The following specification particularly describes the invention and the methodology to be
performed.
INTRODUCTION
The present application relates to a commercially viable process technique for in-vitro production of AMF (Arbuscular Mycorrhizal Fungus) spores. More specifically, the present invention relates to large scale commercially viable process for in-vitro production of AMF spores wherein optimization of large scale production of Mycorrhizal spores (AMF spores) involved a unique split plate method in containers, which is carried out in the following steps: i). Proliferating callus root clumps explanted in three individual container;
ii). each container containing 80-110 ml of three different media like MW medium, M medium and MSR medium respectively;
iii). assess the root colonization by Rhizophagus irregularis using magnified grid line intersection method;
iv). number of spores counted and
v). whole root length from each container examined to calculate the intra-radical structures of AMF.
The AMF spores produced by the process of the present invention are useful in re-establishing the mycorrhizal populations in the soil for uplifting the soil health, plant growth and yield.
The arbuscular mycorrhizal spore (AMF) fungus plays crucial role to attain plant health and soil fertility. The plant root association with the mycorrhizal fungi overcome the challenges like water stress (Liu et al., 2015; Bompadre et al.,,2014), heavy metal resistance against aluminium and lead in in vitro (Gavito et al., 2014) chromium immobilization (Wu et al., 2016), mercury contaminated soils, copper stress (Almeida-Rodriguez et al., 2015; Ambrosini et al., 2015), arsenate and arsenite stressed soils (de Andrade et al., 2015; Cattani et al., 2015), against oxidative stress (Driai et al., 2015) . In association with soil microbial flora AMF supports plant systems on nutrient availability (Tekaya et al., 2016), bioremediation (Ingrid et al., 2016; Fester, 2013) and heavy metal resistance (Dhawi et al., 2016). AMF association also gives resistance against many diseases like black sigatoka in in vitro banana (Anda et al., 2015) and various soil borne plant pathogens (Tahat et al., 2010).

In sustainable agriculture, AMF has great potential in crop production and environment preservation (Oruru and Njeru, 2016; Aggarwal et al., 2011). Unfortunate dependency on toxic chemical pesticides and synthetic fertilizers in farming practices leads to the degradation of soil health, water pollution, natural pollution and ultimately human and animal health (Aktar et al., 2009). By using the AMF in farming as biofertilizer, can minimize the usage of toxic chemical pesticides and synthetic fertilizers (Oruru and Njeru, 2016). In recent research findings, Rhizophagus irregularis plays key role in reclamation of saline soils and heavy metal —r.nntaminatedrsoils.(Lenoir_et_al.,_2_0_16; Lone et al„ 2008). Natural presence of these AMF in soils was disappeared, with the continuous application of these synthetic chemicals and deep tillage (Berruti et-al., 2014). For sustainable agriculture, it is mandate to re-establish the mycorrhizal population in soils. But for farmers it is very expensive, to get uncontaminated pure mycorrhizal inoculum because of its obligate symbiotic nature (Ceballos et al., 2013).
To achieve sustainable agriculture system, it is essential to re-establishing the mycorrhizal populations in the soil for uplifting the soil health, plant growth and yield (Wahbi et al., 2015; Ceballos et al., 2013). But because of its obligate symbiotic nature of reproducibility and duration of its life cycle, makes major challenge for large scale production (Berruti et al., 2016). High levels of phosphorous and sugars are also major limiting factors to develop the AMF inoculum (Habibzadeh, 2015; St-Arnaud etal., 1996).
The development of arbuscular mycorrhizal fungus (AMF) in in vitro root organ cultures has greatly influenced by the potential for research and large scale production of uncontaminated inoculum production. In vitro root organ culture system was first reported by White (1943). AM fungal culture development under in vitro root organ culture process is the most promising way to obtain good amount of extraradical spores in a short span of time with contamination free inoculum (Binondo et al., 2012). The excised roots on synthetic mineral media supplemented with vitamins and a sugar source profuse root proliferation, characterized by the formation of numerous braches with small hairy roots with few plant species. The formation of hairy roots is essential for the development of vigorous root biomass and the establishment of continuous AMF cultures. In plant growth regulators, cytokinins, gibberellins (Takeda et al., 2015) may influence the AMF colonization and symbiotic association. Mosse and Hepper (1.975) were first developed this root organ culture system by using Lycopersicum esculentum Mill.
AMF cultivation techniques based on Agrobacterium rhizogenes transformed roots provide the development of the AMF inoculum in vitro (Becard and Fortin, 1988; Adholeya et al., 2005; Ijdo et al., 2011; Schultze, 2013). After the development of monoxenic cultures of AM fungus
through root organ culture made possible for the continuous observations of fungal colonization
4 T OFFICE fMP M W & T i: A - ,■ n o , o ~ -* -, « ^ . ^ ^

^nd mycelium development as well as the sporulation. Minimal mineral media (M) and modified Strullu-Romand (MSR) media were successfully used for AM fungal colonization on non-transformed tomato root cultures (Diop et al., 1994a; Bago et al., 1996). These root organ culture studies giving better knowledge in mycelium development (Bago et al., 1998a), functional aspects of the mycorrhizal root symbiosis (Debiane et al., 2009), dynamics of sporulation in in vitro host roots (Declerck et al., 2001; Declerck et al., 2004; Voets et al., 2009; Ijdo et al., 2011), AMF spore ontogeny (De souza et al., 2005), study of species level reproduction cycles and nutritional requirements (Labidi et al., 2011; Ijdo etal:, 2011) and large "scale production of viable contamination free inoculum (Tiwari and Adholeya, 2002; Voets et al., 2009; Ijdo et al., 2011) with Solanum tuberosum (Puri and Adholeya, 2013).
Only a few species of AMF strains have been successfully established in vitro through root organ culture technique (Tiwari and Adholeya, 2002; Ijdo et al., 2011). Although having the potential use of the in vitro root organ cultures, have obvious limitations in many aspects the AM symbiosis. Importantly the plant host is replaced by a genetically transformed root organ, affected by the absence of regular photosynthetic organs and normal hormonal effects. Transformed root organs may not be active for too many subculturings for continuous production. Sucrose is added to the culture medium to compensate photosynthates were effect the root-fungus interface. Sugars at this interface may modify the biochemistry of the plant fungal interaction. According to Fortin et al., (2002) these sugar concentrations might affect the arbuscules and vesicles development in in vitro transformed carrot roots. This study mainly focuses on selection of media and host for large scale production of AMF inoculum through root organ culture and also to increase the productivity of AMF inoculum.
SUMMARY OF INVENTION
The present invention relates to a commercially viable process for in-vitro production of AMF spores comprising the steps of
a), preparing hairy root culture of roots selected from plants of Tomato, Amaranthus, Carrot, Potato, Sweet potato and Soybean;
b). transformation of roots using Agrobacterium rhizogenes by direct inoculation in a growth medium;
c). development of the regenerative root clumps of the transformed roots in a growth medium; d). isolating and culturing callus root clumps .for large scale production in a growth medium;

^e). inoculation of Rhizophagus irregularis spores in the transformed hairy root clumps in M-medium; •
f). optimization of large scale production of Mycorrhizal spores (AMF spores) by split plate method in containers and
g). isolation of AMF spores in the commercial packs.
The invention further relates to commercially viable process for in-vitro production of AMF
spores wherein isolating and culturing callus root clumps for large scale production in a growing
medium-is-carriedout in the following steps: ~
i). cut the callus root clumps in the size of atleast -1cm;
ii). Grow in the M medium;
iii). allow till multiples of actively growing white hairy roots visible in M-medium and
iv). sub-cultured the sections of clumps in MS medium to get more callus root clumps. The invention also relates to commercially viable process for in-vitro production of AMF spores large scale production of Mycorrhizal spores (AMF spores) by split plate method in containers is carried out in the following steps:
i). Proliferating callus root clumps explanted in three individual container;
ii). each containing 80-110 ml of three different media like MW medium, M medium and MSR medium respectively;
iii). magnified grid line intersection method used to assess the root colonization by Rhizophagus irregularis;
iv). number of spores counted and
v). whole root length from each container examined to calculate the intra-radical structures of AMF.
BRIEF DESCRIPTION OF THE DRAWINGS:
Fig. 1 : Carrot root transformation and AMF development
A) Transformation of carrot roots in MS medium B) Sterilized transformed root bits with lateral roots in MS medium C) Transformed callus root clumps on MW medium D) 1 cm transformed callus root clump cuttings E) Regeneration of root clump suttings in MS medium F) Germination of Rhizophagus irregularis spores under sterilized conditions G) Hyphal developmental stage extending from roots H) Aborted spore in the development of mass inoculum in containers I) Browning of transformed roots after 4 months

Wig- 2 Large scale in vitro production of Rhizopltagus irregularis. A) Large scale production in containers with M medium B) Sieved extraradical spores under stereo microscope C) Intraradical spores in transformed hairy roots D) Modified split plate method in container with MSR medium E) Magnified view of extra radical spores developed in containers with MW medium F) In vitro grown Rhizophagus irregularis well developed spore in M medium.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a commercially viable process for in-vitro production of AMF spores, which are useful in re-establishing the mycorrhizal populations in the soil for uplifting the soil health, plant growth and yield.
In one embodiments according to present invention, it provides a commercially viable process for in-vitro production of AMF spores comprising the steps of-
a). preparing hairy root culture of roots selected from plants of Tomato, Amaranthus, Carrot, Potato, Sweet potato and Soybean;
b). transformation of roots using Agrobacterium rhizogenes by direct inoculation in a growth medium;
c). development of the regenerative root clumps of the transformed roots in a growth medium; d). isolating and culturing callus root clumps for large scale production in a growth medium; e). inoculation of Rhizophagus irregularis spores in the transformed hairy root clumps in M-medium;
f). optimization of large scale production of Mycorrhizal spores (AMF spores) by split plate method in containers and g). isolation of AMF spores in the commercial packs.
In another embodiment, according to present invention, it provides a commercially viable process for in-vitro production of AMF spores wherein step a) of preparing hairy root culture is carried out in the following steps:
i). roots to be washed with water and surface sterilised;
ii). sterilised roots of step i) dipped in 30-70 % ethanol and
iii). roots are sliced into size ranging between 2 - 6 mm thick discs and placed in the MS medium.
In a particular embodiment according to present invention, wherein root sliced in step iii). are placed as basal sides faced upright position.

^ In yet another embodiment, according to present invention, it provides a wherein step b)
of transformation of roots using Agrobacterium rhizogenes by direct inoculation in a growth medium is carried out in the following steps:
i). Agrobacterium rhizogenes in YMB broth was directly used for inoculation of root slices;
ii). inoculation of root slices carried out in MS medium at 26±2°C and
iii). antibiotics selected from carbenicillin, cefotoxime (250 mg/l) and gentamycin (100
mg/l) utilized to get Agrobacterium-free transformed-roots.— — — —
In yet further another embodiment according to present invention, it provides commercially viable process for in-vitro production of AMF spores wherein step c) of development of the regenerative root clumps of the transformed roots in a growth medium is carried out in the following steps:
i). root clumps taken for development in the MS medium; ii). aggregates of root cells collected from 90 days and
iii). inoculation of the aggregates of root cells of step ii) into the MS medium in dark at 26±2°C;
In another embodiment according to present invention, it provides a wherein step d) of isolating and culturing callus root clumps for large scale production in a growing medium is carried out in the following steps:
i). cut the callus root clumps in the size of atleast ~lcm; ii). Grow in the M medium;
iii). allow till multiples of actively growing white hairy roots visible in M-medium and iv). sub-cultured the sections of clumps in MS medium to get more callus root clumps.
In a particular embodiment according to present invention, a wherein step e) of inoculation of Rhizophagus irregularis spores in the transformed hairy root clumps is carried out in M- medium and at 26±2°C in dark.
In further embodiment according to present invention, a wherein step f) of optimization of large scale production of Mycorrhizal spores (AMF spores) by split plate method in containers is carried out in the following steps:
i). Proliferating callus root clumps explanted in three individual container; ii) each containing 80-110 ml of three different media like MW medium, M medium and MSR medium respectively;
iii). magnified grid line intersection method used to assess the root colonization by k, ^Rhizophagusirregularis;

9 iv). number of spores counted and
v). whole root length from each container examined to calculate the intra-radical structures of AMF.
In yet another embodiment according to present invention, a wherein step g) isolation of AMF spores is carried out in following steps:
i. mass production of AMF in 250 ml containers, filled with 100 ml of M medium without sucrose solidified with 0.4% CleriGel (Phytagel in Himedia, India) and auloclaved for sterilization; ii. after solidification of M medium in the containers, inoculate with mycorrhizal infected active root clump and partially immersed with about 20 ml of M medium made up with 1 % agar and sucrose (1%-C1, 2%-C2 and 3%-C3 concentrations) at 40±2°C; iii. the prepared bottles were incubated at 26±2°C in dark for 90 days iv. each bottle yield spores ranging between 80-100 AMF per gm of medium solution (please check whether it should be 800 -1000AMF per gm or ml) For in vitro mass production of endomycorrhizae, actively growing host was selected based on predetermined criteria. For getting Ri T-DNA transformed roots in in vitro, virulent bacterial strain as Agrobactehum rhizogenes was selected for the transformation process.
Bacterial strain propagation
Agrobactehum rhizogenes slant culture MTCC-532 was bought from the Microbial Type Culture Collection (MTCC), Chandigarh, India. A bacterial suspension of this strain was prepared and inoculated into various media like Lauria-Bertani (LB) broth, Yeast Mannitol Broth (YMB), Nutrient broth and Yeast Extract Peptone (YEP) medium incubating overnight on a incubating shaker (ACMAS, New Delhi, India) at 28±2°C at 150 rpm. Almost all media gave good growth but YMB medium showed excellent turbidity at pH 7.0 (0.739±0.004 O.D at 600nm (U.V spectrophotometer, ELICO, India)). YMB medium (1.0 g/litre yeast extract, 2 g/litre mannitol, 3 g/litre glucose, 5 g/litre sucrose, 0.5 g/litre K2HP04, 0.1 g/litre NaCl, 0.2 g/litre MgS04.7H20, 0.1 g/litre NH4C1, 0.05 g/litre CaS04.2H2O) was used for further experiments for subsequent sub-culturing of Agrobactehum rhizogenes in every month. Agrobactehum rhizogenes cultured in 250 ml Erlenmeyer flask and incubated at in darkness on rotary shaker at 150 rpm.

"Carrot hairy root culture:
Carrot roots were found that the easiest source to transform and regenerate among Tomato hypocotyles regions (Solanum lycopersicum), Amaranthus hypocotyle regions (Amaranthus caudatus), Carrot horizontal root slices (Daucuscarota sub sps. sativus), Potato discs (Solatium tuberosum) (Puri and adholeya, 2013), Sweet potato root slices (Ipomea babatas) and Soybean cotyledons (Glycine max). Freshly harvested (greater than I inch width) healthy carrots from polyhouse were taken for easy transformation with Agrobacterium rhizogenes. Carrots were washed thoroughly with tap water and surface sterilised in 1 % sodium hypochlorite for 15 min (Becard and Fortin, 1988), 30 % H202 for 5 min, 0.1 % HgC12 for 10 min with continuous stirring and further rinsed three times (5 min each) in sterile distilled water. Prior to cutting into discs they were dipped in 70 % ethanol and flamed for 3-4 seconds. Then the carrots were sliced into 4-6 mm thick discs and placed on the MS (Murashige and Skoog, 1962) medium, basal sides faced upright position.
Transformation of carrots (Daucuscarota L.)
Agrobacterium rhizogenes (24-48 hours old) in YMB broth was directly used for inoculation of carrot root slices. A loop full of suspension was applied on the surface of carrot root discs aseptically in petri-dishes containing MS medium with 3% sugars and incubated at 26±2°C in dark. After 3-4 weeks of incubation, callus and roots were initiated from the cambium (more than 6 cm length) were excised and placed on freshly prepared MS medium plates. Healthy white root tips with profuse lateral roots showing negative geotropism on the surface of synthetic MS medium. To get Agrobacterium free transformed carrot roots, weekly sub-culturing were done on MS medium plates with antibiotic carbenicillin 500 mg / litre for 3-4 weeks with decreasing concentrations of antibiotic (400, 300, 200, 100 mg/1). The antibiotics like carbenicillin, cefotoxime (250 mg/1) and gentamycin (100 mg/1) were used to control the bacterial growth in transformed hairy roots. To establish bacterial free clone culture 3-4 subsequent subcultures were required.
Development of Root clumps and maintenance
Subsequently sub-cultured transformed carrot hairy roots in M- medium were gradually lost their regeneration capability for continuous development of hairy roots in the fresh medium also. To get the fresh transformed roots all the time was difficult and time consuming. To overcome this continuously reproducing host must be available all the time. For this, these carrot root clumps-were-develppedjri MSTmedjum. After prolonged,incubation-of transformed roots in

''"■media, some of the root tips were shown 3-4 sub roots. The aggregates of root cells were carefully collected from the 90 days old containers and inoculated into the fresh MS medium with 3% of sucrose and incubated in dark at 26±2°C (BOD incubator, Bio Technics India, India). After 45-60 days of incubation, the fully grown callus root clumps were sectioned and were used for the multiplication of several root clumps in MS medium. The - 1 cm sections of these callus root clumps were gave multiples of actively growing white hairy roots in fresh M-medium. To get more callus root clumps for large scale production, sections of clumps were further sub-cultured into the MS medium with 3% sucrose. Thesecallus root clumps with hairy roots were used as regenerative hosts for the endomycorrhizal infection and mass production of mycorrhizal spores.
Spores inoculation and culture development in petri plates
Inoculums ofRhizophagus irregularis were isolated from the Narsapur Forest, Telangana, India, identified and developed in the R and D Centre for Conservation Biology and Plant Biotechnology, Shiva Shakti Biotechnologies Limited, India. The Rhizophagus irregularis spores (8-10) were inoculated into a Petri dish with actively growing transformed hairy root clumps in M- medium and incubated in BOD incubator. The inoculated petri plates were incubated at 26±2°C in dark and monitored daily under compound microscope for spore germination and root infection.
Optimization of Mass production in containers
For large scale production of mycorrhizal spores, 250 ml containers were used for providing the more nutrients and to get the more extraradical spores. Proliferating callus root clumps were explanted in to individual container containing 100 ml of three different media like MW medium, M medium and MSR medium with 1% sucrose concentration in containers and incubated for 90 days 26±2°C in dark growth rooms. The spores formed in the medium were counted and root colonization by Rhizophagus irregularis was assessed by magnified grid line intersection method (McGongle et al., 1990) after roots were stained. The whole root length from each container was examined to calculate the intraradical structures of AMF.
Modified split plate method in containers
Bi-compartment plate system was introduced by St-Arnaud et al., 1996, to study the impact of sucrose on spore and achieved 10 fold increased spore count. By implementing this
method fo.r_ mass produc.tiQn1>of1AMF.)in,l250i,rn] containers, filljed^with L00 ml of M medium
hi \ OrrilrS IHi; i>i w h J. \r ** >' y i *' J. U % / i *? *• U- P

■"without sucrose solidified with 0.4% CleriGel (Phytagel in Himedia, India) and autoclaved for sterilization. After solidification of M medium in the containers, it was inoculated with mycorrhizal infected active root clump and partially immersed with 20 ml of M medium made up with 1 % agar and sucrose (1%-C1, 2%-C2 and 3%-C3 concentrations) at 40±2°C. The prepared bottles were incubated at 26±2°C in dark for 90 days. Modified split plate method was also tested with 3% sucrose concentration with different media MW medium, M medium and MSR medium in containers and incubated at 26±2°C.
After 90 days of incubation in dark the spores were assessed after deionization of phytagel with 10 mM citrate buffer. To count extraradical spores formed outside of the root were counted under stereo zoom microscope (Olympus SZ61 TR, Japan) and intraradical spores formed inside of the root were stained and counted under compound microscope (Olympus CX41 RF, Japan). GraphPadlnStat 3.10 version was used for the statistical analysis.
EXPERIMENTAL RESULTS AND DISCUSSION
Cultivation of Transformed roots
Sections of carrot roots were provided as the potential donor explants for Ri T-DNA mediated transformation with A. rhizogenes strain MTCC-532 in YMB medium. YMB medium was selected out of the LB medium, Nutrient medium and YEP medium based on the optical density at 600 nm. Among different media YMB medium was got 0.739 as highest optical density within 24 hours of incubation in dark at 28±2°C and 150 rpm. Among Tomato (40 % of hypocotyles), Amarathus (10% of hypocotyles), carrot (70% of root slices), Potato (40% of potato discs), Sweet potato (30% of discs) and soybean (40% of cotyledons), carrots were transformed easily and shown maximum percentage of transformation. After 7 days of inoculation, successful transformation was shown by the maximum callus formation at cambial regions and direct emergence of roots from endodermal sites.
70 % of root slices were shown the callus formation by placing the basal sides of the slices in upright position on the MS medium. Some of the carrot root discs were rotten and became brown due to over growth of Agrobacterium rhizogenes. The full-fledged transformed roots were mostly emerged from endodermal regions of the carrot sections after 3-4 weeks period (Figure 1A). With the exposure of cambium in carrot root discs, the hairy root initiation made easy to generate healthy hairy roots. The vigorously grown thicker roots with more lateral roots, greater than 5 cm size were cut off and transferred to MS medium with 3 % sugar and specific .. antibiotics Jo get-bacterjal-fcee, ,ro.ots. Carbenicillin and .eefotoxi me .-were^ successfully found to

roots in MS medium B) Sterilized transformed root bits with lateral roots in MS medium C) Transformed callus root clumps on MW. medium D) 1 cm transformed callus root clump cuttings E) Regeneration of root clump cuttings in MS medium F) Germination of Rhizophagus irregularis spores under sterilized conditions G) Hyphal developmental stage extending from roots H) Browning of transformed roots after 4 months

"control the Agrobacterium growth in transformed hairy roots. These Agrobacterium-free roots were vigorously grown in hormone free medium with negative geotropism (Figure 1B).
Development of callus root clumps
The continuously sub cultured transformed carrot roots were gradually lost their reproducibility. These roots became brown and inactive to reproduce. These aggregates placed in the fresh MS medium gave the opaque, massive callus root clumps within 45 to 60 days (Figure lC) with multiple fresh-hairy roots/The Tern" sectioriTof these~eallus roofclurhps again in fresh MS medium with 3% sucrose were giving the massive callus root clumps in subsequent sub culturing also (Figure 1D). These callus root clumps in M medium produced the actively growing white hairy roots vigorously (Figure 1E). These root clumps were also found inactivated slowly in the fresh M medium with continuous subcultures. But it was found that, - 1 cm callus root clumps were very active in the MS medium (3% sucrose) with number of subcultures. By maintaining in MS medium root clumps were maintained during the entire cycle of production.
Inoculation and development of mycorrhizal inoculum
The spores of Rhizophagus irregularis was successfully germinated and colonized the hairy roots within a week (Figure 1F). The monoxenic spores were germinated within 3-4 days on M medium with subtending hyphae and spreaded all the directions randomly (Figure 1G). Within 2-3 days hyphae contacted the extensively grown hairy roots from callus root clumps. Hyphae proliferated vigorously on the surface of roots forming various shapes of appressoria. Extraradical spores were started developing after 30 days, with incubation in 26±2°C temperature in BOD incubator. In prolonged incubation (4 months) sporulation was randomly increased and roots turned orange to brown colour. But 3-4 % of monoxenically developed spores were aborted by leakage and spilled around the medium (Figure 1H). With prolonged incubation, roots were turned brown due to shortage of nutrients and root exudates (Figure 1 I). Establishment of AMF in petri plates were supported the observation of mycelial growth, spore formation and hyphal network. But for mass multiplication of extraradical and intraradical spores of Rhizophagus irregularis, containers (bottles or jars) were opted.
Large scale production in containers
After 5 days of inoculation in 100 ml containers, mycorrhizal hyphae were spreaded in to the medium and new whi,te-hajry coots w.ere. developed-from the callus joo.t .clump. By increased

Figure 2: Large scale in vitro production of Rhizophagus irregularis. A) Large scale production in containers with M medium B) Sieved extraradical spores under stereo microscope C) Intraradical spores in transformed hairy roots D) Modified split plate method in container with MSR medium E) Magnified view of extra radical spores developed in containers with MW medium F) In vitro grown Rhizophagus irregularis well developed spore in M medium

growth of transformed hairy roots from the callus root clump, density of mycelial network was increased in the medium. Because of negative geotropism in transformed carrot roots, hairy roots were mostly developed on the surface of medium by hosting the AMF. In 60 days to 90 days time period, sporulation was vigorously developed in the containers (Figure 2 A). The density of mycelial threads and spores was very high near roots and diluted at the bottom of the container, After 90 days of incubation, the roots were separated from the container and phytagel based medium was de-ionized with (10 mM) citrate buffer. The medium was sieved with mesh size 300, 150 and 60 standard test sieves and spores counted under stereomicroscope with gridline intersect method (Figure 2 B).
Roots were extensively grown in all the three media and shown almost similar percentage of root colonization as 88% (Figure 3). The ~1 cm chopped and stained root bits were made possible to count intraradical spores. The intraradical spores were slightly higher in MW medium (21077±2096.30) than in the MSR medium (19639±752.40) but significantly varied from M medium (16236± 1186.70) (Figure 2 C). The extraradical spore count was significantly higher in M medium (39458±1098.00) than the other two media, MW medium (21077±2096.30) and MSR medium (19639±752.40) (Figure 4). M medium was supported to develop good number of Rhizophagus irregularis spores than MW medium and MSR medium with 1% of sucrose concentration. Implementation of split plate method in containers
By the implementation of split plate method in containers, within 10 days of incubation partially immersed root clumps were developed several new white hairy roots with extended lateral roots in 3% sucrose concentrated bottles than 2% and 1% concentrations. The extended hyphae were developed in the all the three media, but extensive mycelial network was developed in the containers, having 3% of sucrose than the other concentrations (Figure 2 E). But the sporulation was developed slowly 40 days onwards in bottom phase (M medium without sucrose). After 40 days of incubation, establishment of spores was randomly increased.
The de-ionised and sieved M medium was filled with 88271 ±965.14 total spores in C3, 76374±758.49 in C2 and 40665±1730.40 in CI respectively. The percentage of root colonization was significantly decreased with the increase of sucrose concentration (Figure 5). C3 (72329±664.00) containers were shown highest extraradical spore count than the C2 (59023±875.27) and CI (22941± 1244.60). The intraradical spore count was observed almost same in C2 (17351±720.73) and CI (17724± 1044.00) but lowest intraradical spore count was recorded in C3 (15972±679.66) (Figure 6). The entrapped colonized callus root clumps with

-different sugar concentrations were gave significant variations in the development of Rhizophagus irregularis spores and development of fresh hairy roots from the clumps.
During the incubation, C3 containers were responded early with fresh hairy roots from the callus root clump and showed the hyphal development towards sugar-free M medium within 2-3 days. Other media (C2 and CI) were also simultaneously showed the positive results with the difference of 7 to 10 days. Within 20 days mycelial density was increased tremendously in C3 M medium. In 90 days of incubation massive sporulation was observed in the sugar-free phase of C3 (Figure 2 F) solidified with phytagel and good regenerative root clumps were developed in the sugar containing C3 M medium solidified with agar.
The comparison study with three different media with modified split plate method, MW medium was shown significant higher spore production (99204±1438.10) than the M medium (89514±2272.10) and MSR medium (82142± 1059.3). The extraradical spore development was very high in sugar free MW medium (82067± 1343.30) than M medium (72886± 1440.30) and MSR medium (63501±1358.70) as shown in figure 3. The intraradical spore count was shown almost same as their percentage of root colonization (Figure 7) in MW medium (99204±1438.10, 75.4±2.50), M medium (89514±2272.10, 75.5±2.37) and MSR medium (82142± 1059.30, 74.7±2.21) (Figure 8).
For large scale production of AMF inoculum in in vitro, so much of research has been conducting from last two decades. The present study is also to develop a protocol for continuous large scale production of AMF spores, by modifying the root organ culture method with split plate method. To get the successful transformation, selection and identification of virulent Agrobacterium rhizogenes bacterial strain is important. MTCC-532 (Chandigarh, India) strain was successfully transformed the carrot roots. For continuous maintenance of this bacterial strain in YMA (Yeast Mannitol agar) medium in every 30 days intervals is also important.
Adholeya et al., (2005) developed in vitro protocol for large scale production of AMF through carrot root organ culture. Transformation with Tomato, Amaranthus, Carrot, Potato, Sweet potato and Soybean parts, carrot roots were gave very good success rate of transformation with Agrobacterium rhizogenes. The source of carrots was also place an important role to get maximum success rate in transformation. Freshly harvested and actively growing carrots were shown almost 70 % of success rate than the carrots from local markets. By placing the basal sides in upright position carrot slices shows very good response due to their endogenous auxin levels (Fusconi, 2014). Transformed carrot slices were formed the fresh white callus and hairy roots at cambial regions. To get bacterial free transformed roots carbenicillin (500 mg) was ., -rShown-immediate re.sp.qnse.wjthjnr3-4 subcultures (Nauutila.et al., 1.995). _

The host reproducibility plays an important role in the continuous large scale production of AMF culture. For the continuous production of AMF inoculum callus root clumps (active host) were developed and continuously maintained in MS medium without losing their reproducibility. Rhizophagus irregularis culture from R and D Centre for Conservation Biology and Plant Biotechnology was proliferated and inoculated easily to callus root clumps. For the continuous maintenance of active callus root clumps 3% of sucrose in MS medium was needed. With continuous subculturings in M medium, MSR medium and MW medium with 1 % sugars got necrosis and lost the reproducibility "of hairy roots.
The mass production protocol achieved by Puri and Adholeya, (2013) in jars made possible to reduce the cost and available to the farmers. St-Arnaud et al. (1996) was studied the variation in spore development in bi-compartment plate system. With the inspiration of split plate method container was separated by 0.4% CleriGel and 1% agar, it was made possible after solidification of phytagel covered with agar used MW medium (Figure 2 E). St-Arnaud et al., (1996) got the tenfold higher spore in the sucrose free medium. In the present study, with the implementation of these two methods, 99204 total spore counts were achieved within 90 days of incubation in MW medium.
Certain specific aspects and embodiments of the present application as explained in more detail with reference to the aforementioned examples, which are provided by way of illustration only and should not be construed as limiting the scope of the invention in any mariner.

1. A commercially viable process for in-vitro production of AMF spores comprising the
steps of
a), preparing hairy root culture of roots selected from plants of Tomato, Amaranthus,
Carrot, Potato, Sweet potato and Soybean;
b). transformation of roots using Agrobacterium rhizogenes by direct inoculation in a
_-__RrQwth.mediuni; . —- —
c). development of the regenerative root clumps of the transformed roots in a growth
medium;
d). isolating and culturing callus root clumps for large scale production in a growth
medium;
e). inoculation of Rhizophagus irregularis spores in the transformed hairy root clumps in
M-medium;
f). optimization of large scale production of Mycorrhizal spores (AMF spores) by split
plate method in containers and
2. A commercially viable process for in-vitro production of AMF spores according to claim 1
wherein step a) of preparing hairy root culture is carried out in the following steps:
i). roots to be washed with water and surface sterilised; ii). sterilised roots of step i) dipped in 70 % ethanol and
iii). roots are sliced into size ranging between 2-3 mm thick discs and placed in the MS medium.
3. A process for preparing hairy root culture according to claim 2 wherein root sliced in step iii). are placed as basal sides faced upright position.
4. A commercially viable process for in-vitro production of AMF spores according to claim 1 wherein step b) of transformation of roots using Agrobacterium rhizogenes by direct inoculation in a growth medium is carried out in the following steps:
i). Agrobacterium rhizogenes in YMB broth directly used for inoculation of root slices; ii), inoculation of root slices carried out in MS medium at 26±2°C and iii). antibiotics selected from carbenicillin, cefotoxime (250 mg/1) and gentamycin (100 mg/1) utilized to get Agrobacterium free transformed roots.

.^5. A commercially viable process for in-vitro production of AMF spores according to claim 1 wherein step c) of development of the regenerative root clumps of the transformed roots in a growth medium is carried out in the following steps:
i). root clumps taken for development in the MS medium; ii). aggregates of root cells collected from 90 days and
iii). inoculation of the aggregates of root cells of step ii) into the MS medium in dark at 26±2T;
6. A commercially viable process for in-vitro production of AMF spores according to claim 1
wherein step d) of isolating and culturing callus root clumps for large scale production in a
growing medium is carried out in the following steps:
i). cut the callus root clumps in the size of atleast ~lcm;
ii). Grow them in the M medium;
iii). allow till multiples of actively growing white hairy roots visible in M-medium and
iv). sub-cultured the sections of clumps in MS medium to get more callus root clumps.
7. A commercially viable process for in-vitro production of AMF spores wherein step e) of inoculation of Rhizophagus irregularis spores in the transformed hairy root clumps is carried out in M- medium and at 26±2°C in dark.
8. A commercially viable process for in-vitro production of AMF spores according to claim 1 wherein step f) of optimization of large scale production of Mycorrhizal spores (AMF spores) by split plate method in containers is carried out in the following steps:
i). Proliferating callus root clumps explanted in three individual container; ii). each container containing 80-110 ml of three different media like MW medium, M medium and MSR medium respectively;
iii). assess the root colonization by Rhizophagus irregularis using magnified grid line intersection method; iv). number of spores counted and
v). whole root length from each container examined to calculate the intra-radical structures of AMF.
9. A commercially viable process for in-vitro production of AMF spores according to claim 1
wherein step g) isolation of AMF spores is carried out in following steps:

mass production of AMF in 250 ml containers, filled with 100 ml of M medium
without sucrose solidified with 0.4% CleriGel (Phytagel in Himedia, India) and
autoclaved for sterilization;
after solidification of M medium in the containers, inoculate with mycorrhizal infected
active root clump and partially immersed with about 20 ml of M medium made up with
1 % agar and sucrose (1%-C1, 2%-C2 and 3%-C3 concentrations) at 40±2°C;
the prepared bottles were incubated at 26±2°C in dark for 90 days
each bottle yield spores ranging between 500-1000 AMF per gm of medium