FEMS Microbiology Letters 248 (2005) 111–118
www.fems-microbiology.org
Development of an autotrophic culture system for the in vitro
mycorrhization of potato plantlets
Liesbeth Voets a, Hervé Dupré de Boulois a, Laurent Renard a,
Désiré-Georges Strullu b, Stéphane Declerck c,*
a
c
Université catholique de Louvain, Unité de Microbiologie, Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium
b
Laboratoire de Phytonique, Université dÕAngers, 2 Bd. Lavoisier, 49045 Angers Cedex, France
Université catholique de Louvain, Mycothèque de lÕUniversité catholique de Louvain (MUCL1), Unité de Microbiologie, Croix du Sud 3,
1348 Louvain-la-Neuve, Belgium
Received 25 February 2005; received in revised form 13 May 2005; accepted 16 May 2005
First published online 31 May 2005
Edited by Y. Okon
Abstract
An autotrophic culture system was developed for the in vitro mycorrhization of potato plantlets. Roots of micropropagated
plantlets were associated to an arbuscular mycorrhizal fungus under in vitro conditions, while shoots developed under open air conditions. Several thousand spores, an extensive extraradical mycelium and an abundant root colonization were obtained. Spores were
able to colonize new plantlets under the same conditions. These results support the capacity of the autotrophic culture system to
continuously culture arbuscular mycorrhizal fungi and may serve as a powerful tool to investigate various aspects of the symbiosis
for which a source–sink relationship and photosynthetic active tissues are necessary.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Arbuscular mycorrhizal fungi; Continuous culture; Monoxenic; Solanum tuberosum; Sporulation dynamics
1. Introduction
The technique of in vitro cultivation of root organs
has been developed over the last decades [1–3] opening
important avenues for studying plant–fungi interactions.
It is found especially useful for the study of arbuscular
mycorrhizal (AM) fungi since these obligate biotrophs
rely on plant tissues to complete their life cycle. All areas
of the AM fungi biology per se, as well as of the biology
of the symbiotic relationship, have been revisited using
in vitro cultures [4].
1
Part of the Belgian Coordinated Collections of Micro-organisms
(BCCM).
*
Corresponding author. Tel.: +32 10 47 46 44; fax: +32 10 45 15 01.
E-mail address: [email protected] (S. Declerck).
Despite the large array of applications, the use of excised root organs to study the AM symbiosis presents
some major limitations, materialized by the absence of
photosynthetic tissues, a normal hormone balance and
physiological source–sink relationships [5]. For instance
physiological studies, such as those involving the mechanisms of transport of minerals and carbohydrates from
fungus to plant shoots and inversely, may suffer the absence of photosynthetic tissues. Identically, the addition
of sucrose to the growth medium, to compensate for the
absence of photosynthates, may modify the biochemistry of the plant–fungal interaction [5]. In this context,
the development of in vitro cultivation systems in which
autotrophic plants are associated to AM fungi appears
essential.
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.05.025
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L. Voets et al. / FEMS Microbiology Letters 248 (2005) 111–118
The first in vitro system associating an AM fungus
with a whole plant dates back to the early sixties. The
fungus Endogone sp. was associated to different plantlets
(white clover, corn, onion, cucumber, orchard grass) on
an inorganic salt medium [6]. In the following two decades several authors experienced the in vitro association
of an AM fungus with a host plant in liquid or agar
medium [7–11]. Plants originating from somatic embryos were also demonstrated to form typical AM fungal structures in vitro [12]. Unfortunately, and perhaps
due to the difficulty to reproduce and utilize these systems and to the limited quantitative and qualitative data
generated on the growth and development of the symbiotic partners, little attention has been paid to these in vitro culture systems.
In the mid-nineties a tripartite (strawberry plant –
mycorrhizal carrot root) [13] and a bipartite (strawberry
– AM fungal isolated spores) [14] in vitro culture system
were developed. With these systems, studies were performed on the water uptake and stomatal conductance
[15,16], on the effect of light and CO2 on the growth
and photosynthesis of mycorrhizal plants [17] and on
the use of mycorrhizal vitroplants in the micropropagation industry [18]. However, these systems presented
some drawbacks. In the tripartite culture system [13],
three partners were associated (the vitroplant, the AM
fungus and the root organ culture). Therefore, the strict
one-to-one organism interaction could be disturbed. In
the bipartite culture system on polyurethane foam [14]
the development of the extraradical mycelium as well
as its micro-morphology, i.e., the architecture of the colony [19] is difficult to investigate non-destructively.
In the present study we developed a novel and easy to
use culture system, in which the plant roots are associated to an AM fungus and develop on a gelled medium
under strict in vitro conditions, while the photosynthetic
shoot develops under open air conditions. Spore production dynamics, intraradical root colonization, germination capacity of the newly produced spores and their
capacity to reproduce the fungal life cycle were assessed.
2. Materials and methods
2.1. Potato stock plantlets
In vitro propagated potato plantlets of Solanum
tuberosum L. cvs. Désirée, Kennebec, Pink Fir Apple
and Rosa were supplied by the Station de Haute Belgique in Libramont, Belgium and S. tuberosum andigena
cv. Waycha by the Scottish Agricultural Science Agency,
Scotland. The cultivar Solanum phureja (CGN 18281)
was supplied as true potato seeds by the Center for
Genetic Resources, Wageningen, The Netherlands. The
true potato seeds were surface sterilized with NaClO
for 10 min, then rinsed 3 times during 10 min with
deionized sterilized (121 C for 15 min) water. After surface sterilization, the seeds were plated in Petri plates
(90 mm diameter, 10 seeds per plate) containing the
Modified Strullu Romand (MSR) medium ([20] modified from [11]) and were kept in the dark at 27 C. After
germination, nodal cuttings were placed in sterile culture
boxes (90 · 60 · 50 mm, 20 cuttings per box), filled with
50 ml of 4.412 g l 1 Murashige and Skoog (MS) medium
[21], supplemented with 20 g l 1 sucrose, 3 g l 1 Gel
Groe (ICN, Biomedicals, Inc., Irvine, CA, USA) and
adjusted to pH 5.9 before sterilization (121 C for
15 min). Plantlets were kept in a growth chamber at a
constant temperature of 22 C and illuminated for
16 h d 1 under a photosynthetic photon flux of
15 lmol m 2 s 1. The plantlets of the other cultivars
were also sub-cultured in vitro using nodal cuttings as
described above. The process was repeated every 6–8
weeks.
2.2. Arbuscular mycorrhizal fungi cultures
Transformed carrot roots (Daucus carota L.) colonized with Glomus intraradices Schenck & Smith
(MUCL 43194) were purchased from GINCO (http://
www.mbla.ucl.ac.be/ginco-bel). Cultures were provided
in Petri plates (90 mm diameter) on the MSR medium.
The Petri plates were incubated 4 months in an inverted
position in the dark at 27 C. Several thousand spores
were produced during this period.
2.3. Autotrophic culture system for the in vitro
mycorrhization of potato plantlets
A hole (±2 mm diameter) was made in the side and
the lid of Petri plates (90 mm diameter) containing
40 ml of MSR medium lacking sucrose and vitamins,
and solidified with 4 g l 1 Gel Groe. Seven days-old
rooted nodal explants, cultured on the MS medium, as
described above, were transferred to the Petri plates.
One plantlet was inserted in each Petri plate, with the
roots placed on the surface of the medium and the shoot
extending beyond the hole (Figs. 1 and 2).
Spores of G. intraradices from the AM fungal cultures
described above were isolated by solubilization of the
medium [22] and maintained in deionized sterilized
(121 C for 15 min) water before inoculation. An
approximate of 150 spores were placed in the vicinity
of the roots of the potato plantlets in each Petri plate.
The Petri plates were then closed and the hole cautiously
plastered with sterilized (121 C for 15 min) silicon
grease (VWR International, Belgium) to avoid contaminations. The Petri plates were then sealed with Parafilm
(Pechiney, Plastic Packaging, Chicago, IL 60631),
covered with an opaque plastic bag and incubated vertically in a growth chamber set at 22/18 C (day/night)
with 70% relative humidity, a 16 h photoperiod and a
L. Voets et al. / FEMS Microbiology Letters 248 (2005) 111–118
Spores
113
Extraradical
mycelium
Transformed
carrot root
G. intraradices culture in
association with a transformed
carrot root on the Modified
Strullu-Romand medium (Petri
plate 90 mm diam.)
Isolated spores of G.
intraradices after
solubilisation of the medium
Potato plantlet
Hole plastered with
silicon grease
In vitro culture of potato plantlets on
Murashige and Skoog medium (Box size
90 × 60 × 50 mm)
Newly produced
spores and mycelium
Mycorrized potato
root
Autotrophic culture system
Modified StrulluRomand medium
lacking sucrose
and vitamins
Fig. 1. Diagrammatic representation of the autotrophic culture system for the in vitro mycorrhization of potato plantlets.
photosynthetic photon flux (PPF) of 300 lmol m 2 s 1.
The lamps used in the growth chamber covered a spectrum from k = 400–700 nm. At week 4, 9 and 15
(according to the duration of the experiments described
below), 10 ml of sterilized (121 C for 15 min) MSR
medium lacking sucrose and vitamins (cooled to 40 C
in a water bath) was added to the Petri plates to maintain an adequate level of MSR medium and to supply
the plantlets with renewed nutrients. All the operations
described above were conducted under a horizontal laminar hood.
Each autotrophic culture system was considered as an
experimental unit in the experiments described below.
2.4. Experiment 1: Dynamics of spore production and
development of plant and fungus
Eight experimental units consisting of S. tuberosum
L. cv. Rosa plantlets inoculated with G. intraradices
were non-destructively monitored to establish the
dynamics of spore production. The total number of
newly produced spores was counted 5, 6, 7, 8, 10, 13,
17 and 22 weeks after the association of the AM fungal
propagules with the potato plantlets. The extraradical
hyphal length was estimated at week 22. Spore number
and hyphal length were estimated under a stereo microscope at 10–40· magnification. A grid of lines was
marked on the bottom of each Petri plate. Spores were
counted in each cell formed by the grid and summed
over the entire Petri plate [23]. For the estimation of
the hyphal length [24], the presence of hyphae was recorded at each point where they intersected a line and
the total number of intersects between hyphae and gridlines was included in the formula of Newman [25], used
to relate the total length of roots (or hyphae) [26] in a
given area to the number of times they intersected a
number of straight lines placed within this area. Plantlets were harvested at week 22. Shoot height was measured and microtubers counted. Roots were cleared in
10% KOH and stained with 0.2% Trypan blue [27]. Sixty
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L. Voets et al. / FEMS Microbiology Letters 248 (2005) 111–118
2.6. Experiment 3: suitability of the autotrophic culture
system for the in vitro mycorrhization of different potato
cultivars
Five cultivars were tested: S. tuberosum L. cvs. Désirée, Kennebec, Pink Fir Apple, S. tuberosum andigena
cv. Waycha and S. phureja. Eight experimental units
were considered for each cultivar. The newly produced
spores were counted after a period of 6 weeks as described above.
2.7. Statistical analysis
Data analysis was performed with the statistical
package Statistica for Windows [28]. Data that were
normally distributed and had homogeneous variances
were subjected to an analysis of variance (ANOVA).
The Tukey HSD (honest significant difference) test was
used to identify the significant differences (p 6 0.05).
Fig. 2. Autotrophic culture system associating a potato plantlet (S.
tuberosum cv. Rosa) with isolated spores of G. intraradices. Four weeks
old culture.
randomly selected root pieces (10 mm length) were
mounted on microscope slides and examined under a
bright-field light microscope at 40 or 125· magnification. The frequency (%F) and intensity (%I) of AM fungal root colonization were estimated [23].
The experiment was repeated with the same cultivar
and fungal inoculum and under the same growth conditions for a period of 6 weeks in order to assess the reproducibility of the results obtained. Eight experimental
units were considered. Spore production and hyphal
length were estimated as above.
2.5. Experiment 2: spore germination and continuous
culture
Newly produced spores, isolated from experiment 1,
were tested for their capacity to germinate and ability
to reproduce the fungal life cycle. Fifty spores were isolated from the Petri plates with forceps under laminar
flow hood and placed singly in Petri plates (90 mm
diameter) containing the MSR medium without sucrose
and vitamins. Germination was observed every 4 days
during 16 days under stereo microscope at 10–40· magnification. An approximate of 50 spores were also associated to S. tuberosum cv. Rosa plantlets in the
autotrophic culture system to test their ability to reproduce the fungal life cycle. Eight experimental units were
considered. After 6 weeks of culture the number of newly
produced spores, extraradical hyphal length and %F and
%I of root colonization were estimated as above.
3. Results
3.1. Experiment 1: dynamics of spore production and
development of plant and fungus
Spores started to germinate within 7 days following
their association with the potato plantlets. Hyphal
growth was observed emerging through the lumen of
the subtending hyphae and extended straight in the medium with several short ramifications formed. After 2
weeks, the first contact points between hyphae and roots
were observed. At that time, hyphae started to spread
more profusely into the medium. The first newly produced spores were observed at week 3. The number of
spores produced was low during the 5 initial weeks of
culture and increased thereafter (Fig. 3). After 4–6
weeks of culture, a network of hyphae covered the whole
volume of the medium. Numerous branched absorbing
structures were observed and anastomoses between primary hyphae and between secondary and primary hyphae were noticed. Arbuscules and vesicles (Fig. 4)
were detectable in the roots starting from week 6. Culture medium was added in the Petri plates at week 4, 9
and 15 but did not impact spore production dynamics.
A mean number of 940 ± 334 spores per week was
formed during the last 5 weeks of the experiment, i.e.,
from week 17 to week 22. At the end of the experiment,
an average of 12,264 ± 4745 spores was produced on a
mean hyphal length of 1300 ± 244 cm. Fully developed
spores were yellow-brown and contained numerous lipid
droplets. Their mean diameter averaged 60–90 lm.
Spores were terminal or intercalary and appeared single
or in clusters containing up to 15 individuals. Some
empty spores were detected at all observation times.
At the end of the experiment, no plateau phase was
L. Voets et al. / FEMS Microbiology Letters 248 (2005) 111–118
115
20000
Number of spores per Petri plate
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
0
2
4
6
↑
8
10
↑
12
Time (weeks)
14
16
18
20
22
↑
Fig. 3. Dynamics of spore production of G. intraradices associated to autotrophic potato plantlets (S. tuberosum cv. Rosa) grown on the MSR
medium lacking sucrose and vitamins. Each curve illustrates the evolution of the number of spores in one experimental unit. The dotted line
represents the mean spore production of the eight replicates. Arrows indicate the time of addition of culture medium to the experimental units.
was occupied by thin roots. After 10 weeks of culture,
plantlets started to form stolons with microtubers produced inside and outside the Petri plates. At the end of
the experiment, plant shoots were about 15–20 cm high
with 0–3 microtubers (8–10 mm diameter) produced.
The experiment was repeated under the same growth
conditions as above for a period of 6 weeks. Spore production reached a value of 404 ± 378 which was statistically not different (p = 0.70) to the number of spores
(483 ± 421) produced after 6 weeks in the first part of
the experiment (see Fig. 3). The extraradical hyphal
length reached a value of 125 ± 96 cm. The architecture
of the extraradical mycelium as well as the growth characteristics of the plantlets was similar as above.
Fig. 4. Potato root (S. tuberosum cv. Rosa) colonized by G. intraradices in the autotrophic culture system. The vesicles (arrowhead) are
clearly visible in the root. Note the production of spores (double
arrowhead) on the extraradical hyphae. Scale bar = 150 lm.
reached and spores were still in the phase of abundant
production (Fig. 3).
Root colonization at week 22, estimated by %F and
%I, reached a value of 74 ± 7% and 29 ± 4%, respectively. The roots contained many hyphae, vesicles and
arbuscules. The vesicles and arbuscules were observed
in the cortex of the main and lateral roots.
After transfer to the autotrophic culture system, the
potato plantlets continued their growth. Primary roots
developed on the surface and into the culture medium.
They ramified profusely after a few days and, within
4–5 weeks, nearly the complete volume of the medium
3.2. Experiment 2: spore viability and continuous culture
Ninety-eight percent of the newly produced spores
germinated within 4 days. No subsequent germination
was noted during the 16 days observation. Hyphae
growth was observed emerging through the lumen of
the subtending hyphae. Long hyphae were formed with
several short ramifications. The spores were able to form
associations with the roots of the potato plantlets in the
autotrophic culture system and to produce new spores.
An average of 944 ± 815 spores were produced after 6
weeks. This value did not significantly differ (p = 0.12
and p = 0.11, respectively) from the number of spores
produced in experiment 1.
A network of extraradical hyphae covered the whole
Petri plate with the formation of numerous branched
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L. Voets et al. / FEMS Microbiology Letters 248 (2005) 111–118
3500
3000
Spores production
2500
2000
1500
b
1000
ab
500
ab
ab
Kennebec
Cultivar
Waycha
a
0
Phureja
Pink Fir Apple
Desirée
Fig. 5. Spore production of G. intraradices, 6 weeks after association to the roots of different potato cultivars in autotrophic, in vitro conditions.
Histograms represent means of eight replicates ± SE. Statistically significant differences (p 6 0.05) are indicated by different letters within the
histograms.
absorbing structures and anastomoses. Hyphal length
averaged 255 ± 200 cm. This value did not significantly
differ (p = 0.12) from the hyphal length measured in
the first experiment. The internal root colonization
was estimated at %F = 23 ± 19% and %I = 11 ± 2%.
3.3. Experiment 3: suitability of the autotrophic culture
system for the in vitro mycorrhization of different potato
cultivars
Whichever the potato cultivar considered, spores
were able to germinate, producing long hyphae that
grew into the medium with several ramifications formed.
The production of new spores followed the same dynamic as in experiment 1 with few spores produced until
week 4 followed by a fast increase thereafter. The number of newly produced spores varied between the different cultivars (Fig. 5). The highest number was obtained
with S. tuberosum cv. Désirée as host and the lowest
with S. phureja. Differences were significant between
these two cultivars. No differences in spore production
were further observed between the other cultivars.
4. Discussion
In the present study, we developed an easy to use and
reproducible autotrophic culture system for the in vitro
mycorrhization of potato plantlets.
The mineral composition of the growth medium was
identical to the MSR medium used to grow AM fungi
on excised transformed carrot roots [20], but without
sucrose and vitamins supplemented to the medium.
Shoots developed under high light intensity (PPF =
300 lmol m 2 s 1), allowing plant photosynthesis [29]
and therefore production of carbon and vitamins, while
roots developed under dark growth conditions. As with
the classical MS medium, the potato plantlets belonging
to six different cultivars, grown on the MSR medium
lacking sucrose and vitamins, developed actively [30].
The duration of experiment 1 (22 weeks) also supported
the formation of stolons and microtubers.
Spore production exceeded 12,000 individuals after
22 weeks of culture. Data reported with the same AM
fungal strain (MUCL 43194) [31] and with two different
strains, MUCL 41833 [32] and MUCL 43204 [31] of the
same AM fungal species, associated to excised transformed carrot roots on the MSR medium, gave comparable results. Only one study, to our knowledge,
reported higher spore production with G. intraradices
(MUCL 41833) in mono-compartmental Petri plates
[33]. However in all these experiments, spore productions were recorded at the plateau phase [32], while in
our experiment spores were still in the phase of heavy
production with a weekly increase of 940 ± 334 individuals during the last 5 weeks. The absence of a plateau
phase during this 22 weeks experiment could be related
to the addition of MSR medium at fixed intervals, supplying the potato host plantlets with nutrients and therefore the fungal symbiont with carbon issued from the
photosynthesis. Addition of growth medium was earlier
tested [34] with G. intraradices associated to excised
transformed carrot roots on the Minimal medium [35].
This author observed that the addition of glucose but
also of mineral nutrients increased the spore production
as compared to cultures not receiving supplemental C
and/or minerals.
Whichever the experiment considered, spore production highly varied between replicates of the same cultivar. The coefficient of variation was 39% after 22
L. Voets et al. / FEMS Microbiology Letters 248 (2005) 111–118
weeks of culture in experiment 1 and even higher in
experiments 2 and 3. Such observation confirmed previous results obtained with in vitro cultures of various
AM fungi [36,26]. Specifically focusing on G. intraradices, coefficients of variation calculated from different
experiments ranked from 11% [37] to 22% [31], 38%
[38] and even 61% [33]. The high variability observed
in our experiment could be attributed to the host–fungus
interaction and to both the fungal and plant physiology.
Indeed, partner communication in the pre-symbiotic
and symbiotic phase is a complex multi-step process under genetic control, regulated by an intimate molecular
dialog between the host root and the obligate symbiont
[39]. Such multi-step process could impact root colonization and subsequent spore production. Initiation of
the symbiotic phase (i.e., root colonization) in our
experiment varied noticeably between replicates. In
some experimental units, AM fungi colonized roots
within 3 weeks while in others it took more than 4
weeks. As a result, and specially for the experiments restricted to 6 weeks, the spore counts highly varied between the experimental units. It is assumed that such
variability could partly level off at the later stage of culture development and probably when cultures reach the
plateau phase. Such assumption should be considered in
further experiments.
The length of extraradical hyphae measured in our
study was similar to the values recorded in experiments
conducted on excised transformed carrot roots with
three different strains of G. intraradices [31,33]. Identically, the intraradical root colonization values estimated
in our system were equivalent with the ones obtained on
excised transformed carrot roots associated with G.
intraradices, using the same method of estimation [33].
The newly produced spores were able to germinate,
colonize a new host and reproduce the fungal life cycle.
Spore production was close to the number obtained in
the mother generation. Hyphal length was important
and root colonization abundant. This supports the
capacity of the autotrophic culture system to maintain
AM fungi under continuous culture.
Spore production was obtained on four European
and two Andean potato cultivars demonstrating the
suitability of the system to cultivars differing in genotype
and phenotype. Spores numbers, however, differed between the cultivars as earlier reported with tomato excised root lines [40] and different cultivars of carrot
and clover excised roots [41]. This suggested that
responsiveness of AM fungi varied between cultivars
of a same species. Further experimentations are expected to continue in order to decipher the factors, at
the level of the cultivar, which result in high rates of
spore production.
The autotrophic culture system developed in the present study allowed the AM fungus to develop, under in
vitro conditions, typical colonization structures and to
117
produce an abundant extraradical mycelium with spores
morphologically similar to those observed in the classical monoxenic culture systems on excised root organs
[20] and in pot cultures. Spore production, extraradical
hyphal length and intraradical root colonization were
close to the values recorded with the same strain or different strains of the same species cultured in association
with excised transformed carrot roots [31–33]. The newly
produced spores were capable to form associations with
a new host in vitro, following sub-cultivation, indicating
that the AM fungus is able to complete its life cycle
through successive generations. The autotrophic culture
system was successfully applied to different potato cultivars (several European and Andean cultivars) and to
Medicago truncatula L. (data not shown). Such system
facilitates the study of various aspects regarding biochemistry, genetics and physiology of the symbiosis for
which a source–sink relationship and photosynthetic
active tissues are necessary.
Acknowledgments
This work was supported by the EC contract ICA4CT2002-10016 entitled ‘‘Sustainable potato production
in Andean urban and peri-urban areas by combining
bio-composting and microbial inoculants’’. The authors
thank the Station de Haute Belgique in Libramont, Belgium (Mr. J.L. Rolot) for providing some of the in vitro
propagated potato cultivars. S.D. gratefully acknowledges the financial support from the Belgian Federal Science Policy Office (contract BCCM C3/10/003).
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