DESC:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of plant molecular biology and genetic engineering, and in specific, relates to a method for producing human insulin in an edible plant system through genetic modification using a plant expression vector and Agrobacterium tumefaciens-mediated transformation, enabling direct consumption of the harvested plant material as a food-grade product under contained conditions.
Background of the invention:
[0002] Diabetes mellitus is a chronic metabolic disorder caused by insufficient production or impaired utilization of insulin, resulting in elevated blood glucose levels and leading to complications such as cardiovascular disease, neuropathy, nephropathy, and retinopathy. Conventional management typically involves the administration of exogenous insulin through subcutaneous injections or insulin pumps, which is inconvenient, costly, and associated with patient discomfort, thereby affecting long-term treatment compliance.

[0003] Edible plant-based production of therapeutic proteins, which include insulin, has been explored as a potential alternative to injectable formulations. This approach offers advantages such as reduced production costs, improved patient compliance, and simplified distribution, as the therapeutic protein is consumed in a food-compatible form.

[0004] In existing approaches, various plant species, which include lettuce, rice, and tobacco, have been genetically engineered to express human insulin or insulin-like peptides. For example, prior art WO2004091995A2 discloses methods for expressing proinsulin in transgenic lettuce plants, while CN102237753A describes the expression of recombinant human insulin in rice endosperm. These methods demonstrate the feasibility of producing insulin in plant systems; however, the use of non-edible plant parts, low expression yields, and the requirement for downstream purification remain common limitations.

[0005] Non-patent literature also reports the expression of therapeutic proteins in edible plant systems, but often highlights challenges such as achieving sufficient protein stability during plant growth and storage, ensuring bioactivity after consumption, and preventing gene flow to non-transgenic crops. While these studies contribute valuable insights, there remains a need for approaches that combine high-yield insulin expression in edible plant parts with practical considerations for consumption and scalability.

[0006] By addressing all the above-mentioned problems, there is a need for a method for producing human insulin in an edible plant system through genetic modification using a plant expression vector and Agrobacterium tumefaciens-mediated transformation, enabling direct consumption of the harvested plant material as a food-grade product under contained conditions. There is also a need for a method that allows the cultivation of transgenic edible plantlets under controlled environmental conditions to ensure bioactive insulin production and stability. There is also a need for a method that yields insulin-containing edible plant material with an expected insulin content of more than 0.5 grams per gram of plant tissue. There is also a need for a method that ensures the produced insulin-containing plant material remains stable and safe for consumption when stored under standard vegetable storage conditions.

[0007] Therefore, there is also a need for a method that is adaptable to multiple edible plant species capable of expressing bioactive human insulin. There is also a need for a method that enables cultivation under greenhouse or contained conditions to prevent cross-pollination and gene flow. There is also a need for a method that is scaled from laboratory prototype to industrial production while maintaining insulin yield and stability. There is also a need for a method that preserves the organoleptic qualities of the edible plant material, ensuring safety and consumer acceptability. There is also a need for a method that avoids downstream extraction, purification, and formulation steps, thereby simplifying production and reducing costs.
Objectives of the invention:
[0008] The primary objective of the present invention is to provide a method for producing human insulin in an edible plant system through genetic modification using a plant expression vector and Agrobacterium tumefaciens-mediated transformation, enabling direct consumption of the harvested plant material as a food-grade product under contained conditions.

[0009] Another objective of the present invention is to provide a method that employs a synthesized human insulin cDNA sequence for integration into the plant genome, eliminating the need for mRNA extraction and reducing process complexity.

[0010] The other objective of the present invention is to provide a method that utilizes Agrobacterium tumefaciens-mediated transformation for the stable transfer of the human insulin gene into edible plant tissues.

[0011] The other objective of the present invention is to provide a method that allows the cultivation of transgenic edible plantlets under controlled environmental conditions to ensure bioactive insulin production and stability.

[0012] The other objective of the present invention is to provide a method that yields insulin-containing edible plant material with an expected insulin content of more than 0.5 grams per gram of plant tissue.

[0013] The other objective of the present invention is to provide a method that is adaptable to multiple edible plant species capable of expressing bioactive human insulin.

[0014] The other objective of the present invention is to provide a method that enables cultivation under greenhouse or contained conditions to prevent cross-pollination and gene flow.

[0015] The other objective of the present invention is to provide a method that is scaled from laboratory prototype to industrial production while maintaining insulin yield and stability.

[0016] The other objective of the present invention is to provide a method that preserves the organoleptic qualities of the edible plant material, ensuring safety and consumer acceptability.

[0017] Yet another objective of the present invention is to provide a method that avoids downstream extraction, purification, and formulation steps, thereby simplifying production and reducing costs.

[0018] Further objective of the present invention is to provide a method that ensures the produced insulin-containing plant material remains stable and safe for consumption when stored under standard vegetable storage conditions.
Summary of the invention:
[0019] The present disclosure proposes a method for producing human insulin in an edible plant system. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0020] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method for producing human insulin in an edible plant system through genetic modification using a plant expression vector and Agrobacterium tumefaciens-mediated transformation, enabling direct consumption of the harvested plant material as a food-grade product under contained conditions.

[0021] According to one aspect, the invention provides a method for preparing genetically modified edible plant material expressing bioactive human insulin. At one step, a human insulin complementary deoxyribonucleic acid (cDNA) sequence is synthesized. At one step, the synthesized human insulin cDNA sequence is cloned into a pCAMBIA-based plant expression vector. At one step, Agrobacterium tumefaciens is transformed with the recombinant plant expression vector via electroporation.

[0022] At one step, edible plant tissue is contacted with the transformed Agrobacterium tumefaciens under conditions effective for T-DNA transfer into the plant genome. At one step, the transformed plant tissue is cultivated in a contained environment using Murashige and Skoog (MS) medium with clerigel to obtain edible plant material containing human insulin. The edible plant material is processed for direct consumption as a food-grade product.

[0023] In one embodiment herein, the plant expression vector is pCAMBIA1302, which comprises a lac promoter, a terminator, a kanamycin-resistance selection marker, and a T-DNA region flanked by left border (LB) and right border (RB) sequences for targeted genomic integration. The human insulin cDNA sequence, approximately 250 base pairs, is inserted into the multiple cloning site between EcoRI and HindIII restriction enzyme recognition sites. The edible plant tissue is selected from the group consisting of vegetative buds of Brassica oleracea var. capitata, seeds of Vigna radiata, seeds of Triticum aestivum, and seeds of Arachis hypogaea.

[0024] In one embodiment herein, the transformed plant tissue is cultivated in petri plates or glass tubes under controlled laboratory conditions, which include a temperature of 20–25°C, humidity of at least 50%, and a photoperiod of at least 12 hours of light followed by 12 hours of darkness, resulting in noticeable growth of plantlets within 1 to 2 weeks. The cultivation is performed under containment to prevent cross-pollination and unintended gene flow.

[0025] In one embodiment herein, the edible plant material is harvested and processed by washing with water to produce a consumable raw edible form containing human insulin, or optionally ground into a powder form, and is obtained from locally sourced vegetables after verifying tissue culture compatibility in Murashige and Skoog (MS) medium before transformation, ensuring compliance with biosafety regulations.

[0026] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0027] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0028] FIG. 1 illustrates a method for preparing genetically modified edible plant material expressing bioactive human insulin, in accordance to an exemplary embodiment of the invention.

[0029] FIG. 2A illustrates the tissue culture compatibility of Vigna radiata seeds grown in MS medium with clerigel, in accordance with an exemplary embodiment of the invention.

[0030] FIG. 2B illustrates the tissue culture compatibility of Triticum aestivum seeds grown in MS medium with clerigel, in accordance with an exemplary embodiment of the invention.

[0031] FIG. 2C illustrates the tissue culture compatibility of Arachis hypogaea seeds grown in MS medium with clerigel, in accordance with an exemplary embodiment of the invention.

[0032] FIG. 3 illustrates the map of the recombinant plasmid vector pCAMBIA1302 containing the human insulin cDNA sequence, in accordance with an exemplary embodiment of the invention.

[0033] FIG. 4 illustrates a schematic view of the preparation of the genetically modified edible plant material expressing the bioactive human insulin, in accordance with an exemplary embodiment of the invention.
Detailed invention disclosure:
[0034] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0035] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method for producing human insulin in an edible plant system through genetic modification using a plant expression vector and Agrobacterium tumefaciens-mediated transformation, enabling direct consumption of the harvested plant material as a food-grade product under contained conditions.

[0036] According to one exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for preparing genetically modified edible plant material expressing bioactive human insulin. At step 102, a human insulin complementary deoxyribonucleic acid (cDNA) sequence is synthesized. At step 104, the synthesized human insulin cDNA sequence is cloned into a pCAMBIA-based plant expression vector. At step 106, Agrobacterium tumefaciens is transformed with the recombinant plant expression vector via electroporation.

[0037] At step 108, edible plant tissue is contacted with the transformed Agrobacterium tumefaciens under conditions effective for T-DNA transfer into the plant genome. At step 110, the transformed plant tissue is cultivated in a contained environment using Murashige and Skoog (MS) medium with clerigel to obtain edible plant material containing human insulin. The edible plant material is processed for direct consumption as a food-grade product.

[0038] In one embodiment herein, the plant expression vector is pCAMBIA1302, which comprises a lac promoter, a terminator, a kanamycin-resistance selection marker, and a T-DNA region flanked by left border (LB) and right border (RB) sequences for targeted genomic integration. The human insulin cDNA sequence, approximately 250 base pairs, is inserted into the multiple cloning site between EcoRI and HindIII restriction enzyme recognition sites. The edible plant tissue is selected from the group consisting of vegetative buds of Brassica oleracea var. capitata, seeds of Vigna radiata, seeds of Triticum aestivum, and seeds of Arachis hypogaea.

[0039] In one embodiment herein, the transformed plant tissue is cultivated in petri plates or glass tubes under controlled laboratory conditions, which include a temperature of 20–25°C, humidity of at least 50%, and a photoperiod of at least 12 hours of light followed by 12 hours of darkness, resulting in noticeable growth of plantlets within 1 to 2 weeks. The cultivation is performed under containment to prevent cross-pollination and unintended gene flow.

[0040] In one embodiment herein, the edible plant material is harvested and processed by washing with water to produce a consumable raw edible form containing human insulin, or optionally ground into a powder form, and is obtained from locally sourced vegetables after verifying tissue culture compatibility in Murashige and Skoog (MS) medium before transformation, ensuring compliance with biosafety regulations.

[0041] According to another exemplary embodiment of the invention, FIGs. 2A-2C refer to the tissue culture compatibility of Vigna radiata seeds, Triticum aestivum seeds, and Arachis hypogaea seeds grown in Murashige and Skoog (MS) medium with clerigel, respectively. In one embodiment herein, FIG. 2A illustrates Vigna radiata seeds germinating under aseptic conditions in MS medium containing clerigel, showing successful differentiation of root and shoot systems in vitro, demonstrating compatibility of the species for transformation and edible consumption without cooking.

[0042] In one embodiment herein, FIG. 2B illustrates Triticum aestivum seeds cultured in MS medium with clerigel, showing germination and healthy seedling development, the seeds being suitable for powdering post-harvest and consumption by mixing with juice or water, offering an alternative edible format.

[0043] In one embodiment herein, FIG. 2C illustrates Arachis hypogaea seeds germinating in MS medium with clerigel, showing root and shoot system development in vitro, indicating suitability for the intended transformation process and direct consumption as raw seeds.

[0044] In one embodiment, the in vitro culture of all three plant species is maintained under controlled conditions that comprise a temperature of 20–25 °C, a relative humidity of at least 50%, and a photoperiod that includes a minimum of 12 hr of light followed by at least 12 hr of darkness. These conditions demonstrate the adaptability of the plants for recombinant human insulin gene expression via Agrobacterium-mediated transformation and confirm their feasibility as edible carriers of bioactive insulin.

[0045] According to another exemplary embodiment of the invention, FIG. 3 refers to the map of the recombinant plasmid vector pCAMBIA1302 containing the human insulin cDNA sequence. In one embodiment herein, the pCAMBIA1302 vector has an overall size of at least 10.55 kilobase pairs without the human insulin gene and includes a multiple cloning site (MCS) positioned between EcoRI and HindIII restriction enzyme recognition sites, a lac promoter for controlling the expression of the inserted gene, and a kanamycin-resistance selection marker in the vector backbone.

[0046] In one embodiment herein, the synthesized human insulin cDNA sequence, having a size of at least 250 base pairs, integrates into the MCS immediately adjacent to the lac operator sequence to enable regulated expression under the lac promoter. In one embodiment herein, the selection of EcoRI and HindIII sites allows complete deletion of the MCS to prevent any frame shifts in the gene, thereby ensuring accurate protein expression.

[0047] In one embodiment herein, the T-DNA region of the vector is flanked by left border (LB) and right border (RB) sequences to facilitate precise transfer of the entire gene cassette, which includes the human insulin cDNA sequence and associated vector components, into the plant genome through Agrobacterium tumefaciens-mediated transformation. In one embodiment herein, the recombinant plasmid has a final size of at least 10.8 kilobase pairs after insertion of the human insulin cDNA sequence, enabling stable incorporation into edible plant tissues for bioactive insulin production.

[0048] According to another exemplary embodiment of the invention, FIG. 4 refers to a schematic view 400 of the preparation of human insulin in an edible plant system. In one embodiment herein, the process initiates with the synthesis of a human insulin complementary deoxyribonucleic acid (cDNA) sequence, which is synthesized for use in the method.

[0049] In one embodiment herein, the synthesized human insulin cDNA sequence is cloned into a pCAMBIA-based plant expression vector, specifically pCAMBIA1302, with the sequence inserted into the multiple cloning site (MCS) between EcoRI and HindIII restriction enzyme recognition sites, adjacent to a lac promoter, and the vector includes a kanamycin-resistance selection marker and a T-DNA region flanked by left border (LB) and right border (RB) sequences for targeted genomic integration.

[0050] In one another embodiment herein, the recombinant plasmid vector is introduced into Agrobacterium tumefaciens using an electroporation technique to obtain a recombinant bacterial strain capable of delivering the human insulin cDNA sequence into edible plant tissue.

[0051] In one embodiment herein, edible plant tissue, selected from the group consisting of vegetative buds of Brassica oleracea var. capitata, seeds of Vigna radiata, seeds of Triticum aestivum, and seeds of Arachis hypogaea, is contacted with the transformed Agrobacterium tumefaciens under conditions effective for T-DNA transfer into the plant genome, and the transformed tissue is cultivated in a contained environment using Murashige and Skoog (MS) medium with clerigel under sterile conditions.

[0052] In one embodiment herein, the transformed plant tissue is cultivated in petri plates or glass tubes under controlled laboratory conditions, which include a temperature of 20–25°C, humidity of at least 50%, and a photoperiod of at least 12 hours of light followed by 12 hours of darkness, resulting in noticeable growth of plantlets within 1 to 2 weeks.

[0053] In one embodiment herein, the cultivated plant material is harvested as edible plant material containing human insulin, with the process performed under containment to prevent cross-pollination and unintended gene flow.

[0054] In one embodiment herein, the harvested edible plant material is processed by washing with water to produce a consumable raw edible form containing human insulin, or optionally ground into a powder form, and is obtained from locally sourced vegetables after verifying tissue culture compatibility in Murashige and Skoog (MS) medium before transformation, ensuring compliance with biosafety regulations.

[0055] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a method for producing human insulin in an edible plant system is disclosed. The proposed method for producing human insulin in an edible plant system through genetic modification using a plant expression vector and Agrobacterium tumefaciens-mediated transformation, enabling direct consumption of the harvested plant material as a food-grade product under contained conditions. The method allows the cultivation of transgenic edible plantlets under controlled environmental conditions to ensure bioactive insulin production and stability. The method that yields insulin-containing edible plant material with an expected insulin content of more than 0.5 grams per gram of plant tissue. The method that ensures the produced insulin-containing plant material remains stable and safe for consumption when stored under standard vegetable storage conditions.

[0056] The method is adaptable to multiple edible plant species capable of expressing bioactive human insulin. The method that enables cultivation under greenhouse or contained conditions to prevent cross-pollination and gene flow. The method is scaled from laboratory prototype to industrial production while maintaining insulin yield and stability. The method preserves the organoleptic qualities of the edible plant material, ensuring safety and consumer acceptability. The method avoids downstream extraction, purification, and formulation steps, thereby simplifying production and reducing costs.

[0057] It will readily be apparent that numerous modifications and alterations is made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
,CLAIMS:CLAIMS:
I/We Claim:
1. A method for producing human insulin in an edible plant system, comprising:
synthesizing a human insulin complementary deoxyribonucleic acid (cDNA) sequence;
cloning the synthesized human insulin cDNA sequence into a pCAMBIA-based plant expression vector, which comprises a lac promoter, a terminator, and a kanamycin-resistance selection marker;
introducing the recombinant plant expression vector into agrobacterium tumefaciens via electroporation;
contacting edible plant tissue with the transformed agrobacterium tumefaciens under conditions effective for T-DNA transfer into the plant genome; and
cultivating the transformed plant tissue in a contained environment using murashige and skoog (MS) medium with clerigel at a temperature varies between 20 °C to 25 °C, humidity of at least 50%, and a photoperiod of at least 12 hr of light followed by at least 12 hr of darkness to obtain an edible plant material containing human insulin,
wherein the method is performed under containment to prevent cross-pollination and does not involve propagation of a plant variety as such.
2. The method as claimed in claim 1, wherein the pCAMBIA-based plant expression vector is pCAMBIA1302, which comprises a T-DNA region flanked by left border (LB) and right border (RB) sequences for targeted genomic integration, and the human insulin cDNA sequence of at least 250 base pairs is inserted into the multiple cloning site between EcoRI and HindIII restriction enzyme recognition sites.
3. The method as claimed in claim 1, wherein the edible plant tissue is selected from the group consisting of vegetative buds of Brassica oleracea var. capitata, seeds of Vigna radiata, seeds of Triticum aestivum, and seeds of Arachis hypogaea.
4. The method as claimed in claim 1, wherein the transformed plant tissue is cultivated in petri plates or glass tubes under controlled laboratory conditions.
5. The method as claimed in claim 1, wherein the cultivation of the transformed plant tissue results in noticeable growth of plantlets within 1 to 2 weeks in the MS medium.
6. The method as claimed in claim 1, wherein the edible plant material is harvested and processed by washing with water to produce a consumable raw edible form containing human insulin.
7. The method as claimed in claim 6, wherein the harvested edible plant material is optionally ground into a powder form.
8. The method as claimed in claim 1, wherein the edible plant material is produced as a food-grade material under biosafety regulations to ensure containment and prevent unintended gene flow.
9. The method as claimed in claim 2, wherein the recombinant pCAMBIA1302 vector has a size of at least 10.8 kilobases after insertion of the human insulin cDNA sequence.
10. The method as claimed in claim 1, wherein the edible plant material is obtained from locally sourced vegetables, and the method includes verifying tissue culture compatibility of the plant tissue in murashige and skoog (MS) medium before transformation.