Title of Invention	"A NOVEL PROCESS OF GENITIC TRANSFORMATIOHN IN CHICKPEA USING AGROBACTERIUM"
Abstract	A process of genetic transformation in chickpea seed using Agrobacterium comprising culturing of Agro bacterium tumefaciens strain EHA105 harbouring pBINIAa3, Bkb having known insect resistance cry IAa3 gene with 35S CaMV promoter overnight in liquid Agro Bacterium minimal medium as herein described, supplemented with kanamejcin 56 mg/1, rifamicin 10 mg/L at 26 to 30°C on a shaker at 100 to 130 rpm wherein the O.D. at 600 A° is 0.4 to 0.8 taken and soaking sterilized seeds for overnight on a shaker.

Title of Invention

"A NOVEL PROCESS OF GENITIC TRANSFORMATIOHN IN CHICKPEA USING AGROBACTERIUM"

Abstract

A process of genetic transformation in chickpea seed using Agrobacterium comprising culturing of Agro bacterium tumefaciens strain EHA105 harbouring pBINIAa3, Bkb having known insect resistance cry IAa3 gene with 35S CaMV promoter overnight in liquid Agro Bacterium minimal medium as herein described, supplemented with kanamejcin 56 mg/1, rifamicin 10 mg/L at 26 to 30°C on a shaker at 100 to 130 rpm wherein the O.D. at 600 A° is 0.4 to 0.8 taken and soaking sterilized seeds for overnight on a shaker.

Full Text	Field of Invention; This invention relates to the novel method of using bacterial culture to transform the chickpea seed material with the gene of interest and storing it for generations without using tissue culture technique. Background of the Invention; Previous researches have reported the development of Insect resistant transgenic lines of different plant materials using standard transformation protocol followed by tissue culture technique. Subsequently these tissue culture grown plants are being transferred to the field for further growth. The major problem being the poor survival rate of these transgenic plants. Objects of the Invention; The object of this invention is develop a novel protocol for storage of the gene of interest in chickpea through generations. Other object is to develop a process without involving tissue culture technique, wherein the rate of survival is poor. Another object is to produce a simple method using Agrobacterium strain. Detailed description of Invention; A novel protocol has been developed for the storage of insect resistance gene in chickpea which is applicable to other crop species too. Insect resistant transgenic lines of cultivar C-235 have been developed. Bacterial strain Agrobacterium tumefaciens strain EHA 105 harbouring pBINIAa3 (13 kb) having crylAa3 gene with 35S CaMV promoter was used. Plant material Seeds of chickpea cultivar C-235. Methods The Agrobacterium bacterial culture was grown overnight in liquid AB minimal medium (Annexure A) supplemented with kanamycin (50mg/I) and rifampicin (10mg/I) at different temperatures from 26-30 preferably 28°C on a shaker at different revolution 100-130 rpm preferably 120 rpm. O.D. of the culture was recorded at 600 A°. Seeds of chickpea cv.235 were sterilized in 0.1% HgCh solution for 10 minutes and then washed with sterilized distilled water 5-6 times to remove traces of mercuric chloride. The surface sterilized seeds were soaked in the above bacterial culture taken from OD 0.4 to 0.8 preferably 0.6 and kept overnight on a shaker. These seeds were germinated on germination medium (20g agar-agar in one liter water) containing 250mg/I cefotaxime. Fifteen day old seedlings were transferred to potted soil. Genomic DNA was isolated from these plants and its PCR amplification was done using crylAa3 gene specific primers: Forward Primer 5' GGGATGGCTAACAACCCAAACATC3' Reverse Primer 5' GGCAAACTCTGGTCCAGAGAAACC3' Two plants (no. 11 and 13) showed the presence of the transgene as is clear from the photograph of the gel. Seed was collected from these plants (To) and next generation (Ti) was raised next year. Again PCR was done and the presence of the gene was confirmed. Cut leaf bioassay was also conducted on second instar larvae of Helicoverpa armigera. Leaf pieces from transgenic and non-transgenic (control) plants were taken separately in Petri dishes. The larvae were starved for two hours and then fed on the leaf pieces. The larvae died which were fed leaves of transgenic plants while those on control plant leaves survived. This confirmed the expression of the inserted transgene. PCR amplification of genomic DNA of to transgenic gave an amplified product of 380 pp (plate 1) confirming the presence of crylAa3. Seed from TO plants were collected and TI generation was raised. Insect larvae bioassay was conducted using leaves of these Tl plants. The bioassay (plate 2) confirmed the presence of the gene. Annexure A Composition of AB minimal medium; AB salts (20X): Ammonium Chloride (20g/I) Magnesium sulphate heptahydrate (6.0 g/I) Potassium chloride (3.0 g/I) Calcium chloride dihydrate (0.20 g/I) Ferrous sulphate heptahydrate (0.05 g/I). AB Buffer (20X): Dipotassium hydrogen phosphate (78.6 g/I) Sodium dihydrogen phosphate (23.0 g/I). Glucose (0.5 g/I) Sterile water 90 ml For preparation of 100ml AB minimal medium, 5 ml of AB salts (20X), 5ml of AB buffer (20X) was added to 90 ml of sterile distilled water containing 0.5 g glucose. WE CLAIM; 1. A process of genetic transformation in chickpea seed using Agrobacterium comprising culturing of Agro bacterium tumefaciens strain EHA105 harbouring pBINIAa3, Bkb having known insect resistance cry IAa3 gene with 35S CaMV promoter overnight in liquid Agro Bacterium minimal medium as herein described, supplemented with kanamejcin 56 mg/1, rifamicin 10 mg/L at 26 to 30°C on a shaker at 100 to 130 rpm wherein the O.D. at 600 A° is 0.4 to 0.8 taken and soaking sterilized seeds for overnight on a shaker. 2. A process of genetic transformation in chickpea seed using Agrobacterium as claimed in claim 1, wherein the temperature of supplement is 28°C on a shaker at 120 rpm, wherein the O.D. is 0.6. 3. A process of genetic transformation in chickpea seed using Agrobacterium as claimed in claim 1, wherein the genomic DNA is detected by PCR amplification using Cry/Aa3 gene specific primers.

Full Text

Field of Invention;
This invention relates to the novel method of using bacterial culture to transform the chickpea seed material with the gene of interest and storing it for generations without using tissue culture technique.
Background of the Invention;
Previous researches have reported the development of Insect resistant transgenic lines of different plant materials using standard transformation protocol followed by tissue culture technique. Subsequently these tissue culture grown plants are being transferred to the field for further growth. The major problem being the poor survival rate of these transgenic plants.
Objects of the Invention;
The object of this invention is develop a novel protocol for storage of the gene of interest in chickpea through generations.
Other object is to develop a process without involving tissue culture technique, wherein the rate of survival is poor.
Another object is to produce a simple method using Agrobacterium strain.
Detailed description of Invention;
A novel protocol has been developed for the storage of insect resistance gene in chickpea which is applicable to other crop species too.
Insect resistant transgenic lines of cultivar C-235 have been developed.
Bacterial strain
Agrobacterium tumefaciens strain EHA 105 harbouring pBINIAa3 (13 kb) having crylAa3 gene with 35S CaMV promoter was used.
Plant material
Seeds of chickpea cultivar C-235. Methods
The Agrobacterium bacterial culture was grown overnight in liquid AB minimal medium (Annexure A) supplemented with kanamycin (50mg/I) and rifampicin (10mg/I) at different temperatures from 26-30 preferably 28°C on a shaker at different revolution 100-130 rpm preferably 120 rpm. O.D. of the culture was recorded at 600 A°.
Seeds of chickpea cv.235 were sterilized in 0.1% HgCh solution for 10 minutes and then washed with sterilized distilled water 5-6 times to remove traces of mercuric chloride. The surface sterilized seeds were soaked in the above bacterial culture taken from OD 0.4 to 0.8 preferably 0.6 and kept overnight on a shaker. These seeds were germinated on
germination medium (20g agar-agar in one liter water) containing 250mg/I cefotaxime. Fifteen day old seedlings were transferred to potted soil. Genomic DNA was isolated from these plants and its PCR amplification was done using crylAa3 gene specific primers:
Forward Primer 5' GGGATGGCTAACAACCCAAACATC3'
Reverse Primer 5' GGCAAACTCTGGTCCAGAGAAACC3'
Two plants (no. 11 and 13) showed the presence of the transgene as is clear from the photograph of the gel.
Seed was collected from these plants (To) and next generation (Ti) was raised next year. Again PCR was done and the presence of the gene was confirmed. Cut leaf bioassay was also conducted on second instar larvae of Helicoverpa armigera. Leaf pieces from transgenic and non-transgenic (control) plants were taken separately in Petri dishes. The larvae were starved for two hours and then fed on the leaf pieces. The larvae died which were fed leaves of transgenic plants while those on control plant leaves survived. This confirmed the expression of the inserted transgene.
PCR amplification of genomic DNA of to transgenic gave an amplified product of 380 pp (plate 1) confirming the presence of crylAa3. Seed from TO plants were collected and TI generation was raised. Insect larvae bioassay was conducted using leaves of these Tl plants. The bioassay (plate 2) confirmed the presence of the gene.
Annexure A
Composition of AB minimal medium; AB salts (20X): Ammonium Chloride (20g/I) Magnesium sulphate heptahydrate (6.0 g/I) Potassium chloride (3.0 g/I) Calcium chloride dihydrate (0.20 g/I) Ferrous sulphate heptahydrate (0.05 g/I). AB Buffer (20X): Dipotassium hydrogen phosphate (78.6 g/I) Sodium dihydrogen phosphate (23.0 g/I). Glucose (0.5 g/I) Sterile water 90 ml
For preparation of 100ml AB minimal medium, 5 ml of AB salts (20X), 5ml of AB buffer (20X) was added to 90 ml of sterile distilled water containing 0.5 g glucose.

WE CLAIM;
1. A process of genetic transformation in chickpea seed using Agrobacterium comprising culturing of Agro bacterium tumefaciens strain EHA105 harbouring pBINIAa3, Bkb having known insect resistance cry IAa3 gene with 35S CaMV promoter overnight in liquid Agro Bacterium minimal medium as herein described, supplemented with kanamejcin 56 mg/1, rifamicin 10 mg/L at 26 to 30°C on a shaker at 100 to 130 rpm wherein the O.D. at 600 A° is 0.4 to 0.8 taken and soaking sterilized seeds for overnight on a shaker.
2. A process of genetic transformation in chickpea seed using Agrobacterium as claimed in claim 1, wherein the temperature of supplement is 28°C on a shaker at 120 rpm, wherein the O.D. is 0.6.
3. A process of genetic transformation in chickpea seed using Agrobacterium as claimed in claim 1, wherein the genomic DNA is detected by PCR amplification using Cry/Aa3 gene specific primers.

Documents:

76-DEL-2007-Abstract-(06-07-2011).pdf

76-DEL-2007-Abstract-(18-04-2012).pdf

76-DEL-2007-Abstract.pdf

76-DEL-2007-Assignment.pdf

76-DEL-2007-Claims-(06-07-2011).pdf

76-DEL-2007-Claims-(18-04-2012).pdf

76-DEL-2007-Claims-(23-04-2012).pdf

76-DEL-2007-Claims.pdf

76-del-2007-Correspondence Others-(06-07-2011).pdf

76-DEL-2007-Correspondence Others-(18-04-2012).pdf

76-DEL-2007-Correspondence Others-(23-04-2012).pdf

76-del-2007-correspondence-ohers-1.pdf

76-DEL-2007-Correspondence-Others.pdf

76-DEL-2007-Description (Complete).pdf

76-DEL-2007-Drawings.pdf

76-del-2007-Form-1-(06-07-2011).pdf

76-DEL-2007-Form-1.pdf

76-del-2007-form-18.pdf

76-del-2007-Form-2-(06-07-2011).pdf

76-DEL-2007-Form-2.pdf

76-DEL-2007-Form-6.pdf

76-del-2007-GPA-(06-07-2011).pdf

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Patent Number

252590

Indian Patent Application Number

76/DEL/2007

PG Journal Number

21/2012

Publication Date

25-May-2012

Grant Date

23-May-2012

Date of Filing

11-Jan-2007

Name of Patentee

NATIONAL RESEARCH DEVELOPMENT CORPORATION

Applicant Address

"ANUSANDHAN VIKAS", 20-22, ZAMROODPUR COMMUNITY CENTRE, KAILASH COLONY EXTENSION, NEW DELHI-110 048.

Inventors:

#	Inventor's Name	Inventor's Address
1	PRAVEEN BATRA	DEPARTMENT OF BIOTECHNOLOGY, CCS HARYANA AGRICULTURE UNIVERSITY, HISAR-125 004
2	NA	NA
3	PUSHPA KHARB	DEPARTMENT OF BIOTECHNOLOGY, CCS HARYANA AGRICULTURE UNIVERSITY, HISAR-125 004
4	VIJAY CHOWDHURY	DEPARTMENT OF BIOTECHNOLOGY, CCS HARYANA AGRICULTURE UNIVERSITY, HISAR-125 004

PCT International Classification Number

C12N 1/20

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA