Title of Invention

"A BIOFERTILIZER AND A PROCESS FOR THE PREPARATION THEREOF"

Abstract

This invention relates to a process for the preparation of a biofertilizer comprising steps of a crude inoculum of vesicular-arbuscular mycorrhizal fungi (VAMF) obtaining from the conventional mode of production, purifying the obtained VAMF spores from said crude inoculum by using an agent Eurographin-60 for density gradient centrifugate, subjecting said purified spores to the step of surface sterilization/decontamination as herein defined, developing root organ culture as herein described by using a substrate for example carrot, redeveloping culture on developed root organ culture and sterilized/decontaminated spores thereof, producing continuous VAMF spores as herein defined, subjecting the same to the step of harvesting, and mixing said harvested spores with the carrier such as pond fly ash pre-sterlised to get the said biofertilizer.

Full Text

FIELD OF INVENTION This invention relates to a bioferti1izer and a process for the preparation thereof. BACKGROUND OF INVENTION Vesicu1ar-arbuscu1ar mycorrhizal fungi (VAMF) are obligate biotrophs which resist all attempts to be cultivated exenically (on semi synthetic medium). This lack of independent growth has prevented vesicular-arbuscular mycorrhizal fungi from being advantageously employed as a symbiotic partner of most vascular plants, under a wide variety of pedologic and climatic conditions. Simultaneously, numerous studies have suggested the beneficial role of a vesicu1ar-arbuscu1ar mycorrhizal fungi in improving plant growth by increasing resistance to drought and disease, as well as by enhancing nutrient absorption efficiency. OBJECTS OF THE INVENTION An object of this invention is to propose a novel process for the preparation of a bioferti1izer. Another object of this invention is to propose a process for the preparation of a biofertilizer which is no longer specific to certain plants. According to this invention there is provided a process for the preparation of a biofertilizer comprising steps of: i. a crude inoculum of vesicular-arbuscular mycorrhizal fungi (VAMF) obtaining from the conventional mode of production, ii. purifying said VAMF spores as herein defined, iii. subjecting said purified spores to the step of surface sterilization/decontamination as herein defined, iv. developing root organ culture as herein defined, v. developing dual culture of root organ culture and sterilized/decontaminated spores, vi. producing continuous VAMF spores as herein defined, vii. subjecting the same to the step of harvesting, and viii. mixing said harvested spores with the carrier to get the said biofertilizer. In accordance with this invention the process is performed as per the following steps; Step 1: Crude inoculum containing soil and mycorrhiza propogules of Glomus 1ntraradices obtained from conventional mode of production processed using wet sieving and decanting method. The process allows removal of large amount of soil debris from the crude to obtain Vesicular arbuscular mycorrhiaal fungi (VAMF} spores of Glorous Intraradices alongwith some amount of debris obtained and kept for step 2. Step 2: The spores alongwith smal1 fraction of debris so obtained is further processed for density gradient centrifugation using Eurographin 60 (a contrast agent used for medical diagnostic purposes) for different gradient layers performed and layer of purified spores after centrifugation on 60% gradient is removed and kept for step 3. Step 3: The spores so obtained are processed for surface sterilization using 0.03 to 0.7% detergent (Twen 20) and (0.5 to 20%), a bleaching agent (chloramine T) and antibiotic solution of gentamicine sulphate and streptomycin sulphate (75-15 mg per litre and 20-60 mg per litre respectively) and then the spores were washed and kept for step 5. Step 4: A carrot (Daucass carrota var. pusa kesar) is chosen for transformation of DNA from Agrobectarium rhizogenes using standard protocol. The roots those emerged from transformed explants were transferred for further growth and screened on the media as herein described. The clonol cultures so obtained are maintained on medium for use in Step 5. Step 5: The surface sterilized spores are kept for germination on the media as herein described in dark between 27- o 30 C. Germinated spores between 3-8 days is transferred on a fresh media in a container containing alongwith a growing root tip from Step 4 and both kept closer. The development of colonization in roots of VAMF species is observed microscopically and succesful wal cultures are further processed for step 6. Step 6 The grown dual culture between 10-20 weeks old subjected to make multiple copies by cutting a medium block containing root and daughter spores and placed on fresh media in container for mass production. The selected root clone posses property to regrow on a fresh medium and help VAMF to grow along and produce large quantities of spores. The container containing spores produced between 12-24 weeks are processed for Step 7. Step 7: A media containing spores and roots from Step 6 are subjected to deionization using sodium citrate buffer (8-15 mM). The liquid phase so obtained after this treatment processed for wet sieving and decanting methodology using 75 and 500 mesh sieves in order to separate spores and colonized roots from deionized media under sterile environment. The material obtained is kept for further process under Step 8. Step 8 : The material containing colonized roots and spores are mixed in appropriate quantities with respect to type of application such as forestry, agriculture, horticulture, etc, with pond fly ash pre-steri1ized. The finished product in this form is ready for application. Many quality checks in this process are essential in order to confirm viability of produced spores and genetic stability using standard DMA fingerprinting protocols are also used. The media used comprises MgSO .7H 0 (ø.ø6-ø.ø8% 4 2 w/v), KNO (ø.øø6-ø.ø1% w/v), KCI (ø.øø4-ø.øø8% w/v), 3 KH PO (ø.ø2-ø.øøø6% w/v), Ca(NO ) .4H 0(ø.ø1 to ø.ø3% 24 322 w/v), Sucrose (1-3% w/v), NaFeEDTA (ø.øøø6-ø.øø1% w/v), K1(ø,øøb-ø.øø8% w/v), MnCl .H 0 (ø.øøø4-ø,øøø7% w/v), 2 2 ZnSO .7H 0 (ø.øøø1-ø.øøø4% w/v), H BO (ø,øøøø5-ø.øøø2% 42 33 w/v), CuS0 .5H 0 (ø.øøøøø5-ø.øøøø2% w/v), Na MoO .2H 0 42 242 (ø.øøøøøø1-ø.øøøøøø4% w/v), Glycine (ø.øøø1 to ø .øøø5% w/v), Thiamine hydroch1 oride (ø.øøøøø5-ø.øøøø4% w/v), Pyridoxine hydroch1 oride (ø.øøøøø5-ø.øøøø4% w/v), Nicotinic acid (ø.øøøø2-ø.øøøø7% w/v),. Myo inositol (ø.øø2-ø.øøø7% w/v), Phytagel (ø.2-ø.5% w/v). The other chemical such as Na S'U . 1øH 0 24 2 (ø.ø3-ø.ø6% w/v) and NaH PO .2H 0 (ø.øø1-ø,øøø3% w/v) 242 may also be added into said media. WE CLAIM; 1. A process for the preparation of a biofertilizer comprising steps of: 1. a crude inoculum of vesicular-arbuscular mycorrhizal fungi (VAMF) obtaining from the conventional mode of production, 2. purifying the obtained VAMF spores from said crude inoculum by using an agent Eurographin-60 for density gradient centrifugate, 3. subjecting said purified spores to the step of surface sterilization/decontamination as herein defined, 4. developing root organ culture as herein described by using a substrate for example carrot, 5. redeveloping culture on developed root organ culture and sterilized/decontaminated spores thereof, 6. producing continuous VAMF spores as herein defined, 7. subjecting the same to the step of harvesting, and 8. mixing said harvested spores with the carrier such as pond fly ash pre-sterlised to get the said biofertilizer. 2. A process as claimed in claims 1 and 2 wherein said the purified spores are sterilized/decontaminated by using 0.03 to 0.07% detergent (Twen 20) and 0.5 to 20% bleaching agent like chloramine T (75-15mg/litre) and antibiotic solution of gentamicine sulphate (20-6 mg/litre) and subsequently washing the spores with water. 3. A process as claimed in claim 1 wherein said media used for development of root organ culture and spores comprises MgSO4 . 7H2O (0.06-0.08% w/v), KNO3 (0.006-0.01% w/v), KCI (0.004- 0.008% w/v), KH2PO4 (0.02-0.0006% w/v), Ca(NO3)2.4H2O(0.01 to 0.03% w/v), Sucrose (1-3% w/v), NaFeEDTA (0.0006-0.001% w/v), KI(0.005-0.008% w/v), MnCl2.H2O (0.0004-0.0007% w/v), ZnSo4-7H20 (0.0001-0.0004% w/v), H3BO3 (0.00005-0.0002% w/v), CuSO4.5H2O (0.000005-0.00002% w/v), Na2Mo04.2H20 (0.0000001-0.0000004% w/v), Glycine (0.0001 to 0.0005% w/v), Thiamine hydrochlride (0.000005-0.00004% w/v), Pyridoxine hydrochloride (0.000005-0.00004% w/v), Nicotinic acid (0.00002- 0.00007% w/v), Myo inositol (0.0002-0.0007% w/v), Phytagel (0.2- 0.5% w/v). 4. A process as claimed in claim 4 wherein said media containing the chemical such as Na2S04.10H20 (0.03-0.06% w/v) and NaH2P04.2H2)0 (0.001-0.0003% w/v) can be added into said media. 5. A process as claimed in claim 1, step 5, wherein said culture is developed by keeping the spores in a media at the temperature of 27-30°C, the germinated spores are then transferred on a fresh media alongwith a growing root tip after 3-8 days of germination. 6. A process as claimed in claim 1 wherein the step of continuous production of VAMF spores comprises cutting a media block after 10-20 weeks of said dual culture and placing the same in the cut portion of the media of the same size. The cut portion of the media is put to the cut portion of the dual culture to provide fresh media thereto. 7. A process as claimed in claim 1 wherein said step of harvesting comprises subjecting the VAMF spores grown with the media to the step of deionization using sodium citrate buffer (8-15 mM), the liquid phase so obtained is subjected to the step of wet sieving and decanting using 75 and 500 mesh sieves in order to separate spores and colonized roots 8. A biofertilizer and a process for the preparation thereof substantially as herein described and illustrated.

Documents:

857-del-1999-abstract.pdf

857-del-1999-claims.pdf

857-del-1999-Correspondence-Others-(14-02-2011).pdf

857-del-1999-correspondence-others.pdf

857-del-1999-correspondence-po.pdf

857-del-1999-description (complete).pdf

857-del-1999-form-1.pdf

857-del-1999-Form-15-(14-02-2011).pdf

857-del-1999-form-19.pdf

857-del-1999-form-2.pdf

857-del-1999-form-3.pdf

857-del-1999-form-4.pdf

857-del-1999-form-5.pdf

857-del-1999-gpa.pdf

857-del-1999-petition-138.pdf