Title of Invention

"A PROCESS FOR THE STORAGE OF A VAM (VESCICULAR ARBUSCULAR MYCORRHIZA) BIOFERTILIZER"

Abstract

This invention relates to a process for the storage of a VAM (Vescicular Arbuscular Mycorrhiza) biofertilizer characterized by the steps of subjecting colonized roots of the same to the step of pre freezing with cryoprotective agent so as to protect the roots from freeze injury wherein the step of prefreezirig consists of slow cooling the roots to a temperature of -30±2°C at a rate of 1-3 °C/min, subjecting the colonized roots to a primary step of freeze drying wherein primary freeze drying is carried out at -100 + 2°C for a period of 10-12 hours and then to a secondary step of freeze drying which is carried out in a lyphollizer and till the chamber pressure is approximate or equal to that of condenser pressure which is below 150 psi units.

Full Text

FIELD OF INVENTION This invention relates to a process for the storage of a VAM (Vescicular Arbuscular Mycorrhiza) biofertilizer. PRIOR ART It is generally known that mycorrhiza is a special typ" of fungus, and depending upon the type of symbiosis, there are 4 types of mycorrhizal fungi. Endomycorrhizae O, Ectomycorrhizae O, Orchid mycorrhizae O and Ericoid mycorrhizae. Endomycorrhizae, which are otherwise known as vesicular arbuscular mycorrhizae are ubiquitous in nature and found harboring most of the herbaceous plant species. There are six genera and about VA mycorrhizal species of known to literature. OBJECTS OF THE INVENTION An object of this invention is to propose a process for the preparation and storage of a fertilizer, and in particular to a VAM inoculum. Another object of this invention is to propose a process for the preparation and storage of a fertilizer, and in particular to a VAM inoculum, which is efficient. Further objects and advantages of this invention will be more apparent from the ensuing description. At the outset of the description which follows, it is to be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only an exemplary embodiment, and without having to imply and limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and reading of the invention and not intended to be taken restrictively. STATEMENT OF INVENTION According to this invention there is provided a process for the storage of a VAM (Vescicular Arbuscular Mycorrhiza) biofertilizer characterized by the steps of subjecting colonized roots of the same to the step of pre freezing with cryoprotective agent so as to protect the roots from freeze injury wherein the step of prefreezing consists of slow cooling the roots to a temperature of -30±2°C at a rate of 1-3 °C/min, subjecting the colonized roots to a primary step of freeze drying wherein primary freeze drying is carried out at -100 ± 2°C for a period of 10-12 hours and then to a secondary step of freeze drying which is carried out in a lyphollizer and till the chamber pressure is approximate or equal to that of condenser pressure which is below 150 psi units. Strangely, the present invention reflects the benefit of using root-based mycorrhizal incoulum over the soil based mycorrhizal inoculum. Further, root-based inoculum is lighter and easy to exhange over soil-based inoculum. Reference is now made to a working example with reference to the roots as obtained and described hereinabove. EXAMPLE Colonized roots were cut into equal sized bits of approx. 1cm. Root bit inoculum of 0.5 gm was weighed and placed in pre sterilized vacutainers (10 ml capacity). The vacutainers were placed in a cooling water bath O with methanol as solvent. The temperature of the cooling bath was gradually brought down from 28°C (room temperature) to -30°C. the temperature in the cooling bath was maintained at -30°C for a period of 2 hours. This pre freezing of the root inoculum with the cryoprotectant gives a solid substrate to proceed with freeze drying. 4ml of 10% DMSO was added to each vacutainer with root inoculum and loosely plugged. Dimethyl sulphoxide is a cryoprotective agent which when added to biologically active cell suspension helps in minimizing the possible damage experienced during freezing and freeze drying. The pre freezed tubes were loaded on to the shelves of a freeze drying chamber (Acrylic chamber) filled with crushed ice mixture (ice+NaCl+CaCl2). The purpose of adding crushed ice is to minimize the temperature gradient between the sample temperature and the chamber temperature (room temperature). Prior loading the chamber on to the freeze drying equipment, the condenser temperature was brought down to below -70°C and the system evacuated to a pressure below 150 psi units. Freeze drying is carried out for 10-12 hours. Thereafter when the samples appeared visibily dry, the vacutainers were removed and replaced on clean shelves without crushed ice mixture and proceeded for secondary freeze drying. Secondary freeze drying is done to remove the bound water. As the sample mainly consists of colonized roots with spores attached, secondary freeze drying becomes essential. In this step, by avoiding the ice mixture in the shelves, the temperature gradient between the condenser and the freeze drying chamber is increased with facilitates efficient removal of bound water from the sample. At the end of secondary freeze drying, determined by equalizing the condenser pressure and chamber pressure (which corresponds almost to the sample pressure), the samples were removed and the vacutainers were closed tight and immediately sealed using liquid wax. The inoculum used for freeze drying is obtained by raising pot cultures. Indigenous VAM colonized soil from Saccharum mungae field, and colonized roots from the same S.mungae site were collected. The soil inoculum was concentrated by using conventional wet sieving and decanting method (Daniels & Skipper 1982). The concentrated soil inoculum and the colonized roots were used as inoculum in the pots. The inoculum was placed by following the layering method (Jackson et al. 1972. Menge & Timmer 1982, Afek et al. 1990). In each pot, pre germinated seedlings of Maize and Sorghum were individually placed The pots were watered regularly and care was taken to prevent flooding/higher moisture levels. The plants were grown for about three and a half months and harvested at the end of the life cycle. The colonized roots of Sorghum and Maize were chopped off from the shoot and the roots collected avoiding the substrate soil. Pre-freezing Colonized roots are cut into equal size bits of for example approximately 1 cm. 0.5 gm of root bit inoculum and placed in pre sterilized vacutainers is added to each vacutainers with root inoculum and loosely plugged. The vacuta'.ners are placed in a cooling water bath with methanol as solvent. The temperature of the cooling bath is gradually brought down from room temperature to -30° +2°_C. The temperature in the cooling bath is maintained at -30° + 2°_C for a period of 2 to 2-1/2 hours. Primary freeze-drying The pre freezed tubes are loaded on to the shelves of a freeze drying chamber filled with crushed ice mixture (ice + NaCl + CaCl2). Prior loading the samples on to the chamber of freeze drying equipment, the condenser temperature was brought down to below -70°C and the system evacuated to a pressure below 150 units. Freeze drying was carried out for ten hours at a temperature of -100° + 2°_C for a period of 10-12 hours. Secondary freeze-drying The vacutainers are removed and replaced on clean shelves without crushed ice mixture and proceeded for second round of freeze drying. The end point of secondary freeze-drying is determined by equalizing the condenser pressure and chamber pressure. The samples were removed and the vacutainers were closed tight and immediately sealed using liquid wax. Samples in triplicate were used to determine The roots thus obtained by secondary freeze drying is subjected to rehydration which comprises in pouring sterile and distilled water/deionized/water purified by reverse osmosis (RO water) into the sealed vacutainer till the roots are submerged wherein the temperature of water is same as that of the roots with the variation of ±2°C. The storage is carried out for 10 minutes -2 hours depending upon the volume of roots. Determination of percentage end moisture: The KF titration followed was a colorimetric titration using one component reagent (Qualigens, Bombay) containing imidagole as a base (devoid of pyridine). The stock vessel of the instrument (KF titrator, Orion) was filled with the one component reagent. Care was taken that the stock vessel and their inter connections were absolutely dry. A pre-titration was carried out with anhydrous methanol as the solvent. After the initial calibration of the instrument to 100% moisture using pure water, a determined quantity of the sample was administered to the pre-dried working medium. In order to add the sample, the cell was opened for as short a time as necessary and then closed again immediately. The inclusion of atmosphere moisture was thereby reduced to an absolute minimum. Titration of water content in the sample was proceeded until a stable end-point is achieved. The water content of the sample was digitally displayed as percentage moisture of the sample. Results: Freeze-dried root samples rehydration showed an increase in IP over r.on-lyopilized (control) root samples of both maize and Sorghum (fig. 1 & 2). When freeze-dried root samples were used as inoculum for IP determination without rehydration a reduction in IP values was observed (figs. 1, 2). Similar trend was continued even after storage. The increased IP of freeze-dried and rehydration root samples was prominent until six months of storage (figs. 3, 4). When IP was observed during the eight month of storage, there was a decrease in IP value irrespective of the fact that the rocts were rehydrated or not prior to inoculation in IP experiment. This decrease was however small in case of the viability of the sample immediately after freeze drying. A set of sealed samples (plate I) were kept at room temperature and harvested at definite intervals to assess the affect of storage upon freeze-dried root inoculum. Viability test: The viability of the samples was assessed by determining the Infectivity Potential (IP) of the root-bit inoculum. Pre-sterilized soil of weight 80 gms. was taken in small plastic pots of 7 X 5 cm size. The root inoculum ( 1 gm fresh weight) was placed as a layer on top of the pre sterilized soil. A layer of pre sterilized soil was placed covering the root bit inoculum. Pre sterilized seedlings of Sarghum vulgare were placed in each pot. Each pot consisted fixed number of seedlings (8 seedlings per pot). The pot were maintained in a green house for 11 days. The pots were harvested on the 12th day. Plants were carefully removed and washed clean of adhering soil particles. The roots were chopped into fine bits of approx. 1 cm length and stained. The root staining was done following the method developed by Philip & Hayman (1970). The stained roots were observed using the compound microscope (Gallen III, Leica, Switzerland) at 200x and the number of primary entry points (PEP) counted. Similar procedure was followed using the freeze dried root samples also. In one set of experiment, the freeze-dried root-bits were rehydrated or reconstituted in sterile distilled water prior placing them in pots. In another set of experiment, the freeze-dried root-bits were directly used as inoculum without prior reconstitution step. The IP of roots calculated by above mentioned formula and IP values of all three (i.e. non freeze dried, freeze dried and non rehydrated and freeze dried and rehydrated) were compared. Similar procedure was repeated for every sample that was harvested at definite intervals over a storage period of one year. Table 1 : Affect of freeze-drying, rehydration and time of storage upon the infectivity potential of VA mycorrhizal root inoculum as compared to that of non-lyophilized inoculum. (Table Removed) lyophilized and rehydrated root samples (table 1) and more pronounced in lyophilized and non-rehydrated root samples. The statistical means of IP along with the standard errors were plotted (figs. 1-4) using the soft ware 'sigma plot' (Jandel Scientific) with IP expressed per gm root inoculum. The figures 3 & 4 show uniform decrease in IP for freeze-dried and rehydrated root samples while a non-uniform decrease was exhibited by freeze-dried and non-rehydrated root samples. The karlfisher's test revealed less than 1% (0.256) moisture in the root samples. WE CLAIM; 1. A process for the storage of a VAM (Vescicular Arbuscular Mycorrhiza) biofertilizer characterized by the steps of subjecting colonized roots of the same to the step of pre freezing with cryoprotective agent so as to protect the roots from freeze injury wherein the step of prefreezing consists of slow cooling the roots to a temperature of -30±2°C at a rate of 1-3 °C/min, subjecting the colonized roots to a primary step of freeze drying wherein primary freeze drying is carried out at -100 ± 2°C for a period of 10-12 hours and then to a secondary step of freeze drying which is carried out in a lyphollizer and till the chamber pressure is approximate or equal to that of condenser pressure which is below 150 psi units. 2. A process as claimed in claim I wherein the step of freeze drying is carried out in the presence of a cyroprotective agent which is dimethyl sulphoxide. 3. A process as claimed in claim 1 or 2 wherein the colonized root is rehydrated with water such as herein described. 4. A process for the storage of a VAM (Vescicular Arbuscular Mycorrhiza) biofertilizer substantially as herein described and illustrated in the example.

Documents:

457-del-1998-abstract.pdf

457-del-1998-claims.pdf

457-del-1998-correspondence-others.pdf

457-del-1998-correspondence-po.pdf

457-del-1998-description (complete).pdf

457-del-1998-form-1.pdf

457-del-1998-form-13.pdf

457-del-1998-form-19.pdf

457-del-1998-form-2.pdf

457-del-1998-form-3.pdf

457-del-1998-form-5.pdf

457-del-1998-form-6.pdf

457-del-1998-gpa.pdf