US4520020A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method of preventing virus increase in plants
Assignee: Ministry of Agriculture and Rural Development
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Filing: 1982-07-15
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Granted: 1985-05-28
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Expires: 2002-07-15
Abstract
Abstract A method of isolating material which inhibits virus replication in plants comprising isolating protoplasts from a local lesion-responding tobacco plant having an N gene, preferably a Samsun NN tobacco plant, inoculating the protoplasts with Tobacco Mosaic Virus, and either removing the protoplasts after a predetermined amount of time and isolating the desired material from the protoplast incubation medium, or removing the virus after a predetermined amount of time and isolating the desired material from the preparation. Also provided is a method of isolating material which inhibits virus replication in plants comprising isolating tissue from a "green island" area of a tobacco plant which responds systemically to Cucumber Mosaic Virus, preferably a Samsun NN, a Xanthi-nc or Samsun plant, homogenizing the tissue and isolating the desired material from the homogenate, as well as a method of immunizing plants against virus replication comprising applying thereto the material isolated as above.
Inventors
- 👤 Gad Loebenstein
- 👤 Abed Gera
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 05:05
CPC Classifications
A; A01; A01N; A01N65/00; A01N65/08; A01N65/38; A01N65/385; C; C12; C12N; C12N1/00; C12N15/00; C12N15/09; C12N15/11; C12P; C12P1/00; C12R; C12R2001/00; C12R2001/91
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
65 words
A method of isolating a material which inhibits replication of TMV, CMV and PVX comprising the steps of: isolating protoplasts of a plant of the genus Nicotiana tabacum L. having an N gene; inoculating the protoplasts with Tobacco Mosaic Virus; incubating the inoculated protoplasts under continuous illumination for between 24 and 96 hours; removing the protoplasts from the incubation medium; and sterilizing the incubation medium.
Claim 2
Independent Claim
65 words
A method of isolating a material which inhibits replication of TMV, CMV and PVX comprising the steps of: isolating protoplasts of a plant of the genus Nicotiana tabacum L. having an N gene; inoculating the protoplasts with Tobacco Mosaic Virus; incubating the inoculated protoplasts under continuous illumination for between 24 and 96 hours; removing the protoplasts from the incubation medium; and sterilizing the incubation medium.
Claim 3
Dependent Claim
58 words
The method of claim 1 wherein said step of isolating protoplasts comprises the steps of: incubating leaf portions of a plant of the genus Nicotiana tabacum L. having an N gene with pectinolytic and cellulolytic enzymes to produce protoplasts; washing the protoplasts with mannitol; floating the washed protoplasts on 23% sucrose; and washing the floated protoplasts with mannitol.
Claim 4
Dependent Claim
16 words
The method of claim 1 wherein said incubating step comprises incubating said protoplasts for 72 hours.
Claim 5
Dependent Claim
56 words
The method of claim 1 further including, following the step of sterilizing, the steps of: adding ZnAc 2 to the sterilized supernatant to a final concentration of 0.02M; after about three hours, centrifuging the solution; dissolving the precipitate by bringing the solution to between pH 2.5-2.6; centrifuging the solution; and dialyzing the supernatant against phosphate buffer.
Claim 6
Dependent Claim
18 words
The method of claim 4 further including the step of lyophilizing the supernatant after said step of dialyzing.
Claim 7
Dependent Claim
38 words
The method of claim 1 wherein said step of isolating protoplasts comprises the step of isolating protoplasts from a plant selected from the group consisting of Nicotiana tabacum L. cv. Samsun NN and Nicotiana tabacum L. cv. Xanthi-nc.
Claim 8
Independent Claim
80 words
A method of isolating a material which inhibits replication of TMV, CMV and PVX comprising the steps of: isolating protoplasts of a plant of the genus Nicotiana tabacum L. having an N gene; inoculating the protoplasts with Tobacco Mosaic Virus; incubating the inoculated protoplasts under continuous illumination for between 24 and 96 hours; removing the protoplasts from the incubation medium; homogenizing the removed protoplasts in phosphate buffer; centrifuging the homogenate; and ultracentrifuging the supernate to remove the Tobacco Mosaic Virus.
Claim 9
Independent Claim
80 words
A method of isolating a material which inhibits replication of TMV, CMV and PVX comprising the steps of: isolating protoplasts of a plant of the genus Nicotiana tabacum L. having an N gene; inoculating the protoplasts with Tobacco Mosaic Virus; incubating the inoculated protoplasts under continuous illumination for between 24 and 96 hours; removing the protoplasts from the incubation medium; homogenizing the removed protoplasts in phosphate buffer; centrifuging the homogenate; and ultracentrifuging the supernate to remove the Tobacco Mosaic Virus.
Claim 10
Independent Claim
107 words
A method of isolating a material which inhibits replication of TMV, CMV and PVX comprising the steps of: inoculating with Cucumber Mosaic Virus a plant of the genus Nicotiana tabacum L. which responds systemically to Cucumber Mosaic Virus; after about four weeks, separating "green islands" tissue from the remainder of the leaf; homogenizing the "green islands" tissue; centrifuging the homogenate; dialyzing the supernate against phosphate buffer; adding ZnAc 2 to the dialyzed supernate to a final concentration of 0.02M; after about three hours, centrifuging the solution; dissolving the precipitate by bringing the solution to between pH 2.5-2.6; centrifuging the solution; and dialyzing the supernate against phosphate buffer.
Claim 11
Independent Claim
107 words
A method of isolating a material which inhibits replication of TMV, CMV and PVX comprising the steps of: inoculating with Cucumber Mosaic Virus a plant of the genus Nicotiana tabacum L. which responds systemically to Cucumber Mosaic Virus; after about four weeks, separating "green islands" tissue from the remainder of the leaf; homogenizing the "green islands" tissue; centrifuging the homogenate; dialyzing the supernate against phosphate buffer; adding ZnAc 2 to the dialyzed supernate to a final concentration of 0.02M; after about three hours, centrifuging the solution; dissolving the precipitate by bringing the solution to between pH 2.5-2.6; centrifuging the solution; and dialyzing the supernate against phosphate buffer.
Claim 12
Dependent Claim
44 words
A method according to claim 8 and wherein said step of inoculating a plant comprises the step of inoculating a plant selected from the group consisting of Nicotiana tabacum L. cv. Xanthi-nc, Nicotiana tabacum L. cv. Samsun NN and Nicotiana tabacum L. cv. Samsun.
Claim 13
Independent Claim
103 words
A method of isolating a material which inhibits replication of TMV, CMV and PVX comprising the steps of: inoculating with Cucumber Mosaic Virus a plant of the genus Nicotiana tabacum L. which responds systemically to Cucumber Mosaic Virus; after about four weeks, separating "green islands" tissue from the remainder of the leaf; isolating protoplasts from said "green islands" tissue; inoculating the protoplasts with a virus selected from the group consisting of Tobacco Mosaic Virus and Cucumber Mosaic Virus; incubating the inoculated protoplasts under continuous illumination for between 24 and 96 hours; removing the protoplasts from the incubation medium; and sterilizing the incubation medium.
Claim 14
Independent Claim
103 words
A method of isolating a material which inhibits replication of TMV, CMV and PVX comprising the steps of: inoculating with Cucumber Mosaic Virus a plant of the genus Nicotiana tabacum L. which responds systemically to Cucumber Mosaic Virus; after about four weeks, separating "green islands" tissue from the remainder of the leaf; isolating protoplasts from said "green islands" tissue; inoculating the protoplasts with a virus selected from the group consisting of Tobacco Mosaic Virus and Cucumber Mosaic Virus; incubating the inoculated protoplasts under continuous illumination for between 24 and 96 hours; removing the protoplasts from the incubation medium; and sterilizing the incubation medium.
Claim 15
Dependent Claim
46 words
A method according to claim 10 wherein said step of isolating protoplasts comprises the steps of: incubating said tissue with pectinolytic and cellulolytic enzymes to produce protoplasts; washing the protoplasts with mannitol; floating the washed protoplasts on 23% sucrose; and washing the floated protoplasts with mannitol.
Claim 16
Dependent Claim
40 words
A method according to claim 10 wherein said step of inoculating a plant comprises the step of inoculating a Nicotiana tabacum L. cv. Xanthi-nc plant and said step of inoculating the protoplasts comprises inoculating said protoplasts with Cucumber Mosaic Virus.
Claim 17
Dependent Claim
41 words
A method of immunizing a plant susceptible to TMV, CMV and PVX against the replication of any of said viruses comprising the step of introducing material isolated according to the method of either claim 1 or claim 5 into the plant.
Claim 18
Dependent Claim
29 words
The method of claim 13 wherein said step of introducing comprises introducing said material between about 5 and about 12 hours after infection of the plant by said virus.
Claim 19
Dependent Claim
27 words
The method of claim 13 wherein said step of introducing comprises the step of introducing the material through a cut stem of the plant to be immunized.
Claim 20
Dependent Claim
26 words
The method of claim 13 wherein said step of introducing comprises the step of spraying the material onto the leaves of the plant to be immunized.
Claim 21
Dependent Claim
24 words
The method of claim 13 wherein said plant is selected from the group consisting of tobacco, cucumber, sweet and bell pepper, and tomato plants.
Claim 22
Dependent Claim
35 words
A method of immunizing a plant susceptible to TMV, CMV and PVX against the replication of said viruses comprising the step of introducing material isolated according to the method of claim 8 into the plant.
Claim 23
Dependent Claim
29 words
The method of claim 18 wherein said step of introducing comprises introducing said material between about 5 and about 12 hours after infection of the plant by said virus.
Claim 24
Dependent Claim
27 words
The method of claim 18 wherein said step of introducing comprises the step of introducing the material through a cut stem of the plant to be immunized.
Claim 25
Dependent Claim
26 words
The method of claim 18 wherein said step of introducing comprises the step of spraying the material onto the leaves of the plant to be immunized.
Claim 26
Dependent Claim
24 words
The method of claim 18 wherein said plant is selected from the group consisting of tobacco, cucumber, sweet and bell pepper, and tomato plants.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-05-27 | 1988-11-26 | 1989-05-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-05-27 | 1992-11-26 | 1993-05-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-05-27 | 1996-11-26 | 1997-05-28 | $7700.00 | Window Closed / Lapsed |