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Advances in Mass Production Technology of Arbuscular Mycorrhiza 43

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Advances in Mass Production Technology of Arbuscular Mycorrhiza
A. Khaliq, D.J. Bagyaraj1 and M. Alam
Central Institute of Medicinal and Aromatic Plants (CIMAP) P.O. CIMAP, Lucknow 226015, India 1 Centre for Natural Biological Resources and Community Development 41 RBI Colony, Anand Nagar, Bangalore 560024, India

Introduction
The term mycorrhiza, derived from two words ‘mycos’ and ‘rhiza’, literally means ‘fungus root’. Frank (1885) for the first time proposed this term to describe the mutualistic association occurring between plant root and fungi [1]. Mycorrhizal associations are categorized into two classes: Ectomycorrhiza and Endomycorrhiza. The ectomycorrhiza are, generally, associated with the roots of higher woody perennials. These fungi form intercellular hyphal ramification with the host cortex i.e., hartig net and dense hyphal covering to roots (i.e., mantle). Endomycorrhiza is characterized by fungal penetration of the host cell where three categories are recognized. These are orchidaceous, ericoid and arbuscular. The associated endophyte in first two belongs to higher fungi with septate hyphae. An intermediate group with septate, inter- and intracellular mycelium was placed in a separate group called Ectendomycorrhiza [2]. Arbuscular mycorrhizal (AM) symbiosis, also referred as vesiculararbuscular mycorrhizal symbiosis [3], is the most common and has been estimated to occur in more than 80% of flowering plant species [2]. These associations are extremely ancient and AM fungi have even been identified in fossils of early Devonian land plants [4]. The AM fungi are obligatory biotrophic in nature, colonizes the cells of the roots in order to obtain nutrition from the host plant. In addition to growth
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inert substrates such as vermiculite. aquatic conditions [15]. pot culture may include selection of some strains favoured by the particular growth conditions. production of healthy plants. They make association with the plants found mostly in all soils and weather conditions like desert [14]. The potential utilization of AM fungi highly depends on a suitable inoculum that can be produced in a simple way and be spread on agricultural land with traditional equipments. which is inoculated with pure stock culture of a particular AM isolate in a living root system [18. Gradually. therefore. Khaliq et al.44 A. Existence of different physiological strains within a single morphological species has been reported [20] but such strain differences are difficult to accommodate in a culture collection based on morphological criteria. © 2010 by Taylor & Francis Group. the product is so bulky and heavy to maintain and transport them to the site of its application. Firstly. Thirdly. This might be due to the obligate biotrophic nature of the fungus. they must be grown in the presence of living host root system. 19]. Progress in the development of inoculum of AM fungi has been severely hampered by inability to grow them in axenic culture. which are discussed in this review. there is a risk of cross contamination of adjacent pots of endophyte. The inoculum so produced consists of a mixture of soil. pieces of hyphae and infected root pieces and its production generally takes 3-4 months. it has several drawbacks. Classical Methods Soil-Root Inoculum This is the standard and conventional way of maintaining AM culture around the world. or a mixture of these replaced the soil. Secondly. which are the site for translocation of minerals between plant and roots. sand. very limited amount of inoculum can be produced by this method. The most common medium used so far for this purpose is sterilized soil. Several techniques of inoculum production have been developed time to time by different mycorrhizologists for the sake of its practical utilization and understanding its physiological. Similarly. spores. they form a network of external hyphae which absorbs phosphates and other mineral nutrients from the soil and translocate to the root. within the roots by forming intracellular dichotomously branched structures. LLC . soil stabilization and development of resistance against soil-borne plant pathogens due to AM infection reveal that these fungi play an important role in the ecosystem [5-13]. the arbuscules. biochemical and genetic characteristics. Although this practice is most common and widely used by the workers. salt marsh [16] and highly alkaline usar soil [17]. impurity of inoculum containing other microorganisms and lack of genetic stability under these conditions. perlite. Improvement in plant growth and biomass.

Dehne and Backhaus used light expanded clay material as a carrier material in a hydroponic culture system [23]. The pellets measured 12×12×6 mm and had an average dry weight of 1. The inoculum produced by this method provides easily harvestable solid mat of roots with more concentrated and bulky form of inoculum than that produced by plants grown in soil or other solid media. the plant roots lie in a shallow layer of floating nutrient solution [25].000 pellets in a day. and sporocarps. Nutrient Film Culture In this technique of inoculum production. Soil-Free Inoculum Hewitt introduced a new type of inoculum production technique where author used calcined montmorillonite clay as supporting medium with supplementation of Long Ashton mineral solution [22]. They used polymeric hydrogel product ‘Water-Works’ (USA) as a soil-free substrate for producing AM inoculum. Vermiculite was used as a co-substrate together with the dehydrated polymer crystals.55 g. as claimed by the workers. These dry pellets are glued with seeds by gum arabic and then used. for the study of legume-rhizobia interaction [27]. Vestburg and Sukainen described a new method for producing inoculum in a soil-free substrate [24]. Bone meal was used as a light fertilizer. sophisticated equipments have been designed for continuous adjustment of pH and nutrient concentration. The pellets can easily be broadcasted like other fertilizers and may be spread during seed sowing/transplantation. It proved to be a good substrate for plant growth and abundant mycorrhizal infection and eliminated the need for soil in the growth medium. They obtained upto 60% of mycorrhized roots of beans with attached mycelium. The technique involves the production of © 2010 by Taylor & Francis Group. In deep flowing solution culture. two persons can comfortably make 10. LLC . This technique had a potential for the production of contamination free inoculum. Mosse and Thompson carefully manipulated the nutrient concentration in this culture and used Rhizobium nodules for nitrogen supplementation in medium [26]. Aeroponic Culture This culture was for the first time introduced by Zobel et al. spores. Using this method. Most time consuming step in that manufacture was ensuring that the clay-binding agent was evenly distributed throughout the soil inoculum.Advances in Mass Production Technology of Arbuscular Mycorrhiza 45 Inoculum Rich Soil-Pellets Hall and Kelson introduced a new technique for AM inoculation [21]. They prepared soil pellets enriched with the inoculum.

Fast-growing roots are cloned by repeated subcultures. respectively. inoculated roots in a chamber providing nutrient solution in the form of mist. The A. They get abundant arbuscules and vesicles formed in the roots. Lack of physical substrate in aeroponic culture makes it ideal system for obtaining sufficient amount of clear propagules. Isolated root can be propagated continuously in different solid and liquid media with high reproducibility. G. for germination. rhizogenes. Hung and Sylvia [28] refined the technique of Zobel et al. Root Organ Culture It is really a challenging goal for modern plant biologists to culture the AM fungi under axenic conditions. control of cultural conditions and sampling of mycorrhiza and associated nutrient solution.46 A. The pH of the medium is adjusted to 5. etunicatum inocula retained their infectivity after cold storage (4°C) in either sterile water or moist vermiculite for at least four and nine months. gram negative bacterium produces a neoplastic plant disease syndrome known as ‘Hairy Root’ which has ability to grow rapidly showing two-fold multiplication within 45© 2010 by Taylor & Francis Group.5 before autoclaving. deserticola and G. Initiation of isolated root requires pre-germination of seeds previously surface sterilized with classical disinfectants (sodium hypochlorite. but also useful for critical physiological and genetic studies of this obligate biotroph. a soil dwelling. the use of genetically transformed roots by Ri plasmid of Agrobacterium rhizogenes has given a new impetus to the culture production of these obligate symbionts. intraradices. and G. hydrogen peroxide). Mosse in 1962. Clonal roots of some 15 plants have been established and this list has enlarged during the last decades. Germination of seeds occurs after 48 hr at 28°C in the dark on water agar or moistened filter papers. into the root. etunicatum. Later Mosse and Hepper [30] and Miller-Wideman and Watrud [31] opened the path of in vitro culture when they separately used growing roots in vitro and succeeded in establishing vesicular-arbuscular mycorrhizal symbiosis. Major advantages of this system over others are lack of physical substrate. LLC . She demonstrated the necessity of Pseudomonas sp. Khaliq et al. for the first time. and then thoroughly washed in sterile distilled water. appresorium formation and even penetration of Endogone sp. useful not only for inoculation. Culture Establishment on Transformed Roots Nowadays. established the culture by inoculating the germinating resting spores of Endogone sp. [29]. The tips (2 cm) of emerged roots can be transferred to a rich medium such as modified White medium [32] or Strullu and Romand medium [33]. They also found that aeroponically produced G. [27] and used bahia grass (Paspalum notatum) and sweet potato (Ipomoea batatas) and colonized these roots with Glomus deserticola.

cuccumopine and mikimopine.. mannopine. because this bacterium has a higher Figure 1: Agrobacterium rhizogenes induced clonal culture of tomato roots. Production of Transformed Roots Hairy roots produced by A. old in nutrient broth) at distal surface. Stem segments of 25-30-day-old tomato plant. This characteristic is due to presence of integrated copies of transfer DNA (T-DNA) which occurs in Ri (root inducing) plasmid. Similarly loci encoding for auxin and other loci responsible for the production of roots are still the areas of further investigations [36]. which is a large plasmid [35]. they are repeatedly treated with suitable antibiotics like carbenicillin or cephalexin to make the roots free from original bacterial inoculum and thereby establish the isolation of a single root piece clonal culture. © 2010 by Taylor & Francis Group. which is indicative of transformation. The TR-DNA locus of T-DNA of Ri plasmid also carry a gene responsible for the production of certain amino-acid derivatives called opines like agropine. An example of the production of such culture from tomato stem tissues [37] is described (Figure 2). after surface sterilization with sodium hypochlorite (2% available chlorine) for 5 min. On the solid culture media. The rapid and stable root growth due to modification by A.Advances in Mass Production Technology of Arbuscular Mycorrhiza 47 48 hr in Atropa belladona and Nicotiana tabaccum [34]. LLC . They are also highly stable and capable of secondary metabolite production. rhizogenes infection can be sub-cultured as excised roots (Figure 1). were inoculated with freshly grown bacterium (48 hrs. rhizogenes infection is very important and favourable for the mass production of arbuscular-mycorrhizal culture.

standards [(a) agropinic acid. and (c) agropine]. non-transformed roots. © 2010 by Taylor & Francis Group. (b) mannopine + mannopinic acid. and Lanes C. Khaliq et al. respectively [37]. LLC . A4 and LBA 9402. rhizogenes strain ATCC 15834. D and E are transformed roots incited by A. Figure 3: Arbuscules and mycelia produced in the cortical tissues of Agrobacterium rhizogenes mediated transformed roots of tomato after inoculation of Gigaspora margarita (× 400) [37]. Lane B. Lane A and F.48 A. Figure 2: Electrophoretic analysis of opines in Agrobacterium rhizogenes mediated transformed roots of tomato.

which was simple. [43]. easy and highly effective than the processes described by earlier workers. LLC . The latter technique removes dead spores and other soil debris. which is the main source of contamination. Mosse and Hepper used sporocarp of Glomus mosseae [30]. Attempts have also been made for successful production of hairy root culture from different plant parts like leaf. In the process. Strullu and Romand [33] as well as Zhipeng and Shiuchien [40] used infected root segments colonised with mycorrhiza. Mosse and Hepper [30] stated that since sporocarps are big in size and contain several chlamydospores. A thorough perusal of literature reveals that different workers have used different techniques of spore sterilization [29. 43-47]. Surface Sterilization of Mycorrhizal Propagules Surface sterilization of AM fungal spores is a very important step for establishing infection on the host. Successful transformation was later confirmed either by the detection of opines in hairy root tissues [39] (Figure 3) or by molecular hybridization [35].05% tween-20 in an injection vial under light vacuum © 2010 by Taylor & Francis Group. viable and contamination-free. The inoculum should be healthy. they have plenty of chance to take infection in comparison to single chlamydospore. followed by density gradient centrifugation in order to further purify the spores [42]. few transformed roots were found to be proliferated which were later excised and maintained as a clonal culture.Advances in Mass Production Technology of Arbuscular Mycorrhiza 49 virulence on the distal surface than apical part due to the higher endogenous auxin level [38]. Abdul-Khaliq and Bagyaraj [37] mixed a light orange stain (edible) in the middle gradient of sucrose in order to make clear visual separation of different gradients prior to and after the centrifugation. the spores were treated with 2% chloramine-T containing 0. After 8-10 days. cotyledon. Becard and Fortin [32] and Abdul-Khaliq and Bagyaraj [37] used azygospores of Gigaspora margarita. the segments were transferred to MS medium amended with cephalexin (500 mgL-1) and again incubated under the same conditions. Establishment of Infection by Mycorrhizal Propagules on Transformed Roots Selection of Mycorrhizal Propagules Selection of the type of mycorrhizal propagules is very important for infecting hairy roots. Different workers have used different types of inocula. gentamycin and streptomycin sulphate. Most of them have used chloramine-T (2%). anther etc. Abdul-Khaliq and Bagyaraj [37] modified the procedure of sterilization of Mertz et al. Thereafter. While using this technique. The treated stem segments were placed in Murashige and Skoog (MS) medium and incubated in dark for two days at 25ºC. The isolation of chlamydospores generally involves wet sieving and decanting [41].

They also used bicompartmental culture system. three days after inoculation. Roots growing from modified Murashige and Skoog (MS) nutrient medium and sugar into a compartment containing water agar and neutralized peat were infected with germinated spores of Glomus mosseae. they needed treatment of chloramine-T and antibiotic solution prior to inoculation on hairy roots. later. Becard and Fortin [32]. developed by taking out air from the solution by injection syringe [43]. Based on their experiment. understanding the negatively geotropic nature of the germ tube. strongly branched. the fungus showed some directional growth towards the root. Accordingly. Inoculation of Mycorrhizal Propagules Solid nutrient media are good substrate for the establishment of dual culture of root and fungus. one part was lacking sucrose. The spores were then treated with sterile antibiotic solution (gentamycin 100 µg mL-1 + streptomycin sulphate 200 µg mL-1) for 20 min. above 2 mM total N. and the hyphae became attached. Thereafter. Since the germ tube growth is negatively geotropic in nature. Mosse and Hepper [30] for the first time established the root organ culture of AM. They also established the dual culture on liquid medium. If the spores were stored for few months. Mugnier and Mosse [46] established the root organ culture of Ri T-DNA transformed roots of Convolvulus sepium and established the mycorrhizal infection in these roots. germ tube of G. The fungus spread intercellularly and arbuscules began to form by day 10. 1 filter paper placed in a funnel fitted with rubber tube and stop cock. They began to form the characteristic. essentiality of EDTA and acidic range of pH (below 6) for better germination of spore and penetration of mycelium to the roots. so that germ tube could grow towards the surface of the medium and come in contact with properly placed roots [48]. inserted a single spore into the medium at the bottom of Petri plate. A single spore © 2010 by Taylor & Francis Group. The spores were washed thoroughly with sterile distilled water to remove tween-20 and chloramine-T. This was followed by formation of appresorium and root penetration by day 5. improved the methodology and adopted it for the study of initial events of AM ontogenesis. They divided the medium into two parts. germ tubes developed but passed across root without becoming attached to the surface.50 A. the process of inoculation was simplified.2 mM N. They suggest the importance of concentration of N in the medium. In range of 1 to 2 mM N. Khaliq et al. and other was a complete medium. They were either utilized immediately or were stored at 4°C for 3 to 4 months in a sealed Petri plate. Thereafter. septate fan-like structure on the root surface. Light vacuum was able to remove liquefied gases and air droplets adhered to the spore surface. When the nutrient medium contained less than 0. the spores were removed on sterile Whatmann no. They directly inoculated a single ungerminated spore of Gigaspora margarita on a single root system. LLC . Later. mosseae stopped growing. they suggested lesser sugar (less than 20 g L-1).

They are also generally used by different workers for co-cultivating roots and AM propagules. © 2010 by Taylor & Francis Group. Miller-Wideman and Watrud [31] used 1/10 diluted MS medium for co-culturing G. was found suitable for hairy root culture preparation.Advances in Mass Production Technology of Arbuscular Mycorrhiza 51 was placed near the single transformed root in the same Petri plate and incubated the whole Petri plate vertically in such a way that germ tube grow upward and immediately come in contact with the root [37. St. intraradices with transformed roots and/or normal roots of carrot [55] as well as for G. LLC . presence of ammonium ions (in MS medium) was found to be detrimental to root growth because of steep fall in pH of the medium due to this additive. Mosse and Philips [52] reported detrimental effect of sodium on AM establishment as they observed internal development of Endogone mosseae in the roots of Trifolium parviflorum decrease when sodium was present in the medium. High P level in soil has also been shown to decrease or eliminate mycorrhizal infection [53]. Moreover. P. 49]. It has also been convenient for dual culture of G. since this medium has been developed only for root organ culture. [56] grew G. The most frequently used media for plant tissue culture are White’s [50] and Murashige and Skoog (MS) medium [51]. Reducing the concentration of sucrose in the medium also benefits the mycorrhizal colony and arbuscules development. there must be such a balance in the nutrient medium that the growth of the host as well as fungal symbionts should progress during dual culture establishment. Since the root. Arnaud et al. On the contrary. In this direction the medium modified by Becad and Fortin [32]. margarita with transformed roots of tomato [37]. in White’s medium. Mosse and Hepper [30] and by Abdul-Khaliq and Bagyaraj [37] with less in sucrose and P. and sucrose in the culture medium. although the root diameter is reduced. with no or least ammonium salts. they had success in establishing the symbiosis. Likewise. they felt difficulty in root growth evaluation due to poor nutritional status of culture medium. margarita on tomato seedling explant. intraradices but simultaneously suppressed mycorrhizal infection of transformed carrot roots. establishment of mycorrhizae greatly depends upon presence/absence and concentration of sodium sulphate. It is more appropriate to use White’s medium for root initiation. sometime also replacing ammonium salts with nitrate salts. provided nutritional conditions to stimulate “independent” growth of G. rich in sucrose and P. Nutrient Medium The most important factor for successful arbuscular mycorrhiza formation in root organ culture is the adjustment of appropriate culture medium for dual cultivation of both the components. which needs rich nutrient medium for its growth and the AM fungi grows normally in relatively poor nutrient conditions. although. Although. keeping in mind the delicacy of obligate biotrophic nature of the AM propagules. The media used by Mosse [54]. the additive nitrate counteracts the acidification in the culture medium following root growth that buffers the culture medium and retains pH at 6 for several months.

versiforme associated with excised tomato roots. margarita was greatly stimulated by a synergistic interaction between volatile and exudated factors produced by © 2010 by Taylor & Francis Group. the production of 3-5 newly formed G. but as sporocarps in the environment. In 1984. margarita spores from Petri plate of tomato root culture was reported [31]. intraradices in transformed roots of modified White’s medium [32] in a double compartment system of Petri plate.5 µM. Khaliq et al. Hyphal growth of G. modifying the host and growth parameter. The upper half compartment had sucrose only where transformed mycorrhizal root system was growing. They counted up to 34. During the same year St. Similarly. The G. [58].000 spores’ production per Petri plate. [62] reported that three flavonoids—apigenin. LLC . [61] reported for the first time the establishment of an arbuscular mycorrhizal association between G. Later Diop et al. sinuosum sporocarps survived not as single spores. [59] reported production of an average of 9500 spores of G. Arnaud et al. Only the endosymbiont was permitted to the second (lower) compartment containing the same medium devoid of sucrose.000 spores with a mean of 15. while working with G. Gianinazzi-Pearson et al. The experiment has added G. [56] made a record of 34. Colonization of the lower compartment by the mycelium took place between six and eight weeks after sub-culturing the mycorrhizal roots in the upper compartment.) roots in monoxenic culture. versiforme per Petri plate during transformed carrot root coculturing in modified Strullu-Romand (MSR) medium [57]. intraradices and G. hesperitin and naringerin—promoted spore germination and hyphal growth of G. Becard and Fortin [32] reported 100 spores per Petri plate of the same AM species and this number was further increased to 450 [57]. Declerck et al. There has been an interest for the production of aseptic spores of AM fungus. mainly for research purpose for almost two decades. Bi et al. sinuosum (= Sclerocystis sinuosa) and transformed Ri T-DNA carrot (Daucus carota L.15-1.000 mostly viable spores per Petri plate in the distal compartment. Quercetin was also found as a highly stimulatory flavonoid which had a synergistic effect with carbon dioxide. Four years later. fasciculatum [45]. margarita at concentrations of 0. Root Exudates and Flavonoids Role of plant exudates and flavonoids is also very important in stimulating the germination as well as growth of AM fungi in dual culture system. sinuosum to the list of fungi by using monoxenic culture with transformed Ri T-DNA carrot roots.52 A. A small but growing number of papers have reported the successful establishment of AM associations using a number of AM fungal species [60]. It was shown that white clover root-exudate stimulates hyphal elongation of G. Monoxenic culture technology is a very powerful tool for the experimental establishment of arbuscular mycorrhizal associations. repeated the production of 100-1000 aseptic mature spores per Petri plate.

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