DESC:F O R M 2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: POSITIVE PRESSURE BASED TREATMENT OF PLANTS AND APPLICATIONS THEREOF

2. APPLICANT (A) NAME:AGRIMAA LIFESCIENCE PVT LTD

(B)ADDRESS: NO.76, BOMMURU AGRAHARA VILLAGE, NAGUVANAHALLI GP, BELAGOLA HOBLI, SRIRANGPATNA TQ, MANDYA DIST 571606

3. NATIONALITY (C)INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
PRIORITY CLAIM
[001] The instant patent application is related to and claims priority from the co-pending India provisional patent application entitled, “POSITIVE PRESSURE BASED TREATMENT OF PLANTS AND APPLICATIONS THEREOF,” Patent Application no.:202241052417, Filed on: 14 September 2022, which is incorporated in its entirety herewith.
[002] BACKGROUND OF THE DISCLOSED EMBODIMENT:
[003] Technical field
[004] The disclosed embodiment is in the technical field of method and composition for the transformation of plants. More particularly, a method of gene transfer or chromosome doubling agents to plants employing pneumatic or hydraulic pressure.
[005] Related art
[006] Last few years many techniques/ methods for the genetic transformation of plants have been developed and utilized.
[007] Prior art methods have been employed to obtain a transgenic plant containing a foreign nucleic acid comprising a gene or a feature of interest. In the case of nucleic acid molecules encoding a transgene are stably integrated into a plants' genome, particularly the nuclear genome, but also into the genome of e.g., plant plastids. The major aims of the preceding transformation process to yield a stable "knock-in", but also "knock-out" of a gene or region of interest.
[008] In addition, prior art methods have been employed to obtain a transient expression of the construct of interest to be transformed or transfected to transiently influence the genetic material of a plant, for example, the genome, or the transcriptome thereof in a controlled way.
[009] At present, there exists a variety of plant transformation methods to introduce genetic material into a plant cell of interest. One of the well-known techniques is transformation with Agrobacterium. Agrobacterium transformation, is better established for dicotyledonous plants than for monocotyledonous plants.
[010] Transformation process for monocot plants such as corn through Agrobacterium mediated transformation of immature embryos is, however, time consuming and expensive.
[011] There are several prior arts about Agrobacterium mediated transformation of both monocot and dicot plants. However, the said approaches, require a cumbersome regeneration of the transformed plant cell or tissue to obtain a transgenic or genetically modified plant.
[012] The floral dip methodologies are regularly applied to Arabidopsis, but such simple method of plant transformation does not exist for any other plant species.
[013] Therefore, there is an urgent need for delivery of gene transfer agents, especially for monocot plant species, and to improve the scalability of recombinant protein production.
[014] SUMMARY OF THE DISCLOSED EMBODIMENT:
[015] The primary objective of the disclosed embodiment is to provide system, method and composition for the transformation of plants.
[016] According to an aspect of the disclosed embodiment, a method for infiltrating a plant with an agent, wherein the method comprising the steps of: (i) providing the plant in a closed container; (ii) providing the agent into the container to completely or partially cover the plant; and (iii) applying a positive pressure ranging from 5-15 psi from top of the container using an airtight piston for about 1 minute to 24 hours, wherein the application of positive pressure facilitates the infiltration of the agent into the intercellular spaces or the inaccessible regions comprising meristem of the plant.
[017] According to an embodiment, wherein the positive pressure is applied using a hydraulic unit or a pneumatic unit.
[018] According to one more embodiment, wherein the agent is a suspension of an Agrobacterium strain comprising a cell density of about 0.1-2.0 OD, wherein the Agrobacterium strain comprising a Ti plasmid bearing a nucleotide sequence encoding a polypeptide.
[019] According to an embodiment, wherein the positive pressure facilitates the delivery of the Agrobacterium strain into the cells through filling intercellular spaces of the plant with the suspension.
[020] According to another embodiment, wherein the polypeptide is a recombinant protein, like any industrial protein like Trypsin or any pharmaceutical protein like Insulin or any antibodies or vaccine proteins etc
[021] According to yet another embodiment, wherein the nucleotide sequence encoding the polypeptide is cloned in a binary vector comprising a T-DNA region that comprises a promoter and a reporter gene. The binary vector comprises pCAMBIA1300, pCAMBIA1305.
[022] According to another embodiment, wherein the agent is a chromosome doubling agent, wherein the chromosome doubling agent is a colchicine solution.
[023] According to yet another embodiment, wherein the colchicine is delivered into the meristem region of the plant.
[024] According to an embodiment, wherein the agent is a gene editing agent or gene silencing agent comprising 10-10,000 oligonucleotides.
[025] According to another embodiment, wherein plant is oriented in an upright or an inverted position.
[026] According to an aspect of the disclosed embodiment, a system for infiltrating a plant with an agent, wherein the system comprises, (i) a closed container that receives the plant to be infiltrated with the agent, wherein the agent is filled in the container to completely or partially cover the plant part; (ii) an airtight piston configured to apply a positive pressure ranging from 5-15 psi from top of the container for about 1 minute to 24 hours, wherein the application of positive pressure facilitates the infiltration of the agent into the intercellular spaces and inaccessible regions of the plant part.
[027] According to one more embodiment, wherein the positive pressure is applied using a hydraulic unit or a pneumatic unit.
[028] According to another embodiment, wherein the agent is a suspension of an Agrobacterium strain comprising a cell density of about 0.1-2.0 OD. The Agrobacterium strain comprising a Ti plasmid bearing a nucleotide sequence encoding a polypeptide.
[029] According to yet another embodiment, wherein the positive pressure facilitates the transformation of cells with the nucleotide sequence encoding the polypeptide through filling intercellular spaces of the plant with the suspension.
[030] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
[031] BRIEF DESCRIPTION OF THE DRAWINGS:
[032] Example embodiments of the disclosed embodiment will be described with reference to the accompanying drawings briefly described below.
[033] FIG.1 illustrates the vacuum infiltration, according to the aspects of the disclosed embodiment.
[034] FIG.2 illustrates the syringe infiltration, according to the aspects of the disclosed embodiment.
[035] FIG.3 illustrates a monocot shoot structure of sugarcane (as an example of monocot shoot) showing the bases of the leaves whorling around shoot apex where any topically applied agent cannot reach, according to the aspects of the disclosed embodiment.
[036] FIG.4 illustrates the method of infiltration of Agrobacterium suspension into intercellular spaces of plants using positive pressure, according to the aspects of the disclosed embodiment.
[037] FIG.5 illustrates the efficient infiltration of Agrobacterium suspension into intercellular spaces of plants using positive pressure. Note the water-soaked appearance of intact seedlings which were subjected to infiltration, according to the aspects of the disclosed embodiment.
[038] FIG.6 illustrates the transient GUS expression in intact seedlings of different dicot plants, according to the aspects of the disclosed embodiment.
[039] FIG.7 illustrates the transient expression in intact corn seedling. (a) 7-day old corn seedling infiltrated by Agrobacterium (b) GUS expression throughout the all the three leaves of the seedling (c) close up of upper region of the leaf (d) close-up of lower region of leaf (e) GUS expression in root (f) GFP expression in the lower region of leaf, according to the aspects of the disclosed embodiment.
[040] FIG.8 illustrates a commercial ‘hydroponic corn fodder production unit’ in stacked trays, according to the aspects of the disclosed embodiment.
[041] FIG.9(A) illustrate preconditioning plants with a dark period of 1-4 days before Agropress increases extent of Agrobacterium mediated gene transfer, according to the aspects of the disclosed embodiment.
[042] (FIG.9B i, ii) illustrate variability of gene expression in a single experiment, according to the aspects of the disclosed embodiment.
[043] FIG.10 illustrates an Agropressmethod on corn seedlings, according to the aspects of the disclosed embodiment.
[044] FIG.11 illustrates a flowchart of the method, according to the aspects of the disclosed embodiment.
[045] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit (s) in the corresponding reference number.
[046] DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENT:
[047] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
[048] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[049] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
[050] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
[051] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
[052] Example embodiments of the disclosed embodiment are described with reference to the accompanying figures.
[053] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[054] DEFINITIONS:
[055] The term ‘Plant transformation’ is a genetic engineering tool for introducing transgenes into plant genomes. It is now being used for the breeding of commercial crops.
[056] A central feature of transformation is insertion of the transgene into plant chromosomal DNA. Transgene insertion is infrequently, if ever, a precise event.
[057] The term ‘Gene Transfer’ The introduction of new DNA into an existing organism's cell, usually by vectors such as plasmids and modified viruses.
[058] The term polyploidy often is described as “chromosome doubling,” which implies a somatic (nonreproductive) event during formation. A somatic chromosome doubling event could occur in a zygote or in developing seedlings or even in active apical meristematic tissues.
[059] The term ‘Pneumatics’ provides fluid power by means of pressurised air or gases.
[060] The term ‘Hydraulics’ provides fluid power by means of pressurised liquids, such as oil or water.
[061] The term ‘gene knock-in’ therefore can be seen as a gain-of-function mutation and a gene knockout a loss-of-function mutation, but a ‘gene knock-in’ may also involve the substitution of a functional gene locus for a mutant phenotype that results in some loss of function.
[062] The term ‘Dicotyledonous plants’ can be defined as the plants whose seeds consist of two cotyledons. These are the angiosperms that have a true vascular bundle. The root system of the plant is the taproot system. The seeds of the plant can be split into two equal halves.
[063] The term ‘Agrobacterium’ is a phytopathogenic bacterium that causes crown gall disease in plants because of its unique capability of transferring its genetic material to the plant genome.
[064] The term ‘Monocotyledons are plants that have only one cotyledon. They also have other characteristics, such as flower parts in multiples of threes. This means they could have three, six or nine petals, or they could have sepals that make up their flowers. They also have adventitious roots.
[065] The term ‘Arabidopsis thaliana is a small flowering plant that is widely used as a model organism in plant biology. Arabidopsis is a member of the mustard (Brassicaceae) family, which includes cultivated species such as cabbage and radish.
[066] The term ‘vacuum infiltration’ plant leaves are first submerged into an infiltration media which contains the Agrobacterium strain harboring the target recombinant DNA. The submerged plants are then subject to a negative atmospheric pressure in a vacuum chamber.
[067] The term ‘Colchicine’ is an alkaloid derived from Colchicum autumnale, commonly known as autumn crocus. It prevents cells from forming spindles during mitosis, preventing chromosomal segregation during anaphase. As a result, colchicine induces multiple f sets of chromosomes.
[068] The term ‘Transient GUS’ (beta-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells.
[069] The term ‘The green fluorescent protein’ (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.
[070] The term ’CaMV 35S promoter’ is regularly used to drive the expression of recombinant proteins in plants. In angiosperms, several studies showed that reporter genes driven by this promoter display unequal tissue and developmental expression patterns.
[071] The term ‘NOS terminator’ is a 127 bp sequence found in genetically modified plants. In addition to their own chromosomes, plants contain organelles with DNA called chloroplasts.
[072] The term ‘pcambia1300’ Agrobacterium binary vector for plant transformation, with hygromycin- and kanamycin-resistance genes. Sequence Author: Cambia. Explore Over 2.7k Plasmids: Plant Vectors | More Plasmid Sets.
[073] The term ‘Agropress’is the method of infusing Agrobacterium cell suspension into intercellular spaces of plants using a positive pressure.
[074] EMBODIMENTS OF THE DISCLOSURE:
[075] The disclosed embodiment relates to a method and composition for the transformation of plants. More particularly, a method of gene transfer or chromosome doubling agents to plants comprising pneumatic or hydraulic pressure.
[076] Furthermore, an efficient and high throughput delivery of agents to the desired regions of the plants, including inaccessible regions.
[077] METHOD OF THE DISCLOSURE:
[078] A method for infiltrating a plant with an agent, wherein themethod comprising the steps of: (i) providing the plant in a closed container; (402) (ii) providing the agent into the container to completely or partially cover the plant;(404) and (iii) applying a positive pressure ranging from 5-15psi from top of the container using an airtight piston (406) for about 1 minute to 24 hours, wherein the application of positive pressure facilitates the integration of the agent into the plant. (408) (FIG.4)
[079] With positive pressure all the intercellular spaces are effectively filled with Agrobacterium solution in intact plants which makes the plant tissue appear water-soaked un-infiltrated (left) and infiltrated okra (right) (502), un-infiltrated (left) and infiltrated cotton seedlings (504).Efficient infiltration of Agrobacterium suspension into intercellular spaces of plants using positive pressure. Note the water-soaked appearance of intact seedlings which were subjected to infiltration. (FIG.5)
[080] Examples of inaccessible regions include meristem or leaf bases of a typical monocot plant as shown in the (FIG.3), a typical monocot shoot structure illustrated with sugarcane stem. Dewlap (302), sheath (304), spindle (306), shoot apex (308) and node (310). It also shows that the bases of the leaves (312)whorling around shoot apex (314) where any topically applied agent cannot reach.
[081] EXPERIMENTAL DATA:
[082] This in turn results in transformation of most of the cells as evidenced through reporter gene expression in the 6-different dicot species testedtomato (602), okra (ladies finger) (604), tobacco (606), cotton (608), chili (hot paper) (610), brinjal (612) (FIG.6).
[083] The method can be used to transiently express proteins in monocots as well, including intact seedlings of corn. Describes the transient expression in intact corn seedling. (a) 7-day old corn seedling infiltrated by Agrobacterium (b) GUS expression throughout the all the three leaves of the seedling (c) close up of upper region of the leaf (d) close-up of lower region of leaf (e) GUS expression in root (f) GFP expression in the lower region of leaf (FIG.7).
[084] Note: As X-gluc does not easily penetrate the cuticular layer of corn, leaf surface was injured using a sand paper before dipping in X-gluc. Hence staining has occurred only around the injured areas and hence does not appear to be uniform.
[085] As example embodiments, following genes and vectors were used for the gene transfer:
[086] GENES:
[087] ß-glucuronidase (GUS)
[088] Green Fluorescent Protein (GFP)
[089] CaMV 35S promoter
[090] NOS terminator
[091] BINARY VECTORS:
[092] pCAMBIA1300- Vector contains kanamycin resistance gene for bacterial selection, hygromycin B resistance gene for plant selection into which inventor cloned eGFP gene under CaMV 35promoter.
[093] pCAMBIA1305 - binary plasmids pCAMBIA1305 is an Agrobacterium binary vector for plant transformation, with hygromycin- and kanamycin-resistance and GUSPlus™ genes.
[094] The preconditioning plants with a dark period of 1- 4 days before Agropress increases extent of Agrobacterium mediated gene transfer. FIG.9(A)
[095] It show that age of seedlings and a treatment of growth in darkness before subjecting them to Agropress greatly influences gene transfer.The efficiency of gene transfer is measured in terms of green fluorescence best infection (High green fluorescence) FIG.9B(i) and least infection (Low green fluorescence) as shown in FIG.9B(ii)
[096] An Agropress method on corn seedlings, wherein the method involve, 50ml syringe is cut and fused/welded on to a 50ml centrifuge tube (FIG.10A)
[097] The resulting unit along with the plunger of 50ml syringe is used as the ‘Infiltration device’.
[098] Three seedlings (grown under 5 days light + 2 days dark conditions) are inserted into the unit (roots inserted first, towards the bottom) (FIG.10B).
[099] Agrobacteria, grown overnight in LB, is poured into the unit and mounted onto a 250ml plastic beaker (FIG.10C). (Note: The rim of the syringe (as marked in FIG.10 A and D) supports the unit on the measuring cylinder.
[0100] A plastic petri-plate is kept inverted on the plunger to support a weight (FIG.10D).
[0101] A weight of 10 Kg is placed on the plastic petri-plate (FIG.10E) and held in place for 3 mins. After the 3 mins infiltration, the device is dismantled in reverse of the order mentioned above. (FIG.10)
[0102] The term ‘Agropress’ is the method of infusing Agrobacterium cell suspension into intercellular spaces of plants using a positive pressure.
[0103] FLOWCHART OF THE METHOD:
[0104] Placing target plants in a container (1102), Pouring overnight grown Agrobacterium culture suspension to completely cover the plants (1104), Applying positive pressure from the top using an airtight piston for a duration of 1-4 minutes (1106), Filling intercellular spaces with Agrobacterium solution (1108), Resulting in the plant tissue appearing water-soaked, Leading to the transformation of most cells (1110), With evidence of reporter gene expression in six dicot species (1112), Allowing for transient protein expression in monocots, including corn seedlings (1114).
[0105] HYDRAULIC PRESSURE VS CENTRIFUGATION TO DELIVER COLCHICINE:
[0106] COLCHICINE: Colchicine treatment is the most common procedure used for chromosome doubling by applying colchicine, an antimicrotubule agent. This treatment has proved especially successful with barley, maize, and wheat where careful consideration has been given to the length of colchicine treatment and to additional compounds that improve and facilitate the doubling effect. Colchicine can also be used to haploid plants obtained from the anther culture technique.
[0107] Is considered an effective chemical mutagen to induce diploid gametes in plants. It inhibits microtubule polymerization by binding to tubulin, and thus it inhibits the mitotic spindle formation resulting in the development of a polyploid cell.
[0108] THE PRESSURE EFFICACY, AND PERCENTAGE OF SUCCESS:
[0109] 100% of plants subjected to Agropress at 12-15 psi for 1 to 3 mins show successfulrecombinant protein expression.
[0110] AMOUNT OF PRESSURE:
[0111] Applying a positive pressure within the range of 5 to 15 pounds per square inch (psi).
[0112] Table 1 shows the comparison of colchicine treatment methods in corn dihaploid production.
Present Disclosure Centrifugation
Hydraulic press or pneumatic press used to push agent Centrifugal force used to push agent
Growth of plant was found to be not affected even by 20Kg/sq cm positive pressure Centrifugation at higher speed and longer duration is known to adversely affect plant survival and growth (e.g. more than 300rpm for 1 minute or 150rpm for 10 minutes adversely affect corn seedling growth).
Designing hydraulic machines is easy for any size of plant with minimal expense Building centrifuges for larger plants is extremely difficult and expensive
Hydraulic pressure is uniform in all directions. (This helps in delivery of the agent only to the roots or shoots possible by orienting the plants appropriately) Centrifugal force is unidirectional. Only one orientation of plants is possible and agent cannot be differentially applied to shoots and roots.

[0113] Table 2 illustrates the comparison of recombinant protein production systems using plants
Magnifection®
Icon Genetics HyperTrans®
Leaf Expression Proposed
Plant species Tobacco (C3 plant) Tobacco (C3 plant) Corn (C4 plant)
Agrobacterium infection process Negative pressure Negative pressure Positive pressure
Biomass 50000Kg/ Ha / year 50000Kg / Ha / year 7 million Kg / Ha / year
Source of viral elements Tobacco Mosaic Virus Cowpea Mosaic Virus Maize Streak Virus or any monocot infectable virus
[0114] The following examples are offered by way of illustration and not by way of limitation.
[0115] EXAMPLE 1: MAIZE GROWTH SYSTEM
[0116] Availability of ‘hydroponic fodder maize growth system’ is widely adopted by farmers (FIG.8). It enables production of enormous biomass in small area (7000 Tons fresh weight/ Ha / year) and thus ideally suited for recombinant protein production in a ‘small contained facility’.
[0117] Further, these corn seedlings can be infected with Agrobacterium in HTP manner if a hydraulic pressure machine is employed (FIG.8) shows a ‘hydroponic corn fodder production unit’ in stacked trays.
[0118] EXAMPLE 2: PRODUCTION OF RECOMBINANAT PROTEIN IN PLANT
[0119] Recombinant protein production in plants is well known to be cost effective and very easy to scale up at short notice. However, transgenic plants are highly controversial. The blue and the fluorescent green color shown in FIG.6 and FIG.7 are the result of the production of recombinant protein in corn seedlings transiently. Our process of recombinant protein expression does not involve creation of transgenic plants or GM plants and we can make the corn plants produce our proteins in a short period of time - just for a few days. The reproductive parts in these seedlings like the pollen, egg or the seed are not involved and that is the difference from permanently altered GM plants.
[0120] Such transient expression is well known in tobacco. The tobacco system recombinant protein production has been commercialized in western countries. However, transient expression has never been shown in monocot plants like corn.
[0121] The new method we have developed is based on our discovery that the base of leaves of corn seedlings, hidden inside the leaf whorls are highly competent for Agrobacterium mediated transformation. Doing so, a normal non-transgenic seedling temporarily begins to produce the protein of our interest.
[0122] POSITIVE PRESSURE VS NEGATIVE PRESSURE TO DELIVER AGROBACTERIUM:
[0123] Use of negative pressure to deliver Agrobacterium to dicot plants, specifically tobacco leaves, is widely used (FIG.1 and 2). This new method contrasts with the existing, widely practiced usage of vacuum infiltration (negative pressure) for Agrobacterium mediated transient expression of foreign proteins in plants which is less efficient and works only in a few plants like Nicotiana (tobacco). At present the several prior arts use this method for pharmaceutical protein production in plants (Table 2). Syringe infusion of Agrobacterium is practiced in academic labs for small scale expression in plants. However, this is labour intensive and not suitable for industrial scale production of desired proteins.
[0124] SYSTEM FOR INFILTRATING A PLANT WITH AN AGENT:
[0125] According to an aspect of the disclosed embodiment, a system for infiltrating a plant with an agent, wherein the system comprises, (i) a closed container that receives the plant to be infiltrated with the agent, wherein the agent is filled in the container to completely or partially cover the plant part; (ii) an airtight piston configured to applying a positive pressure ranging from 5-15 psi from top of the container for about 1 minute to 24 hours, wherein the application of positive pressure facilitates the integration of the agent into the plant part.
[0126] According to one more embodiment, wherein the positive pressure is applied using a hydraulic unit or a pneumatic unit.
[0127] According to another embodiment, wherein the agent is a suspension of an Agrobacterium strain comprising a cell density of about 0.1-2.0 OD, wherein the Agrobacterium strain comprising a nucleotide sequence encoding the polypeptide.
[0128] According to yet another embodiment, wherein the positive pressure facilitates the transformation of cells with the nucleotide sequence encoding the polypeptide through filling intercellular spaces of the plant with the suspension.
[0129] ADVANTAGES OF THE DISCLOSED EMBODIMENT:
[0130] The advantage of corn seedlings system is the possibility of massive biomass production in a small area up to 7 million Kgs / Ha / year. This is possible because, seedlings are raised at very high density in multiple racks as shown in the Figure 6 and each cycle merely takes 2-weeks. This intensive biomass production system is being used by farmers to produce fodder.
[0131] Further, one can boost recombinant protein production level through employing viral genetic elements viz., the replicase and the movement proteins. This viral genetic element strategy has shown to result in > 30-fold increase in recombinant protein production in tobacco using Tobacco Mosaic Viral elements. We intend to use Maize Streak Virus elements or other virus elements to boost protein production.
[0132] USES, APPLICATIONS AND BENEFITS OF THE DISCLOSED EMBODIMENT:
[0133] Efficient and high throughput delivery of agent to the desired but inaccessible regions of the plants. Particularly, the method can be used for the following different applications:
[0134] Gene transfer to plants.
[0135] Transient gene expression in monocot plants (promoter studies, flower induction).
[0136] Recombinant protein production platform.
[0137] Colchicine application to the meristem of monocot plants for chromosome doubling specially in di-haploid production.
[0138] BEST MODE TO PRACTICE:
[0139] Examples of the modes to practice in commercial sense are listed below
[0140] High throughput (HTP) inoculation of plants specifically monocot plants where no method currently exists for recombinant protein production.
[0141] Delivery of gene editing agents or gene silencing agents (like oligonucleotides) to the meristem or to other regenerable cells of plants.
[0142] HTP Agrobacterium infection for gene or genetic element functional analysis
[0143] Efficient doubling of haploid plants e.g., corn and rice through delivery of colchicine to the meristem.
[0144] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[0145] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0146] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosed embodiment. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0147] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the disclosed embodiment are presented for example purposes only. The disclosed embodiment is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
[0148] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
[0149] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[0150] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0151] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosed embodiment. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0152] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the disclosed embodiment are presented for example purposes only. The disclosed embodiment is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
[0153] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
,CLAIMS:I/WE CLAIM:
1. A method for infiltrating a plant with an agent, wherein the method comprising the steps of: (i) providing the plant in a closed container; (ii) providing the agent into the container to completely or partially cover the plant; and (iii) applying a positive pressure ranging from 5-15 psi from top of the container using an airtight piston for about 1 minute to 24 hours, wherein the application of positive pressure facilitates the infiltration of the agent into the intercellular spaces or the inaccessible regions comprising meristem of the plant.

2. The method as claimed in claim 1, wherein the positive pressure is applied using a hydraulic unit or a pneumatic unit.

3. The method as claimed in claim 1, wherein the agent is a suspension of an Agrobacterium strain comprising a cell density of about 0.1-2.0 OD, wherein the Agrobacterium strain comprising a Ti plasmid bearing a nucleotide sequence encoding a polypeptide.

4. The method as claimed in claim 3, wherein the positive pressure facilitates the delivery of the Agrobacterium strain into the cells through filling intercellular spaces of the plant with the suspension.

5. The method as claimed in claim 3, wherein the polypeptide is a recombinant protein comprising trypsin, insulin, antibodies or vaccine proteins.

6. The method as claimed in claim 3, wherein the nucleotide sequence encoding the polypeptide is cloned in a binary vector comprising a T-DNA region that comprises a promoter and a reporter gene, wherein the binary vector comprises pCAMBIA1300, pCAMBIA1305.

7. The method as claimed in claim 1, wherein the agent is a chromosome doubling agent, wherein the chromosome doubling agent is a colchicine solution.

8. The method as claimed in claim 8, wherein the colchicine is delivered into the meristem region of the plant.

9. The method as claimed in claim 1, wherein the agent is a gene editing agent or gene silencing agent comprising 10-10,000 oligonucleotides.
10. The method as claimed in claim 1, wherein plant is oriented in an upright or an inverted position.

11. A system for infiltrating a plant with an agent, wherein the system comprises, (i) a closed container that receives the plant to be infiltrated with the agent, wherein the agent is filled in the container to completely or partially cover the plant part; (ii) an airtight piston configured to applying a positive pressure ranging from 5- 15 psi from top of the container for about 1 minute to 24 hours, wherein the application of positive pressure facilitates infiltration of the agent into the intercellular spaces and inaccessible regions comprising meristem of the plant part.

12. The system as claimed in claim 11, wherein the positive pressure is applied using a hydraulic unit or a pneumatic unit.

13. The system as claimed in claim 11, wherein the agent is a suspension of an Agrobacterium strain comprising a cell density of about 0.1-2.0 OD, wherein the Agrobacterium strain comprising a Ti plasmid bearing a nucleotide sequence encoding the polypeptide.

14. The system as claimed in claim 11, wherein the positive pressure facilitates the delivery of the Agrobacterium strain into the cells through filling intercellular spaces of the plant with the suspension.

Dated this 14th day of September, 2023
Number range CHAPTER Signature.......................................................
(LIPIKA SAHOO)
Registration Number: IN/PA-2467
Agent for Applicant
This document is signed with the digital signature of Patent Agent for the Applicant
LIPIKA SAHOO (IN/PA-2467)