Title of Invention

A PROCESS FOR PREPARATION OF A BIO-REAGENT FOR USE FOR ELIMINATION OF PLANT VIRUS.

Abstract The present invention relates to a process for preparation of a bio-reagent for use in elimination of plant virus. The process comprises of drying the roots of the plant in hot air oven, powdering of plant material, first refluxing the plant material at least thrice with organic solvent on boiling water bath, filtration, drying and second refluxing with organic solvent at least three times and finally vacuum drying. A culture medium comprises of Murashige & Skoog's salt, benzyl amino purine (BAP), oc-naphthalene acetic acid (NAA), gibberlic acid, sucrose and meso-inositol is prepared and the plant extract as obtained above is mixed with the culture medium thus prepared.
Full Text FIELD OF INVENTION
PRIOR ART
Plant extracts are known to be used to inhibit the activity of certain viruses. The crude extract of pokeweed (Phytolacca decandroL), has reported (by Duggar and Armstrong in 'Annals of Missouri Botanical Garden' 12:359-366,1925) to markedly inhibit the activity of ordinary Tobacco Mosaic Virus (TMV). The extracts of Phytolacca rigida is reported (by Johnson, in 'Phytopathology' 31:679-701, 1941) to inhibit cornpletery Tobacco Mosaic Virus, even after aging in vitro for long periods. The extracts of leaves of spinach, garden beet, sugar beet and chard when mixed in equal parts with the incula of Tobacco Mosaic Virus and Cabbage .Mosaic Virus, completely or almost completely inhibited their activity. When spinach extract was mixed in equal parts with the incula of Tobacco Ring Spot Virus, Latent Potato Ring Virus and Cucumber Mosaic Virus, the infectivity of these incula was also almost nearly completely inhibited (Kuntz and Walker,'Phytopathology'37:561579, 1947).
The main drawback of the above known plant extracts is that these extracts and method of use thereof enable inhibition of viruses but are not effective enough to achieve complete retrieval.
Another drawback of the above known plant extracts is that the known plant extracts are of no use in producing virus free plant material through tissue culture techniques, especially in plants where such procedures are becoming standard procedures for mass multiplication.
OBJECTS OF THE PRESENT INVENTION
The main object of the present invention is to provide a process for preparation of plant extract of Asparagus adscendens and in vitro method of use thereof for plant virus eliminaton.
Another object of the present invention is to propose a process of retrieve different viruses from plant systems.
SUMMARY OF PRESENT INVENTION
preparation of plant extract of Asparagus species comprising hot air oven drying of the roots of the plant at temperature around 45°C, powdering the plant material, refluxing plant material at least thrice with an organic solvent taking plant material and organic solvent in the ratio of 2:5 (w/v), followed by at least three times refluxing with another organic solvent like acetone after drying, taking dried plant material and solvent in the ratio of 2:5(w/v);
Chrysanthemum virus "B' free plants have been produced using the plant extract based on Asparagus species. The present process provides an environment friendly approach to eliminate virus from plant systems.
The process of present invention involves collection of roots of Asparagus adscendens Roxb, refluxing with benzene on boiling water bath, filtration of the contents, drying of spent plant roots, refluxing roots with acetone thrice, addition of acetone extracts of roots in the liquid multiplication medium of Chrysanthemum in different concentration, culturing explants from Chrysanthemum virus B infected plants of Chrysanthemum cv Jugli in the supplemented media and regeneration of complete Chrysanthemum virus B free plants.
DESCRIPTION OF PROCESS
According to this invention there is provided a process for preparation of extract of the plant of Asparagus species for virus elimination particularly of Chrysanthemum Virus B wherein the process comprises of drying the roots of the plant in hot air oven, powdering of plant material, first refluxing the plant material at least thrice with organic solvent such as benzene on boiling water bath, filtration, drying and second refluxing with organic solvent such as acetone at least three times and finally vacuum drying.
In accordance with the present invention, the process for preparation of plant extract of Asparagus adscendens, comprises following steps:
(a) Drying
Roots of plants of Asparagus adscendens Roxb are collected and dried in hot air oven around 45°C and powdered.
(b) Extraction With Organic Solvents
The air dried plant material is refluxed at least thrice, with organic solvents preferably benzene, on boiling water bath for two hours, taking plant material and organic solvent in the preferred ratio of 2:5. The contents are filtered and spent roots are dried. The roots are then refluxed again with acetone taking dried spent roots and organic solvent in the preferred w/v ratio of 2:5. The final extracts are dried in vacuum.
METHOD OF USE
(a) Preparation of Culture Medium
A culture medium for carnation multiplication is prepared by taking MS (Murashige and Skoog's) salts, 1.5 to 1.25 mg/1, preferably 2.0 mg/1 BAP (Benzyle Amino Purine); 0.75 to 1.25 mg/1 preferably 1.0 mg/1 of NAA (a-napthlene acetic acid); 0.75 to 1.25 mg/1 preferably 1.0 mg/1 gibbrellic acid (GAs); 24-35 g/1, preferably 30 g/1 sucrose, and 75 to 125 mg/1 preferably 100 mg/1 meso inositol.
(b) Preparation of Multiplication Medium
The plant extract prepared by the process described above is added to the culture medium prepared as above and finally multiplication medium having four different concentrations namely 1.0 mg/1, 2.5 mg/1, 5.0 mg/1 and 7.5 mg/1, are prepared.
(c) Elinination of Virus
Shoot tips as well as auxiliary buds from Chrysanthemum Virus B infected plants of Chrysanthemum cv Jugali or from their established cultures are sub-cultured on the media prepared as above and complete plants of Chrysanthemum regenerated.
(d) Evaluation .
Virus status of regenerated plants is judged using Alkine phosphate.based direct
antigen coating indirect ELISA (Enzyine Link Immunosorbent. Assay) system against
Chrysanthemum Virus B. The root extracts of Acetone were able to eliminate the virus
at the concentration level of 7.5mg/l.
The invention will now be illustrated with working examples which are intended
to be a typical example to illustrate the working of the invention and is not intended to
imply any limitation on the scope of the present invention.
WORKING EXAMPLES
Example 1
Roots of plants of Asparagus adscendens are collected and dried in hotair oven at 45°C and powered, 100 g of which was refluxed thrice with 250 ml of benzene on boiling water bath for 2 hrs and contents were filtered. Spent plant roots were dried. The roots were then refluxed with 250 ml of acetone thrice and the extract so obtained was used as ingredient in media along with other hormones after final drying in vacuum.
Stock solutions of four different concentrations of the extracts viz. 1.0, 2.5, 5.0
and 7.5 mg/1 obtained as above was prepared in liquid culture medium for
Chrysanthemum containing MS salts, 2.0 mg/1 benzyle amino purine (BAP), 1.0 mg/1 (cc-
napthlene acetic acid (NAA), 1.0 mg/1 gibbrellic acid (GAs), 30 g/1 sucrose, and 100 mg/1
meso inositol.
Nodal explants from Chrysanthemum Virus B infected plants of Chrysanthemum cv Jugali or from their established cultures were subcultured on these media and their effect on virus concentration was recorded using Alkaline phosphatase based direct antigen coating indirect enzyme linked immunosorbent assay (ELISA) system against Chrysanthemum Virus B.
The root fractions in Asparagus adscendens gave a mild reaction between 1.0 and
2.5 mg/1 concentrations and a strong negative reaction between 5.0 and 7.5 mg/1
concentration.
Example. 2
Roots of plants' of Asparagus officinalis are collected and dried in hot air oven at 45°C and powered, 100 g of which was refluxed thrice with 250 ml of benzene on boiling water bath for 2 hrs and contents were filtered. Spent plant roots were dried. The roots were then refluxed with 250 ml of acetone thrice and the extract so obtained was used as ingredient in media along with other hormones after final drying in vacuum.
. Stock solutions of four different concentrations of the extracts viz. 1.0, 2.5, 5.0 and 7,5 mg/1 obtained as above was prepared in liquid culture medium for Chrysanthemum containing MS salts, 2.0!mg/l benzyle amino purine (BAP), 1.0 mg/1 (a-napthlene acetic acid (NAA), 1.0 mg/1 gibbrellic acid (GAs), 30 g/1 sucrose and 100 mg/1 meso inositol finally multiplication medium having different concentration viz, 1.0, 2,5,'" 5.0 and 7.5 mg/1 of each extracts was prepared.
Nodal explants from Chrysanthemum Virus B infected plants of Carnation cv
Scania or from their established cultures were subcultured on these media and their effect
on virus concentration was recorded using Alkaline phosphatase based direct antigen
coating indirect enzyme linked immunosorbent assay (ELISA) system against
Chrysanthemum Virus B. -
The root fractions in Asparagus officinalis gave a mild reaction between 1.0 and
2.5 mg/1 concentrations and a strong negative reaction between 5.0 and 7.5 mg/1
concentration. .
It is to be understood that the process of the present invention is susceptible to modifications, changes and adaptations by those skilled in the art. Such modifications, changes, adaptations are intended to be within the scope of the present invention which is further set forth under the following claims:-







WE CLAIM:
1. A process for preparation of extract of the plant of Asparagus species for virus
elimination sparticularly of Chrysanthemum Virus B wherein the process
comprises of drying the roots of the plant in hot air oven, powdering of plant
material, first refluxing the plant material at least thrice with organic solvent
such as benzene on boiling water bath, filtration, drying and second refluxing
with organic solvent such as acetone at least three times and finally vacuum
drying.
2. A process as claimed in claim 1 wherein said hot air oven heating of plant
material is carried preferably at temperature around 45°C.
3. A process as claimed in claim 1 wherein weight to volume ratio of plant
material to organic solvent during first refluxing is preferably 2:5.
4. A process as claimed in claim 1 wherein the weight to volume ratio of dried
spent plant material to organic solvent during second refluxing is also 2:5.
5. An in vitro method for use of an extract of the plant of Asparagus species for
virus elimination particularly of Chrysanthemum virus B comprising of
preparation of culture medium of different concentrations by addition of plant
extract of Asparagus species to culture medium and then sub culturing
Chrysanthemum Virus B infected plants of Chrysanthemum in the
multiplication medium of different concentrations thus prepared and
regenerating Chrysanthemum Virus B free plants.

6. A process as claimed in claim 5 wherein said culture medium comprises of
preferably MS (Murashige and Skoog's) salts, benzyle amino purine (BAP),
a-napthlene acetic acid (NAA), gibbrellic acid (GAs) sucrose and meso
inositol.
7. A process as claimed in claim 5 wherein said culture medium has 1.5 to 2.5
g/1 preferably 2.0 mg/1 of BAP.
8. A process as claimed in claim 5 wherein said culture medium has 0.75 to 1.25
mg/1 preferably 1.0 mg/1 of NAA.
9. A process as claimed in claim 5 wherein said culture medium has 0.75 to 1.25
mg/I preferably 1.0 mg/1 of gibberbic acid (GAs).
10. A process as claimed in claim 7 wherein said culture medium has 24-35 g/1
preferably 30 g/1 of sucrose.
11. A process as claimed in claim 5 wherein said culture medium has 75 to 125
mg/1 preferably 100 mg/1 of meso inositol.
12. A process as claimed in claim 5 wherein the preferred concentration of
multiplication medium is from 5.0 mg/1 to 10.0 mg/1 for virus elimination.
13. The plant extract based on Asparagus species and in vitro method of use
thereof for plant virus elimination as substantially described and illustrated
herein.


Documents:

144-del-2002-abstract.pdf

144-del-2002-claims.pdf

144-del-2002-correspondence-others.pdf

144-del-2002-correspondence-po.pdf

144-del-2002-description (complete).pdf

144-del-2002-form-1.pdf

144-del-2002-form-2.pdf

144-del-2002-form-3.pdf

144-del-2002-form-4.pdf

144-del-2002-gpa.pdf


Patent Number 199706
Indian Patent Application Number 144/DEL/2002
PG Journal Number 42/2008
Publication Date 17-Oct-2008
Grant Date 22-Dec-2006
Date of Filing 25-Feb-2002
Name of Patentee DEFENCE RESEARCH & DEVELOPMENT ORGANISATION.
Applicant Address MINISTRY OF DEFENCE, GOVT. OF INDIA, B-341, SENA BHAWAN, DHQ P.O., NEW DELHI-110 011.
Inventors:
# Inventor's Name Inventor's Address
1 BHARDWAJ SATYA VRAT DEPTT. OF BIOTECHNOLOGY, DR. Y.S. PARMAR UNIVERSITY OF HORTICULTURE & FORESTRY, NAUNI-SOLAN (HP) 173230.
2 ROY SHUBHR JYOTSNA DEPTT. OF BIOTECHNOLOGY, DR. Y.S. PARMAR UNIVERSITY OF HORTICULTURE & FORESTRY, NAUNI-SOLAN (HP) 173230.
3 MANGAL MANISHA DEPTT. OF BIOTECHNOLOGY, DR. Y.S. PARMAR UNIVERSITY OF HORTICULTURE & FORESTRY, NAUNI-SOLAN (HP) 173230.
4 HANDA ANIL DEPTT. OF MYCOLOGY & PLANT PATHOLOGY, DR. Y.S. PARMAR UNIVERSITY OF HORTICULTURE & FORESTRY, NAUNI-SOLAN (HP) 173230.
PCT International Classification Number A61K 35/78
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA