DESC:FORM 2
The Patent Act 1970
(39 of 1970)
&
The Patent Rules, 2005

COMPLETE SPECIFICATION
(SEE SECTION 10 AND RULE 13)

TITLE OF THE INVENTION

“Non-tissue culture mediated, marker free gene transfer methods”

APPLICANT:

Name : Camson Biotechnologies Ltd

Nationality : India

Address : C-7, 7th Floor, Corporate Block, Golden
Enclave, Airport Road, Bangalore 560 017

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed:-

FIELD OF INVENTION

[001] The present invention generally relates to genetic transformation methods, and, more particularly, to in-planta method of gene transfer.

BACKGROUND OF INVENTION
[002] Genetic modification of plants to introduce new traits such as disease resistance, pest resistance, draught resistance, etc, into plants is a widely adopted technology. The introduction of new traits in genetically modified plants (GM plants) is done by adding one or foreign genes, capable of providing the desired characters, into the plant’s genome.
[003] Various gene transfer techniques are known. The generally used transformation methods include Electroporation, Micro-injection, Biolistics, PEG mediated gene transfer, Agrobacterium mediated gene transfer, etc. Agrobacterium Tumefaciens is a soil bacteria which is a very good example of natural transformation system. A. Tumefaciens gain entry into a plant through a wound site. It further infects the plant through the integration of the T-DNA, which is present in the Ti-plasmid (Tumor inducing plasmid) of A. Tumefaciens, into the chromosomal DNA of the plant. In addition to the T-DNA, the Ti-plasmid contains all the other genes which facilitate the gene transfer. The engineered Ti-plasmid is generally used to facilitate the desired gene transfer.
[004] Generally, the approach taken in genetic transformation of plants is to transform an individual plant cell with the desired gene which is further cultured and regenerated into a whole plant. These approaches of transformation involve tissue culture and generation of sterile plant which have certain drawbacks. The tissue culture dependent approach involve huge investments, are laborious, decreased transformation efficiency, decreased plant survivability, etc.
[005] In recent times, in-planta transformation approach is widely implemented to overcome drawbacks posed by the conventional tissue culture dependent approach. In-planta transformation involves direct inoculation of the plant with a medium containing the gene of interest. The in-planta methods are highly preferred as they do not involve the step of regeneration of the plant. Standardized methods of Agrobacterium mediated in-planta transformation of rice, corn, buckwheat, etc through wounding of flower, seedling, etc are already known. However, there is a need for simple, efficient and commercially viable in-planta transformation methods.

OBJECT OF INVENTION
[001] The principal object of the invention is to provide non-tissue culture mediated, marker free method of transforming plants.
[002] Another object of the invention is to provide a method of producing antibiotic marker free genetically modified plants.
[003] These and other objects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

DETAILED DESCRIPTION OF INVENTION
[004] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[005] The embodiments herein disclose in-planta gene transfer methods. In an embodiment, the in-planta gene transfer is achieved by meristem injury. In another embodiment, the in-planta gene transformation is achieved by way of pollen transfer. Further, embodiments herein disclose methods of producing antibiotic marker free genetically modified plants through Agrobacterium mediated in-planta gene transformation. In an embodiment, the method of producing antibiotic marker free genetically modified plants includes inoculation or treatment with Agrobacterium culture.
[006] Genes
[007] The genes used for transformation, in various embodiments herein, may be genes which may be introduced into the plant in order to confer a desired trait in the plant. The desired traits may include traits such as increased quality of plant product, draught resistance, disease resistance, pest resistance, promote growth of plant, etc. In an embodiment, the gene to be transferred into the plant may be such that it confers better quality of plant product. In another embodiment, the gene to be transferred into the plant may be such that it provides pest resistance.
[008] The gene used for transformation of plants may be any gene sequence. In an embodiment, the gene for transformation may comprise of DNA or RNA sequence. In an embodiment, the gene that is to be transferred into a plant may be inserted into a vector which may be constructed through generally known methods in the field.
Vector
[009] The vector used to transform plants, in various embodiments herein, may be any vector generally known in the field. In an embodiment the vector may be engineered plasmid of Agrobacterium. In another embodiment, the vector is engineered Ti-plasmid of A. Tumefaciens. In an embodiment, the gene that is to be transferred into the plant (i.e. the gene of interest) is inserted into the Ti-plasmid of A. Tumefaciens. The gene of interest may be inserted into the Ti-plasmid by generally known methods in the field.
[0010] Further, the Agrobacterium carrying the gene of interest is cultured in an appropriate medium. In an embodiment, the appropriate medium is such that it facilitates gene transfer by meristem injury. In another embodiment, the appropriate medium is such that it facilitates gene transfer by way of pollen grains.
Agrobacterium culture for meristem injury
[0011] In an embodiment, the Agrobacterium carrying the gene of interest may be cultured in any appropriate media, generally known in the field, containing plant growth hormones such as 6-Benzylaminopurine (6-BAP) and indole-3-acetic acid (IAA).
[0012] In an embodiment, the Agrobacterium culture carrying the gene of interest is prepared in Luria Broth (LB) medium containing antibiotics such as streptomycin and kanamycin. Further, this starter culture is inoculated, preferably in a proportion of 1:100, into a predetermined volume of LB medium and placed in a shaker, preferably, for 12 hours at 28°C. The culture is then centrifuged, preferably at 6000 rpm for 5 minutes, to pellet the cells. The pellet is further separated and re-suspended in Agrobacterium (AB) media containing auxins and cytokinins. In an embodiment, the cytokinins and auxins are 6-BAP and IAA, respectively. Further, Acetosyringone is added to the AB media containing the pellet and vortexed to re-suspend the pellet followed by incubation in dark room, preferably for 10 to 30 mins, with gentle shaking.
[0013] Experiments providing the in-planta transformation method according to various embodiments herein are provided herein by way of examples only and should not be construed to limit the scope of the present invention.
Example 1:
[0014] Agrobacterium culture carrying gene construct of interest was prepared in 5ml of Luria Broth (LB) medium containing 100ppm of Streptomycin and 50ppm of Kanamycin. Further, starter culture was inoculated in a proportion of 1:100 in 50ml LB medium and placed on a shaker for 12 hours at 28°C. The culture was then transferred into a 25ml centrifuge tube and centrifuged at 6000rpm for 5 min
[0015] The supernatant was discarded and the pellet was re-suspended in 2 ml of AB solution containing 0.5-1mg/L of BAP and 0.02-2.5mg/L of IAA hormones. Further, 100uM of Acetosyringone was added to the pellet in AB solution and vortexed to resuspend the pellet. The suspension was then incubated in dark room for 10-30 min with gentle shaking.
[0016] In an embodiment the AB solution comprises of a mixture of equal proportion of AB salt solution and AB buffer, and glucose (preferably in the range of 0.5 to 5%). In an embodiment, the composition of AB salt solution comprises of Ammonium Chloride (NH4Cl), Magnesium Sulphate (MgSO4.7H2O), Potassium Chloride KCl, Calcium Chloride (CaCl2) and Ferric Sulphate (FeSO4.7 H2O). In another embodiment, the composition of AB buffer comprises of Potassium hydrogen phosphate (KH2PO4) and Sodium hydrogen phosphate (NaH2PO4).

Example 2:
AB salt: (5x in 100ml)
Sl no. Ingredient Concentration
1 NH4Cl 0.2% to 5%
2 MgSO4.7H2O 5mM to 25mM
3 KCl 50mM to 100mM
4 CaCl2 25mM to 50mM
5 FeSO4.7 H2O 0.001% to 0.5%

Example 3:
AB buffer: (5x in 100ml)
Sl no. Ingredient Quantity
1 KH2PO4 0.5 to 10.5%
2 NaH2PO4 0.1 to 5.8%

Agrobacterium culture for pollen transfer
[0017] In an embodiment, the Agrobacterium carrying the gene of interest may be cultured in any appropriate medium generally known in the field containing sucrose.
[0018] In an embodiment, the Agrobacterium culture carrying the gene of interest is prepared in Luria Broth (LB) medium containing antibiotics such as streptomycin and kanamycin. Further, this starter culture is inoculated, preferably in a proportion of 1:100, into a predetermined volume of LB medium and placed in a shaker, preferably, for 12 hours at 28°C. The culture is then centrifuged, preferably at 6000 rpm for 5 minutes, to pellet the cells. The pellet is further separated and re-suspended in pollen germination medium (PGM) containing sucrose. Further, Acetosyringone is added to PGM containing the pellet and vortexed to re-suspend the pellet followed by incubation in dark room, preferably for 10 to 30 mins, with gentle shaking.
Example 4:
[0019] Agrobacterium culture carrying gene construct of interest was prepared in 5ml of Luria Broth (LB) medium containing 100ppm of Streptomycin and 50ppm of Kanamycin. Further, starter culture was inoculated in a proportion of 1:100 in 50ml LB medium and placed on a shaker for 12 hours at 28°C. The culture was then transferred into a 25ml centrifuge tube and centrifuged at 6000rpm for 5 min
[0020] The supernatant was discarded and the pellet was re-suspended in PGM solution containing 5 to 25 % of sucrose. Further, 100uM of Acetosyringone was added to the pellet in PGM solution, and vortexed to resuspend the pellet. The suspension was then incubated in dark room for 10-30 min with gentle shaking.
[0021] In an embodiment, the PGM composition comprises of Potassium Nitrate (KNO3), Manganese Sulphate (MnSO4), Boric Acid (H3BO4), Magnesium sulphate (MgSO4.7H2O) and Gibberellic acid (GA3).
[0022] Example 5:
PGM composition: (5x in 100ml)
Sl no. Ingredient Concentration (mM)
1 KNO3 0.1-2.52 mM
2 MnSO4 0.5-6.06mM
3 H3BO4 0.3-4.66mM
4 MgSO4.7H2O 0.01-0.82mM
5 GA3 0.001-0.01uM

Transformation by meristem injury
[0023] In an embodiment, gene transformation of plant is by meristem injury of a standing crop (in a pot or field). In an embodiment, the in-planta gene transfer is by inoculation of injured meristem of a standing crop with Agrobacterium culture. In an embodiment, the Agrobacterium culture used to inoculate injured meristem is prepared as per the methods disclosed in the various embodiments herein.
[0024] In an embodiment, gene transformation is by injury of meristem at the nodal region. In another embodiment, gene transfer by injury of meristem involves breaking apical dominance to stimulate axillary meristem growth. In an embodiment, breaking apical dominance involves removal of all growing tips to boost induction of nodal branches.
[0025] The number of gene that may be transferred into a plant, by way of various embodiments herein, may depend on the number of nodes in a plant. In an embodiment, 1 to 5 genes may be transferred into an individual plant containing 4 to 5 nodes.
[0026] In an embodiment, in-planta gene transfer method by meristem injury involves removal of growing tips of a plant, preferably around 24 hours prior to introduction of gene. Further, using a sterile syringe needle, injury is made at the nodal region of the plant by pricking gently, preferably to about 4 to 5 mm deep for around 9 to 10 times. The Agrobacterium culture containing the gene of interest is then added to the injury site with continued pricking. The plant is then placed in dark for infection. The plant is then transferred to the growth chamber providing controlled exposure to light. Further, Cefatoxime was introduced to the injured site, to eliminate Agrobacterium contamination. Once new shoots are observed at the injured site, the plant may be shifted to a green house to facilitate further development of the branch.
Example 6:
[0027] The growing tips of the plant selected for transformation was removed to boost induction of nodal branches (breaking apical dominance), around 24 hours prior to introduction of gene. Injury was then made at the nodal region using a 5 ml sterile syringe needle by pricking to gently about 4 to 5 mm deep for about 9 to 10 times. Further, 20 ul of induced Agrobacterium culture was added on the injured site with continued pricking for 4 to 5 timed. The plant was kept in dark for 24 hours for infection to occur. The plant was then transferred to growth chamber maintained at 25°C wherein it was provided with 16 hours of light and 8 hours of dark. Further, 20ul of Cefatoxime (200 ppm) was added on the injured site (to eliminate the Agrobacterium contamination). The plant was shifted to green house once new shoots from the injured site was observed.
Transformation by pollen transfer
[0028] In an embodiment, gene transformation of plant is by pollen transfer where in the pollen is treated with a culture containing the gene of interest. In an embodiment, the Agrobacterium culture used to inoculate treat pollen is prepared as per the methods disclosed in the various embodiments herein.
Example 7:
[0029] The unopened flowers were emasculated to remove the male part around 0 to 2 hours prior to transfer. The pollens were then collected 2 to 3 hours before and incubated at around 28°C to 42°C to achieve maturation. The pollens were then mixed with the culture in PGM solution containing 5 to 25 % of sucrose and then incubated for 0 to 30 minutes at room temperature in dark. The culture solution carrying the pollens in PGM solution was applied on to the stigma and the stigma was covered to avoid cross pollination. The treated pollen maybe used to pollinate the same plant from which the pollens were collected or a different plant.
[0030] After pollination by the pollens in PGM solution, the flower may be allowed to continue their development. The seeds may be then harvested and analyzed to check transformation.
Plant
[0031] The method of transformation disclosed in the various embodiments herein may be used to transform plants of any species. In an embodiment, the plant is a monocot plant such as maize, rice, wheat, barley, oats, etc. In another embodiment, the plant is a dicot plant such as sunflower, cotton, pepper, peanuts, fruits, etc.
[0032] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

,CLAIMS:CLAIMS
We claim:
1. A method for genetic transformation of a plant, the method comprising;
removing growing tips of the plant selected for transformation,
wounding of plant meristem,
applying Agrobacterium culture comprising gene of interest on the wound; wherein the Agrobacterium culture is in Agrobacterium (AB) solution comprising 6-Benzylaminopurine (6-BAP) in the range of 0.5-1mg/L and indole-3-acetic acid (IAA) in the range of 0.02-2.5mg/L, and
applying Cefatoxime on the wound.

2. The method for genetic transformation of a plant as claimed in claim 1, wherein the AB solution further comprises of Ammonium Chloride (NH4Cl), Magnesium Sulphate (MgSO4.7H2O), Potassium Chloride KCl, Calcium Chloride (CaCl2) , Ferric Sulphate (FeSO4.7 H2O), Potassium hydrogen phosphate (KH2PO4), Sodium hydrogen phosphate (NaH2PO4) and glucose .

3. The method for genetic transformation of a plant as claimed in claim 1, wherein the AB solution further comprises of Ammonium Chloride (NH4Cl) in the range of 0.2% to 5%, Magnesium Sulphate (MgSO4.7H2O) in the range of 5mM to 25mM, Potassium Chloride KCl in the range of 50mM to 100mM, Calcium Chloride (CaCl2) in the range of 25mM to 50mM, Ferric Sulphate (FeSO4.7 H2O) in the range of 0.001% to 0.5% , Potassium hydrogen phosphate (KH2PO4) in the range of 0.5% to 10.5% , Sodium hydrogen phosphate (NaH2PO4) in the range of 0.1% to 5.8% and glucose is in the range of 0.5 to 5%.

4. A method for genetic transformation of a plant, the method comprising;
treating pollen with Agrobacterium culture comprising gene of interest; wherein the Agrobacterium culture is in pollen germination medium (PGM) comprising sucrose in the range of 5 to 25 %, Potassium Nitrate (KNO3) in the range of 0.1-2.52 mM, Manganese Sulphate (MnSO4) in the range of 0.5-6.06mM, Boric Acid (H3BO4) in the range of 0.3-4.66mM, Magnesium sulphate (MgSO4.7H2O) in the range of 0.01-0.82mM and Gibberellic acid (GA3) in the range of 0.001-0.01uM,
pollinating with the treated pollen,
obtaining transformed seeds from the pollinated plant, and
germinating the transformed seeds to obtain a transformed plant.

5. The method for genetic transformation of a plant as claimed in claim¬¬¬¬ 4, wherein the treated pollen maybe used to pollinate the same plant from which the pollens were collected or a different plant.

Date 22nd November 2013 Signature
Mr. Vikram Pratap Singh Thakur
Patent Agent