CA2767844A1 - Transformation of dicotyledonous plants by an isolated shoot apex meristem method - Google Patents
Transformation of dicotyledonous plants by an isolated shoot apex meristem method Download PDFInfo
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- CA2767844A1 CA2767844A1 CA2767844A CA2767844A CA2767844A1 CA 2767844 A1 CA2767844 A1 CA 2767844A1 CA 2767844 A CA2767844 A CA 2767844A CA 2767844 A CA2767844 A CA 2767844A CA 2767844 A1 CA2767844 A1 CA 2767844A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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Abstract
Method of transforming dicotyledonous plants, the method comprising: (a) isolating a shoot apex from a seedling; (b) dividing the shoot apex into at least two pieces such that the apical meristem is exposed; (c) making an incision into a meristematic region of the apical meristem; (d) exposing the incised meristematic region to a transformation vector and regenerating a plant from the incised shoot apex.
Description
TRANSFORMATION OF DICOTYLEDONOUS PLANTS BY AN ISOLATED SHOOT APEX MERISTEM
METHOD
TECHNICAL FIELD
The present invention relates to a method for transforming plants. In particular, the present invention relates to a method of transforming dicotyledons via isolated shoot apices.
BACKGROUND TO THE INVENTION
Transgenesis, or genetic transformation, allows for the generation of plants with improved traits significantly faster than conventional breeding practices.
This technology is based on the delivery of genes of interest, from a broad range of sources, into the plant genome. Transformation of dicots is primarily performed utilizing Agrobacterium-mediated DNA delivery which relies on the ability of Agrobacterium tumefaciens to transfer a portion of its DNA, called T-DNA, into plant cells.
For various reasons, Agrobacterium-mediated transformations generally have relatively low success rates. For example, there is a high potential for somaclonal variation in the resultant transforms. Also, certain commercially important cultivars are recalcitrant to transformation by Agrobacterium.
It has been suggested that a way of overcoming some of the issues with Agrobacterium-mediated transformation would be to excise shoot apex tissue from the target plant and transform this tissue with an Agrobacterium vector. Shoot apex tissue permits rapid propagation of transformed plants as most dicots can be regenerated into intact plants from the shoot apex explants. See, for example, US Patent Numbers 5,164,310; 7,122,722; 6,858,777; and US Published Patent Application Number 2003/0208795.
The shoot apex comprises meristematic tissue which contains undifferentiated cells (meristematic cells) and is found in zones of the plant where growth occurs. Apical I
meristems (or growing tips) are found in the buds and growing tips of roots and shoots in plants. An apical meristem is usually a dome-shaped structure that comprises several layers. The number of layers varies according to plant type. In general the outermost layer is called the tunica and is comprised of epidermal (L 1) and sub-epidermal (L2) layers. The innermost layers (L3) are known as the corpus. The corpus and tunica play a critical part in plant physical appearances as all plant cells are formed from the meristems. Cells in the L1 and L2 layers divide in a sideways fashion which keeps these layers distinct, while the L3 layer divides in a more random fashion.
Brassica species include a large group of agriculturally important crops including canola. Canada and the United States produce between seven and ten million tonnes of canola seed per year. Annual Canadian exports alone total three to four million tonnes of the seed, seven hundred thousand tonnes of canola oil and one million tonnes of canola meal. Canola oil has been claimed to promote good health due to its very low saturated fat and high monounsaturated fat content, and beneficial omega-3 fatty acid profile.
Canola was developed through conventional plant breeding but the cultivars grown today are mostly varieties that have been genetically engineered. For example, approximately 80% of canola planted in Canada has been modified to be herbicide-tolerant.
However, some of the most commercially important brassica cultivars are recalcitrant to Agrobacterium transformation.
SUMMARY OF THE INVENTION
The present invention relates to a method of transforming dicotyledonous plants with a vector comprising the genetic material of interest. The present method comprises:
(a) isolating the shoot apices from seedlings and optionally, culturing the isolated apices on an appropriate medium;
(b) dividing a selected shoot apex into at least two, preferably asymmetric, pieces so that a portion of the apical meristem is exposed;
(c) making an incision into the meristematic region of the apical meristem;
METHOD
TECHNICAL FIELD
The present invention relates to a method for transforming plants. In particular, the present invention relates to a method of transforming dicotyledons via isolated shoot apices.
BACKGROUND TO THE INVENTION
Transgenesis, or genetic transformation, allows for the generation of plants with improved traits significantly faster than conventional breeding practices.
This technology is based on the delivery of genes of interest, from a broad range of sources, into the plant genome. Transformation of dicots is primarily performed utilizing Agrobacterium-mediated DNA delivery which relies on the ability of Agrobacterium tumefaciens to transfer a portion of its DNA, called T-DNA, into plant cells.
For various reasons, Agrobacterium-mediated transformations generally have relatively low success rates. For example, there is a high potential for somaclonal variation in the resultant transforms. Also, certain commercially important cultivars are recalcitrant to transformation by Agrobacterium.
It has been suggested that a way of overcoming some of the issues with Agrobacterium-mediated transformation would be to excise shoot apex tissue from the target plant and transform this tissue with an Agrobacterium vector. Shoot apex tissue permits rapid propagation of transformed plants as most dicots can be regenerated into intact plants from the shoot apex explants. See, for example, US Patent Numbers 5,164,310; 7,122,722; 6,858,777; and US Published Patent Application Number 2003/0208795.
The shoot apex comprises meristematic tissue which contains undifferentiated cells (meristematic cells) and is found in zones of the plant where growth occurs. Apical I
meristems (or growing tips) are found in the buds and growing tips of roots and shoots in plants. An apical meristem is usually a dome-shaped structure that comprises several layers. The number of layers varies according to plant type. In general the outermost layer is called the tunica and is comprised of epidermal (L 1) and sub-epidermal (L2) layers. The innermost layers (L3) are known as the corpus. The corpus and tunica play a critical part in plant physical appearances as all plant cells are formed from the meristems. Cells in the L1 and L2 layers divide in a sideways fashion which keeps these layers distinct, while the L3 layer divides in a more random fashion.
Brassica species include a large group of agriculturally important crops including canola. Canada and the United States produce between seven and ten million tonnes of canola seed per year. Annual Canadian exports alone total three to four million tonnes of the seed, seven hundred thousand tonnes of canola oil and one million tonnes of canola meal. Canola oil has been claimed to promote good health due to its very low saturated fat and high monounsaturated fat content, and beneficial omega-3 fatty acid profile.
Canola was developed through conventional plant breeding but the cultivars grown today are mostly varieties that have been genetically engineered. For example, approximately 80% of canola planted in Canada has been modified to be herbicide-tolerant.
However, some of the most commercially important brassica cultivars are recalcitrant to Agrobacterium transformation.
SUMMARY OF THE INVENTION
The present invention relates to a method of transforming dicotyledonous plants with a vector comprising the genetic material of interest. The present method comprises:
(a) isolating the shoot apices from seedlings and optionally, culturing the isolated apices on an appropriate medium;
(b) dividing a selected shoot apex into at least two, preferably asymmetric, pieces so that a portion of the apical meristem is exposed;
(c) making an incision into the meristematic region of the apical meristem;
(d) exposing the incised meristematic region to a transformation vector; and (e) optionally, culturing the incised shoot apex.
The present method provides effective transformation of dicotyledonous plants with a high frequency of success and reduced incidence of somaclonal variation. The present method permits regeneration of transformants in a reasonable time period. The present method can be performed without using selection media which can negatively impact the regeneration efficiency in tissue culture.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be described in conjunction with reference to the following drawings, in which:
Fig. 1 shows a schematic representation of a typical apical meristem;
Fig. 2 is a micrograph of a Brassica sp. shoot tip; and Fig.3 shows a schematic representation of a shoot tip.
DETAILED DESCRIPTION OF THE INVENTION
The present method comprises isolating shoot apices from seedlings by, for example, excision. Preferably the seedlings are at least three days old, more preferably at least five days old, even more preferably at least seven days old. Preferably, the seedlings are fifteen days old or less, more preferably ten days old or less.
The isolated apicies are preferably incubated for a least one day, preferably two days, prior to further processing. This period of incubation may conveniently be performed in a culture medium such as basal Murashige and Skoog medium (MS), Shoot Induction Medium (SIM), Callus Induction Medium (CIM), or mixtures thereof.
Preferably, the pre-culture is done on CIM. Pre-culture of the explants seems to enable them to better cope with the stress of exposure to Agrobacterium.
The present method provides effective transformation of dicotyledonous plants with a high frequency of success and reduced incidence of somaclonal variation. The present method permits regeneration of transformants in a reasonable time period. The present method can be performed without using selection media which can negatively impact the regeneration efficiency in tissue culture.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be described in conjunction with reference to the following drawings, in which:
Fig. 1 shows a schematic representation of a typical apical meristem;
Fig. 2 is a micrograph of a Brassica sp. shoot tip; and Fig.3 shows a schematic representation of a shoot tip.
DETAILED DESCRIPTION OF THE INVENTION
The present method comprises isolating shoot apices from seedlings by, for example, excision. Preferably the seedlings are at least three days old, more preferably at least five days old, even more preferably at least seven days old. Preferably, the seedlings are fifteen days old or less, more preferably ten days old or less.
The isolated apicies are preferably incubated for a least one day, preferably two days, prior to further processing. This period of incubation may conveniently be performed in a culture medium such as basal Murashige and Skoog medium (MS), Shoot Induction Medium (SIM), Callus Induction Medium (CIM), or mixtures thereof.
Preferably, the pre-culture is done on CIM. Pre-culture of the explants seems to enable them to better cope with the stress of exposure to Agrobacterium.
In the present method the apex is divided into at least two pieces. Preferably the pieces are asymmetrical. The division may be performed in any suitable manner that exposes the apical meristematic tissue. Preferably, the lateral face of the meristem is exposed. In one preferred embodiment, one of the cotyledonary leaves is peeled off lengthwise so as to expose the lateral face of meristem.
The present method comprises making at least one incision into the exposed meristematic tissue. It is highly preferred that the incision penetrates to and exposes the L2 layer. Preferably, at least two incisions are made. These incisions may be made in any suitable portion of the exposed apical meristem but preferably they are made on either side of the apical dome. While not wishing to be bound by theory, it is believed that exposing the L2 layer improves the efficacy of the transformation method because the L2 layer contributes most of the mesophyll tissue of the resultant plant.
The present method comprises exposing the isolated shoot tip to a plant transformation vector. The vector may take any form suitable for transforming dicotyledons. It is highly preferred that the present transformation is mediated by Agrobacterium lumefaciens.
The shoot tip explants may be exposed to Agrobacterium harbouring the DNA of interest by any suitable means. Preferably the explants are added to an Agrobacterium suspension for a suitable period of time such as 5 minutes. The suspension may be shaken in order to maximize the chances of a successful infection.
After exposure to the vector, the explants may then be regenerated. For example, the explants may be placed shoot induction medium and incubated for a suitable period.
The explants may be transferred to selection media for selection of transformants therefrom. When the shoots are of an appropriate size, they may be transferred to root induction media for root regeneration. Rooted plants can be grown under suitable conditions known to those skilled in these arts.
Preferred targets for the present method include canola and other commercially useful dicots. The present method is particularly useful for those cultivars that are recalcitrant to traditional transformation methods, such as for example TOPAS;
TOPAS
4079, Zarfam, Modena, and Opera.
Referring now to the figures, Fig. 1 shows a schematic representation of an apical dome. An apical meristem, which includes the apical dome, is composed of the epidermal LI layer (1), the subepidermal L2 layer (2), and the corpus L3 layer (3).
Fig. 2 is a micrograph of a typical Brassica sp, shoot tip showing the leaf primordia (10), the apical meristem (11), the apical dome (12), auxiliary buds (13), and the preferred locations for the incisions (14).
Fig. 3 shows a schematic representation of a shoot tip showing the leaf primordial (10), the apical meristem (11), the apical dome (12), auxiliary buds (13), and the preferred locations for the incisions (14).
EXAMPLE
Hypocotyl explants were obtained from 8-day-old canola seedlings (Invigor 5020) obtained by culturing sterilized seeds in '/2 Murashige and Skoog (MS) supplemented with 2% sucrose, pH 5.8 (seed germination medium) with incubation under a 16/8 hour photoperiod. The explants were then transferred aseptically on callus induction medium (CIM) and incubated for 2 days.
Agrobacterium suspension in MS (pH 5.6-5.8) containing 100 mM
acetosyringone was obtained by pelleting bacterial cells by centrifugation at 4000 rpm for 15 min at room temperature.
The pre-cultured shoot tip explants were dissected into two asymmetrical pieces.
The smaller piece was discarded. An incision was made on both sides of the meristematic region in the larger half of the bisected explants so as to expose the germ line cells.
Excess tissues i.e hypocotyls and cotyledonary leaves were trimmed off. Care was taken not to remove any shoot primordia.
The present method comprises making at least one incision into the exposed meristematic tissue. It is highly preferred that the incision penetrates to and exposes the L2 layer. Preferably, at least two incisions are made. These incisions may be made in any suitable portion of the exposed apical meristem but preferably they are made on either side of the apical dome. While not wishing to be bound by theory, it is believed that exposing the L2 layer improves the efficacy of the transformation method because the L2 layer contributes most of the mesophyll tissue of the resultant plant.
The present method comprises exposing the isolated shoot tip to a plant transformation vector. The vector may take any form suitable for transforming dicotyledons. It is highly preferred that the present transformation is mediated by Agrobacterium lumefaciens.
The shoot tip explants may be exposed to Agrobacterium harbouring the DNA of interest by any suitable means. Preferably the explants are added to an Agrobacterium suspension for a suitable period of time such as 5 minutes. The suspension may be shaken in order to maximize the chances of a successful infection.
After exposure to the vector, the explants may then be regenerated. For example, the explants may be placed shoot induction medium and incubated for a suitable period.
The explants may be transferred to selection media for selection of transformants therefrom. When the shoots are of an appropriate size, they may be transferred to root induction media for root regeneration. Rooted plants can be grown under suitable conditions known to those skilled in these arts.
Preferred targets for the present method include canola and other commercially useful dicots. The present method is particularly useful for those cultivars that are recalcitrant to traditional transformation methods, such as for example TOPAS;
TOPAS
4079, Zarfam, Modena, and Opera.
Referring now to the figures, Fig. 1 shows a schematic representation of an apical dome. An apical meristem, which includes the apical dome, is composed of the epidermal LI layer (1), the subepidermal L2 layer (2), and the corpus L3 layer (3).
Fig. 2 is a micrograph of a typical Brassica sp, shoot tip showing the leaf primordia (10), the apical meristem (11), the apical dome (12), auxiliary buds (13), and the preferred locations for the incisions (14).
Fig. 3 shows a schematic representation of a shoot tip showing the leaf primordial (10), the apical meristem (11), the apical dome (12), auxiliary buds (13), and the preferred locations for the incisions (14).
EXAMPLE
Hypocotyl explants were obtained from 8-day-old canola seedlings (Invigor 5020) obtained by culturing sterilized seeds in '/2 Murashige and Skoog (MS) supplemented with 2% sucrose, pH 5.8 (seed germination medium) with incubation under a 16/8 hour photoperiod. The explants were then transferred aseptically on callus induction medium (CIM) and incubated for 2 days.
Agrobacterium suspension in MS (pH 5.6-5.8) containing 100 mM
acetosyringone was obtained by pelleting bacterial cells by centrifugation at 4000 rpm for 15 min at room temperature.
The pre-cultured shoot tip explants were dissected into two asymmetrical pieces.
The smaller piece was discarded. An incision was made on both sides of the meristematic region in the larger half of the bisected explants so as to expose the germ line cells.
Excess tissues i.e hypocotyls and cotyledonary leaves were trimmed off. Care was taken not to remove any shoot primordia.
The trimmed tips were inoculated with the Agrobacterium suspension for 10 min with constant shaking. The explants were then transferred to plates containing CIM.
After incubation for 72 hours, the tissues were washed and transferred again to CIM
containing 250 mg/1 carbenicillin. The explants were further incubated at 22 +
2 C under a 16/8 h photoperiod.
After 2 weeks of incubation, the explants comprising hypocotyls were transferred to shoot induction medium (SIM) containing 250 mg/1 carbenicillin.
Regenerated shoots were transferred to shoot elongation medium (SEM). Shoots elongated readily in both media within 2 weeks. When the size of the shoots reached about 1 cm to about 1.5 cm, they were transferred to root induction medium (RIM) for regeneration of roots. Once rooted, the plantlets were transferred to a soil-less growth medium for further growth, and were checked for gene expression.
The following media were used:
Callus Induction Medium (CIM):
MS basal medium supplemented with 3% sucrose, 0.1 g/1 myo-inositol, 0.8%
agar, 1 mg/12,4-D, 5 mg/I silver nitrate, 0.5 g/l MES; pH 5.8.
Shoot induction Medium (SIM):
MS basal medium supplemented with 3% sucrose, 0.1 g/l myo-inositol, 0.8%
agar, 2 mg/1 BA, 5 mg/1 silver nitrate, 0.5 g/1 MES; pH5.8.
Shoot Elongation medium (SEM):
MS basal medium supplemented with 1% sucrose, 0.1 g/1 myo-inositol, 0.8%
agar, 0.5 g/1 MES, 0.05 mg/I BA; pH 5.8.
Root Induction Medium (RIM):
V2 MS basal medium supplemented with 1% sucrose, 0.8% agar, 0.5 g/1 MES; pH
5.8.
After incubation for 72 hours, the tissues were washed and transferred again to CIM
containing 250 mg/1 carbenicillin. The explants were further incubated at 22 +
2 C under a 16/8 h photoperiod.
After 2 weeks of incubation, the explants comprising hypocotyls were transferred to shoot induction medium (SIM) containing 250 mg/1 carbenicillin.
Regenerated shoots were transferred to shoot elongation medium (SEM). Shoots elongated readily in both media within 2 weeks. When the size of the shoots reached about 1 cm to about 1.5 cm, they were transferred to root induction medium (RIM) for regeneration of roots. Once rooted, the plantlets were transferred to a soil-less growth medium for further growth, and were checked for gene expression.
The following media were used:
Callus Induction Medium (CIM):
MS basal medium supplemented with 3% sucrose, 0.1 g/1 myo-inositol, 0.8%
agar, 1 mg/12,4-D, 5 mg/I silver nitrate, 0.5 g/l MES; pH 5.8.
Shoot induction Medium (SIM):
MS basal medium supplemented with 3% sucrose, 0.1 g/l myo-inositol, 0.8%
agar, 2 mg/1 BA, 5 mg/1 silver nitrate, 0.5 g/1 MES; pH5.8.
Shoot Elongation medium (SEM):
MS basal medium supplemented with 1% sucrose, 0.1 g/1 myo-inositol, 0.8%
agar, 0.5 g/1 MES, 0.05 mg/I BA; pH 5.8.
Root Induction Medium (RIM):
V2 MS basal medium supplemented with 1% sucrose, 0.8% agar, 0.5 g/1 MES; pH
5.8.
Claims (13)
1. A method of transforming dicotyledonous plants, the method comprising:
(a) isolating a shoot apex from a seedling;
(b) dividing the shoot apex into at least two pieces such that the apical meristem is exposed;
(c) making an incision into a meristematic region of the apical meristem; and (d) exposing the incised meristematic region to a transformation vector.
(a) isolating a shoot apex from a seedling;
(b) dividing the shoot apex into at least two pieces such that the apical meristem is exposed;
(c) making an incision into a meristematic region of the apical meristem; and (d) exposing the incised meristematic region to a transformation vector.
2. A method according to claim 1, wherein the division of the shoot apex exposes the lateral meristem.
3. A method according to claim 1, wherein the incision penetrates to, and exposes, the L2 layer.
4. A method according to claim 1, wherein two incision are made into the meristomatic region.
5. A method according to claim 1, wherein transformation with the transformation vector is mediated by Agrobacterium tumefaciens.
6. A method according to claim 1, wherein the isolated shoot apex is pre-cultured prior to division.
7. A method according to claim 1, wherein the method further comprises culturing the incised shoot apex.
8. A method according to claim 1, wherein the method further comprises regenerating a plant from the incised shoot apex.
9. A method of transforming canola wherein the method comprises:
(a) isolating a plurality of shoot apices from a plurality of canola seedlings;
(b) selecting a shoot apex from the plurality of shoot apices;
(c) dividing the shoot apex into at least two pieces thereby exposing a portion of the apical meristem therein;
(d) making an incision into a meristematic region of theexposed apical meristem thereby exposing a portion of the L2 layer therein;
(e) exposing the incised meristematic region to a transformation vector; and (f) regenerating a plant from the incised shoot apex.
(a) isolating a plurality of shoot apices from a plurality of canola seedlings;
(b) selecting a shoot apex from the plurality of shoot apices;
(c) dividing the shoot apex into at least two pieces thereby exposing a portion of the apical meristem therein;
(d) making an incision into a meristematic region of theexposed apical meristem thereby exposing a portion of the L2 layer therein;
(e) exposing the incised meristematic region to a transformation vector; and (f) regenerating a plant from the incised shoot apex.
10. A method according to claim 9, wherein two incision are made into the meristomatic region.
11. A method according to claim 9, wherein transformation with the transformation vector is mediated by Agrobacterium tumefaciens.
12. A method according to claim 9, wherein the isolated shoot apex is pre-cultured prior to division.
13. A method according to claim 9, wherein the method further comprises culturing the incised shoot apex.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US23321309P | 2009-08-12 | 2009-08-12 | |
| US61/233,213 | 2009-08-12 | ||
| PCT/CA2010/001242 WO2011017808A1 (en) | 2009-08-12 | 2010-08-12 | Transformation of dicotyledonous plants by an isolated shoot apex meristem method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2767844A1 true CA2767844A1 (en) | 2011-02-17 |
Family
ID=43585820
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2767844A Abandoned CA2767844A1 (en) | 2009-08-12 | 2010-08-12 | Transformation of dicotyledonous plants by an isolated shoot apex meristem method |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2767844A1 (en) |
| WO (1) | WO2011017808A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2015133652A1 (en) | 2014-03-06 | 2017-04-06 | 国立研究開発法人理化学研究所 | Plant transformation methods |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU747514B2 (en) * | 1998-02-19 | 2002-05-16 | Cotton Incorporated | A method for the production of transgenic plants using apical shoot tips |
-
2010
- 2010-08-12 CA CA2767844A patent/CA2767844A1/en not_active Abandoned
- 2010-08-12 WO PCT/CA2010/001242 patent/WO2011017808A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2011017808A1 (en) | 2011-02-17 |
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