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Journal of Applied and Natural Science 6 (1): 290-293 (2014)
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Establishing monoxenic culture of arbuscular mycorrhizal fungus Glomus

intraradices through root organ culture
M. Srinivasan*, K. Kumar, K. Kumutha and P. Marimuthu
Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore-641003 (Tamil Nadu),
INDIA
*Corresponding author. E-mail: srinimicrotech@gmail.com
Received: April 9, 2014; Revised received: May 5, 2014; Accepted: June 10, 2014

Abstract: Arbuscular mycorrhizal fungi are soil fungi distributed worldwide, forming symbiosis with most of the
vascular plants for their growth and survival, which is used for sustainable agriculture and ecosystem management.
This study investigated the establishment of monoxenic cultures of Glomus intraradices in association with
transformed carrot hairy root. The G.intraradices spores were isolated from sugarcane rhizosphere by wet sieving
and decanting technique and propagated in open pot culture. Transformation in to carrot hairy root was done using
Agrobacterium rhizogenes. Surface sterilization of G.intraradices spores co-cultured with transformed carrot hairy
root in Modified Strulla and Romand (MSR) medium was found the host root growth as well as for germination AM
spores. After three months of incubation in dark condition, significant production of extensive hyphal growth on
MSR medium and an average of 8500-9000 spores per petri dish was observed. The in vitro inoculum exhibited
higher potential of root colonization due to numerous intraradices mycelium with extensive spore load. The
produced monoxenic inoculum can be used in place of traditional system where it has a advantage of producing
contaminant free propagulas. Thus the monoxenic culture system, a powerful tool, of AM sporulation, can be used
for the mass production of monoxenic inoculum of AM fungi besides studying its biology.

Keywords: Carrot hairy root induction, Glomus intraradices, Monoxenic inoculum, MSR medium, Sugarcane
rhizosphere

INTRODUCTION first reported, the in vitro association of an Endogone

Continued increase in global population, with the species with plant. In the mid-1970s, Mosse and
limitations in the world's supply of natural resources, Hepper (1975) successfully established a culture of an
extensive use of chemical fertilizers and degeneration AM fungus associated with excised roots of tomato
of the environment is a major challenge to the agricul- (Lycopersicum esculentum Mill) and red clover
tural production today. Contrary to the chemical (T. pratense L.) in gelled medium. After this
fertilizers, organic manures and bioinoculants are less primlinary work, ten years later the first observation of
expensive and achieving high productivity without in vitro AM inoculum production using carrot hairy
harming the environment. In order to implement such a root was developed by Becard and Fortin in 1988.
plan, the judicious use of nature's own biofertilizers Similarly Chabot et al. (1992) developed monoxenic
such as arbuscular mycorrhizal fungi (AMF) (Frank, culture of AM using a mono-compartmental method.
1885) are Obligate symbionts behavior belonging to Using another approach Declerck et al., 1996,
the phylum Glomeromycota (Schuessler et al., 2001) established in vitro AM inoculum by dual culture sys-
that cannot complete their life cycle without establish- tem having sterilized mycorrhizal root segments and
ing a functional symbiosis with host plant. AMF Agrobacterium transformed carrot root. Fortin et al.
enhances the nutrient availability especially phospho- (2002) used split-plate method of monoxenic culture,
rus, augment water uptake and induces resistant separating a proximal compartment containing the
against diseases and boost the crop yield (Lekberg and carrot root and AM fungus from a distal compartment
Koids, 2005). Conventional methods available for to develop high density AM spore inoculum. Many
large scale production of AM fungi are pot cultivation different strains of AM fungi have been developed in
with sterilized soil, aeroponics, hydroponics and green monoxenic culture system. However, (Ijdo et al.,
house based in vivo methods (Ijdo et al., 2011). 2011) reported that only Glomus intraradices
However these methods have limitation in high quality species complex are fast colonizers that are able to
inoculum production. Production of monoxenic AM multiply around ten thousand in vitro propagules in
culture under in vitro conditions is one of the most 5-7 months of incubation. However, all the above
promising ways to obtain high number of spore reported studies have taken a long period (around 7
propagules in a shortest time with contamination monthes) to produce monoxenic AM spores using
free inoculum (Binondo et al., 2012). Mosse (1962) different explants. This sporulation time when
ISSN : 0974-9411 (Print), 2231-5209 (Online) All Rights Reserved © Applied and Natural Science Foundation www.ansfoundation.org
291 M. Srinivasan et al. / J. Appl. & Nat. Sci. 6 (1): 290-293 (2014)

compare to conventional methods is much longer and

difficulty to commercialization. In order to produce
high quality inoculum with higher number of spores in
a relatively shorter period of time it is important to
develop suitable method for inoculum mass produc-
tion. Studies related to this area are less hence our
present study aims to mass produce high potential
monoxenic G. intraradices inoculum with help of
transformed carrot hairy root in shorter time span. Figs.1& 2. Hairy root induction from carrot after 1-2 week
incubation on the MS medium.
MATERIALS AND METHODS
Fungal inoculum propagation: G. intraradices fungi
used for this experiment was isolated from the
rhizosphere of sugarcane and multiplied in pot culture
of maize (Zea mays L. var-NK6240). The soil-sand
mix (2:1 w/w) substrates were sterilized in an
autoclave at 15 lb for half an hour to kill the
indigenones AMF propagules and to avoid cross
Fig. 3. Mass multiplication of Fig. 4. Co –Cultivation
contamination. After 60 of days incubation the soil transformed hairy root on the through Root organ culture.
samples were collected from pot, spores were isolated MSR medium.
by wet-sieving and decanting technique (Daniels and
Skipper, 1982). The mycorrhizal root were removed
from pot culture, estimation of AM colonization
was done by root clearing and staining technique
(Phillips and Hayman, 1970)
Sterilization of spores: The G. intraradices spores
were surface sterilized according to Becard and Piche
(1992) by showing then for 10 min in a 2 % w/v
Chloramine-T with Tween 20 (0.1 % v/v) which then Fig.5. In vitro G. intraradi- Fig.6. Extensive hyphal
ces spore germination and network growth.
followed by 30 min in antibiotic solution containg germ tube growth..
(Streptomycin 200 mg/lit, Ampicillin 200 mg/lit. Then
spores were rinsed for several times with sterile dis-
tilled water and store at 40C.
Carrot hairy root culture: Freshly harvested carrots
were surface sterilized using 0.1 % HgCl2 for 10 min
with continuous stirring. They were further rinsed
three times (each for 5 min) in sterile distilled water
and then dipped in 70 % ethanol for 30 sec and
superficially flamed and peeled out. Each carrot was Fig.7. Formation of new G. Fig. 8. In vitro mass produc-
sliced into 0.5 cm thick discs and were placed on 0.5 % intraradices spores from tion G. intraradices spores on
MS (Murashige and Skoog1962) plates with the basal germ tube. the MSR medium.
sides facing upwards. A loopful of 48 hours old
A. rhizogenes (MTCC-532) strain was pricked
manually for wounding on carrot surface and incubated
at 28ºC in dark for 2-3 weeks.
Monoxenic culture of AM inoculum production by
root organ culture (ROC): The surface sterilized G.
intraradices spores, and Ri-t-DNA transformed carrot
hairy root were associated routinely in Modified
Strulla and Romand (MSR) medium (Strulla and
Fig.9. Cluster of mature in Fig.10. Presence of internal
Romand 1986). The MSR mediun composed of (g/lit)
vitro G. intraradices hyphae and vesicles with in
MgSO47H2O – 73.9, KNO3-7.6, KCl -6.5, KH2PO4- spores. mycorrizal infected carrot
0.41 Ca(NO3)2.4 H2O-35.9, NaFeEDTA-0.16, root.
microelements-MnSO4.4H2O-1.225 CuSO4.5H20-1.1,
adding gallon gum (3g/lit) after that the medium was
ZnSO4.7H2O-0.14, H3BO3-0.925, Na2MOO4.-2H2O-
sterilized @1210C for 15 mins. 10-15 spores of AM
0.12 (NH4)6 Mo7O24.4H2O-1.7, Vitamins-Calcium
fungi were placed in different location near the emerg-
panthotenate-0.09, Biotin-0.0001, Nicotinic acid-0.1,
ing growth tip of pre grown carrot hairy root. Then
Pyridoxine-0.09, Thiamine-0.1 and Cyanocobalamine-
plates were incubated in inverted position at 27 oC in
0.04 Sucrose- 10. The pH was adjusted to 5.5 before
the dark condition.
 M. Srinivasan et al. / J. Appl. & Nat. Sci. 6 (1): 290-293 (2014) 292

RESULTS darkness at 27oC provided a suitable condition for

bacterial strains to insert their copies of Ri t-DNA.
Assessment of colonization potential of These findings were pointed out by Mugnier and
G. intraradices in pot culture: The root colonization Mosse, (1987) who observed that transformation
potential of isolated G. intraradices AM fungus under efficiency is highly dependent on the bacterial strain
green house condition was 45 percent and the used, which is related to the type of vir plasmid, binary
maximum spore count (140 /100g of soil) was plasmid or bacterial chromosomal background as a
observed in sand mixture substrate after 60 days of factor influencing hairy root initiation. The
inoculation. Plants subsequently grown without AMF co-cultivation of G. intraradices spores with carrot
had no sign of AM colonization. The results clearly hairy root was used in present study, because of their
indicated that the isolated G. intraradices spore is comfort of propagation and better adaption in culture
alive and viable with ability to colonize roots and than normal root. This result is supported by Douds
multiply quickly at sixty days of inoculation. (2002) and Gadkar et al. (2006), where transformed
Agrobacterium mediated- transformation: After 10 Daucus carota DC1 and DC2 hairy roots respectively
to 15 days of inoculation with Agrobacterium have been successfully used to initiate monoxenic
rhizogenes, callus induction was observed on the culture of AM fungi through root organ culture. In our
surface of carrot discs, followed by appearance of the study 3-7 days Co- Cultivation with transformed carrot
transformed roots on the side wall of discs (Fig. 1). root and G. intraradices spores, the hyphal growth was
Hairy root initiation continued to occur upto 2-3 moves towards host root. Desouza and Berbara, 1999
weeks (Fig. 2). A typical hairy root growth was observed that 80 % of spore germination and hyphal
observed the appearance of numerous lateral roots growth, on the 14th day of incubation. Hyphal growth
(Fig. 3) and its characteristic exhibiting the negative lengths of over 10 mm from germination spores have
geotrophic growth habit. These transformed hairy roots been reported under the best experimental conditions
were subsequently subcultured in fresh MS medium (Douds and schenck,1991) After that germ tube
containing antibiotic solution, cefotaxime at 250 mg/l branched and auxiliary cells grew in all directions
(HiMedia,Mumbai, India) to make it free from followed by hyphae proliferation numerously near the
A. rhizogenes. The bacterial free hairy roots (around 6 root and density of hyphae on the surface of the
cm long) were cut, transferred to a MSR medium and medium. The same result was noted by Costa et al.
incubated for 5 days at 27 oC in the dark condition. (2013) who observed that in in vitro culture of
In vitro propagation G. intraradices: After seven Gigaspora decipiens and Clomus clarum produced
days of co-cultivation, (Fig.4) the spore germination typical structures like branched asorbing structure
and hyphal growth was observed from surface (BAS) after spores germination. Pratap chandran and
sterilized G. intraradices spores (Fig. 5). with potty (2010) also reported that in vitro co-culture of
simultaneous growth of germ tube towards hairy root . AM fungi C. microcarpum with Vigna vexilate hairy
After the initial contact between the germ tube and the root on the 12-20 days of incubation 60 % of media
root, the intercellular colonization took place on the surface was covered with heavy mycelial network
12th day . During 18-20 days of incubation germ tube growth and fan-like structures were observed. After 30
produced, multiply laterial branches on the root and days of incubation, it was observed that in this study
media surface by extensive hyphal proliferation heavy sporulation took place with extensive hyphal
(Fig.6). After that, rapid enlargement of mycelium in network. We observed numerous vegetative spores of
the form of clusters and the formatiom if new spores G. intraradices were produced in the medium after two
(Fig.7) was observed within 30 days. The rate of spore months and spores matured to light brown colour to a
formation was slow during next 30 Days which was dark brown colour (Fig. 9). In our investigation, the
followed by a rapid increase in spore number on 70th rate of sporulation was higher after 70 days
days of incubation was observed. An average of 8500- incubation. The reason might be due to the decrease of
9000 spores per petri dish was produced after 3 months sucrose and other mineral component level in the
of incubation (Figs. 8,9) medium. Similar trend was observed by James et al.
Three month old roots were harvested from prtidish, in (2013) who showed that the rate of sporulation
vitro AM colonization was observed by root depended on the sucrose concentration of the media.
clearing and staining technique, the stained roots Medium supplement with sucrose had less sporulation
showed numerous internal hyphae, arbuscules and than the medium without sucrose. This result was also
vesicles. The percentage of mycorrhizal colonization supported by the study of Diop (1994) and Clark
around 75-80 % was recorded (Fig. 10) (1997) on Water agar medium without supplement
DISCUSSION mineral also showing support to sporulation of AM
spores. In the present study MSR medium was most
Carrot is one of the most suitable and well known appropriate medium, through which approximately
model plant species for hairy root production (Bidondo 8500 to 9000 spores can be produced per plate after 3
et al., 2012).In this present study, it was observed that months of root organ culture. Ijdo et al. (2011) also
inoculation of carrot discs with 48 hours old reported that M medium (Becard and Fortin 1988) and
A. rhizogenes (MTCC-532) culture and incubated in MSR medium (Strullu and Romand 1986) are
293 M. Srinivasan et al. / J. Appl. & Nat. Sci. 6 (1): 290-293 (2014)

frequently used for culture AM fungi on ROC. Diop, T.A., Plenchette, C. and Strullu, D. G. (1994a). Dual
Declarck et al. (1996) developed MSR medium to axenic culture of sheared-rood inocula of vesicular-
optimize the growth of intracellular mycelium and arbuscular mycorrhizal fungi associated with tomato
expensive sporulation of the fugues under in vitro roots. Mycorrhiza, 5: 17-22.
Douds, D. D. and Schenck, N.C. (1991). Germination and
condition. In event of sporulation Declerck et al. hyphal growth of VAM fungi during and after storage
(2001) found same trends when carrot was used as host in soil at five matric potentials. Soil Biology and
plant to produce 8,400 Glomus intraradices spores per Biochemistry, 23: 177-183.
petri plate after 12 weeks of incubation. Douds, D.D. (2002). Increased spore production by Glomus
intraradices in the split-plate monoxenic culture system
Conclusion by repeated harvest, gel replacement, and resupply of
The root organ cultural method produced mycorrhizal glucose to the mycorrhiza. Mycorrhiza, 12:163–167.
Fortin, J.A., Becard, G., Declerck, S., Dalpe, Y., StArnaud,
root segments holding G. intraradices spore with
M., Coughlan, A.P. and Piche, Y. (2002). Arbuscular
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factors released from hyphae of arbuscular mycorrhizal
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