1. A system for enhancing abiotic stress tolerance in crops, said system comprising: a genetic modification module configured to introduce one or more abiotic stress tolerance genes into crop plants; a transformation module designed to insert said abiotic stress tolerance genes into target crop plant cells; a selection module for isolating transformed crop plant cells exhibiting abiotic stress tolerance; a regeneration module for cultivating whole plants from said transformed crop plant cells; a verification module for assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants; and a monitoring module configured to evaluate the abiotic stress tolerance of said regenerated plants under controlled and field conditions.

2. The system of claim 1, wherein said genetic modification module comprises CRISPR-Cas9 technology for targeted gene editing.

3. The system of claim 1, wherein said genetic modification module is configured to introduce multiple abiotic stress tolerance genes simultaneously.

4. The system of claim 1, wherein said transformation module employs Agrobacterium-mediated transformation techniques.

5. The system of claim 1, wherein said transformation module utilizes biolistic transformation methods for gene insertion.

6. The system of claim 1, wherein said selection module employs antibiotic or herbicide resistance markers for isolating said transformed crop plant cells.

7. The system of claim 1, wherein said regeneration module includes tissue culture techniques for regenerating whole plants from said transformed crop plant cells.

8. The system of claim 1, wherein said verification module utilizes quantitative PCR and RNA sequencing for assessing said gene expression.

9. The system of claim 1, wherein said monitoring module is integrated with remote sensing technology for real-time assessment of abiotic stress tolerance in field conditions.

10. A method for enhancing abiotic stress tolerance in crops, said method comprising the steps of: introducing one or more abiotic stress tolerance genes into target crop plant cells; transforming said target crop plant cells with said abiotic stress tolerance genes; isolating transformed crop plant cells exhibiting abiotic stress tolerance; regenerating whole plants from said transformed crop plant cells; assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants; and evaluating the abiotic stress tolerance of said regenerated plants under controlled and field conditions. SYSTEM FOR ENHANCING ABIOTIC STRESS TOLERANCE IN CROPS Abstract The present disclosure provides a system for enhancing abiotic stress tolerance in crops, said system comprising a genetic modification module configured to introduce one or more abiotic stress tolerance genes into crop plants, a transformation module designed to insert said abiotic stress tolerance genes into target crop plant cells, a selection module for isolating transformed crop plant cells exhibiting abiotic stress tolerance, a regeneration module for cultivating whole plants from said transformed crop plant cells, a verification module for assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants, and a monitoring module configured to evaluate the abiotic stress tolerance of said regenerated plants under controlled and field conditions. Fig. 1 , Claims:Claims :

1. A system for enhancing abiotic stress tolerance in crops, said system comprising: a genetic modification module configured to introduce one or more abiotic stress tolerance genes into crop plants; a transformation module designed to insert said abiotic stress tolerance genes into target crop plant cells; a selection module for isolating transformed crop plant cells exhibiting abiotic stress tolerance; a regeneration module for cultivating whole plants from said transformed crop plant cells; a verification module for assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants; and a monitoring module configured to evaluate the abiotic stress tolerance of said regenerated plants under controlled and field conditions.

2. The system of claim 1, wherein said genetic modification module comprises CRISPR-Cas9 technology for targeted gene editing.

3. The system of claim 1, wherein said genetic modification module is configured to introduce multiple abiotic stress tolerance genes simultaneously.

4. The system of claim 1, wherein said transformation module employs Agrobacterium-mediated transformation techniques.

5. The system of claim 1, wherein said transformation module utilizes biolistic transformation methods for gene insertion.

6. The system of claim 1, wherein said selection module employs antibiotic or herbicide resistance markers for isolating said transformed crop plant cells.

7. The system of claim 1, wherein said regeneration module includes tissue culture techniques for regenerating whole plants from said transformed crop plant cells.

8. The system of claim 1, wherein said verification module utilizes quantitative PCR and RNA sequencing for assessing said gene expression.

9. The system of claim 1, wherein said monitoring module is integrated with remote sensing technology for real-time assessment of abiotic stress tolerance in field conditions.

10. A method for enhancing abiotic stress tolerance in crops, said method comprising the steps of: introducing one or more abiotic stress tolerance genes into target crop plant cells; transforming said target crop plant cells with said abiotic stress tolerance genes; isolating transformed crop plant cells exhibiting abiotic stress tolerance; regenerating whole plants from said transformed crop plant cells; assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants; and evaluating the abiotic stress tolerance of said regenerated plants under controlled and field conditions.

Description:

SYSTEM FOR ENHANCING ABIOTIC STRESS TOLERANCE IN CROPS
Field of the Invention
[0001] The present disclosure generally relates to agricultural biotechnology. Further, the present disclosure particularly relates to a system for enhancing abiotic stress tolerance in crops.
Background
[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] In recent years, advancements in agricultural biotechnology have become increasingly important. Such advancements address various challenges faced by modern agriculture. Among these challenges, abiotic stress in crops is a significant concern. Abiotic stress refers to the negative impact of non-living factors such as drought, salinity, extreme temperatures, and nutrient deficiencies on crop productivity. The effects of abiotic stress on crops lead to reduced yield, poor quality, and compromised food security. Enhancing the abiotic stress tolerance of crops is a crucial area of research and development.
[0004] Various conventional methods have been utilized to enhance abiotic stress tolerance in crops. Traditional breeding techniques involve selecting and crossbreeding crop varieties exhibiting desirable traits. Such methods, however, are time-consuming and may not always yield satisfactory results due to the complex nature of stress tolerance traits. Additionally, the unpredictability of environmental conditions further complicates the effectiveness of traditional breeding methods in conferring stress tolerance.
[0005] Another well-known technique involves the application of chemical treatments to enhance stress tolerance in crops. The use of plant growth regulators, osmoprotectants, and anti-transpirants has been explored. However, chemical treatments often result in limited success and can have adverse environmental and health effects. The reliance on chemical treatments is also not sustainable in the long term, prompting the need for more reliable and eco-friendly approaches.
[0006] The other most popular approach for enhance abiotic stress tolerance in crops is Marker Assisted Selection (MAS). Abiotic stresses such as heat, drought, salinity etc. are complex in nature and they are controlled by multiple genes (Polygenic traits, QTLs). Multiple genes are distributed on different chromosomes. For QTLs mapping, there is requirement of creation of mapping population, availability of DNA based molecular markers and recording of phenotypic traits by precisely manner.
[0007] Genes and molecular markers associated with stress tolerance mechanisms are crucial for improving crop productivity under high temperature, drought, salinity and waterlogging environments. However, the precision of identification of these markers through genomic tools largely depend on the phenotypic data on plant responses to stress which is often influenced by various environmental factors under field conditions.
[0008] Further, genetic modification techniques have been developed to introduce abiotic stress tolerance genes into crop plants. For instance, the utilization of transgenic approaches to incorporate genes responsible for stress-responsive pathways has shown promise. However, the integration of such genes into crop genomes often encounters challenges related to gene expression stability, off-target effects, and regulatory concerns. Moreover, public acceptance of genetically modified crops remains a contentious issue, affecting the widespread adoption of such technologies.
[0009] Moreover, advancements in plant tissue culture techniques have enabled the regeneration of whole plants from transformed cells. Plant tissue culture involves the cultivation of plant cells, tissues, or organs under controlled conditions to develop complete plants. This technique facilitates the propagation of genetically modified plants. However, the efficiency of plant regeneration and the stability of introduced traits remain critical challenges. Additionally, the verification of gene expression and trait manifestation in regenerated plants necessitates rigorous testing and validation protocols.
[00010] Furthermore, high-throughput screening methods have been employed to evaluate the abiotic stress tolerance of genetically modified crops. Such methods involve the use of automated systems to assess various physiological and biochemical parameters under controlled and field conditions. While high-throughput screening provides valuable insights, the accuracy and reliability of such evaluations can be influenced by environmental variability and experimental limitations. The need for comprehensive monitoring and evaluation systems to ensure the consistent performance of stress-tolerant crops is evident.
[00011] In light of the above discussion, there exists an urgent need for solutions that overcome the problems associated with conventional systems and/or techniques for enhancing abiotic stress tolerance in crops.

Summary
[00012] The following presents a simplified summary of various aspects of this disclosure in order to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects, and is intended to neither identify key or critical elements nor delineate the scope of such aspects. Its purpose is to present some concepts of this disclosure in a simplified form as a prelude to the more detailed description that is presented later.
[00013] The following paragraphs provide additional support for the claims of the subject application.
[00014] In a first aspect, the present disclosure provides a system for enhancing abiotic stress tolerance in crops, said system comprising a genetic modification module configured to introduce one or more abiotic stress tolerance genes into crop plants, a transformation module designed to insert said abiotic stress tolerance genes into target crop plant cells, a selection module for isolating transformed crop plant cells exhibiting abiotic stress tolerance, a regeneration module for cultivating whole plants from said transformed crop plant cells, a verification module for assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants, and a monitoring module configured to evaluate the abiotic stress tolerance of said regenerated plants under controlled and field conditions. Such a system enables enhanced crop resilience under abiotic stress conditions, leading to improved crop survival and yield. Furthermore, said system facilitates precise gene insertion and efficient selection of transformed plants.
[00015] The system of the present disclosure, wherein said genetic modification module comprises CRISPR-Cas9 technology for targeted gene editing. Such a system enables accurate and efficient editing of specific genes associated with abiotic stress tolerance. Additionally, said system reduces off-target effects, thereby increasing the precision of genetic modifications.
[00016] The system of the present disclosure, wherein said genetic modification module is configured to introduce multiple abiotic stress tolerance genes simultaneously. Such a system enables the simultaneous enhancement of multiple stress tolerance traits in crops, improving overall plant resilience. Furthermore, said system reduces the time required for developing stress-tolerant crop varieties.
[00017] The system of the present disclosure, wherein said transformation module employs Agrobacterium-mediated transformation techniques. Such a system enables the stable integration of abiotic stress tolerance genes into the crop plant genome. Moreover, said system is well-suited for dicotyledonous plants.
[00018] The system of the present disclosure, wherein said transformation module utilizes biolistic transformation methods for gene insertion. Such a system enables the direct delivery of abiotic stress tolerance genes into plant cells. Furthermore, said system is effective for monocotyledonous plants and plants recalcitrant to Agrobacterium-mediated transformation.
[00019] The system of the present disclosure, wherein said selection module employs antibiotic or herbicide resistance markers for isolating said transformed crop plant cells. Such a system enables the efficient identification and selection of transformed cells exhibiting abiotic stress tolerance. Moreover, said system reduces the time and resources required for screening transformed cells.
[00020] The system of the present disclosure, wherein said regeneration module includes tissue culture techniques for regenerating whole plants from said transformed crop plant cells. Such a system enables the efficient regeneration of whole plants from single transformed cells. Furthermore, said system ensures the development of healthy and viable plants.
[00021] The system of the present disclosure, wherein said verification module utilizes quantitative PCR and RNA sequencing for assessing said gene expression. Such a system enables the accurate quantification of gene expression levels in regenerated plants. Additionally, said system provides insights into the molecular mechanisms underlying abiotic stress tolerance.
[00022] The system of the present disclosure, wherein said monitoring module is integrated with remote sensing technology for real-time assessment of abiotic stress tolerance in field conditions. Such a system enables continuous and non-destructive monitoring of plant stress responses. Furthermore, said system facilitates timely interventions to mitigate stress impacts on crop performance.
[00023] In a second aspect, the present disclosure provides a method for enhancing abiotic stress tolerance in crops, said method comprising the steps of introducing one or more abiotic stress tolerance genes into target crop plant cells, transforming said target crop plant cells with said abiotic stress tolerance genes, isolating transformed crop plant cells exhibiting abiotic stress tolerance, regenerating whole plants from said transformed crop plant cells, assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants, and evaluating the abiotic stress tolerance of said regenerated plants under controlled and field conditions. Such a method enables the development of crop plants with improved tolerance to abiotic stresses, thereby enhancing crop productivity and sustainability. Moreover, said method provides a systematic approach to integrating and validating stress tolerance traits in crops.
Brief Description of the Drawings
[00024] The features and advantages of the present disclosure would be more clearly understood from the following description taken in conjunction with the accompanying drawings in which:
[00025] FIG. 1 illustrates a system for enhancing abiotic stress tolerance in crops, in accordance with the embodiments of the present disclosure.
[00026] FIG. 2 illustrates a method for enhancing abiotic stress tolerance in crops, in accordance with the embodiments of the present disclosure.
Detailed Description
[00027] In the following detailed description of the invention, reference is made to the accompanying drawings that form a part hereof, and in which is shown, by way of illustration, specific embodiments in which the invention may be practiced. In the drawings, like numerals describe substantially similar components throughout the several views. These embodiments are described in sufficient detail to claim those skilled in the art to practice the invention. Other embodiments may be utilized and structural, logical, and electrical changes may be made without departing from the scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims and equivalents thereof.
[00028] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[00029] Pursuant to the "Detailed Description" section herein, whenever an element is explicitly associated with a specific numeral for the first time, such association shall be deemed consistent and applicable throughout the entirety of the "Detailed Description" section, unless otherwise expressly stated or contradicted by the context.
[00030] FIG. 1 illustrates a system for enhancing abiotic stress tolerance in crops, in accordance with the embodiments of the present disclosure. The system for enhancing abiotic stress tolerance in crops comprises several interconnected components designed to introduce and evaluate abiotic stress tolerance genes within crop plants. The genetic modification module is configured to introduce one or more abiotic stress tolerance genes into crop plants. This genetic modification module employs a series of genetic engineering techniques, including the use of vectors, promoters, and other regulatory elements, to ensure the proper insertion and expression of the target genes within the plant genome. The genetic modification module involves the preparation of gene constructs that contain the desired abiotic stress tolerance genes, along with appropriate regulatory sequences to drive their expression in crop plants. The genetic constructs are carefully designed to include elements such as promoters, enhancers, terminators, and selectable markers to facilitate the identification and selection of successfully transformed cells. The genetic modification module ensures that the target genes are accurately and stably integrated into the plant genome, enabling the crop plants to express the desired abiotic stress tolerance traits.
[00031] The transformation module is designed to insert said abiotic stress tolerance genes into target crop plant cells. This transformation module utilizes various techniques, such as Agrobacterium-mediated transformation, particle bombardment, and electroporation, to introduce the genetic constructs containing the abiotic stress tolerance genes into the crop plant cells. The transformation process involves the preparation of plant tissues or cells, such as leaf discs, embryogenic calli, or protoplasts, which are then exposed to the genetic constructs in the presence of a suitable transformation medium. The transformation module ensures that the target genes are effectively delivered into the plant cells, where they can be integrated into the plant genome. The transformation module includes optimization of transformation conditions, such as the concentration of the genetic constructs, the duration of exposure, and the selection of appropriate plant tissues, to maximize the efficiency of gene transfer and minimize damage to the plant cells.
[00032] The selection module is responsible for isolating transformed crop plant cells exhibiting abiotic stress tolerance. This selection module employs a series of selection processes to identify and isolate plant cells that have successfully incorporated the abiotic stress tolerance genes and are expressing the desired traits. The selection process typically involves the use of selectable markers, such as antibiotic or herbicide resistance genes, which allow the transformed cells to survive and proliferate in the presence of the corresponding selection agents. The selection module includes the preparation of selection media containing the appropriate selection agents and the cultivation of the transformed cells on such media. The selection module also involves the screening of transformed cells for the presence and expression of the abiotic stress tolerance genes using molecular techniques, such as PCR, Southern blotting, and qRT-PCR. The selection module ensures that only the transformed cells exhibiting the desired abiotic stress tolerance traits are selected for further regeneration and analysis.
[00033] The regeneration module is designed for cultivating whole plants from said transformed crop plant cells. This regeneration module involves the use of tissue culture techniques to induce the transformed cells to develop into complete plants. The regeneration process includes the preparation of suitable culture media containing the necessary nutrients, hormones, and growth regulators to support the development and differentiation of the transformed cells. The regeneration module involves the transfer of the selected transformed cells to regeneration media, where they are induced to form shoots, roots, and ultimately whole plants. The regeneration module also includes the optimization of culture conditions, such as temperature, light, and humidity, to promote the healthy growth and development of the regenerated plants. The regeneration module ensures that the transformed cells are able to develop into viable, fertile plants that can be evaluated for their abiotic stress tolerance.
[00034] The verification module is responsible for assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants. This verification module employs a series of molecular and biochemical techniques to confirm that the regenerated plants contain the introduced genes and are expressing the desired abiotic stress tolerance traits. The verification process includes the extraction of DNA, RNA, and proteins from the regenerated plants, followed by the analysis of these biomolecules using techniques such as PCR, Southern blotting, qRT-PCR, and Western blotting. The verification module also involves the assessment of the physiological and biochemical responses of the regenerated plants to abiotic stress conditions, such as drought, salinity, and extreme temperatures. The verification module ensures that the regenerated plants exhibit the desired abiotic stress tolerance traits and that the introduced genes are functioning as intended.
[00035] The monitoring module is configured to evaluate the abiotic stress tolerance of said regenerated plants under controlled and field conditions. This monitoring module involves the systematic assessment of the performance of the regenerated plants under various abiotic stress conditions in both controlled environments, such as growth chambers and greenhouses, and in field trials. The monitoring process includes the design and implementation of experimental protocols to evaluate the growth, development, and yield of the regenerated plants under abiotic stress conditions. The monitoring module involves the collection and analysis of data on various physiological, biochemical, and agronomic parameters, such as plant height, biomass, chlorophyll content, osmolyte accumulation, and yield components. The monitoring module also includes the statistical analysis of the data to determine the significance of the observed differences between the transformed and non-transformed plants. The monitoring module ensures that the regenerated plants are thoroughly evaluated for their abiotic stress tolerance and that their performance is validated under real-world conditions.
[00036] In an embodiment, the genetic modification module comprises CRISPR-Cas9 technology for targeted gene editing. CRISPR-Cas9 is a precise tool for editing genomes, allowing researchers to alter DNA sequences and modify gene function. The technology utilizes a guide RNA (gRNA) to direct the Cas9 enzyme to a specific location within the genome, where it creates a double-strand break. The cell's natural repair mechanisms then repair the break, enabling the introduction of desired genetic changes. The genetic modification module includes the design and synthesis of specific gRNAs that target the abiotic stress tolerance genes. Once the CRISPR-Cas9 components are delivered into the crop plant cells,

the Cas9 enzyme, guided by the gRNA, induces targeted cuts in the DNA. This allows for the insertion, deletion, or modification of the abiotic stress tolerance genes, resulting in the enhancement of stress tolerance traits in the crop plants. The use of CRISPR-Cas9 technology within the genetic modification module significantly increases the precision and efficiency of gene editing, ensuring that the target genes are accurately modified and integrated within the plant genome.
[00037] In an embodiment, the genetic modification module is configured to introduce multiple abiotic stress tolerance genes simultaneously. This configuration involves the use of multi-gene constructs or stacked gene cassettes that contain several abiotic stress tolerance genes. Each gene within the construct is flanked by appropriate regulatory elements, such as promoters and terminators, to ensure their independent expression within the plant cells. The multi-gene constructs are designed to confer a broad spectrum of stress tolerance traits, such as drought, salinity, and temperature resistance, by incorporating genes that are known to confer tolerance to these specific stresses. The genetic modification module employs techniques such as plasmid-based transformation, viral vectors, or CRISPR-based multiplexing to deliver the multi-gene constructs into the crop plant cells. The simultaneous introduction of multiple abiotic stress tolerance genes enhances the overall stress resilience of the plants by enabling them to withstand various environmental stresses concurrently. This multi-gene approach within the genetic modification module allows for the development of crop plants with comprehensive abiotic stress tolerance capabilities, thereby improving their growth and yield under challenging environmental conditions.
[00038] In an embodiment, the transformation module employs Agrobacterium-mediated transformation techniques. Agrobacterium tumefaciens, a soil bacterium, is used to transfer genetic material into plant cells. The transformation process begins with the preparation of Agrobacterium strains containing the desired abiotic stress tolerance genes within a T-DNA region of a binary vector. Plant tissues, such as leaf discs or embryogenic calli, are then co-cultivated with the Agrobacterium strains under suitable conditions that promote the transfer of the T-DNA into the plant genome. During co-cultivation, the Agrobacterium attaches to the plant cell surfaces and transfers the T-DNA, which integrates into the plant genome, carrying the abiotic stress tolerance genes. Following the co-cultivation period, the plant tissues are transferred to a selection medium containing specific antibiotics or herbicides to select for successfully transformed cells. The use of Agrobacterium-mediated transformation in the transformation module is advantageous due to its high efficiency, the stable integration of transgenes, and the ability to transform a wide variety of crop species. This method is particularly effective for dicotyledonous plants and certain monocotyledonous plants, providing a robust and reliable means of introducing abiotic stress tolerance genes into crop plants.
[00039] In an embodiment, the transformation module utilizes biolistic transformation methods for gene insertion. Biolistic transformation, also known as particle bombardment, involves the delivery of genetic material into plant cells using high-velocity microprojectiles. The process begins with the coating of gold or tungsten particles with the DNA constructs containing the abiotic stress tolerance genes. These DNA-coated particles are then accelerated using a gene gun or particle delivery system to penetrate the plant cell walls and membranes, delivering the genetic material into the cells. The plant tissues, such as leaves, embryogenic calli, or meristems, are placed on a target platform within the biolistic device, and the particle bombardment process is carried out under vacuum conditions to enhance particle penetration. Once inside the plant cells, the DNA constructs integrate into the plant genome, leading to the expression of the abiotic stress tolerance genes. The biolistic transformation method is versatile and can be used for a wide range of plant species, including those that are recalcitrant to Agrobacterium-mediated transformation. This method is particularly useful for monocotyledonous plants, such as cereals and grasses, providing an alternative and effective means of introducing abiotic stress tolerance genes into crop plants.
[00040] In an embodiment, the selection module employs antibiotic or herbicide resistance markers for isolating transformed crop plant cells. The selection process begins with the introduction of selectable marker genes, which confer resistance to specific antibiotics or herbicides, into the genetic constructs alongside the abiotic stress tolerance genes. After the transformation process, the plant tissues or cells are cultured on a selection medium containing the corresponding antibiotic or herbicide. Only those cells that have successfully integrated the genetic constructs, including the selectable marker genes, are able to survive and proliferate on the selection medium. Commonly used selectable markers include genes conferring resistance to antibiotics such as kanamycin, hygromycin, or spectinomycin, and herbicides such as glyphosate or phosphinothricin. The selection module involves the careful preparation of selection media with appropriate concentrations of the selection agents to ensure effective selection of transformed cells while minimizing non-specific effects on plant growth. The selected cells are then further cultured and propagated to regenerate whole plants. The use of antibiotic or herbicide resistance markers in the selection module provides a reliable and efficient means of isolating transformed crop plant cells that contain the desired abiotic stress tolerance genes.
[00041] In an embodiment, the regeneration module includes tissue culture techniques for regenerating whole plants from said transformed crop plant cells. Tissue culture techniques involve the cultivation of plant cells, tissues, or organs under sterile conditions on a nutrient medium. The regeneration process starts with the transfer of selected transformed cells to a suitable culture medium that supports cell division and differentiation. The medium typically contains a balanced mix of macronutrients, micronutrients, vitamins, hormones, and growth regulators. The transformed cells first undergo callus formation, where they proliferate and form an undifferentiated mass of cells. Subsequently, the calli are transferred to a shoot induction medium containing cytokinin hormones that promote the formation of shoots. Once shoots develop, they are transferred to a root induction medium containing auxin hormones to facilitate root development. The regenerated plantlets are then acclimatized to ex vitro conditions by gradually exposing them to the natural environment. Tissue culture techniques ensure that the regenerated plants retain the genetic modifications introduced during the transformation process and exhibit the desired abiotic stress tolerance traits. The regeneration module provides a controlled and reproducible method for developing whole plants from transformed cells, enabling the evaluation and utilization of genetically modified crops.
[00042] In an embodiment, the verification module utilizes quantitative PCR (qPCR) and RNA sequencing for assessing gene expression. Quantitative PCR is a sensitive technique used to quantify the amount of specific DNA or RNA sequences in a sample. In this module, qPCR is employed to measure the expression levels of the abiotic stress tolerance genes in the regenerated plants. RNA is first extracted from the plant tissues, converted into complementary DNA (cDNA) using reverse transcription, and then subjected to qPCR analysis using gene-specific primers. The qPCR results provide quantitative data on the expression levels of the target genes, allowing for the assessment of their transcriptional activity. Additionally, RNA sequencing (RNA-seq) is utilized to analyze the entire transcriptome of the regenerated plants. RNA-seq involves the extraction of total RNA, preparation of RNA libraries, and sequencing using high-throughput sequencing technologies. The resulting data is analyzed to identify differentially expressed genes and to provide a comprehensive overview of the gene expression profiles. The verification module ensures that the introduced abiotic stress tolerance genes are actively expressed in the regenerated plants and that the expected molecular responses to abiotic stress are observed.
[00043] In an embodiment, the monitoring module is integrated with remote sensing technology for real-time assessment of abiotic stress tolerance in field conditions. Remote sensing technology involves the use of sensors and imaging devices to collect data on various plant physiological and morphological traits from a distance. In this module, remote sensing tools such as multispectral and hyperspectral cameras, thermal cameras, and LiDAR sensors are deployed to monitor the performance of the regenerated plants under field conditions. These sensors capture data on parameters such as canopy temperature, chlorophyll fluorescence, spectral reflectance, and plant height, which are indicative of the plants' abiotic stress tolerance. The collected data is transmitted to a central processing unit, where it is analyzed using advanced algorithms to assess the stress tolerance levels of the plants in real-time. The integration of remote sensing technology in the monitoring module allows for continuous, non-destructive monitoring of large-scale field trials, providing valuable insights into the plants' responses to environmental stresses. This approach enables the rapid identification of high-performing genotypes and supports the development of stress-resilient crop varieties for agricultural applications.
[00044] FIG. 2 illustrates a method 200 for enhancing abiotic stress tolerance in crops, in accordance with the embodiments of the present disclosure. At step 202, one or more abiotic stress tolerance genes are introduced into target crop plant cells, wherein such genes are selected based on their ability to confer resistance to abiotic stress factors such as drought, salinity, and extreme temperatures. At step 204, the target crop plant cells are transformed with the introduced abiotic stress tolerance genes through techniques such as Agrobacterium-mediated transformation, gene gun, or CRISPR-Cas9, ensuring stable integration into the plant genome. At step 206, transformed crop plant cells exhibiting abiotic stress tolerance are isolated by using selective markers or screening assays, thereby enabling the identification of successfully modified cells. At step 208, whole plants are regenerated from the isolated transformed crop plant cells through tissue culture methods, including callus induction, shoot regeneration, and root formation, ensuring the development of fully functional plants. At step 210, the presence and expression of abiotic stress tolerance genes in regenerated plants are assessed using molecular techniques such as PCR, qRT-PCR, and Southern blotting, confirming successful genetic modification. At step 212, the abiotic stress tolerance of regenerated plants is evaluated under controlled and field conditions by exposing the plants to various abiotic stress factors and measuring physiological, biochemical, and yield-related parameters to determine enhanced tolerance.
[00045] Example embodiments herein have been described above with reference to block diagrams and flowchart illustrations of methods and apparatuses. It will be understood that each block of the block diagrams and flowchart illustrations, and combinations of blocks in the block diagrams and flowchart illustrations, respectively, can be implemented by various means including hardware, software, firmware, and a combination thereof. For example, in one embodiment, each block of the block diagrams and flowchart illustrations, and combinations of blocks in the block diagrams and flowchart illustrations can be implemented by computer program instructions. These computer program instructions may be loaded onto a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions which execute on the computer or other programmable data processing apparatus create means for implementing the functions specified in the flowchart block or blocks.
[00046] Throughout the present disclosure, the term ‘processing means’ or ‘microprocessor’ or ‘processor’ or ‘processors’ includes, but is not limited to, a general purpose processor (such as, for example, a complex instruction set computing (CISC) microprocessor, a reduced instruction set computing (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, a microprocessor implementing other types of instruction sets, or a microprocessor implementing a combination of types of instruction sets) or a specialized processor (such as, for example, an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a digital signal processor (DSP), or a network processor).
[00047] The term “non-transitory storage device” or “storage” or “memory,” as used herein relates to a random access memory, read only memory and variants thereof, in which a computer can store data or software for any duration.
[00048] Operations in accordance with a variety of aspects of the disclosure is described above would not have to be performed in the precise order described. Rather, various steps can be handled in reverse order or simultaneously or not at all.
[00049] While several implementations have been described and illustrated herein, a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein may be utilized, and each of such variations and/or modifications is deemed to be within the scope of the implementations described herein. More generally, all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific implementations described herein. It is, therefore, to be understood that the foregoing implementations are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, implementations may be practiced otherwise than as specifically described and claimed. Implementations of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

Claims
I/We Claim:
1. A system for enhancing abiotic stress tolerance in crops, said system comprising:
a genetic modification module configured to introduce one or more abiotic stress tolerance genes into crop plants;
a transformation module designed to insert said abiotic stress tolerance genes into target crop plant cells;
a selection module for isolating transformed crop plant cells exhibiting abiotic stress tolerance;
a regeneration module for cultivating whole plants from said transformed crop plant cells;
a verification module for assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants; and
a monitoring module configured to evaluate the abiotic stress tolerance of said regenerated plants under controlled and field conditions.
2. The system of claim 1, wherein said genetic modification module comprises CRISPR-Cas9 technology for targeted gene editing.
3. The system of claim 1, wherein said genetic modification module is configured to introduce multiple abiotic stress tolerance genes simultaneously.
4. The system of claim 1, wherein said transformation module employs Agrobacterium-mediated transformation techniques.
5. The system of claim 1, wherein said transformation module utilizes biolistic transformation methods for gene insertion.
6. The system of claim 1, wherein said selection module employs antibiotic or herbicide resistance markers for isolating said transformed crop plant cells.
7. The system of claim 1, wherein said regeneration module includes tissue culture techniques for regenerating whole plants from said transformed crop plant cells.
8. The system of claim 1, wherein said verification module utilizes quantitative PCR and RNA sequencing for assessing said gene expression.
9. The system of claim 1, wherein said monitoring module is integrated with remote sensing technology for real-time assessment of abiotic stress tolerance in field conditions.
10. A method for enhancing abiotic stress tolerance in crops, said method comprising the steps of:
introducing one or more abiotic stress tolerance genes into target crop plant cells;
transforming said target crop plant cells with said abiotic stress tolerance genes;
isolating transformed crop plant cells exhibiting abiotic stress tolerance;
regenerating whole plants from said transformed crop plant cells;
assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants; and
evaluating the abiotic stress tolerance of said regenerated plants under controlled and field conditions.

SYSTEM FOR ENHANCING ABIOTIC STRESS TOLERANCE IN CROPS
Abstract
The present disclosure provides a system for enhancing abiotic stress tolerance in crops, said system comprising a genetic modification module configured to introduce one or more abiotic stress tolerance genes into crop plants, a transformation module designed to insert said abiotic stress tolerance genes into target crop plant cells, a selection module for isolating transformed crop plant cells exhibiting abiotic stress tolerance, a regeneration module for cultivating whole plants from said transformed crop plant cells, a verification module for assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants, and a monitoring module configured to evaluate the abiotic stress tolerance of said regenerated plants under controlled and field conditions.
Fig. 1

, Claims:Claims
I/We Claim:
1. A system for enhancing abiotic stress tolerance in crops, said system comprising:
a genetic modification module configured to introduce one or more abiotic stress tolerance genes into crop plants;
a transformation module designed to insert said abiotic stress tolerance genes into target crop plant cells;
a selection module for isolating transformed crop plant cells exhibiting abiotic stress tolerance;
a regeneration module for cultivating whole plants from said transformed crop plant cells;
a verification module for assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants; and
a monitoring module configured to evaluate the abiotic stress tolerance of said regenerated plants under controlled and field conditions.
2. The system of claim 1, wherein said genetic modification module comprises CRISPR-Cas9 technology for targeted gene editing.
3. The system of claim 1, wherein said genetic modification module is configured to introduce multiple abiotic stress tolerance genes simultaneously.
4. The system of claim 1, wherein said transformation module employs Agrobacterium-mediated transformation techniques.
5. The system of claim 1, wherein said transformation module utilizes biolistic transformation methods for gene insertion.
6. The system of claim 1, wherein said selection module employs antibiotic or herbicide resistance markers for isolating said transformed crop plant cells.
7. The system of claim 1, wherein said regeneration module includes tissue culture techniques for regenerating whole plants from said transformed crop plant cells.
8. The system of claim 1, wherein said verification module utilizes quantitative PCR and RNA sequencing for assessing said gene expression.
9. The system of claim 1, wherein said monitoring module is integrated with remote sensing technology for real-time assessment of abiotic stress tolerance in field conditions.
10. A method for enhancing abiotic stress tolerance in crops, said method comprising the steps of:
introducing one or more abiotic stress tolerance genes into target crop plant cells;
transforming said target crop plant cells with said abiotic stress tolerance genes;
isolating transformed crop plant cells exhibiting abiotic stress tolerance;
regenerating whole plants from said transformed crop plant cells;
assessing the presence and expression of said abiotic stress tolerance genes in said regenerated plants; and
evaluating the abiotic stress tolerance of said regenerated plants under controlled and field conditions.