-
derived microplants during the acclimatization period,
Kalopanax septemlobus was uninoculated or inoculated
with mycorrhizal fungi coded as AMM6 (a mixture of
unidentified species of Glomus and Acaulospora collected in
a closed mine tailing site in Bonghwa, Korea) during ex vitro
and grown in oven-sterilized peat vermiculite medium.
After 2 months, treated microplants were transferred into
pots filled with the same medium amended with phosphorus
fertilizer {0, 2, 4, 8, 16 and 32 mg P [as Ca(H2PO4)2

H2O] kg medium-1 coded as P0, P2, P4, P8, P16 and P32,
respectively}. At this stage, inoculated plants were greener,
with broader leaves and well-developed root systems
and had higher survival than the uninoculated ones. After
6 months, inoculated plants were 54 % heavier than the
uninoculated counterpart. In sterile medium, total dry
weight of uninoculated plants was promoted at P8 and
highest at P16. Total dry weight at P16 by uninoculated
plants was attained at P4 by the mycorrhiza-inoculated
counterpart. In non-sterile medium, total dry weight of
inoculated plants was increased at P8. By contrast, unino-
culated plants did not respond to the applied P rates. In
conclusion, more SE-derived microplants survived and grew
N. S. Aggangan
H.-K. Moon
Division of Biotechnology, Korea Forest Research Institute,
Suwon 441-350, Korea
H.-K. Moon
e-mail: hkmoon@forest.go.kr
N. S. Aggangan (&)
National Institute of Molecular Biology and Biotechnology
(BIOTECH), University of the Philippines, 4031 College Los
Baños, Laguna, Philippines
e-mail: nelly_aggangan@yahoo.com
better in sterile medium. Maximum benefits from AMM6
was attained with applied 4 and 8 mg P kg medium-1
(P4–P8) in sterile and non-sterile medium, respectively.
Keywords Acaulospora
Ex vitro
Glomus
In vitro

Kalopanax septemlobus
Somatic embryo
Introduction
Kalopanax septemlobus (Thunb. Ex A. Murr) Koidz.
[Synonym Kalopanax pictus (Thunb.) Nakai], is a deci-
duous hardwood tree in the family Araliaceae, and is found
growing in the north, northeastern, central, and southwest
China (Liu and Zheng 1998) and in Korea (Moon et al.
2008). This tree species is important with multiple uses and
has nutritional and medicinal purposes (Park et al. 1998;
Choi et al. 2002). The seeds contain 38 % oil used in the
soap-making industry (Liu and Zheng 1998). Young buds
during springtime are eaten as fresh vegetables and many
farmers cultivate the tree as a cash crop (Moon et al. 2005).
Propagation of this tree species has been a problem due to
the prolonged (2 years) seed dormancy. Cuttings and
grafting are possible but not practical because of the thorny
trunk and branches which make it difficult to handle for
cultivation. Micropropagation has been extensively used
for rapid multiplication of many important plant species
including K. septemlobus. However, its widespread use is
restricted by the high percentage of plants lost or damaged
during transfer from in vitro to ex vitro conditions.
Micropropagated plants have been continuously exposed to
a unique microenvironment that has been selected to pro-
vide minimal stress and nearly optimal conditions for
plant multiplication. These conditions lead to a phenotype
which is more fragile than greenhouse-grown plants due
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to different anatomical, morphological, and physiological
factors (Wang et al. 1993; Azcón-Aguilar et al. 1997a).
After transfer to ex vitro conditions, the plantlets have to
correct the above-mentioned abnormalities to become
adapted to a natural environment.
An acclimatization process before transfer to the nursery
is required to improve survival and growth of the plantlets.
Micropropagated K. septemlobus are incubated in an
acclimatization room prior to transfer to the glasshouse but
still the survival is low, coupled with very slow rate of
growth during the acclimatization period (Moon et al.
2005, 2008). The low percentage of surviving plantlets and
slow growth is a problem in expanding the commercial
planting of clonal K. septemlobus. Early inoculation and
colonization of in vitro-propagated plantlets by selected
mycorrhizal fungi has been reported to reduce transplant
shock during acclimatization (Chavez and Ferrera-Cerrato
1990; Azcón-Aguilar et al. 1997b; Monticelli et al. 2000).
K. septemlobus is in nature associated with arbuscular
mycorrhizal (AM) fungi. The benefits of AM for in vitro
propagated plantlets to enhance the survival rate and
improve the quality of in vitro rootless and rooted micro-
plants have been reported for many crops such as Annona
cherimola (Azcón-Aguilar et al. 1997a), guava (Estrada-
Luna et al. 2000), cassava (Azcón-Aguilar et al. 1997b),
apples and plum (Fortuna et al. 1996), and cherry and
common ash (Lovato et al. 1994).
AM fungi are the most extensively studied type of
mycorrhizae because they are present in most agricultural
and natural ecosystems and play an important role in plant
growth, health, and productivity (Smith and Read 1997).
AM fungi belong to the class Zygomycotina, order Glo-
males, with about 150 species of the genera Acaulospora,
Enthrophosphora, Gigaspora, Glomus, Sclerocystis, and
Scutellospora (Morton et al. 1993). These fungi form
morphologically distinct resting spores in the medium and
can be multiplied in the presence of a host plant. The
presence of roots allows development under favorable
conditions of vegetative mycelium which can colonize
60–90 % of the length of the root system (Bianciotto et al.
1995). Colonized plants are better able to obtain their
nourishment from the soil and resist environmental stres-
ses, including deterrence of infection by root pathogens.
The application of AM fungi also offers the opportunity to
reduce fertilizer inputs (mainly of phosphorus) (Hooker
et al. 1994). Phosphorus (P) is second to nitrogen as the
most limiting nutrient in the soil affecting plant growth.
High concentration of soil P as a result of intensive agri-
culture can decrease populations of AM fungi (Smith and
Read 1997). Likewise, high soil P concentrations have
inhibitory effects on spore germination and development of
mycorrhizal fungi (Smith and Read 1997). The substrates
used for ex vitro growth of micropropagated plants are
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important for growth and development of AM symbiosis.
The most common substrates used in plant tissue culture
are peat-based without soil and synthetic substrates like
perlite or vermiculite. Different substrates have varying
effects on the development of AM fungi and mycorrhiza
formation is favored in fumigated substrates depending
on the AM partner (Gianinazzi et al. 1990; Lee and Kim
1994; Aggangan et al. 1999, 2009a). Field performance
of micropropagated plants can be greatly improved by
ensuring a suitable mycorrhizal establishment prior to
outplanting. To our knowledge, there are no published
reports on cultural practices that may improve the survival
and facilitate growth of SE-derived K. septemlobus mi-
croplants during the acclimatization period. Moreover,
mycorrhiza inoculation studies on this tree species are not
available. Thus, this study was conducted to determine the
growth response of SE-derived K. septemlobus microplants
to soil sterilization, mycorrhizal inoculation, and rates of P
fertilizer, and to determine the rates of P fertilizer that can
maximize the benefits from mycorrhizal inoculation.
Materials and methods
Experimental design
The experiment had two parts. Part 1 used two factors (soil
sterilization and mycorrhiza inoculation) in a Complete
Randomized Design with five replicates and Part 2 had a
three-factors (soil sterilization, mycorrhiza inoculation and
rates of P fertilizer) factorial in Randomized Complete
Block Design with five replicates. The experiment was
established in an acclimatization room of the Korea Forest
Research Institute (KFRI), Suwon, Korea, from March to
September 2009.
Production of somatic embryogenetic plantlets
Somatic embryo (SE)-derived K. septemlobus microplants
were produced following the protocol developed by Moon
et al. (2008). Explants were obtained from young buds of a
rejuvenated mature tree. Explants were surface-sterilized in
70 % ethanol for 1 min, 2 % NaOCl for 20 min and rinsed
three times in sterile distilled water. The surface-sterilized
explants were cultured on MS medium (Murashige and
Skoog 1962) containing 3 % sucrose, 3 g gelrite L-1,
4.4 lM 2,4-dichlorophenoxyacetic acid (2,4-D), and
0.5 lM thidiazuron (TDZ) to induce embryogenic callus.
Induced embryogenic calli were transferred onto MS
medium containing 2 % sucrose and 0.05 % activated
charcoal and cultured in the dark for 4 weeks maintained at
24 ± 2 °C. Somatic embryos were then transferred onto ‘
MS with 1 % sucrose for germination and incubated for
Plant Biotechnol Rep (2013) 7:71–82
6 weeks under light (40 lmol m-2 s-1) maintained at
24 ± 2 °C.
Mycorrhiza inoculant and inoculation
The AMM6 used in this study was selected from among 26
rhizosphere medium samples because it has the highest
spore count (863 spores per 30 g air dried medium, n = 3).
Spores were extracted following the wet sieving and
decanting techniques (Brundrett et al. 1996) and counted
under a microscope. After counting, the spores were sur-
face-sterilized with 10 % chlorox, rinsed several times in
sterile water, and inoculated into pots filled with auto-
claved sand planted with bahia (Paspalum notatum) grass
as trap plant. After five (June–October 2008) months
incubation in a glasshouse of KFRI, Suwon, AMM6 was
produced and consisted of chopped colonized roots of
bahia grass, spores, and other infective propagules plus
sand (the growing medium). AMM6 gave the highest
sporulation (1,474 ± 220 spores per 15 g dry soil) among
the 26 rhizosphere samples after 5 months growth in pot
culture. AMM6 is a mixture of unidentified species of
Glomus and Acaulospora collected in May 2008 in a closed
mine tailing site in Bonghwa, Korea. AM fungi native in
this site has been studied for the site’s immediate rehabil-
itation as well as other heavy metals-laden sites in Korea.
In an earlier experiment, AMM6 dominated the other
native AM fungi in promoting growth and total dry weight
of Acacia mangium seedlings under glasshouse conditions
(Aggangan et al. 2009b). The effectiveness of AMM6 on
non-transgenic and transgenic clones (with cadmium-,
zinc-, lead-, and arsenic-tolerant genes) of Populus alba 9
glandulosa for phytoremediation purposes were concur-
rently established in the glasshouse of KFRI, Suwon.
Uniform-sized (2–3 cm length, with two to three leaves)
microplants freshly taken from the bioreactor were washed
in water and planted in rectangular boxes (40 cm 9
70 cm 9 20 cm) filled with 500 g (dry weight) autoclaved
or non-sterile peat perlite vermiculite (1:1:1 v/v/v) medium.
Prior to planting, 250 g AMM6 (containing 2,500–5,000
spores) was drenched in rows (2–3 cm depth) where the
microplants were inserted. In each box, there were seven
rows and each row was planted with 5 microplants (a total of
35 microplants per box). Uninoculated treatment received
the same amount of soil inoculant cultured in a similar
manner as the AMM6 but with no mycorrhizal fungi. The
absence of spores of mycorrhizal fungi in the soil or colo-
nization in the roots was confirmed prior to use. Rectangular
boxes were covered with clear polyvinyl and placed in the
acclimatization room maintained at 24 ± 2 °C at 16 h
photoperiod (40 lmol m-2 s-1). The box cover was grad-
ually opened 1 week after transplanting and acclimatized
plants were fully uncovered at week 3.
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P fertilizer application
Pre-weighed P fertilizers for the different rates {0, 2, 4, 8,
16, and 32 mg P [as Ca(H2PO4)2
H2O] kg medium21
coded as P0, P2, P4, P8, P16 and P32, respectively} were
added into the respective plastic pots (9.14 cm top dia-
meter 9 7.14 cm bottom diameter 9 7.14 cm height) with
undrained polythene bag liners, filled with 150 g (oven dry
weight) non-sterile or sterile peat perlite vermiculite
medium (referred to as soil in this paper). The P fertilizers
were added evenly on the soil surface. The soil was then
mixed thoroughly and watered to field capacity (10 %
w/w) and incubated on benches inside an acclimatization
room for 1 week prior to planting.
Maintenance
Each rectangular box in Part 1 received 15 ml  strength
Hoagland’s solution (Bonner and Galston 1952) once a
week, started 1 month after planting. Each plant in Part 2
was given with 5 ml  strength Hoagland’s solution
without P, once a week for 1 month and 10 ml pot-1
onward. Each pot was covered with aluminum foil to
prevent or minimize contamination and watering was done
carefully. Plants were watered by weight when necessary.
Pots were re-randomized once a week in order to avoid a
possible lack of homogeneity of light in the glasshouse that
might alter plant responses to the treatments.
Assessment of plant growth and mycorrhizal root
colonization
Height and diameter were measured at once a month until
harvest (4 months after P application). At harvest, shoots
were cut 1 cm above the soil surface and the root systems
were gently washed under running water. Randomly
selected fine (diameter less than 0.5 mm) root sub-samples
(0.2 g fresh weight) were chopped into 2–3 mm lengths
and fixed in 70 % ethanol for counting mycorrhizal root
colonization. The roots were cleared and stained with try-
pan blue (Brundrett et al. 1996) and mycorrhizal coloni-
zation was determined using the gridline intersection
method (Giovannetti and Mosse 1980). Root colonization
was expressed as the percentage of roots colonized by AM
fungi over the total number of roots counted multiplied by
100. The total plant fresh weight was measured immedi-
ately after harvest and dry weight of roots, leaves and stem
were obtained after 3 days at 65 °C.
Phosphorus concentration and uptake
Plant samples were dried at 70 °C for 48 h, ground, and
stored at 60 °C until analysis. Total P was analyzed by the
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colorimetric method described by Murphy and Riley

(1962). P uptake was computed as the product of plant total
dry weight and P concentration.

Statistical analysis

All data gathered in Part 1 were analyzed using a two-

way analysis of variance (ANOVA) in CRD, whereas in
Part 2, all data were analyzed using a three-way ANOVA
in RCBD. Treatment means were compared using Dun-
can’s multiple range test (DMRT) at p \ 0.05. Mycor-
rhizal root colonization data were arcsine-transformed
before being subjected to ANOVA analysis (Gomez and
Gomez 1984). Correlation between percentage mycorrhi-
zal infection and the parameters measured was also
determined. Statistical analyses were done using
MSTATC statistical computer program [Michigan State
University (MSU) 1989].

Results

General observation

The uninoculated plants had stunted growth with yellowish

leaves (Fig. 1a, b). Mycorrhizal plants on the other hand,
had greener and broader leaves (Fig. 1c, d) than the non-
mycorrhizal counterpart. At 2 months (Part 1), mycorrhizal
plants were 43 and 23 % taller than the uninoculated plants
when grown in sterile and non-sterile soil, respectively
(Fig. 2a). AMM6 increased stem diameter by 13 % in non-
sterile soil and 7 % in sterile soil relative to the uninocu-
lated ones (Fig. 2b). Survival or microplant recovery was
generally better in sterile soil than in non-sterile medium
especially if inoculated with mycorrhizal fungi (Fig. 2c).
At harvest, more fine roots was formed by the non-
mycorrhizal plants in sterile than in non-sterile medium
(Fig. 3a, b). AMM6 inoculated plants had a well developed
root systems with numerous fibrous roots than the
uninoculated counterpart irrespective of soil sterilization
treatment (Fig. 3c, d).
Mycorrhizal root colonization
Root colonization by AMM6 2 months after inoculation
ranged from 5 to 9 % in sterile medium and \5 % in non-
sterile counterpart (data not shown). Four months after P
fertilizer application, root colonization was 4–20 % in
sterile medium and \5 % in non-sterile medium (Fig. 4).
The highest (20 %) root colonization was obtained at P16.
Colonized roots had vesicles and mycelia, but there were
no arbuscules (Fig. 5a, b). The roots of the uninoculated
plants were not colonized by mycorrhizal fungi (Fig. 5c).
Fig. 1 Two-month-old control/uninoculated (a, b) and mycorrhizal
(c, d) Kalopanax septemlobus microplants and grown in nonsterile
(a, c) and sterile (b, d) peat perlite vermiculite medium incubated in
an acclimatization room
Effect of soil sterilization
In general, all the parameters measured were higher in
sterile than in non-sterile medium. Sterilization of the
peat perlite vermiculite soil medium used in this study
improved microplants recovery and early growth after ex
vitro. Sterilization promoted height and diameter growth
of K. septemlobus throughout the 6-month experimenta-
tion period (data not shown). At 2 months, height was
21 % higher in sterile medium than in non-sterile
counterpart (1.4 cm) (Fig. 2a). For stem diameter, a
133 % increase over those in the non-sterile medium
(0.06 cm) was obtained (Fig. 2b). Moreover, survival of
microplants was 87–90 % in sterile soil medium while
that in non-sterile counterpart was 65–80 % (Fig. 2c).
Plant dry weight was likewise increased by 85 % in
sterile medium (Table 1). At harvest, sterilization
increased (p \ 0.001) shoot, root and total dry weight by
115, 69 and 85 %, respectively, over those grown in
non-sterile medium.

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Sterile Non-sterile plants were heavier (50–58 %) than the non-mycorrhizal

a 3.0
counterpart (Table 1).
a
2.5
b Effect of P rates
2.0 bc c
Height (cm)

Irrespective of sterilization and inoculation treatments,

1.5 height and diameter growth of K. septemlobus was improved
only if fertilized with a minimum of 8 mg P kg soil-1 (P8)
1.0 and a maximum of 16 mg P kg soil-1 (P16). Doubling the
rate from 16 to 32 mg P kg soil-1 (P32) did not show further
0.5
beneficial effects on height and diameter (Table 1). Growth
0 obtained at P16 and P32 was comparable to each other.
However, the application of 2 mg P kg soil-1 (P2) gave
b 0.20 significant increases in root, shoot, and total plant dry weight
a
b bc (Table 1). P16 (16 mg P kg soil-1) promoted the heaviest
plant dry weight but similar to those obtained at P32
0.15 d (32 mg P kg soil-1).
Diameter (cm)

0.10 Interaction effects of soil sterilization, mycorrhizal

inoculation and P rates

0.05 Two- and three-way ANOVA revealed significant interac-

tion between soil sterilization, mycorrhizal inoculation and
P rates. The results of the three-way (three factors) are
0 presented. Significant interaction between soil sterilization,
a b mycorrhizal inoculation, and P rates was observed on stem
c 100 ab
diameter, plant fresh and dry weight, and P uptake
(Tables 2, 3, 4, 5, 6, 7). In sterile soil, stem diameter of
80 c uninoculated plants were increased (p \ 0.001) when sup-
plied with 16 mg P kg soil-1 (P16) (Table 2). Doubling the
Survival (%)

60 rate had no additional benefit on the uninoculated plants.

40
20
0 and with the uninoculated counterpart grown also in sterile
Control AMM6 Control AMM6
Fig. 2 Height (a), stem diameter (b), and survival (c) of Kalopanax
septemlobus microplants 2 months after inoculation with mycorrhizal
fungi AMM6 and grown in sterile or non-sterile peat perlite
vermiculite medium. Bars SE; those with the same letters are not
significant from each other using DMRT at p \ 0.05, n = 5
Effect of mycorrhizal inoculation
Inoculation with AMM6 promoted plant height and dia-
meter of 2 months old K. septemlobus (Figs. 1a, b, 2a, b).
At harvest, height of the uninoculated microplants was
taller than the AMM6 inoculated ones and there was no
difference in terms of stem diameter (Table 1). On the
other hand, shoot, root, and total dry weight of mycorrhizal
The mycorrhizal counterpart had a significant increase in
stem diameter when supplied with 8 mg P kg soil-1 (P8) as
compared with the unfertilized counterpart. Stem diameter
obtained by mycorrhizal plants grown in sterile soil sup-
plied with P8, P16 and P32 was comparable from each other
medium and fertilized with P16 and P32. In non-sterile soil,
stem diameter of the uninoculated plants was not affected
by the increasing rates of P fertilizer. By contrast, a sig-
nificant increase in stem diameter due to inoculation with
AMM6 was obtained at P32. At this P rate, mycorrhizal
plants had 32 % higher stem diameter than those of the
uninoculated (1.32 ± 0.3 mm) counterpart supplied the
same amount of P.
Total fresh weight of uninoculated plants grown in non-
sterile soil was not affected by the increasing P rates
(Table 3). By contrast, inoculation with AMM6 increased
(p \ 0.001) total fresh weight (4.70 g plant-1) when sup-
plied with 16 mg P kg soil-1 (P16) and grown in non-
sterile medium (Table 3). Doubling the P rate to 32 mg
P kg soil-1 gave a slightly heavier (5.11 g plant-1) bio-
mass but comparable with that at P16. Total fresh weight of

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Fig. 3 Six-month-old control/

non-mycorrhizal (a, b) and
mycorrhizal with a mixture of
Glomus and Acaulospora coded
as AMM6 (c, d) Kalopanax
septemlobus in response to
increasing levels
(0–32 mg P kg soil-1) of
phosphorus and grown in sterile
(a, c) and non-sterile peat perlite
vermiculite mixture (b, d)

25
Sterile Non-sterile
Mycorrhizal root infection (%)

20

15

10

Fig. 4 Percentage root colonization by a mixture of unidentified

species of Glomus and Acaulospora (coded as AMM6 in this
experiment) as compared with that of uninoculated control (c) Kalo-
panax septemlobus microplants in sterile and non-sterile medium.
Phosphorus fertilizer levels are: P0 = no fertilizer to
P32 = 32 mg P [as Ca(H2PO4)2
H2O] kg soil-1

mycorrhizal microplants supplemented with 16 and

32 mg P kg soil-1 (P16 and P32, respectively) was 4 times
heavier than the uninoculated counterpart (1.98 g plant-1).
In sterile conditions, total fresh weight was significantly
increased with the addition of 4 mg P kg soil-1 (P4) and
highest at P16 (7.74 g plant-1).
The effect of the three factors on root and shoot of
uninoculated and mycorrhizal K. septemlobus and grown in
sterile or non-sterile soil gave the same trend (Tables 4, 5).
Fig. 5 Roots colonized with a mixture of unidentified species of
Root and shoot dry weight of the uninoculated microplants Glomus and Acaulospora coded as AMM6 (a, b) as compared
grown in sterile soil, was increased (p \ 0.001) when with that of uninoculated control (c) Kalopanax septemlobus
supplied with P8 (Tables 4, 5). On the other hand, root and microplants

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Table 1 Main effects of mycorrhiza inoculation, soil sterilization and rates of phosphorus {0–32 mg P [as Ca(H2PO4)2 kg soil-1]} on height,
diameter, and plant biomass of 6-month-old Kalopanax septemlobus grown in peat perlite vermiculite under growth room conditions (n = 5)
Treatment Height increment Diameter increment Shoot dry weight Root dry weight Total dry weight
(cm) (cm) (g plant-1) (g plant-1) (g plant-1)

Sterile 1.67 ± 0.19a*** 0.303 ± 0.06a*** 0.471 ± 0.03a*** 0.531 ± 0.03a*** 0.988 ± 0.07a***
Non-sterile 1.09 ± 0.15b 0.186 ± 0.01b 0.219 ± 0.01b 0.314 ± 0.02b 0.533 ± 0.03b
Control 1.50 ± 0.25a* 0.239 ± 0.05ans 0.268 ± 0.03b* 0.335 ± 0.03b* 0.597 ± 0.07b*
AMM6 1.26 ± 0.20b 0.250 ± 0.03a 0.423 ± 0.02a 0.509 ± 0.02a 0.895 ± 0.04a
P0 1.08 ± 0.21b** 0.197 ± 0.05c*** 0.152 ± 0.04c*** 0.190 ± 0.04c*** 0.343 ± 0.08c***
P4 1.29 ± 0.24b 0.241 ± 0.05b 0.298 ± 0.03b 0.379 ± 0.03b 0.632 ± 0.06b
P8 1.49 ± 0.52ab 0.246 ± 0.04ab 0.342 ± 0.02b 0.460 ± 0.02ab 0.801 ± 0.04b
P16 1.76 ± 0.63a 0.284 ± 0.01a 0.513 ± 0.04a 0.579 ± 0.03a 1.091 ± 0.07a
P32 1.48 ± 0.25ab 0.268 ± 0.08ab 0.487 ± 0.03a 0.590 ± 0.03a 1.054 ± 0.07a
ns not significant
*, **, *** Significant at 5, 1, and 0.1 %. Treatment mean ± SE with the same letters are not significantly different from each other using DMRT
at p \ 0.05

Table 2 Interaction effects of soil sterilization, mycorrhizal inoculation and P rates (0–32 mg kg soil-1) on stem diameter (mm) of 6-month-old
Kalopanax septemlobus microplants grown in peat perlite vermiculite under acclimatization conditions
P rate Uninoculated AMM6
Sterile Non-sterile Sterile Non-sterile

P0 2.66 ± 0.4c–f*** 1.23 ± 0.6j 1.81 ± 0.8f–j 1.86 ± 0.8f–j

P4 2.98 ± 0.3bcd 2.21 ± 0.2d–i 2.61 ± 0.8c–g 1.89 ± 0.8f–j
P8 2.93 ± 0.5bcd 2.17 ± 0.5d–i 3.48 ± 0.6abc 2.35 ± 0.2d–h
P16 3.87 ± 0.7a 1.69 ± 0.2g–j 3.63 ± 0.6ab 2.18 ± 0.4d–i
P32 3.46 ± 1.2abc 1.32 ± 0.3ij 3.50 ± 0.2abc 2.42 ± 0.5d–h
*** Significant at p \ 0.001. Treatment mean – SE with the same letters are not significantly different from each other using DMRT at
p \ 0.05, n = 5

Table 3 Interaction effects of soil sterilization, mycorrhizal inoculation and P rates (0–32 mg kg soil-1) on total fresh weight (g plant-1) of
6-month-old Kalopanax septemlobus microplants grown in peat perlite vermiculite under acclimatization conditions
P rate Uninoculated AMM6
Sterile Non-sterile Sterile Non-sterile

P0 2.34 ± 0.4e–i*** 0.53 ± 0.1i 1.93 ± 0.4f–i 1.98 ± 0.8e–i

P4 3.58 ± 1.9c–g 1.69 ± 0.6ghi 4.79 ± 1.2bcd 3.74 ± 1.1b–g
P8 3.09 ± 0.7c–h 1.86 ± 0.9ghi 5.08 ± 1.7bc 4.12 ± 2.1b–e
P16 9.04 ± 2.6a 1.24 ± 0.8hi 7.74 ± 1.9a 4.70 ± 1.4bcd
P32 8.01 ± 3.1a 1.33 ± 0.1hi 5.77 ± 1.0b 5.11 ± 1.2bc
*** Significant at p \ 0.001. Treatment mean ± SE with the same letters are not significantly different from each other using DMRT at
p \ 0.05, n = 5

shoot dry weight of mycorrhizal microplants grown in
sterile soil was increased when supplied with P4. Mycor-
rhizal plants grown in sterile soil supplied with P4–P32
gave similar root dry weights (Table 4). Root dry weight
obtained by mycorrhizal plants grown in sterile soil sup-
plied with P8, P16 and P32 were comparable with those of
the uninoculated counterpart as well as mycorrhizal plants
grown in non-sterile soil and fertilized with P8–P32. Root
dry weights obtained by mycorrhizal plants grown in non-
sterile soil supplied with P8 was two times higher than the
mycorrhizal but unfertilized (P0) (0.22 ± 0.1 g plant-1)
ones and 1.2-fold higher than the uninoculated
(0.27 ± 0.1 g plant-1) counterpart grown in non-sterile
soil supplied with P8. Increasing the P rates from P8 to P16

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Table 4 Interaction effects of soil sterilization, mycorrhizal inoculation and P rates (0–32 mg kg soil-1) on root dry weight (g plant-1) of
6-month-old Kalopanax septemlobus microplants grown in peat perlite vermiculite under acclimatization conditions
P rate Uninoculated AMM6
Sterile Non-sterile Sterile Non-sterile

P0 0.27 ± 0.1efg*** 0.05 ± 0g 0.22 ± 0efg 0.22 ± 0.1efg

P4 0.34 ± 0.2c–g 0.22 ± 0.1efg 0.60 ± 0.1a–d 0.37 ± 0.1b–f
P8 0.59 ± 0.1a–d 0.27 ± 0.1efg 0.64 ± 0.2abc 0.59 ± 0.2a–d
P16 0.73 ± 0.4a 0.23 ± 0.1efg 0.83 ± 0.3a 0.53 ± 0.2a–e
P32 0.76 ± 0.2a 0.19 ± 0fg 0.75 ± 0.1a 0.66 ± 0.1ab
*** Significant at p \ 0.001. Treatment mean ± SE with the same letters are not significantly different from each other using DMRT at
p \ 0.05, n = 5

Table 5 Interaction effects of soil sterilization, mycorrhizal inoculation and P rates (0–32 mg kg soil-1) on shoot dry weight (g plant-1) of
6-month-old Kalopanax septemlobus microplants grown in peat perlite vermiculite under acclimatization conditions
P rate Uninoculated AMM6
Sterile Non-sterile Sterile Non-sterile

P0 0.22 ± 0hij*** 0.05 ± 0j 0.18 ± 0hij 0.16 ± 0.1hij

P4 0.26 ± 0.2g–j 0.15 ± 0ij 0.54 ± 0.1b–e 0.28 ± 0.1g–j
P8 0.45 ± 0d–g 0.15 ± 0.1ij 0.52 ± 0.3b–f 0.44 ± 0.2d–g
P16 0.68 ± 0.2abc 0.11 ± 0.1ij 0.87 ± 0.2a 0.38 ± 0.1e–h
P32 0.72 ± 0.2ab 0.09 ± 0ij 0.65 ± 0.1bcd 0.49 ± 0.1c–f
*** Significant at p \ 0.001. Treatment mean ± SE with the same letters are not significantly different from each other using DMRT at
p \ 0.05, n = 5

and P32 did not have additional benefits to both uninocu-
lated and mycorrhizal plants. Root and shoot dry weight of
uninoculated microplants grown in non-sterile soil was not
affected by the applied P rates.
Total dry weights of the uninoculated and mycorrhizal
plants grown in sterile soil were increased (p \ 0.001)
when supplied with P16 and P4, respectively (Table 6).
Total dry weight of mycorrhizal plants obtained at P8 was
comparable with those obtained by the uninoculated plants
grown in sterile soil supplied with P16. In non-sterile soil,
total dry weights of mycorrhizal plants supplied with P8,
P16 and P32 were comparable from each other. In contrast,
total dry weight of uninoculated microplants grown in non-
sterile soil was not affected by the applied P rates. At P8,
total dry weight of mycorrhizal plants grown in non-sterile
soil was higher by 2.5-fold over the uninoculated
(0.42 ± 0.1 g plant-1) counterpart supplied with the same
P rate. Total dry weight of mycorrhizal plants grown in
sterile soil supplied with P4 was higher by 1.7-fold over the
uninoculated (0.68 ± 0.1 g plant-1) counterpart grown in
the same soil and P rate.
Total P uptake of uninoculated and mycorrhizal micro-
plants were increased (p \ 0.05) when grown in sterile soil
and supplied with P16 (Table 7). However, total P uptake in
mycorrhizal microplants was 30 % higher (p \ 0.05) than
the uninoculated microplants (2.33 ± 1.1 mg plant-1). In
the non-sterile soil, mycorrhizal plants had a significant
increase in total P uptake when supplied with P8. Total P
uptake of mycorrhizal plants grown in non-sterile soil sup-
plied with P8 was 2.7-fold higher over the uninoculated
(0.79 ± 0.5 mg plant-1) counterpart grown in the same soil
and P rate. Total P uptakes by uninoculated plants grown in
non-sterile soil were comparable across the P rates and
generally lower than those obtained in sterile soil.
Discussion
Micropropagated plants transferred to ex vitro have used
sterilized substrates whether steamed, pasteurized, or fumi-
gated which resulted to improvement of growth and survival
by mycorrhizal plants (e.g., Salamanca et al. 1992; Lovato
et al. 1994; Yano-Melo et al. 1999; Estrada-Luna et al. 2000).
Sterilization of the substrate kills all microorganisms present
in the soil including the indigenous mycorrhizal fungi, but
this condition will allow the introduced mycorrhizal fungi
free of competitors in colonizing the fine roots. Likewise, the
presence of soil microorganisms may limit mycorrhizal
activity in non-sterile soil (Hetrick et al. 1988). Thus, ster-
ilized potting media should be considered as this treatment

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Table 6 Interaction effects of soil sterilization, mycorrhizal inoculation and P rates (0–32 mg kg soil-1) on total dry weight (g plant-1) of
6-month-old Kalopanax septemlobus microplants grown in peat perlite vermiculite under acclimatization conditions
P rate Uninoculated AMM6
Sterile Non-sterile Sterile Non-sterile

P0 0.49 ± 0.1f–j*** 0.10 ± 0j 0.40 ± 0.1g–j 0.38 ± 0.1g–j

P4 0.68 ± 0.2d–h 0.38 ± 0.1g–j 1.15 ± 0.2bcd 0.64 ± 0.1e–i
P8 0.86 ± 0.1c–g 0.42 ± 0.1f–j 1.70 ± 0.3a 1.04 ± 0.4b–e
P16 1.42 ± 0.6ab 0.34 ± 0.2hij 1.70 ± 0.7a 0.91 ± 0.2c–f
P32 1.47 ± 0.6ab 0.28 ± 0hij 1.31 ± 0.1abc 1.15 ± 0.3bcd
*** Significant at p \ 0.001. Treatment mean ± SE with the same letters are not significantly different from each other using DMRT at
p \ 0.05, n = 5

Table 7 Interaction effects of soil sterilization, mycorrhizal inoculation and P rates (0–32 mg kg soil-1) on total P uptake (mg plant-1) of
6-month-old Kalopanax septemlobus microplants grown in peat perlite vermiculite under acclimatization conditions
P rate Control AMM6
Sterile Non-sterile Sterile Non-sterile

P0 0.89 ± 0.2efg* 0.34 ± 0.1fg 1.32 ± 0.9c–f 0.24 ± 0.1g

P4 1.57 ± 0.4b–e 0.89 ± 0.3efg 1.52 ± 0.6b–e 1.21 ± 0.5d–g
P8 1.35 ± 0.4c–f 0.79 ± 0.5efg 1.27 ± 0.5d–g 2.14 ± 0.7b–d
P16 2.33 ± 1.1bc 0.51 ± 0.3efg 3.02 ± 1.3a 1.29 ± 0.5d–g
P32 2.46 ± 1.6bc 0.36 ± 0fg 2.86 ± 1.0ab 0.66 ± 0.2efg
* Significant at p \ 0.001. Treatment mean ± SE with the same letters are not significantly different from each other using DMRT at p \ 0.05,
n=5

usually improves the arbuscular mycorrhiza formation in
micropropagated plants (Gianinazzi et al. 1990). Timing of
inoculation is also very important. Mycorrhizal colonization
is known to take place only on young, secondary roots before
suberization (Smith and Read 1997). Since these fine, young
roots are produced during ex vitro development, inoculation
is advisable during transplanting in open pots. Early
mycorrhizal inoculation and colonization has been shown to
increase plant survival and growth of tissue cultured grape,
apple, plum pineapple, avocado, strawberry, raspberry, and
cherry reduced transplant shock during acclimatization
(Lovato et al. 1994; Rapparini et al. 1994). Improved sur-
vival rate of micropropagated avocado (Azcón-Aguilar et al.
1992), Anthyllis cystisoides (Salamanca et al. 1992), and in
Leucaena leucocephala (Naqvi and Mukerji 1998) has also
been reported. AM inoculation reduced the acclimatization
period by about 8 weeks in A. cystisoides (Salamanca et al.
1992).
Sterilization of substrates for microplants during ex vitro
acclimatization period is not a practice at the KFRI, Su-
won, South Korea. In this study, soil sterilization generally
resulted in a great difference in growth and biomass yield
of K. septemlobus similar to those previously reported
(Aggangan et al. 1995, 1999, 2009a). Percent root coloni-
zation by AMM6 was higher in sterile than in non-sterile
peat vermiculite medium. In addition, survival, growth, and
biomass yield were generally improved in sterile than in
the non-sterile counterpart. Root colonization of 30 % in
yellow poplar was reported to be the critical level before an
exponential growth rate can occur (Ford and Hay 1987).
Nursery-raised seedlings should have adequate mycorrhi-
zas in their roots before transplanting them in the field.
AMM6 was first used as mycorrhizal inoculant for Acacia
mangium seedlings grown in sterilized substrate similar to
that used in this study (Aggangan et al. 2010). Succeeding
experimentations showed that AMM6 was the most
effective among the seven AM fungi tested in promoting
growth of Liriodendron tulipifera (Aggangan et al. 2011)
and increasing arsenic tolerance of non-transgenic and
transgenic Populus alba 9 P. glandulosa clones (NYCf7,
PCP301CGoR4 and Cd26c11) and Eucalyptus pellita
(manuscripts in preparation and under review, respec-
tively). Percent root colonization in these studies was rel-
atively high (30–60 % after 4 months) as compared to the
root colonization levels (not more than 20 % after
6 months) obtained in this study. The only difference was
that the experiments were conducted in a glasshouse
whereas the present study was established in an acclima-
tization room. Two possible reasons are, firstly, that the
environmental conditions in the glasshouse were more
favorable for mycorrhizal development and root coloniza-
tion, and secondly, the availability of fine roots that could

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be possibly infected by mycorrhizal fungi. The SE-derived
K. septemlobus had single tap roots and no fibrous roots
during ex vitro transplantation, and thus root colonization
may be delayed.
Plant growth and survival-promoting effect of AMM6
was also obtained in non-sterile soil but to a lower degree
as compared with those obtained in sterile soil. Gaur and
Adholeya (1999) reported that inoculation with AM fungi
had a significant and favorable effect on acclimatization of
micropropagated plantlets of Syngonium podophyllum and
Draceana sp. in non-sterile substrate, and the hardening
process was shortened by 15 days. There is a great dif-
ference when comparing the effectiveness of AMM6 in
non-sterile soil with those obtained by the uninoculated
counterpart. A large difference in plant biomass was also
observed between inoculated and uninoculated plum plants
(Fortuna et al. 1996).
The AM inoculants used in this study colonized the roots,
although it was low (\10 % at 2 months and between 15 and
20 % after 4 more months), while growth and biomass were
significantly improved. No relationship between plant dry
weight and percentage root colonization has been reported
previously (Chavez and Ferrera-Cerrato 1990; Aggangan
et al. 2009b). The effects of mycorrhizal inoculation on plant
growth may be due to improved rhizogenesis of the other-
wise poorly rooted transplants and plant hormones produced
by AM fungi (Smith and Read 1997) that may contribute to
improved root formation and development of in vitro-
derived plantlets (Azcón-Aguilar et al. 1997a).
Arbuscular mycorrhizal fungi are an important factor to
mobilize P in soil to roots (Michelsen and Rosendahl 1990).
Higher amounts of P in soil decreased AM activity (Bal-
truschat and Dehne 1988). Faramarzi et al. (2012) evaluated
the effects of mycorrhizal fungus Glomus mosseae and
phosphorus application at a rate of 0, 50, 100, and 150 kg/ha
from ammonium phosphate (46 % source) on some quanti-
tative and qualitative attributes of corn (cv. KSC647). They
found that mycorrhiza significantly affected seed yield,
biomass, harvest index, cob weight, kernel number on cob
rows, 1,000-kernel weight, and seed protein percentage. On
the other hand, phosphorus significantly affected the cob
weight, protein percentage, and cob harvest index. The
highest cob weight and cob harvest index were obtained by
150 kg/ha phosphorus application with mycorrhiza inocu-
lation. Applying phosphorus at 50, 100, and 150 kg/ha levels
increased yield up to 62, 101, and 124 %, respectively, in
comparison to the control, respectively. Mycorrhiza inocu-
lation with 150 kg/ha phosphorus resulted in the highest seed
yield, while no application of mycorrhiza and phosphorus
caused the least seed yield. On the other hand, Swift (2009)
reported that the benefits of mycorrhizae are greatest when
soil phosphorus levels are at or below 50 mg kg-1. Mycor-
rhizal infection of roots declines above this level with little if
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Plant Biotechnol Rep (2013) 7:71–82
any infection occurring above 100 ppm P even when soil is
inoculated with mycorrhizae.
In this study, the effectiveness of AMM6 in promoting
better growth and survival of microplants was realized only
if low levels of P fertilizer was applied. The effectiveness
of AMM6 in promoting plant growth was better if the
growth medium is supplemented with 4–8 mg kg soil-1 in
sterile soil and 8–16 mg kg soil-1 in non-sterile soil.
Without a minimal amount of P fertilizer, AMM6 was not a
good plant promoter for SE-derived K. septemlobus mi-
croplants. The uninoculated plants require at least
16 mg P fertilizer kg soil-1 in sterile soil. In non-sterile
soil, addition of 32 mg kg soil-1 did not affect growth,
biomass yield and P uptake of uninoculated plants. Sur-
prisingly, the addition of P32 had caused slight reductions
in growth and biomass yield in uninoculated microplants.
This may suggests that the applied P fertilizers were not
effectively utilized by the uninoculated plantlets. Fertilizer
inputs can be reduced in mycorrhiza-inoculated micro-
propagated plants to levels considerably lower than those
used in commercial practice and yet plant development was
equivalent to that of uninoculated plants receiving full
fertilizer inputs (Williams et al. 1992).
Infection of the root system of a plant by mycorrhizal
fungi creates a symbiotic (beneficial) relationship between
the plant and fungus. Upon root infection and colonization,
mycorrhizal fungi develop an external mycelium which is a
bridge connecting the root with the surrounding soil (Toro
et al. 1997). One of the most dramatic effects of infection
by mycorrhizal fungi on the host plant is the increase in P
uptake mainly due to the capacity of the mycorrhizal fungi
to absorb phosphate from soil and transfer it to the host
roots (Smith and Read 1997). Formation and function of
AM fungi are affected by levels of fertility in soil or fer-
tilizers applied to the growing medium (Linderman and
Davis 2004; Smith and Read 1997). Phosphorus is the main
element influencing colonization of host plant (Brundrett
et al. 1996). Soil P levels greater than that required for
plant growth eliminated the development of the AM
mycorrhizae (Abbott and Robson 1979; Koide and Li
1990). Linderman and Davis (2004) found that high levels
of phosphorus suppress formation and function of AM
fungi, thus plant growth is reduced. Schubert and Hayman
(1978) found mycorrhizae were no longer effective when
100 mg or more P was added per kilogram of soil while
Swift (2009) reported 50 mg P kg soil-1 or smaller
amount to favor mycorrhizal formation and function. In
this study, 8 mg P kg soil-1 seems to be the optimum level
for the mycorrhiza inoculated plants.
Our results confirm the beneficial effect of inoculation
with mycorrhizal fungi in promoting better plant survival
and growth during acclimatization period. However, the
effectiveness of mycorrhizal fungi such as AMM6 was
Plant Biotechnol Rep (2013) 7:71–82
affected by the rates of P fertilizer and soil sterilization.
Mycorrhizal K. septemlobus microplants grew better in
sterile soil and required smaller (4–8 mg kg soil-1)
amount of P fertilizer than the uninoculated plants
(16 mg kg soil-1). The AMM6 inoculant used in this study
is a mixture of Glomus and Acaulospora and it proved to
have benefited SE-derived K. septemlobus microplants, and
can reduce the amount of P fertilizer required for normal
growth of uninoculated plants. Further experimentations
are needed to verify these initial findings. Field trials
should be done to determine the competitiveness of AMM6
with the indigenous mycorrhizal fungi, pathogens and other
microbes, and soil chemical and physical properties of the
site which contribute to the effectiveness of AMM6. It
is, however, recommended that K. septemlobus for exper-
imentation in the field should already be mycorrhizal
in the nursery. The importance of physiological and
genetic adaptation to the environment should always be
considered; thus, local isolates must be used (Jeffries et al.
2003).
Acknowledgments This study was conducted while the senior
author was on Post-doctoral Fellowship at the Korea Forest Research
Institute, Suwon, South Korea. The authors would like to thank Dr.
Mariechel J. Navarro, Manager of the Global Knowledge Center on
Crop Biotechnology, ISAAA, for her comments and suggestions on
the manuscript.
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