FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention. - "A MOLECULAR TOOL FOR MARKER FREE
TRANSFORMATION OF PLANTS AND METHODS FOR USING SAME"
2. Applicant(s)
(a) NAME : KRISHIDHAN RESEARCH FOUNDATION
PRIVATE LIMITED
(b) NATIONALITY: An Indian Company.
(c) ADDRESS-. 302, Royal House, 11/3, Usha Ganj, Indore-452001
(M.P.), India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
The present invention generally relates to the field of plant transformation and plant genetic engineering. More particularly the invention provides a universal expression cassette for marker free transformation enabling use of any gene with any element in any combination in any plant, plant cells or plant parts by any method of transformation. The invention also extends to a process for marker free transformation of plants using the said expression construct and a transformation kit based on the same.
BACKGROUND AND PRIOR ART OF THE INVENTION
Genetically modified plants have been developed to incorporate various characteristics in plants to meet the demands of the people and to produce best possible products. The various characteristics includes resistance to pest, herbicide, and also to harsh environmental conditions to get an improved yield. This transgenic engineering and genetic modification techniques in plants have a good prospective to improve the crops in a specific way. A number of systems have been developed for introduction of foreign DNA into plant cells. A foreign DNA or a DNA of interest with a desirable gene is introduced into the plant cell, achieved by transformation techniques using Agrobacterium, microinjection, particle gun, protoplast transformation etc.
Depending on the purpose, different genes have been introduced in plants for obtaining the desired features like high yield, improved quality, pest resistance, disease resistance, tolerance to abiotic stress etc. Various selection systems are used for the identification of the transformed cells with the gene of interest wherein a selectable marker gene conferring tolerance to an antibiotic, a herbicide or a hormone etc. is integrated along with the transgene and is introduced into the plant cell.

Further, the transformed cells are screened by growing the cells in regeneration media with applied selection pressure, which allows the transformed cells with selective marker gene to survive and non transformed cells get killed.
However, the presence of antibiotic: resistance gene in the transformed plants is considered undesirable from the ecological point of view. The use of this selectable marker gene can be toxic and allergenic in some cases. The antibiotic resistance genes. present in genetically modified foods, is sometimes, nerceived. to have. probability to get transferred to some pathogenic micro organisms present in the gastrointestinal tract and may render them resistant to treatment with the antibiotics. Due to various concerns raised on the issue of environmental and ecological safety of transgenic/ genetically modified plants, there has been a demand for transgenic engineering technology that produced transgenic plants without the use of selectable antibiotic resistance marker gene, thereby reducing the environmental and health hazard.
Reference may be made to Patent publication U.S. 2005/0097641 which relates to a transformation method for obtaining transgenic plants and plants obtained with the said method. This invention provides a method for transforming a plant cell with a recombinant nucleic acid comprising a T-DNA construct allowing for transfer of said construct into the genome of a plant cell, said construct provided with a foreign nucleic acid that is free of nucleic add encoding a selective marker.
While the above mentioned prior art talks about a transformation method without the use of selectable marker gene it has its own limitations. The border repeats used in the process, if changed, could affect the performance of the method. The border sequences are fixed and they are non replaceable. The polyadenylation signal or terminator is fixed and it cannot be envisaged as replaceable element. The entire

construct is fixed with the vector and cannot be taken out of the vector, and it is also hard to deduce whether the construct works properly with other processes.
There is, therefore, a need for a tool and a method for transformation of plants that circumvent the use of selectable marker gene in plant genetic engineering which will permit user to clone any gene with any element in any combination without going into tedious process of redesigning or assembling of vector de novo for each new application and also to avoid environmental and health hazards and make it eco-friendly.
OBJECT OF THE INVENTION
A primary object of the present invention is to overcome the disadvantages / limitations of the prior art.
Another object of the present invention is to develop a universal expression cassette with high fidelity comprising all the necessary genetic elements required for the marker free transformation of the heterologous gene in plants, plant cells or plant parts.
Another object of the present invention is to develop a universal expression cassette with high fidelity for marker free transformation of plants wherein the genetic elements can be altered as per the requirement of the host plant, allowing the said expression construct to be useful for the transformation of any gene with any element in any combination in any plant.
Another object of the present invention is to develop a method for the marker free transformation of a plant using said expression cassette.

Another object of the present invention is to develop a transformation kit for the marker free transformation of the plant using said expression cassette.
SUMMARY OF THE INVENTION
A simplified summary of the invention in order to provide a basic understanding of some aspects of the invention is provided. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.
The invention provides a universal and high fidelity Linear Expression Cassette (LEC) for marker free transformation of a heterologous gene in plants, plant cells or plant parts comprising genetic elements ordered in 5' to 3' direction: a T-DNA right border, at least a promoter, polycloning sites, transcriptional terminator signal and a T-DNA left border wherein the said genetic elements in said expression cassette comprising restriction sites across the ends joining one element with the other element being capable of altering said genetic elements as per the requirement of the host plant, enabling said expression cassette to be used universally for the transformation of any gene in any plant.
The invention further provides method for marker free transformation of a heterologous gene in a plant, plant cells or plant parts comprising essentially the steps of:

(a) Inserting the said heterologous gene into an expression cassette wherein
the said expression cassette comprising genetic elements ordered in 5' to 3'
direction: a T-DNA right border, at least a promoter, polycloning sites,
transcriptional terminator signal and a T-DNA left border wherein the said
genetic elements in said expression cassette comprising restriction sites across
the ends joining one element with the other element being capable of altering
said genetic elements as per the requirement of the host plant, enabling the
said expression cassette to be used universally for the transformation of any
gene in any plant and wherein said heterologous gene is inserted in said
polycloning site of said expression cassette;
(b) Transferring the recombinant expression cassette comprising the
heterologous gene of step (a) into plant, plant cell or plant part with any of the
compatible or prevalent method of transformation;
(c) Growing plant, plant cell or plant part of step (b) in regeneration media
(d) Regenerating/ propagating of plant or plant cell or plant part of step (c);
(e) Testing the plant or plant cell or plant part of step (d) for the presence and expression of said heterologous gene and identifying the transformed plant or plant cell or plant part; and
(f) Propagating the stably transformed plant for obtaining marker free
transgenic line with said heterologous gene.
The invention also provides a kit for the marker free transformation of a heterologous gene in a plant, plant cells or plant parts essentially comprising:
An expression cassette for marker free transformation of a heterologous gene in plants, plant cells or plant parts comprising genetic elements ordered in 5' to 3' direction: a T-DNA right border, at least a promoter, polycloning sites, transcriptional

terminator signal and a T-DNA left border wherein said genetic elements in said expression cassette comprising restriction sites across the ends joining one element with the other element being capable of altering said genetic elements as per the requirement of the host plant, enabling said expression cassette to be used universally for the transformation of any gene in any plant
These and other advantages of the present invention will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The following drawings are illustrative of particular examples for enabling methods of the present invention, are descriptive of some of the methods, and are not intended to limit the scope of the invention. The drawings are not to scale (unless so stated) and are intended for use in conjunction with the explanations in the following detailed description.
FIG. 1 Schematic illustration of composition of LEC to create marker free tool
DETAILED DESCRIPTION OF THE INVENTION
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Although the embodiments herein are described with various specific embodiments, it will be obvious for a person skilled in the art to practice the embodiments herein with modifications. However, all such modifications are deemed to be within the scope of the claims.

It is also to be understood that the following claims are intended to cover all of the generic and specific features of the embodiments described herein and all the statements of the scope of the embodiments which as a matter of language might be said to fall there between.
The invention provides an expression cassette useful for marker free transformation of plants, plant cells or plant parts. The Linear Expression Cassette (LEC) is designed in-silico and is chemically synthesized by custom DNA synthesis with a combination of genetic elements, with options for replacement, substitution, deletion and /or addition in single or multiple copies, useful for the marker free transformation of plants.
The LEC comprises a synthetic DNA molecule, wherein said synthetic DNA molecule further comprises genetic elements in a particular order from 5' end to 3'end. The synthetic DNA comprising a cohesive end restriction site at 5'end followed by a right border (RB) from T-DNA of any Agrobacterium strain Ti plasmid, preferably from a group comprising octopine, nopaline, succinamopine. The T-DNA Right Border (RB) is followed by a set of cohesive end restriction sites including but not limited to sites for restriction enzymes NotI, Sbfl, PstI, BamHI and combinations thereof.
The cohesive end restriction site is further followed by a promoter capable of efficient expression in plants. The promoter is selected from a group comprising of constitutive, tissue specific and inducible promoters. Promoters like CaMV35S promoter, rice actin promoter, ubiquitin promoter, tobacco PR1 promoter etc. may be used. In one embodiment, present invention is demonstrated with CaMV35S duplicated promoter.

The promoter in the synthetic.DNA is followed by a multiple cloning site (MCS) with a set of cohesive end restriction sites, followed by a transcriptional terminator. In a particular embodiment presented here, the MCS has sites for set of restriction enzymes, including but not limited to Xhol, Ncol, Ndel, Apal, Xbal, Sail and combinations thereof and if required, replaceable with a different synthetic MCS carrying a different set of restriction sites, as per need, for integration of foreign DNA.
The transcriptional terminator is selected from a group comprising terminators capable of transcriptional regulation in eukaryotes. The terminators could be nos terminators, ocs terminators, CaMV35S polyadenylation sequence etc. The terminator sequence is followed by a restriction site, which in turn is followed by a left border (LB) from T-DNA of any Agrobacterium strain Ti plasmid. The Agrobacterium strain Ti plasmid is selected from the group comprising octoptne, nopaline or succinamopine. T-DNA left border is further followed by a cohesive end restriction site at the 3'end.
There are spacers between restriction enzyme sites. Spacers or fillers have been provided to provide space to two different restriction enzymes in double digests for efficient cut. Some of the exemplifying individual purposes of spacers/ fillers are outlined below:
(i) Filler between Kpnl site and RB: In naked DNA transformation method, when LEC along with gene will be excised of the vector for transformation (as demonstrated vide Example 1), this naked DNA with Kpnl overhang may be exposed to exonucleases. The filler has been provided so that it provides stability and integrity of the border repeat which is important for integration of the transferred DNA to genome.

(ii) Filler between RB to Not I site: Similarly this filler is also for preventing RB integrity (where the recombination will start with host genome), to escalate upto start of promoter.
(iii) Filler between Not I and Sbf I site: To provide space between two restriction enzyme sites, so that one can cut at both sites to clone some element between these two sites.
The Right and Left Borders provide site for recombination and/or integration. The inclusion of RB and LB in the expression cassette facilitates for backbone free transformation of the heterologous gene in the host plant.
Different genetic elements in the expression cassette are separated by unique restriction enzyme sites so that other genetic elements can be incorporated or eliminated as per the requirement of the plant in which the gene is to be transformed. The restriction sites used across the components/elements are specifically rare cutters which enables precise activity of restriction enzymes.
The aforesaid molecular tool devoid of selectable marker genes can be universally applied for high fidelity marker free gene transfer, integration and expression in higher plants, preferably cotton, egg plant, maize, rice, tobacco, Arabidopsis, and transformable plant species from diverse group of monocotyledonous and dicotyledonous plants. The expression cassette gives choices of replacing any genetic element in combination, thus directly giving the user a platform to go for replacement wherever needed like between Right border and Promoter, between promoter to terminator, multiple cloning sites / polycloning sites for insertion of gene, between terminator and Left border. Thus one can replace promoter, terminator or any other element in the preferred recombinant situation for achieving marker free transformation of plant. Figure 1 is a schematic illustration of composition of Linear

Expression Cassette to create marker free tool while a sequence based on the same is submitted with the patent application as Sequence Id no. 1.
The said Linear Expression Cassette can be cloned in any compatible high copy number cloning vector or binary vector devoid of any selection marker for plant transformation, DNA transfer, integration, and expression to any higher plant by any appropriate transformation method. This Linear Expression Cassette can be cloned in routine cloning vector (standard molecular biology lab high copy no. cloning vectors, preferably pUC19 or pUC18 or pBlueScript) for the purpose of reproduction or for use in transformation experiments to generate an excised DNA molecule containing a desired gene in the expression cassette of Linear Expression Cassette (LEC) or can be directly used as transforming molecule.
The LEC along with the foreign DNA cloned into any binary vectors devoid of selectable marker gene is transformed into plant species to generate marker free plants through any method like via Agrobacterium mediated in-vitro or through plants pathway like pollen tube pathway or ovary dip method etc. Other methods of transforming LEC along with the foreign DNA can be achieved through conventional methods like particle bombardment, protoplast transformation method etc.
The LEC carrying the foreign gene of interest into the plant species, wherein the foreign gene comprising any heterologous protein gene, antisense DNA, RNAi generating template or any structural gene providing agronomic advantage with relation to biotic stress and abiotic stress.
The expression cassette provides high fidelity in terms of high transformation frequency over wide range of transformation methods, without adding any other extra virulence factor or element.

The invention further provides a method for marker free transformation of a heterologous gene in a plant, plant cells or plant parts comprising essentially the steps of:
(a) Inserting the said heterologous gene into an expression cassette wherein the said expression cassette comprising genetic elements ordered in 5' to 3' direction: a T-DNA right border, at least a promoter, polycloning sites, transcriptional terminator signal and a T-DNA left border wherein the said genetic elements in said expression cassette comprising restriction sites across the ends joining one element with the other element being capable of altering said genetic elements as per the requirement of the host plant, enabling the said expression cassette to be used universally for the transformation of any gene in any plant and wherein the said heterologous gene is inserted in the said polycloning site of said expression cassette;
(b) Transferring the recombinant expression cassette comprising the heterologous gene of step (a) into plant, plant cell or plant part;
(c) Growing plant, plant cell or plant part of step (b) in regeneration media
(d) Regenerating/ propagating of plant or plant cell or plant part of step (c);
(e) Testing the plant or plant cell or plant part of step (d) for the presence and expression of said heterologous gene and identifying the transformed plant or plant cell or plant part; and
(f) Propagating the stably transformed plant for obtaining marker free transgenic line with the said heterologous gene.
The method is advantageous as it does not utilize any selection marker. The genes transformed utilizing construct may be screened by any available method well known in the art like PCR, ELISA or any other available enzymatic assays or bioassays.

The invention furthermore intends to provide a kit for the marker free transformation of a heterologous gene in a plant, plant cells or plant parts. The kit essentially includes:
An expression cassette for marker free transformation of a heterologous gene in plants, plant cells or plant parts comprising genetic elements ordered in 5' to 3' direction: a T-DNA right border, at least a promoter, polycloning sites, transcriptional terminator signal and a T-DNA left border wherein the said genetic elements in said expression cassette comprising restriction sites across the ends joining one element with the other element and is being capable of altering said genetic elements as per the requirement of the host plant, enabling the said expression cassette to be used universally for the transformation of any gene in any plant,
The kit further includes essential reagents for performing the integration and expression of heterologous gene into host plant, plant cells or plant parts. In one embodiment, requisite buffers, reagents and other molecular tools needed to achieve the said objective through this invention may be provided to user for custom oriented use in marker free plant transformation
The present invention will be explained further with reference to non-limiting embodiments of the invention.
An embodiment of the invention provides an in-silico designed and chemically constructed Linear Expression Cassette with unique combination of genetic elements to produce marker free transgenic plants.
In another embodiment of the invention, there is provided a Linear Expression Cassette which is facilitated by unique restriction sites so as to allow the incorporation of new genetic elements or elimination of elements or replacement of

elements depending upon the requirement of the transformation of the heterologous gene in the host plant.
In another embodiment of the invention, the promoter in Linear Expression Cassette is preferably CaMV 35S duplicated promoter.
In another embodiment of the invention, the transcriptional terminators used in the Linear Expression Cassette are preferably nos, ocs, or CaMV 35S polyadenylation sequence/terminators.
In another embodiment of the invention the Agrobacterium strain Ti plasmid used in the right border and left border of Linear Expression Cassette are preferably chosen from a group comprising octopine, nopaline or succinamopine.
In another embodiment of the invention, the Linear Expression Cassette can be cloned in any compatible high copy number vector or binary vector.
In another embodiment of the invention, the Linear Expression Cassette with the foreign DNA can be transformed into plant cells by techniques of particle bombardment, protoplast transformation, Agrobacterium mediated transformation, and also by direct DNA delivery methods like pollen tube pathway method and ovary dip method.
In another embodiment of the invention, there is provided a kit for the marker free transformation of plants or plant cells or plant parts.
The invention will now be explained with the help of following examples. However, the scope of the invention should not be limited to these examples as the person skilled in the art can easily vary the proportion of the ingredients and combinations.

Example 1: Utilization of Pollen tube pathway method of transformation with the marker free tool of the present invention in the form of exogenous naked DNA. A modified CrylAc delta endotoxin encoding gene was cloned in LEC cloned in pUC19 at NcoI-SacI sites utilizing standard molecular biology practices. The LEC containing modified cry 1 Ac gene from this vector was excised as KpnI-SphI fragment and purified through gel elution kit after electrophoresis. The eluted Kpnl-Sphl fragment was mixed with SSC buffer and some other components in two treatments and applied to the prepared flowers of cotton (prepared flowers mentioned here means- flowers were marked on the day of anthesis, 20-24 hrs post pollination, styles were cut to get prepared flowers for transformation utilizing pollen tube pathway). After bolls were set and matured, seeds were harvested and sown and seedlings grown. After 30 days of growth, leaves from all the plants were analyzed for the expression of the modified cry 1 Ac gene utilizing direct ELISA kit method and manufacturer's instructions followed. The number of positive plants scored and frequencies calculated. With regard to two treatments mentioned in Example 1 to treat prepared flowers with excised, naked DNA containing cry 1 Ac modified gene cloned in LEC expression cassette, the details are as under:
Treatment A: A solution containing lA strength MS (1962) basal salts and Gamborg B5 vitamins (1968), 1% (w/v) D-Glucose, 0.1 mg/L (w/v) NAA, 0.1 mg/L (w/v) GA3, 20 raM MES, 2% (w/v) PEG, 0.1 % (v/v) DMSO, 0.1 x SSC buffer (pH 7.2) and containing 50 ug/ml of excised and purified transforming DNA. Treatment B: A solution containing 0.1 x SSC buffer (pH 7.2), 0.1 % (v/v) DMSO, 20mM MES and containing 50 ug/ml of excised and purified transforming DNA. A direct, sandwich ELISA method was used employing Anti CrylAc monoclonal antibody coated plates by Amar Immunodiagnostics, Hyderabad and following manufacturer's instructions for the protocol. The data corresponding to this is presented below.

mod crylAc No. of Flowers No. of bolls No. of bolls No. of seeds No. of seeds
S. No. gene: A Treatment Treated harvested tested sown germinated ELISA + ve
AMFA-31 (26s) AMFA-32 (28s) AMFA-33 (23s) AMFA-34 (29s) AMFA-35 (30s) AMFA-36 (29s) AMFA-37 (26s) AMFA-38 (23$) AMFA-39 (30$) AMFA-42 (26s) AMFA-31 ( 18p) AMFA-32 (9p) AMFA-33 (2p) AMFA-34 (19p) AMFA-35 (18p) AMFA-36 (12p) AMFA-37 (10p) AMFA-38 (18p) AMFA-39 (19p) AMFA-42 (17p) AMFA-31 ( Op) AMFA-32 (Op) AMFA-33 (Op) AMFA-34 (Op) AMFA-35 (lp) AMFA-36 (Op) AMFA-37 (lp) AMFA-38 (Op) AMFA-39 (Op) AMFA-42 (lp)
1 AMF A 72 38 10 Total (270s) Total (142p) Total ( 3p )
2.11%
AMFB-29 (7s) AMFB-30 (16s) AMFB-33 (28s) AMFB-34 {16s) AMFB-35 (27s) AMFB-36 (27$) AMFB-37 (32s) AMFB-39 (17s) AMFB-40 (12s) AMFB-42 (19s) AMF8-29 (2p) AMFB-30 (6p) AMFB-33 (21p) AMFB-34 (6p) AMFB-35 (9p) AMFB-36 (13p) AMFB-37 (21p) AMFB-39 (6p) AMFB-40 (Op) AMFB-42 (6p) AMFB-29 ( Op) AMFB-30 (Op) AMFB-33 (lp) AMFB-34 (Op) AMFB-35 (Op) AMFB-36 (lp) AMFB-37 (Op) AMFB-39 (Op) AMFB-40 -AMFB-42 (Op)
2 AMF B 75 39 10 Total (201s) Total (90p) Total (2p)
2.22%
Example 2: Utilization of Direct DNA delivery based method (like Particle bombardment) using a reporter gene like gusA cloned in LEC in pUC 19 for stable transformation.
A gusA gene from any of the commercially available binary vector ( preferably pBI121, however , others like pCAMBIA 1301 may also be used) is excised and cloned in LEC at Xbal- Sad sites and this total Promoter -gene- terminator expression cassette including right border and left border was cloned in pUC19 utilizing standard molecular biology practices. The pDNA of this vector harbouring gusA gene in LEC in pUC19 cloning vector (referred hereafter as pKRI-942N) was prepared in large quantities and purified. The purified plasmid DNA of pKRI-942N was coated on luM tungsten particles along with spermidine and CaC12 and this tungsten particle coated DNA suspension was bombarded on embryogenic call! cells layer of cotton, utilizing a particle acceleration device, called GenePro. The cells were bombarded at a pressure of 8-10 Kg/cm2 and at a target distance of 10-12 cm. After 48 hrs of bombardment, small groups of cells were taken out from each callus

mound and stained with X-Gluc solution. The cells stained blue and the remaining cells from each mound were preceded for proliferation and regeneration of somatic embryos.
Example 3: Utilization of Direct DNA delivery based method (like Particle bombardment) using a cryl gene cloned in LEC in pUC 19 for transient expression. A synthetic version of crylFa1 gene was excised as Xbal- Sail fragment fr0m pBinAR-CryIF vector ( sourced from NRCPB, IARI New Delhi, gene protected via Indian Patent No. 242768) and cloned in Xbal-Sall digested LEC cloned pUc 19 vector to get the vector pKRI-941N. The pDNA of this vector harbouring synthetic version of crylFa 1 gene in LEC in pUC19 was prepared in large quantities and purified. The purified plasmid DNA of pKRI-941N was coated on luM tungsten particles along with spermidine and CaC12 and this tungsten particle coated DNA suspension was bombarded on tobacco (Nicotiana tabacum) leaves, utilising a particle acceleration device, called GenePro. The leaves were bombarded at a pressure 0f 8-10 Kg/cm2 and at a target distance of 10-12 cm. After 48 hrs of bombardment, leaf area of approximately 2 cm2 were excised from bombarded leaves, cashed in protein extraction buffer and subjected to DAS direct ELISA pfafes coated with Anti-Cry IF antibody. ELISA kit manufacturer instructions were followed to develop the plates and analyze the result. All the bombarded leaves have shown expression of crylF gene (Table 1).
Table 1

Item Mean OD
Bombarded Leaf 1 0.645
Bombarded Leaf 1 0.211
Bombarded Leaf 1 0.501
Non bombarded leaf 0.003

Example 4: Utilization of Agrobacterium mediated transformation using a delta-endotoxin encoding gene like crylFal cloned in part of LEC in CAMBIA DIY vector.
A CaMV 35S promoter between Pstl-Xbal and having Sail site some nucleotide sites apart was cloned as Pstl-Sall fragment in a binary vector pCAMBIA -DIY vector. A synthetic version of cry 1 Fa 1 gene was excised as Xbal- Sail fragment from pBinAR-crylF vector (sourced from NRCPB, IARI New Delhi, gene protected via Indian Patent No. 242768) and cloned in Xbal-Sall digested partial LEC cloned pCAMBIA-D1Y vector to generate crylFal gene in LEC in plant selectable marker free binary vector, referred hereafter as pKRI-1518N. This vector, pKRI-1518N was mobilized in Agrobacterium tumefaciens strain LBA 4404. Brinjal aseptically grown seedlings were used to provide explants, which were transformed by Agrobacterium culture containing vector pKRI-1518N. After co-cultivation, explants were transferred to brinjal regeneration media containing cefotaxime as bactericidal agent. Shoot buds were noticed within 2 months of culture and shoots were transferred to shoot elongation medium. The regenerated shoots were acclimatized and genomic DNA was prepared utilizing standard CTAB procedure. The presence of the crylFal gene in the regenerated plants was screened via PCR. The PCR positive plants were screened for the expression of the transgene by subjecting the crude protein extract to ELISA plates coated with anti-crylF-MAb and high expressing plant lines were selected.

We Claim
1 .An expression cassette for marker free transformation of a heterologous gene in plants, plant cells or plant parts, with genetic elements ordered in 5' to 3' direction, comprising: a T-DNA right border, at least a promoter, atleast one polycloning site, transcriptional terminator signal and a T-DNA left border wherein at least one of said genetic elements has restriction sites across ends joining said genetic elements.
2.The expression cassette as claimed in claim 1, wherein said heterologous gene is inserted in said polycloning site of said expression cassette.
3.The expression cassette as claimed in claim 1, wherein said restriction sites are for rare cutters.
4.The expression cassette as claimed in claim 1 further comprises spacers between different restriction sites for efficient cleaving at said restriction sites.
5.The expression cassette as claimed in claim 1, wherein said promoter is selected from constitutive promoters, tissue specific promoter, inducible promoters and like.
6.the expression cassette as clainied in claim 1, wherein said transcriptional terminator signal is selected from flos terminator, ocs terminator or CaMV 35S polyadenylation sequence and like.
7. The expression cassette as claimed in claim 1, wherein said multiple restriction sites are selected from Xhol, Ncol, Nde I, Apa I, Xba I, Sal I or combinations thereof and are replaceable with a different synthetic polycloning site carrying a different set of restriction sites, as per need, for integration of a foreign DNA.
8. The expression cassette as claimed in claim 1, wherein said T-DNA right border and T-DNA left border is derived frorn the Ti plasmid of an Agrobacterium spp.

9. The expression cassette as claimed in claim 8, wherein said Agrobacterium is A.
tumefaciens.
10. The expression cassette as claimed in claim 8, wherein said Agrobacterium Ti plasmid strain is selected from strains producing octopine, nopaline, succinamopine and like.
11. The expression cassette as claimed in claim 1, wherein said expression cassette is transformed into plant cells via either of Agrobacterium mediated transformation, particle bombardment, electroporation, naked direct DNA delivery methods or viral transformation.
12. A method for obtaining marker free transformation of a heterologous gene in a plant, plant cells or plant parts essentially comprises the steps of:

(a) Inserting the said heterologous gene into an expression cassette wherein said expression cassette/with genetic elements ordered in 5' to 3' direction, comprises: a T-DNA right border, at least a promoter, at least one polycloning site, transcriptional terminator signal and a T-DNA left border wherein at least one of said genetic elements has restriction sites across ends joining said genetic elements ; wherein said heterologous gene is inserted in said polycloning site of said expression cassette;
(b) Transferring said recombinant expression cassette comprising said heterologous gene of step (a) into plant, plant cells or plant part;
(c) Growing said plant, plant cell or plant part of step (b) in
regeneration media;

(d) Regenerating or propagating of said plant or plant cell or plant part of step (c);
(e) Testing said plant or plant cell or plant part of step (d) for presence and expression of said heterologous gene and identifying a stably transformed plant or plant cell or plant part; and
(f) Propagating said stably transformed plant for obtaining marker free transgenic line with said heterologous gene.
13. Method as claimed in claim 12, wherein said restriction sites are for rare cutters.
14. Method as claimed in claim 12 further comprises spacers between different restriction sites for efficient cleaving at said restriction sites.
15. Method as claimed in claim 12, wherein identification of said transformed plant or plant cells or plant parts is done by ELISA, PCR, enzymatic assay method or any other molecular analysis techniques.
16. Method as claimed in claim 12, wherein said promoter is selected from
constitutive promoters, tissue specific promoter, inducible promoters and like.
17. Method as claimed in claim 12, wherein said transcriptional terminator signal is selected from nos terminator, ocs terminator, CaMV 35S polyadenylation sequence and like.
18. Method as claimed in claim 12, wherein said multiple restriction sites are selected from Xhol, Ncol, Nde I, Apa I, Xba I, Sal I or combinations thereof and are replaceable with a different synthetic polycloning site carrying a different set of restriction sites, as per need, for integration of a foreign DNA.

19. Method as claimed in claim 12, wherein said T-DNA right border and T-DNA left border is derived from the Ti plasmid of an Agrobacterium spp.
20. Method as claimed in claim 19, wherein said Agrobacterium is A. tumefaciens.
21. Method as claimed in claim 19, wherein said Agrobacterium Ti plasmid strain is selected from strains producing octopine, nopaline, succinamopine and like.
22. Method as claimed in claim 12, wherein said expression cassette is transformed into plant cells via either of Agrobacterium mediated transformation, particle bombardment, electroporation, naked direct DNA delivery methods or viral transformation.
23. A kit for the marker free transformation of a heterologous gene in a plant, plant cells or plant parts comprising an expression cassette for marker free transformation of a heterologous gene in plants, plant cells or plant parts, with genetic elements ordered in 5' to 3' direction, comprising: a T-DNA right border, at least a promoter, at least one polycloning site, transcriptional terminator signal and a T-DNA left border wherein at least one of said genetic elements has restriction sites across ends joining said genetic elements.
24. The kit as claimed in claim 23, further comprising essential buffers, reagents, enzymes and requisite molecular tools needed for performing integration and expression of heterologous gene into host plants, plant cells or plant parts, preferably the required restriction enzyme (s) to be selected for restriction sites included in polycloning site or combinations thereof.

25. The kit as claimed in Claim 23, wherein genetic elements borders, promoter, polycloning site, terminator, spacer or restriction site of said expression cassette are replaceable with other corresponding genetic elements.
26. The kit as claimed in claim 23 , wherein said promoter is selected from constitutive promoters, tissue specific promoter, inducible promoters and like.
27. The kit as claimed in claim 23, wherein said transcriptional terminator signal is selected from nos terminator, ocs terminator or CaMV 35S polyadenylation sequence and like.
28. The kit as claimed in claim 23, wherein said T-DNA right border and T-DNA left border is derived from the Ti plasmid of an Agrobacterium spp.