“Expression analysis of water stress related genes in Tomato
plants” submitted to the CSIR-NEIST, Jorhat and is a record of an original work done by
me under the guidance of Dr Ratul Saikia, Sr. Principal Scientist of Biological Sciences
And Technology Division(BSTD), CSIR-NEIST.
Expression analysis of water stress related genes in tomato plantRonHazarika
This document summarizes a student's summer training project on analyzing the expression of water stress-related genes in tomato plants. The student isolated RNA from tomato plant leaves under normal and water-stressed conditions, synthesized cDNA, and performed RT-PCR to identify the catalase gene as responsible for the plant's response to water stress. The student gained knowledge about plant stress responses, gene expression analysis techniques, and identified the catalase gene as involved in the tomato plant's response to water logging stress based on differences in gene expression levels under normal and stressed conditions.
Rice stress related gene expression analysis 2019RonHazarika
The study revealed that the proteins seem similar in structure but functionally they are much more diverse. This analysis can help to identify the molecular basis of phenotypic differences and select gene expression targets for in-depth study. The regulation of gene expression in plants, as in other higher eukaryotes, is a subject of daunting complexity.
Rice stress related gene expression analysisRonHazarika
The document summarizes research on stress response proteins in rice (Oryza sativa). It identifies 17 proteins commonly up-regulated and 3 commonly down-regulated in response to drought, heat, and salinity stress. It analyzes the protein with the highest interaction for each group and identifies 10 similar proteins in each family. It examines the proteins' physicochemical properties, 3D structures, and functions in plant defense. The study finds the proteins structurally similar but functionally diverse, concluding they help rice cope with stress through complex regulatory interactions.
Transgenics in biotic stress managementSakthivel R
Transgenic crops can help manage biotic stress by engineering plants to resist pathogens and pests. Bt crops produce Cry toxins from Bacillus thuringiensis that are toxic to insect pests but safe for humans and animals. Bt maize, brinjal, rice and other crops have been engineered with Cry genes to resist key insect pests. Protease inhibitors have also been used to transform plants to interfere with insect digestion. Additionally, genes encoding chitinase and glucanase have been introduced to plants to enhance their resistance to fungal diseases like Rhizoctonia solani. The combined expression of these genes results in more effective prevention of disease development.
Transgenic plants with biotic stress resistanceSakeena Asmi
This document discusses transgenic plants with resistance to biotic stress. It begins by defining biotic stress as damage caused by living organisms like bacteria, viruses, fungi and insects. Developing transgenic plants is presented as a way to improve crop yields by making plants resistant to these stresses. Specific examples of transgenic plants containing genes from Bacillus thuringiensis (Bt) that code for insecticidal proteins are described. Bt genes have been introduced into crops like corn, cotton and potatoes to resist pests like rootworms and Colorado potato beetles. While Bt crops have increased yields, there is a risk of pests developing resistance over time if not managed properly.
APPLICATION OF BIOTECHNOLOGICAL TOOLS IN VEGETABLE IMPROVEMENTshikha singh
This document summarizes M.Sc student Saurabh Singh's seminar presentation on the topic of biotechnology. It defines biotechnology and traces its origins. It describes various biotechnology techniques like tissue culture, genetic engineering, marker assisted selection, and their applications in crop improvement. These techniques help overcome limitations of conventional breeding by allowing precise gene transfer and introducing traits from unrelated species. The document also discusses some challenges of biotechnology like high costs, stability of transgene expression, and potential ecological impacts. It sees opportunities to further develop biotechnology in India with more research investment and scientific capabilities.
This document discusses breeding strategies for abiotic stress tolerance in vegetable crops. It begins by defining different types of environmental stresses plants face, with a focus on abiotic stresses like drought, waterlogging, heat, cold, and salinity. Conventional breeding methods are then outlined, including selection, hybridization, pedigree method, and backcross breeding. Specific strategies for breeding tolerance to drought, salinity, and waterlogging are covered in more detail. Screening criteria and sources of tolerance for different stresses in various vegetable crops are also provided. The document aims to provide an overview of approaches and considerations for developing stress-tolerant vegetable varieties through plant breeding.
This document discusses characteristics and types of mutations, as well as the molecular basis and mechanisms of mutation. Some key points:
1. Mutations are generally recessive and harmful, but a small proportion are beneficial. They are random and recurrent events. Induced mutations often show pleiotropic effects.
2. Types of mutations include point mutations, chromosomal mutations, and cytoplasmic mutations. Chromosomal mutations involve changes in structure like deletions, duplications, inversions, and translocations.
3. Mutation breeding techniques have been used to develop mutants with desirable traits like increased yield, disease resistance, or altered quality attributes in many crop species. Over 2,600 mutant varieties have been developed globally across
Expression analysis of water stress related genes in tomato plantRonHazarika
This document summarizes a student's summer training project on analyzing the expression of water stress-related genes in tomato plants. The student isolated RNA from tomato plant leaves under normal and water-stressed conditions, synthesized cDNA, and performed RT-PCR to identify the catalase gene as responsible for the plant's response to water stress. The student gained knowledge about plant stress responses, gene expression analysis techniques, and identified the catalase gene as involved in the tomato plant's response to water logging stress based on differences in gene expression levels under normal and stressed conditions.
Rice stress related gene expression analysis 2019RonHazarika
The study revealed that the proteins seem similar in structure but functionally they are much more diverse. This analysis can help to identify the molecular basis of phenotypic differences and select gene expression targets for in-depth study. The regulation of gene expression in plants, as in other higher eukaryotes, is a subject of daunting complexity.
Rice stress related gene expression analysisRonHazarika
The document summarizes research on stress response proteins in rice (Oryza sativa). It identifies 17 proteins commonly up-regulated and 3 commonly down-regulated in response to drought, heat, and salinity stress. It analyzes the protein with the highest interaction for each group and identifies 10 similar proteins in each family. It examines the proteins' physicochemical properties, 3D structures, and functions in plant defense. The study finds the proteins structurally similar but functionally diverse, concluding they help rice cope with stress through complex regulatory interactions.
Transgenics in biotic stress managementSakthivel R
Transgenic crops can help manage biotic stress by engineering plants to resist pathogens and pests. Bt crops produce Cry toxins from Bacillus thuringiensis that are toxic to insect pests but safe for humans and animals. Bt maize, brinjal, rice and other crops have been engineered with Cry genes to resist key insect pests. Protease inhibitors have also been used to transform plants to interfere with insect digestion. Additionally, genes encoding chitinase and glucanase have been introduced to plants to enhance their resistance to fungal diseases like Rhizoctonia solani. The combined expression of these genes results in more effective prevention of disease development.
Transgenic plants with biotic stress resistanceSakeena Asmi
This document discusses transgenic plants with resistance to biotic stress. It begins by defining biotic stress as damage caused by living organisms like bacteria, viruses, fungi and insects. Developing transgenic plants is presented as a way to improve crop yields by making plants resistant to these stresses. Specific examples of transgenic plants containing genes from Bacillus thuringiensis (Bt) that code for insecticidal proteins are described. Bt genes have been introduced into crops like corn, cotton and potatoes to resist pests like rootworms and Colorado potato beetles. While Bt crops have increased yields, there is a risk of pests developing resistance over time if not managed properly.
APPLICATION OF BIOTECHNOLOGICAL TOOLS IN VEGETABLE IMPROVEMENTshikha singh
This document summarizes M.Sc student Saurabh Singh's seminar presentation on the topic of biotechnology. It defines biotechnology and traces its origins. It describes various biotechnology techniques like tissue culture, genetic engineering, marker assisted selection, and their applications in crop improvement. These techniques help overcome limitations of conventional breeding by allowing precise gene transfer and introducing traits from unrelated species. The document also discusses some challenges of biotechnology like high costs, stability of transgene expression, and potential ecological impacts. It sees opportunities to further develop biotechnology in India with more research investment and scientific capabilities.
This document discusses breeding strategies for abiotic stress tolerance in vegetable crops. It begins by defining different types of environmental stresses plants face, with a focus on abiotic stresses like drought, waterlogging, heat, cold, and salinity. Conventional breeding methods are then outlined, including selection, hybridization, pedigree method, and backcross breeding. Specific strategies for breeding tolerance to drought, salinity, and waterlogging are covered in more detail. Screening criteria and sources of tolerance for different stresses in various vegetable crops are also provided. The document aims to provide an overview of approaches and considerations for developing stress-tolerant vegetable varieties through plant breeding.
This document discusses characteristics and types of mutations, as well as the molecular basis and mechanisms of mutation. Some key points:
1. Mutations are generally recessive and harmful, but a small proportion are beneficial. They are random and recurrent events. Induced mutations often show pleiotropic effects.
2. Types of mutations include point mutations, chromosomal mutations, and cytoplasmic mutations. Chromosomal mutations involve changes in structure like deletions, duplications, inversions, and translocations.
3. Mutation breeding techniques have been used to develop mutants with desirable traits like increased yield, disease resistance, or altered quality attributes in many crop species. Over 2,600 mutant varieties have been developed globally across
This document summarizes various biotechnological approaches that can be used to improve vegetable crops, including meristem culture, anther culture, embryo rescue, somatic hybridization, and somaclonal variation. Meristem culture is effective for eliminating viruses from plants and can produce virus-free generations. Anther culture can be used for hybrid development, inducing mutations, and generating male plants. Embryo rescue allows the recovery of interspecific hybrids and reduces breeding cycles. Somatic hybridization fuses cells from different species to transfer beneficial traits. Somaclonal variation induces heritable variations during tissue culture that can be selected for traits like stress tolerance. Case studies provide examples of applying each technique for different crops.
The document summarizes a study evaluating 20 cauliflower genotypes for resistance to stalk rot, black rot, and riceyness. The study found that genotypes DC-76 and Pant Shubhra showed the highest resistance to stalk rot, while PSBK-1 and Hermia showed the highest resistance to black rot. Hermia and Pant Shubhra exhibited overall high resistance to both diseases and produced non-ricey curds, making them promising for breeding programs.
This document contains the program information for the 3rd International Conference of Plant Molecular Breeding held from September 5-9, 2010 in Beijing, China. It lists the honorary presidents, organizing committees, program committees, and provides an outline of the conference sessions including plenary lectures on topics like molecular breeding in developing countries, rice functional genomics, and transgenic crop technologies. It also describes concurrent sessions on subjects like molecular breeding for abiotic stress tolerance, gene discovery and function, and molecular breeding for biotic stresses.
i) Breeding crops for resistance to insects, diseases, and abiotic stresses like drought is important to reduce yield losses and costs of control measures.
ii) Mechanisms of resistance include non-preference, antibiosis, tolerance, avoidance, and physiological or biochemical traits like hairiness, toxins, or proline accumulation.
iii) Sources of resistance come from cultivated varieties, germplasm collections, and related wild species, and screening is done under field or controlled conditions.
This document discusses genetically modified drought resistant crops. It begins by defining genetically modified crops as plants modified using genetic engineering to introduce new traits. It then discusses developing drought tolerant crops through conventional breeding and genetic engineering techniques. Conventional breeding is a slow process limited by available genes, while genetic engineering allows introducing genes controlling drought tolerance. The document provides examples of drought tolerance mechanisms in plants and genes introduced through genetic engineering to improve drought resistance in transgenic crops.
Loss due to diseases range from 20 to 30 %, in case of severe infection, total crop may be lost.
Estimated global loss due to insect pests in potential yields of all crops is -14%.
In India losses due to insect pests ranges from 10 to 20 %
Abiotic stresses reduce average yield of crops by upto50% (Bray EA 1997)
Annually about 42% of the crop productivity is lost due to various abiotic stress factors (Oerkeet.al.,1994).
The different types of external stresses that influence the plant growth and development.
These stresses are grouped based on their characters
Biotic
Abiotic
Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress in plants.
This damages the cellular constituents of plants which are associated with a reduction in plant yield.
Crop improvement through genetic engineering can help meet the increasing global demand for food by making crops more resilient to stresses like drought and pests. The process involves isolating genes that confer desirable traits and inserting them into crops using techniques like bacterial infection or particle bombardment. Commercially, early genetically modified crops were made resistant to herbicides or insects. While genetic engineering could boost yields and nutrition, it also raises safety and environmental concerns that need consideration.
1. The seminar discusses developing transgenic plants resistant to insects through the transfer of resistance genes from microorganisms, higher plants, and animals into crop plants.
2. Major objectives of plant biotechnology are to develop plants resistant to biotic and abiotic stresses. Resistance to insects has been achieved by introducing genes encoding Bt toxins from Bacillus thuringiensis and other insecticidal proteins.
3. Useful genes have been isolated from microbes like B. thuringiensis, higher plants like beans and tobacco, and animals like mammals. These genes have been successfully used to engineer insect-resistant crops like cotton, potato, tomato, and tobacco.
The PPT depicts about the biotic and abiotic stresses which are becoming more adverse due to climate change. It breifly discusses about the types of different abiotic and biotic stresses and their impact on agricultural production.
This document discusses genetic engineering for resistance to biotic stress. It defines biotic stress as stress caused by other living organisms that can damage crops. Various techniques for genetically engineering plants for resistance are described, including using genes from Bacillus thuringiensis to make plants resistant to certain insects. Case studies on developing resistance to the European corn borer in Bt corn and developing glyphosate resistance in crops through different strategies are summarized. The development of transgenic crops with traits like insect resistance, herbicide tolerance, and virus resistance are also briefly outlined.
Invitro mutation selection for biotic stresses in Plantsamvannan
In-vitro selection is a somaclonal variation method that uses a selection agent or particular condition to select for somaclones with a desired character. Various mutagens like gamma irradiation, chemicals, and transposons can be used to induce mutations in vitro. Somatic embryogenesis is advantageous for in-vitro selection as it allows treatment of large populations and rapid generation of non-chimeric plants. Chemical mutagens are commonly used for in-vitro selection due to ease of handling. In-vitro selection has been used successfully to obtain disease resistance in various crop species like tobacco, rice, wheat, and potato.
Biotechnological approaches in Host Plant Resistance (HPR)Vinod Pawar
1. The document discusses biotechnological approaches to host plant resistance, including genetic manipulation of secondary plant substances and incorporation of resistant genes in crop varieties.
2. It provides examples of how genetic manipulation can enhance the production of compounds like terpenoids by modifying gene promoters, transcription factors, and subcellular localization.
3. It also gives an example of using marker-assisted backcrossing to introgress three bacterial blight resistance genes into the elite rice variety Samba Mahsuri, resulting in lines with high resistance and yield without compromising quality.
1) Tritrophic interactions involve plants defending against herbivores through direct defenses like trichomes or toxic chemicals, and indirect defenses like emitting volatile compounds to attract natural enemies of the herbivores.
2) Plant traits like trichome density, leaf size, and compactness can impact the ability of rice pests and natural enemies to search for food. Broader leaf width makes it easier for leaf folder larvae to feed and find protection.
3) Rice plants emit volatile compounds in response to damage from insects like stem borers and fall armyworms that attract natural enemies like parasitic wasps to the herbivores.
Genetic Manipulation and Host Plant ResistanceKarl Obispo
This document discusses several topics related to genetically modified crops. It defines transgenic plants as plants that have genes inserted from other species, and cisgenic plants as having genes from the same or closely related species. It provides examples of genetically engineered corn, rice, soybeans, and sugarcane with improved traits like herbicide and pest resistance. The document also discusses methods of genetic engineering like gene guns, electroporation, microinjection, and CRISPR. It outlines benefits of GM crops like increased yields and farmer profits.
Transgenic crops can be used to introduce traits that are difficult or impossible to combine through traditional breeding methods. The document discusses using transgenic methods to introduce virus and insect resistance, herbicide tolerance, drought tolerance, and quality traits like increased nutrients and shelf life into various vegetable crops. Specific examples discussed include brinjal with resistance to fruit and shoot borer, tomato with increased lycopene, zinc, and shelf life. The document compares traditional breeding to transgenic methods and lists the status of development for some GM vegetable crops in India.
This document discusses the role of mutation breeding in crop improvement. It describes how mutation breeding involves inducing mutations using physical or chemical mutagens and exploiting beneficial mutations. Key points covered include:
- Types of mutations include spontaneous and induced mutations. Common mutagens used are radiation, chemicals like EMS, and acridine dyes.
- Procedures for mutation breeding involve selecting a variety, treating plant parts with an optimal mutagen dose, and screening and selecting mutants.
- Achievements using mutation breeding include developing higher yielding varieties of crops like barley, rice, and groundnut with traits like increased size, drought tolerance, and disease resistance.
- Advantages are its low cost and ability
This document discusses using in vitro mutagenesis techniques to induce desirable mutations in flower crops. It describes mutagenesis as a process that changes genetic information, resulting in mutations. Both physical (radiation) and chemical (EMS, DES) mutagens are used. The document presents case studies on inducing mutations in tuberose and rose plants using gamma rays and chemical mutagens. Desirable variations were observed, such as changes in flower shape, color, and stem length. Radiation doses of 40 Gy and 55 Gy were found to successfully induce novel variants in rose. The objective is to develop improved varieties of ornamental plants using these mutation techniques.
Plant and growth development in agronomyUAS Dharwd
The document discusses various aspects of plant growth and development including cell growth, cell division, differentiation, and the role of plant growth regulators. It defines growth and growth rates, and describes the processes of differentiation and dedifferentiation in plant cells. The summary of key points are:
1. Plant growth and development involves processes like cell growth, division, differentiation and is regulated by plant growth regulators.
2. Growth can be arithmetic or geometric, and growth rates can be expressed mathematically.
3. Differentiation is the process by which less specialized cells become more specialized, while dedifferentiation allows cells to regain division capabilities.
4. Plant development results from the complex interactions between growth, differentiation, and
This document provides an overview of plant growth regulators, genetics and inheritance, secondary growth, and environmental issues in gardening. It discusses the main plant growth regulators (auxin, cytokinins, gibberellins, ethylene, and abscisic acid) and their effects. It describes genetics as involving dominant and recessive gene pairs that determine plant characteristics. Secondary growth is explained as thickening stems through vascular cambium and creating growth rings. Environmental issues addressed are the impacts of peat as a growing medium, risks of pest/disease controls, invasive non-native plants, and conserving water usage and runoff.
Trichoderma fungi can enhance drought tolerance in crops through several mechanisms. It colonizes plant roots and produces antioxidants like catalase that reduce reactive oxygen species caused by drought stress. It also produces plant hormones like cytokinins and abscisic acid that regulate stomatal closure and accumulation of solutes to adjust osmotic levels under drought. Studies have shown Trichoderma inoculation improves drought tolerance in various crops like wheat, rice and maize by maintaining higher chlorophyll levels and reducing hydrogen peroxide accumulation compared to non-inoculated plants. The fungi also induce systemic tolerance in plants through increased nutrient uptake and synthesis of growth-promoting substances.
This document summarizes various biotechnological approaches that can be used to improve vegetable crops, including meristem culture, anther culture, embryo rescue, somatic hybridization, and somaclonal variation. Meristem culture is effective for eliminating viruses from plants and can produce virus-free generations. Anther culture can be used for hybrid development, inducing mutations, and generating male plants. Embryo rescue allows the recovery of interspecific hybrids and reduces breeding cycles. Somatic hybridization fuses cells from different species to transfer beneficial traits. Somaclonal variation induces heritable variations during tissue culture that can be selected for traits like stress tolerance. Case studies provide examples of applying each technique for different crops.
The document summarizes a study evaluating 20 cauliflower genotypes for resistance to stalk rot, black rot, and riceyness. The study found that genotypes DC-76 and Pant Shubhra showed the highest resistance to stalk rot, while PSBK-1 and Hermia showed the highest resistance to black rot. Hermia and Pant Shubhra exhibited overall high resistance to both diseases and produced non-ricey curds, making them promising for breeding programs.
This document contains the program information for the 3rd International Conference of Plant Molecular Breeding held from September 5-9, 2010 in Beijing, China. It lists the honorary presidents, organizing committees, program committees, and provides an outline of the conference sessions including plenary lectures on topics like molecular breeding in developing countries, rice functional genomics, and transgenic crop technologies. It also describes concurrent sessions on subjects like molecular breeding for abiotic stress tolerance, gene discovery and function, and molecular breeding for biotic stresses.
i) Breeding crops for resistance to insects, diseases, and abiotic stresses like drought is important to reduce yield losses and costs of control measures.
ii) Mechanisms of resistance include non-preference, antibiosis, tolerance, avoidance, and physiological or biochemical traits like hairiness, toxins, or proline accumulation.
iii) Sources of resistance come from cultivated varieties, germplasm collections, and related wild species, and screening is done under field or controlled conditions.
This document discusses genetically modified drought resistant crops. It begins by defining genetically modified crops as plants modified using genetic engineering to introduce new traits. It then discusses developing drought tolerant crops through conventional breeding and genetic engineering techniques. Conventional breeding is a slow process limited by available genes, while genetic engineering allows introducing genes controlling drought tolerance. The document provides examples of drought tolerance mechanisms in plants and genes introduced through genetic engineering to improve drought resistance in transgenic crops.
Loss due to diseases range from 20 to 30 %, in case of severe infection, total crop may be lost.
Estimated global loss due to insect pests in potential yields of all crops is -14%.
In India losses due to insect pests ranges from 10 to 20 %
Abiotic stresses reduce average yield of crops by upto50% (Bray EA 1997)
Annually about 42% of the crop productivity is lost due to various abiotic stress factors (Oerkeet.al.,1994).
The different types of external stresses that influence the plant growth and development.
These stresses are grouped based on their characters
Biotic
Abiotic
Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress in plants.
This damages the cellular constituents of plants which are associated with a reduction in plant yield.
Crop improvement through genetic engineering can help meet the increasing global demand for food by making crops more resilient to stresses like drought and pests. The process involves isolating genes that confer desirable traits and inserting them into crops using techniques like bacterial infection or particle bombardment. Commercially, early genetically modified crops were made resistant to herbicides or insects. While genetic engineering could boost yields and nutrition, it also raises safety and environmental concerns that need consideration.
1. The seminar discusses developing transgenic plants resistant to insects through the transfer of resistance genes from microorganisms, higher plants, and animals into crop plants.
2. Major objectives of plant biotechnology are to develop plants resistant to biotic and abiotic stresses. Resistance to insects has been achieved by introducing genes encoding Bt toxins from Bacillus thuringiensis and other insecticidal proteins.
3. Useful genes have been isolated from microbes like B. thuringiensis, higher plants like beans and tobacco, and animals like mammals. These genes have been successfully used to engineer insect-resistant crops like cotton, potato, tomato, and tobacco.
The PPT depicts about the biotic and abiotic stresses which are becoming more adverse due to climate change. It breifly discusses about the types of different abiotic and biotic stresses and their impact on agricultural production.
This document discusses genetic engineering for resistance to biotic stress. It defines biotic stress as stress caused by other living organisms that can damage crops. Various techniques for genetically engineering plants for resistance are described, including using genes from Bacillus thuringiensis to make plants resistant to certain insects. Case studies on developing resistance to the European corn borer in Bt corn and developing glyphosate resistance in crops through different strategies are summarized. The development of transgenic crops with traits like insect resistance, herbicide tolerance, and virus resistance are also briefly outlined.
Invitro mutation selection for biotic stresses in Plantsamvannan
In-vitro selection is a somaclonal variation method that uses a selection agent or particular condition to select for somaclones with a desired character. Various mutagens like gamma irradiation, chemicals, and transposons can be used to induce mutations in vitro. Somatic embryogenesis is advantageous for in-vitro selection as it allows treatment of large populations and rapid generation of non-chimeric plants. Chemical mutagens are commonly used for in-vitro selection due to ease of handling. In-vitro selection has been used successfully to obtain disease resistance in various crop species like tobacco, rice, wheat, and potato.
Biotechnological approaches in Host Plant Resistance (HPR)Vinod Pawar
1. The document discusses biotechnological approaches to host plant resistance, including genetic manipulation of secondary plant substances and incorporation of resistant genes in crop varieties.
2. It provides examples of how genetic manipulation can enhance the production of compounds like terpenoids by modifying gene promoters, transcription factors, and subcellular localization.
3. It also gives an example of using marker-assisted backcrossing to introgress three bacterial blight resistance genes into the elite rice variety Samba Mahsuri, resulting in lines with high resistance and yield without compromising quality.
1) Tritrophic interactions involve plants defending against herbivores through direct defenses like trichomes or toxic chemicals, and indirect defenses like emitting volatile compounds to attract natural enemies of the herbivores.
2) Plant traits like trichome density, leaf size, and compactness can impact the ability of rice pests and natural enemies to search for food. Broader leaf width makes it easier for leaf folder larvae to feed and find protection.
3) Rice plants emit volatile compounds in response to damage from insects like stem borers and fall armyworms that attract natural enemies like parasitic wasps to the herbivores.
Genetic Manipulation and Host Plant ResistanceKarl Obispo
This document discusses several topics related to genetically modified crops. It defines transgenic plants as plants that have genes inserted from other species, and cisgenic plants as having genes from the same or closely related species. It provides examples of genetically engineered corn, rice, soybeans, and sugarcane with improved traits like herbicide and pest resistance. The document also discusses methods of genetic engineering like gene guns, electroporation, microinjection, and CRISPR. It outlines benefits of GM crops like increased yields and farmer profits.
Transgenic crops can be used to introduce traits that are difficult or impossible to combine through traditional breeding methods. The document discusses using transgenic methods to introduce virus and insect resistance, herbicide tolerance, drought tolerance, and quality traits like increased nutrients and shelf life into various vegetable crops. Specific examples discussed include brinjal with resistance to fruit and shoot borer, tomato with increased lycopene, zinc, and shelf life. The document compares traditional breeding to transgenic methods and lists the status of development for some GM vegetable crops in India.
This document discusses the role of mutation breeding in crop improvement. It describes how mutation breeding involves inducing mutations using physical or chemical mutagens and exploiting beneficial mutations. Key points covered include:
- Types of mutations include spontaneous and induced mutations. Common mutagens used are radiation, chemicals like EMS, and acridine dyes.
- Procedures for mutation breeding involve selecting a variety, treating plant parts with an optimal mutagen dose, and screening and selecting mutants.
- Achievements using mutation breeding include developing higher yielding varieties of crops like barley, rice, and groundnut with traits like increased size, drought tolerance, and disease resistance.
- Advantages are its low cost and ability
This document discusses using in vitro mutagenesis techniques to induce desirable mutations in flower crops. It describes mutagenesis as a process that changes genetic information, resulting in mutations. Both physical (radiation) and chemical (EMS, DES) mutagens are used. The document presents case studies on inducing mutations in tuberose and rose plants using gamma rays and chemical mutagens. Desirable variations were observed, such as changes in flower shape, color, and stem length. Radiation doses of 40 Gy and 55 Gy were found to successfully induce novel variants in rose. The objective is to develop improved varieties of ornamental plants using these mutation techniques.
Plant and growth development in agronomyUAS Dharwd
The document discusses various aspects of plant growth and development including cell growth, cell division, differentiation, and the role of plant growth regulators. It defines growth and growth rates, and describes the processes of differentiation and dedifferentiation in plant cells. The summary of key points are:
1. Plant growth and development involves processes like cell growth, division, differentiation and is regulated by plant growth regulators.
2. Growth can be arithmetic or geometric, and growth rates can be expressed mathematically.
3. Differentiation is the process by which less specialized cells become more specialized, while dedifferentiation allows cells to regain division capabilities.
4. Plant development results from the complex interactions between growth, differentiation, and
This document provides an overview of plant growth regulators, genetics and inheritance, secondary growth, and environmental issues in gardening. It discusses the main plant growth regulators (auxin, cytokinins, gibberellins, ethylene, and abscisic acid) and their effects. It describes genetics as involving dominant and recessive gene pairs that determine plant characteristics. Secondary growth is explained as thickening stems through vascular cambium and creating growth rings. Environmental issues addressed are the impacts of peat as a growing medium, risks of pest/disease controls, invasive non-native plants, and conserving water usage and runoff.
Trichoderma fungi can enhance drought tolerance in crops through several mechanisms. It colonizes plant roots and produces antioxidants like catalase that reduce reactive oxygen species caused by drought stress. It also produces plant hormones like cytokinins and abscisic acid that regulate stomatal closure and accumulation of solutes to adjust osmotic levels under drought. Studies have shown Trichoderma inoculation improves drought tolerance in various crops like wheat, rice and maize by maintaining higher chlorophyll levels and reducing hydrogen peroxide accumulation compared to non-inoculated plants. The fungi also induce systemic tolerance in plants through increased nutrient uptake and synthesis of growth-promoting substances.
This document discusses plant pathology, which is the scientific study of plant diseases caused by pathogens and environmental conditions. It addresses the causes of plant diseases, including living organisms like fungi, bacteria, viruses and nematodes, as well as non-living factors. The disease cycle and factors affecting disease development are also examined. Plant diseases can cause significant economic losses by reducing crop yields and quality. Understanding plant pathology is important for preventing diseases and maintaining food supply.
Plant pathology is the study of diseases that affect plants. The document outlines key concepts in plant pathology including definitions of plant disease, the disease triangle, classification of diseases, and causes of infectious and non-infectious diseases. It also discusses the objectives and importance of plant pathology, summarizing that plant pathology aims to study the causes and mechanisms of disease, epidemiology, and develop management strategies, in order to reduce losses from diseases and meet global food needs.
This document discusses different types of stress that plants experience and how they deal with it. It defines biotic stress as stress caused by other living organisms like pathogens, insects, weeds etc. and abiotic stress as stress from non-living environmental factors like drought, salinity, temperature etc. Plants have developed different resistance mechanisms to deal with stress, like avoidance through behaviors like ephemerality or deep roots, and tolerance through adaptations like drought-tolerant tissues or cold hardening. Pathogens can damage plants through necrosis or by remaining biotrophic. Plants defend against biotic stress through physical barriers and chemical defenses that can be constitutive or induced upon infection. Stress responses are important in agriculture, ecology and physiology.
The document discusses a seminar on plant pathology. It defines plant pathology as the scientific study of plant diseases caused by pathogens and environmental conditions. Pathogens that cause infectious plant diseases include fungi, oomycetes, bacteria, viruses, viroids, phytoplasmas, protozoa, and nematodes. Plant pathology involves the study of pathogen identification, disease cycles, and management of plant diseases. Common symptoms of plant diseases include color changes, stunted growth, blighted appearances, leaf spots, and wilts. General control measures include quarantine regulations to prevent entry of diseased plants, and field sanitation methods like removing diseased plant debris and crop rotation.
This document provides an introduction to the fundamentals of plant pathology. It discusses key topics including the definition of plant pathology, objectives of plant pathology such as studying the causes and mechanisms of disease, epidemiology, and disease control. It also defines important terminology like pathogen, disease, symptoms, and host. The document outlines the classification of plant diseases and different types of causal agents like fungi, bacteria, viruses. It highlights the importance of plant pathology in reducing crop losses and ensuring food security.
A detailed project on plant diseases,causes, symptoms and control measures with illustrations. The project explains in brief fungal and bacterial and and their control measures.Blast disease, citrus canker and leaf mosaic disease of tapioca are explained in detail. Non - infectious diseases are also mentioned.
MICROORGANISMS ASSOCIATED WITH THE SPOILAGE OF CUCUMBER, GARDEN EGG AND PAWPA...paperpublications3
Abstract: A total of nine cucumbers, each of Garden egg and pawpaw samples were collected from Wurukum, High level and Wadata markets and cultured on appriopate agar, for colony count and isolation of bacteria according to their cultural and biochemical characteristics. The results revealed that garden egg from High Level Market had the highest bacterial count (1.9x105cfu/g) and the least was pawpaw from High Level Market (1.1 x 105 cfu/g). However, it was not statistically significant. The bacteria isolated were; Propianol bacteria (23.3%), Escherichia coli (16.6%), Staphylococcus aureus (36.7%), Bacillus spp (10.0%) and Corynebacteria (13.3%). The fungal isolates were Aspergillus flavus (10%), Aspergillus fumigatus (20%), Aspergillus nidulus (10%), Aspergillus terreus (20%) and mucor (40%). The result of this study shows fruits sold in Wurukum, High Level Market and Wadata Market are contaminated and may cause harm to consumers, so measures such as proper handling should be taken to control the contamination of these fruits.
The document discusses various environmental factors that influence plant growth, including both biotic and abiotic factors. It describes abiotic factors like water, sunlight, temperature, soil composition and topography that affect plant survival and development. It also discusses important biotic factors such as producers, consumers, decomposers and their interactions through parasitism, mutualism, herbivory and allelopathy. Specific environmental conditions required for plant germination, growth and reproduction are also outlined.
This document discusses plant health and different methods of pest and disease control. It explains that gardens are ecosystems with complex relationships between plants, consumers and decomposers. Human activities like harvesting and spraying can disturb the natural balance. Maintaining biodiversity through habitats and plants supports natural predators that control pests. The four main types of control covered are biological, chemical, cultural and integrated pest management. Their benefits and limitations are outlined. Selecting resistant plant varieties and matching plants to conditions can also help avoid health problems. Restoring natural balances through these sustainable practices minimizes pesticide needs.
This document discusses the environmental interactions of weed species. It begins by defining a weed and the environment. It then discusses the interaction of weeds with their environment and how weeds interact with climate factors like light, temperature, water, and wind. It also discusses the interactions of weeds with soil properties such as salinity, texture, fertility, water, pH, and temperature. Finally, it discusses how weeds interact with biotic factors like other plant and animal species and how these interactions can affect weed persistence and distribution.
This document discusses abiotic stresses on plants. It defines abiotic stress as negative impacts from non-living factors like drought, extreme temperatures, salinity, and nutrient deficiencies. Primary abiotic stresses significantly impact global crop yields. The document outlines how these stresses affect plants physiologically by reducing growth, photosynthesis and reproduction. It also explains how plants develop resistance and acclimate to stresses through genetic adaptations like hardening off. Finally, the document notes that abiotic stresses limit biodiversity and agricultural production worldwide.
This document discusses stress in crops caused by both biotic and abiotic factors. It defines stress and provides classifications of stress. Biotic stress results from damage by living organisms like pathogens, insects and weeds. Abiotic stress is caused by non-living environmental factors like drought, salinity, temperature extremes. Drought is a major abiotic stress that limits crop yields globally. The effects of drought on crops are discussed along with mitigation strategies like mulching, fertilizer management and foliar sprays.
Cultivation and collection of medicinal plantMegha Shah
This document discusses the cultivation and collection of drugs from natural sources. It covers advantages and disadvantages of cultivation, methods of propagation including sexual and asexual reproduction, and factors that affect cultivation such as temperature, rainfall, soil properties, pests and weeds. Sexual propagation involves growing plants from seeds while asexual propagation uses vegetative parts like stems or roots. Temperature, rainfall, soil type and fertility all impact plant growth. Pests like fungi, viruses and insects as well as weeds must be controlled for successful cultivation.
Plant diseases significantly impact humans by damaging crops and other plants we rely on for food, clothing, shelter and the environment. They can reduce crop yields and quality, limit what plants can grow in certain areas, make plants unfit for consumption, and cause financial losses. Major diseases have even changed human history, like the Irish potato famine caused by late blight that led to widespread starvation and migration. Plant diseases are typically caused by living organisms like fungi, bacteria and nematodes, non-living environmental factors, or viruses and viroids. Common symptoms include spots, scabs and blotches on plants, while signs are the visible presence of the causal organism itself.
Biotic stress refers to stress caused by living organisms that damage plants, such as fungi, bacteria, insects, and weeds. Fungi cause more plant diseases than any other factor, with over 8,000 fungal species known to cause plant diseases. Plants have various defense mechanisms against biotic stress, including physical defenses like cell walls and waxy cuticles, and chemical defenses like terpenoids, phenolics, and nitrogen compounds that are toxic to pathogens and herbivores. Biotic stress can significantly impact plants and cause pre- and post-harvest losses if not properly managed.
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Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
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Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
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Part II - Body Grief: Losing parts of ourselves and our identity before, duri...
Expression analysis of water stress related genes in tomato plant 2019
1. DECLARATION
I hereby declare that the work is presented in this summer training “Expression analysis of water
stress related genes in Tomato plants” submitted to the CSIR-NEIST, Jorhat is a record of an
original work done by me under the guidance of Dr Ratul Saikia, Sr. Principal Scientist of
Biological Sciences And Technology Division(BSTD), CSIR-NEIST. The results embodied in
this report have not been copied from any other Department/Institute/University.
Ron Hazarika
M.Sc Biotechnology
2nd
semester
2. ACKNOWLEDGEMENT
I would like to express my immense gratitude to my supervisor Dr Ratul Saikia , Sr.
Principal scientist of Biological Sciences And Technology Division(BSTD), CSIR-NEIST for
his motivating guidance and constant encouragement to carry out my work.
I am truly thankful to Dr Hari Prasanna Deka Boruah, Sr. Principal Scientist (HOD) of
BSTD for his support and guidance.
I take this opportunity to put forward my sincere thanks to the Director, Dr G.N. Sastry,
CSIR-NEIST, Jorhat, Assam for giving me this opportunity to undergo summer training at this
institute.
I would like to acknowledge and extend my gratitude to Ms Archana Yadav,Technical
Officer of BSTD for extending her help in performing the experiments.
I would also like to thank Miss Parismita Gogoi and Miss Priyanka Kakoti for their guidance
and support throughout the experiments.
I would like to express my gratitude to Dr. Santanu PalChaudhuri(Professor, Head Of
Division) and Dr. Swatilekha Ghosh, (Associate Professor) of Amity Institute of Biotechnology,
Kolkata (AIBNK), Amity University, Kolkata, West Bengal for their continuous inspirations
during carrying out this training.
Finally I would like to express my warmest thanks to my parents and my friends for their
encouragement, help and support to complete my project successfully.
I express my sincere gratitude to all concerned.
Date: Ron Hazarika
Amity University Kolkata
3. ABSTRACT
Gene expression analysis involves the determination of the pattern of genes expressed at
the level of genetic transcription, under specific circumstances or in a specific cell. The
measurement of gene expression is a critical tool employed across drug discovery, life science
research and the optimization of bioproduction. Expression analysis involves several techniques
ranging from whole genome gene expression analysis such as microarrays or RNA sequencing,
to more specific target gene expression techniques such as qPCR techniques.
Gene expression analysis typically involves the isolation or capture of transcribed RNA
within a sample, followed by amplification and subsequent detection and quantitation.
This project work started with the collection of leaf sample from Tomato plants grown
under stress condition noting the water logging level, isolation of RNA and purification is done.
Extraction of RNA was performed using NucleoSpin® RNA plant isolation kit. Estimation of
cDNA and gene expression analysis is done using RT-PCR.
4. CONTENTS
1. Introduction
i. Plant stress and its cause.
ii. Plants taking in consideration.
iii. Abiotic stress.
iv. Abiotic stress inducible genes.
v. Transcriptional factor genes involved in abiotic stress.
vi. Transcriptional factor involved in response to flooding
stress.
vii. Ribonucleic acid.
viii. Complimentary DNA.
2. Materials and methods.
3. Results and discussion.
4. Conclusion.
5. Implications for future research.
6. References.
5. 1. INTRODUCTION
In recent years, the world has experienced significant challenges from Mother Nature.
Tragic wildfires, severe droughts, heavy rains, massive flooding, hurricanes, and more have
wreaked havoc throughout the states. These environmental threats have ruined crops, harmed
livestock, destroyed vegetations and even normal ecosystem, but especially those in key
agricultural regions. Lately, these natural disasters have taken the news stage due to their
intensity and frequency and represent the impacts of a changing climate. Farmers around the
world often bear the brunt of these disasters and feel the impact of climate change especially
close to home and in their business bank accounts. External environmental impacts like those
plaguing news headlines as of late pose significant risks to plant and crop health and often stress
plants beyond their tolerance limits and can lead to diminished marketable yields. Natural
disasters are an obvious cause of plant stress, even to the naked eye. But, did you ever realize
that plant stress comes in many other forms, some even invisible to the naked eye.
Plant Stress and its causes
Plant stress is a state where a plant is growing in non-ideal growth conditions and has
increased demands put on it. Plant stress refers to any unfavourable condition or substance that
affects a plant’s metabolism, reproduction, root development, or growth. Plant stress can come in
different forms and durations. Some plant stressors are naturally occurring, like drought or wind,
while others may be the result of human activity, like over irrigation or root disturbance.
Plant stress is caused by a variety of factors, some of which are obvious (like natural
disasters), while others occur on a micro scale in the soil. Recent natural disasters represent one
type of plant stress factors, called abiotic factors, which usually occur above ground. A second
type of plant stress factors are called biotic factors, which mostly occur underground, and can
cause plant stress through pathogens and pests.
Abiotic stresses originate from the surrounding environment of the plant. One of the
most important abiotic factors affecting plants is water stress. A plant requires a certain amount
of water for optimal growth, too much water can cause plant cells to swell and burst, whereas too
little water can lead to desiccation. Temperature stresses can also negatively impact a plants
growth and livelihood. Cold weather may affect the amount and rate of uptake of water and
6. nutrients, and hot weather can affect the permeability of plant membranes. Abiotic factors come
in other forms as well such as wind, toxins, and light.
Biotic stresses can cause damage to plants through living organisms that may cause
disease. In agriculture, biotic stresses are most often responsible for pre or post-harvest losses.
Soil is filled with fungi and bacteria – 1 teaspoon of soil can hold billions of microorganisms.
Just like microorganisms found in humans, some can be beneficial, and others can be
detrimental. Healthy soil biological systems showcase an appropriate, harmonious balance
between beneficial microorganisms that protect against biotic stresses and detrimental
microorganisms – that if not held in check can result in biotic plant stresses. Examples of
common biotic plant stress factors include pathogens, insects, and weeds but the exact types of
factors depend on the environment and differ from region to region.
About the plants
1. Tomato - The tomato is the edible, often red, berry of the plant Solanum
lycopersicum, commonly known as a tomato plant. The species originated in
western South America and Central America. Tomatoes are a significant source
of umami flavor. Numerous varieties of the tomato plant are widely grown in temperate
climates across the world, with greenhouses allowing for the production of tomatoes
throughout all seasons of the year. Tomato plants typically grow to 1–3 meters (3–10 ft)
in height. They are vines that have a weak stem that sprawls and typically needs
support. Indeterminate tomato plants are perennials in their native habitat, but are
cultivated as annuals. Determinate, or bush, plants are annuals that stop growing at a
certain height and produce a crop all at once. The size of the tomato varies according to
the cultivar, with a range of 0.5–4 inches (1.3–10.2 cm) in width.
7. 2. Bhut Jolokia: The Bhut jolokia , also known as ghost pepper. ghost chili pepper, ghost
chili and ghost jolokia, is an interspecific hybrid chili pepper cultivated in the Northeast
Indian states of Arunachal Pradesh, Assam, Nagaland and Manipur. It is a hybrid
of Capsicum chinense and Capsicum frutescens and is closely related to the Naga
Morich of Nagaland and Bangladesh. Bhut jolokia mainly belongs to the
species Capsicum chinense Jaqc. It was earlier thought to be a hybrid of Capsicum
frutescens and Capsicum chinense on the basis of randomly amplified polymorphic DNA
(RAPD) analysis. However, it has recently been described as a distinct species (Capsicum
assamicum) on the basis of morphological properties, molecular phylogeny of the internal
transcribed spacer (ITS) region and differential proteomic analysis. Bhut jolokia is a self-
pollinated plant, however, considerable cross pollination (up to 10%) may occur when
insect population is high. It behaves as a semi-perennial herb if grown under optimal
condition. The plant grows to a height of 57-129 cm at 6 months. Under semi perennial
situation it may grow even taller.
ABIOTIC STRESS
Abiotic stress is the negative impact of non-living factors on the living organisms in a specific
environment. The non-living variable must influence the environment beyond its normal range of
variation to adversely affect the population performance or individual physiology of the
organism in a significant way.
Whereas a biotic stress would include living disturbances such as fungi or harmful insects,
abiotic stress factors, or stressors, are naturally occurring, often intangible and inanimate factors
such as intense sunlight, temperature or wind that may cause harm to the plants and animals in
8. the area affected. Abiotic stress is essentially unavoidable. Abiotic stress affects animals, but
plants are especially dependent, if not solely dependent, on environmental factors, so it is
particularly constraining. Abiotic stress is the most harmful factor concerning the growth
and productivity of crops worldwide. Research has also shown that abiotic stressors are at their
most harmful when they occur together, in combinations of abiotic stress factors.
Stress Consequences Plant Responses
Heat stress
High temperature leads to high
evaporation and water deficit.
The consequent increased
turnover of enzymes leads to
plant death.
Efficient protein repair systems
and general protein stability
support survival, temperature can
lead to acclimation.
Chilling and cold stress
Biochemical reactions proceed
at slower rate, photosynthesis
proceeds, carbon dioxide
fixation lags, leading to oxygen
radical damage. Indeed,
freezing lead to ice crystal
formation that can distrupt cells
membranes.
Cessation of growth in adaptable
species may be overcome by
changes in metabolism. Ice
crystal formation can be
prevented by osmolyte
accumulation and synthesis of
hydrophilic proteins.
Drought
Inability to water transport to
leaves leads to photosynthesis
declines.
Leaf rolling and other
morphological adaptations. Stoma
closure reduces evaporative
transpiration induced by ABA.
Accumulation of metabolities,
consequently lower internal water
potential and water attracting
9. Flooding and submergence
Generates anoxic or micro
aerobic conditions
Interfering with mitochondrial
respiration.
Development of cavities mostly
in the roots that facilitate the
exchange of Oxygen and ethylene
between shoot and root
(aerenchyma).
Heavy metal accumulation
and metal stress
In excess, detoxification
reactions may be insufficient or
storage capacity may exceed.
Excess of metal ions may be
countered by export or vacuolar
deposition but metal ions may
also generate oxygen radicals.
High light stress
Excess light can lead to
increased production of highly
reactive intermediates and by-
products that can potentially
cause photo-oxidative damage
and inhibit photosynthesis
Exposure of a plant to light
exceeding what is utilized in
photochemistry leads to
inactivation of photosynthetic
functions and the production of
reactive oxygen species (ROS).
The effects of these ROS can be
the oxidation of lipids, proteins,
and enzymes necessary for the
proper functioning of the
chloroplast and the cell as a
whole.
Abiotic stress-inducible genes
The complex plant response to abiotic stress involves many genes and biochemical
molecular mechanisms. The analyze of the functions of stress-inducible genes is an important
tool not only to understand the molecular mechanisms of stress tolerance and the responses of
higher plants, but also to improve the stress tolerance of crops by gene manipulation. Hundreds
of genes are thought to be involved in abiotic stress responses. Many drought-inducible genes are
10. also induced by salt stress and cold, which suggests the existence of similar mechanisms of stress
responses.
These genes are classified into three major groups:
1.Those that encode products that directly protect plant cells against stresses such as heat stress
proteins (HSPs) or chaperones, LEA proteins, osmoprotectants, antifreeze proteins,
detoxification enzymes and free-radical scavengers.
2.Those that are involved in signalling cascades and in transcriptional control, such as Mitogen-
activated protein kinase (MAPK), Calcium-dependent protein kinase (CDPK) and SOS kinase,
phospholipases and transcriptional factors.
3.Those that are involved in water and ion uptake and transport such as aquaporins and ion
transporters.
Transcriptional factor genes involved in abiotic stress
Plant growth and productivity are under constant threat from environmental changes in
the form of various stress factors. The most common abiotic stresses are drought, flooding or
submergence, salinity, extreme temperatures (heat and freezing) and high light. Furthermore, the
continued modification of the atmosphere by human activities lead to increase in the
concentration of ozone in the troposphere and this can generate oxidative stress, which leads to
the destruction of proteins and cells, premature ageing and reduced crop yields.Tolerance or
susceptibility to these abiotic stresses is a very complex phenomenon, both because stress may
occur at multiple stages of plant development and more than one stress simultaneously affects
the plant. Therefore, the perception of abiotic stresses and signal transduction to switch on
adaptive responses are critical steps in determining the survival and reproduction of plants
exposed to adverse environments.
During the past few years, transcriptome analysis has indicated that distinct environmental
stresses induce similar responses. Overlap between stress responses can explain the phenomenon
known as cross-tolerance, a capability to limit collateral damage inflicted by other stresses
accompanying the primary stress.
Responses to abiotic stresses require the production of important metabolic proteins such as
those involved in synthesis of osmoprotectants and regulatory proteins operating in signal
transduction pathways that are kinases or transcription factors (TFs). The regulation of these
Fig. 1. Transcriptional network of abiotic stress responses.
11. genes requires
proteins operating
in the signal
transduction pathways, such as transcriptional factors, which regulate gene expression by
binding to specific DNA sequences in the promoters of respective target genes. This type of
transcriptional regulatory system is called regulon. At least four different regulons that are active
in response to abiotic stresses have been identified. Dehydration-responsive element binding
protein 1 (DREB1)/C-repeat binding factor (CBF) and DREB2 regulons function in abscisic
acid (ABA)-independent gene expression, whereas the ABA responsive element (ABRE)
binding protein (AREB)/ABRE binding factor (ABF) regulon functions in ABA-dependent gene
expression.
In addition to these major pathways, other regulons, including the NAC (or NAM, No
Apical Meristem) and Myeloblastosis-Myelocytomatosis (MYB/MYC) regulons, are involved
in abiotic stress-responsive gene expression (Fig. 1). Particularly, NAC- type TF OsNAC6 is
induced by abiotic stresses, including cold, drought and high salinity.
Transcriptional factor involved in response to waterlogging stress
12. Flooding and submergence are two conditions that cannot be tolerated by most plants for
periods of time longer than a few days. These stresses lead to anoxic conditions in the root
system. At a critical oxygen pressure, mitochondrial respiration that provides the energy for
growth in the photosynthetically inactive roots will decrease, and then cease and the cells will
die. Recent reviews on gene expression analysis performed by microarray tools reported as the
expression of several transcription factors, such as heat shock factors, ethylene response binding
proteins, MADS-box proteins, AP2 domain, leucine zipper, zinc finger and WRKY factors,
increases in response to various regimes of oxygen deprivation in Arabidopsis and rice. Recently
using a qRT-PCR platform has identified TFs that are differentially expressed by hypoxic
conditions. Among the TFs that have been characterized, members of the AP2 ⁄ ERF-type family
are the most commonly represented in the set of up-regulated TFs, followed by Zinc-finger and
basic helix-loop-helix (bHLH-type) TFs, while TFs belonging to the bHLH family are the most
commonly represented in the set of down-regulated TFs, together with members from the bZIP
and MYB families. In silico experiments and trans-activation assays shown that some TFs active
in flooding stress are able to regulate the expression of hypoxia responsive genes. Particularly,
five hypoxia-induced TFs (At4g29190; LBD41, At3g02550;HRE1, At1g72360; At1g69570;
At5g66980) from different TF families [Zinc Finger, Ligand Binding Domain (LBD) or Lateral
Organ Boundary Domain, ERF, DNA binding with one finger (DOF), ARF] showed this ability.
Accumulation of ROS is a common consequence of biotic and abiotic stresses, including oxygen
deprivation. There is evidence of redox-sensitive TFs, at least one of which might be involved in
the adaptive response to low oxygen. ZAT12, a putative zinc finger-containing TF, is recognized
as a component in the oxidative stress response signalling network of Arabidopsis, promotes
expression of other TFs and the upregulation of cytosolic ascorbate peroxidase 1, a key enzyme
in the removal of H2O2 .
RNA (Ribonucleic
acid )
13. RNA is a polymeric molecule essential in various biological roles
in coding, decoding, regulation and expression of genes. RNA and DNA are nucleic acids, and,
along with lipids, proteins and carbohydrates, constitute the four major macromolecule essential
for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike
DNA it is more often found in nature as a single-strand folded onto itself, rather than a paired
double-strand. Cellular organisms use messenger RNA (mRNA) to convey genetic information
(using the nitrogenous bases of guanine, uracil, adenine, and cytosine, denoted by the letters G,
U, A, and C) that directs synthesis of specific proteins. Many viruses encode their genetic
information using an RNA genome. Some RNA molecules play an active role within cells by
catalyzing biological reactions, controlling gene expression, or sensing and communicating
responses to cellular signals. One of these active processes is protein synthesis, a universal
function in which RNA molecules direct the synthesis of proteins on ribosome. This process
uses transfer RNA (tRNA) molecules to deliver amino acids to the ribosome, where ribosomal
RNA (rRNA) then links amino acids together to form coded proteins.
Structure of RNA
RNA is a ribonucleic acid that helps in the synthesis of proteins in our body. This nucleic acid
is responsible for the production of new cells in the human body. It is usually obtained from the
DNA molecule. RNA resembles same as that of DNA, the only difference being that it has a
single strand unlike the DNA which has two strands and it consists of only single ribose sugar
molecule in it. Hence is the name Ribonucleic acid. RNA is also referred to as a enzyme as it
helps in the process of chemical reactions in the body. The ribonucleic acid has all the
components same to that of the DNA with only 2 main differences within it. RNA has the same
14. nitrogen bases called the adenine, Guanine, Cytosine as that of the DNA except the Thymine
which is replaced by the uracil. Adenine and uracil are considered as the major building blocks
of RNA and both of them form base-pair with the help of 2 hydrogen bonds.
RNA has two major and basic functions as given below-
• Firstly it assists the DNA and acts as a messenger between the DNA and the ribosomes.
• Secondly it helps the ribosomes to choose the right amino acid which is required in
building up of new proteins in the body.
Complementary DNA (cDNA)
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded
RNA (e.g., messenger RNA (mRNA) or microRNA) template in a reaction catalyzed by the
enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes.
When scientists want to express a specific protein in a cell that does not normally express that
protein (i.e., heterologous expression), they will transfer the cDNA that codes for the protein to
the recipient cell. cDNA is also produced naturally by retroviruses(such as HIV-1, HIV-2, simian
immunodeficiency virus, etc.) and then integrated into the host's genome, where it creates
a provirus. The term cDNA is also used, typically in a bioinformatics context, to refer to an
mRNA transcript's sequence, expressed as DNA bases (GCAT) rather than RNA bases (GCAU).
cDNA is derived from mRNA, so it contains only exons but no introns.
1. From the hairpin loop, a DNA polymerase can then use it as a primer to transcribe a
complementary sequence for the ss cDNA.
2. Now, you should be left with a double stranded cDNA with identical sequence as the
mRNA of interest.
15. Applications of cDNA
Complementary DNA is often used in gene cloning or as gene probes or in the creation of
a cDNA library. When scientists transfer a gene from one cell into another cell in order to
express the new genetic material as a protein in the recipient cell, the cDNA will be added to the
recipient (rather than the entire gene), because the DNA for an entire gene may include DNA
that does not code for the protein or that interrupts the coding sequence of the protein
(e.g., introns). Partial sequences of cDNAs are often obtained as expressed sequence
tags (EST).
2.MATERIALS AND METHODS
2.1 SAMPLE PREPARATION
Nutrient Broth was prepared and the three bacterias RJ12, RJ15 and RJ46
was inoculated in the broth and kept at 36o
C for 24 hrs. These bacterial broth was
16. then inoculated to the 10 day old tomato seedlings and allowed to grow. After 10
days waterlogging treatment was performed in the 20 days old seedlings of tomato.
Normal watering was done in the control plants and the waterlogging pots were
filled with water upto 5 cm from the soil level. Then after 7 days of waterlogging
leaves from the plants were collected and further crushed with the help of liquid
nitrogen and RNA was isolated.
Fig: Waterlogging treated plants
Fig: Control plants
2.2 RNA ISOLATION FROM PLANTS
KIT USED- NucleoSpin® RNA plant isolation kit.
PRINCIPLE- One of the most important aspects in the isolation of RNA is to prevent
degredation of RNA during the isolation procedure.
With NucleoSpin RNA plant Method,
17. 1. The cells are first disrupted by grinding in the presence of liquid N2.
2. Complete denaturation is achieved by incubation in a solution containing large
amount of chaotropic ions.
(a chaotropic molecule is a molecule in water solution that can disrupt the
hydrogen bonding network between the water molecules)
3. The lysis buffer immedietly inactivates the RNases- which are present in virtually
all biological materials – creates appropriate binding conditions which favors
adsorption of RNA to silica membrane.
4. Contaminating DNA is removed by an rDNase solution wgich is directly applied
onto the silica membrane during the preparation
5. Washing steps with 2 different buffers removes salts, metabolites and macro
molecular cellular components.
6. Pure RNA is finally eluted under low ionic strength conditions with RNase free
H2O.
PROTOCOL
1. Grind up to 100mg tissue under liquid N2. Grind with mortar and pestle – grind the
sample to a fine powder in the presence of liquid N2. Make sure the sample does not thaw
during or after grinding. Add some frozen powder containing β-marceptaethanol
immediately.
2. Add 350 µl Buffer RA1 + 350 µl β-marceptaethanol to 100mg of tissue and vortex.
(If lysate solidifies upon addition of RA1, use 350 µl of RAP buffer)
3. Reduce the viscosity and clear the lysate by filtration through NucleoSpin filter (violet
ring). Place the filter in 2ml collection tube and centrifuge for 1 min at 11000 rpm.
Transfer the filtrate to new 1.5 m; microcentrifuge tube.
4. Discard the NucleoSpin filter. Add 350 µl ethanol (70 %) and mix it by pipetting up and
down or by vortexing
5. For each preparation, take one NucleoSpin RNA plant column (light blue ring). Place it
in a collection tube and load the lysate. Centrifuge for 30 sec at 11000 rpm.
6. Add 350 µl MDB (membrane desalting buffer) and centrifuge at 11000 rpm for 1 min to
dry the membrane.
7. Preparation of DNase reaction mixture
For each isolation---- 10 µl rDNase +90 µl reaction buffer for rDNase.
18. Now add 95 µl DNase reaction mixtures directly onto the centre of the silica membrane
of the column. Incubate in room temperature for 15 mins.
1st
wash
200 µl buffer RAW2 to the plant column. (Centrifuge for 30sec at 11000rpm). Place the
column in a new collection tube.
2nd
wash
600 µl buffer RA3 to the column. (Centrifuge for 30sec at 11000rpm). Place the column
in a new collection tube.
3rd
wash
250 µl buffer RA3 to the column. (Centrifuge for 2 mins at 11000rpm). Place the column
in a nuclease collection tube.
8. Elute RNA in 60 µl RNase free H2O and centrifuge at 11000 rpm for 1 min. For higher
concentration elute with 40 µl.
20. 2.3 cDNA SYNTHESIS
KIT USED- GeneSure First Strand cDNA Synthesis Kit
The kit uses Reverse Transcriptase, which lowers RNase H activity compared to AMV reverse
transcriptase. The enzyme maintains activity at 42-50° C and is suitable for synthesis of cDNA
up to 13 kb. The recombinant RNase Inhibitor, effectively protects RNA from degredation at
temperatures up to 55° C. First strand cDNA synthesized with this system can be directly used as
a template inPCR or Real-time PCR. It is also ideal for second strand cDNA synthesis . all the
components should be stored at -20° C.
PROTOCOL
1) After thawing, the components of the kits are centrifuged and mixed and stored in ice.
2) The following reagents are added into sterile, nucleasefree tube on ice in indicated order.
Template RNA Total RNA or
specific RNA
0.1 ng- 5 µg
0.01 pg-0.5 µg
Primer Oligo(dT)
primer or
random
Hexamer
Primer or
gene-specific
Primer
1µl
1µl
15-20 pmol
Water nuclease-free To 12 µl
Total volume 12 µl
3) Incubate at 65o
C for 5 mins.
4) Now these components are added in indicated order:
21. 5X Reaction Buffer 4 µl
RNase inhibitor (200/µl) 1 µl
10 mM dNTP Mix 2 µl
M-MuL V RT (200/µl) 1 µl
Total volume 20 µl
5) They are mixed gently and centrifuged.
6) Now the samples are incubated for 60 mins at 42° C followed by termination of the reaction
by heating at 70°C for 5 mins.
2.4 RT-PCR Amplification
The product of the first strand cDNA synthesis can be used directly in RT-PCR or qPCR. was
performed in the Applied Biosystems Step One Plus Real-time PCR System.
Reaction setup- 10pm- 100µm
Preparation of the sample :
22. ACTINE
GENE(housekeeping)
CATALASE GENE
Master Mix 10.00 µL 10.00 µL
Forward Primer 0.20 µL 0.20 µL
Reverse Primer 0.20 µL 0.20 µL
Sample 2.00 µL 2.00 µL
H2O 7.60 µL 7.60 µL
Dilution of the Sample :
cDNA Water Total volume
Control (WL) 9.00 µL 5.63 µL 14.63 µL
RJ12 (WL) 8.00 µL 6.06 µL 14.06 µL
Sample plating
C
actine
C
actine
C
catalase
C
actine
C
catalase
C
catalase
WL
actine
WL
catalase
WL
actine
WL
actine
WL
catalase
WL
catalase
23. 3.RESULTS AND DISCUSION
3.1 Isolation of RNA using NucleoSpin Plant kit of the water logging Bhut Jolokia Leaves
1st
set Concentration Absorbance (A260/A280)
Sample 1 130.3 ng/µl 1.89
Sample 2 225.0 ng/µl 1.90
Sample 3 200 ng/µl 1.95
2nd
set Concentration Absorbance(A260/A280)
Sample 1 150.4 ng/µl 1.70
Sample 2 210.3 ng/µl 1.88
Sample 3 110.6 ng/µl 1.99
3.2 Isolation of RNA using NucleoSpin Plant kit of the water logging Tomato Leaves
Concentration Absorbance (A260/A280)
RJ15 Control 7.7 ng/µl 1.55
RJ15 water logging 55.5 ng/µl 2.06
RJ46 control 92.7 ng/µl 2.05
RJ46 water logging 39.1 ng/µl 2.05
RJ12 control 78.8 ng/µl 2.09
RJ12 water logging 28.9 ng/µl 2.06
Normal control 94.6 ng/µl 2.04
Water logging control 12.3 ng/µl 2.00
24. 3.3 Quantification of the cDNA of the isolated samples of RNA of the Bhut Jolokia with the
help of Takara Kit.
1st
set Concentration Absorbance(A260/A280)
Sample 1 190.5 ng/µl 1.48
Sample 2 149.6 ng/µl 1.70
Sample 3 201.3 ng/µl 1.81
2nd
set Concentration Absorbance (A260/280)
Sample 1 191.7 ng/µl 1.65
Sample 2 155.4 ng/µl 1.83
Sample 3 189.9 ng/µl 1.82
3.4 Quantification of the cDNA of the isolated samples of RNA of the Tomato Plant with the
help of GeneSure Kit.
Concentration Absorbance(A260/280)
RJ15 Control 275.5 ng/µl 1.20
RJ15 water logging 184.7 ng/µl 1.34
RJ46 control 81.2 ng/µl 1.75
RJ46 water logging 131.8 ng/µl 1.64
RJ12 control 81.3 ng/µl 1.74
RJ12 water logging 87.9 ng/µl 1.83
Normal control 93.0 ng/µl 1.72
Water logging control 74.0 ng/µl 1.75
25. 3.5 Results of the samples run in RT-PCR
Applied BiosystemsStepOne Software v2.3ExperimentsWaterlogg.eds
Target
Name
Reporter CT CT Mean CT SD Δ CT
Mean
CT
Threshold
Tm 1
actin SYBR 33.46772003 33.97212601 2.920469 0.043820911 80.65464
actin SYBR 37.11194611 33.97212601 2.920469 0.043820911 80.80377
actin SYBR 31.33671761 33.97212601 2.920469 0.043820911 83.19183
catalase SYBR Undetermine
d
0.530779801 65.14435
catalase SYBR Undetermine
d
0.530779801 66.34305
catalase SYBR Undetermine
d
0.530779801 65.59743
actin SYBR 33.94418716 33.83980942 0.143072 0.043820911 80.65464
actin SYBR 33.89851379 33.83980942 0.143072 0.043820911 80.50551
actin SYBR 33.67671967 33.83980942 0.143072 0.043820911 81.10369
catalase SYBR Undetermine
d
36.96339798 0.530779801 80.05962
catalase SYBR Undetermine
d
36.96339798 0.530779801 97.96591
catalase SYBR 36.96339798 36.96339798 3.123591 0.530779801 68.43078
26. Block Type – 96 well
Chemistry SYBR_GREEN
Experiment D:Applied BiosystemsStepOne Software v2.3ExperimentsWaterlogg.eds
Instrument: StepOnePlus
Sample Name Target Name Task CT Mean
c actin Unknown 33.97213
c catalase Unknown
wl actin Unknown 33.83981
wl catalase Unknown 36.9634
Analysis – SinglePlex
Endogenous gene – actin
RQ Min/ Max – 95.0
Reference - c
27. 4. CONCLUSION
Gene expression profiling simultaneously compares the expression levels of
multiple genes between two or more samples. This analysis can help to identify the
molecular basis of phenotypic differences and select gene expression targets for in-
depth study. Real-time PCR is the gold-standard technique for verification of
differential gene expression profiles. Plant gene expression, in response to stress
cues, is tightly controlled by transcriptional regulators. Posttranslational
modifications are a key mechanism to control the activities of transcription factors
(TFs). The regulation of gene expression in plants, as in other higher eukaryotes, is
a subject of daunting complexity. Nevertheless, even a partial understanding of
how plant genes work, in conjunction with the methods of molecular biology and
plant tissue cultures, opens the door to a dazzling array of techniques for
manipulating various aspects of the phenotypes of plants.
In this project, we had analysed the expression of the water logging gene
that is involved in the water logging stress of the tomato plant. Catalase gene
expression study of the waterlogging plant was performed by keeping Actin gene
as the housekeeping gene. After the extraction of RNA from the leaves of the plant
and estimation of the cDNA , the sample is run in the Real-time PCR system. At
present we are at a very early stage of realizing many of the goals that have been
ambitiously (and, in some cases, unrealistically) proposed for manipulating gene
expression in plants, such as nitrogen-fixing cereals, because the systems are not
sufficiently understood at the molecular and genetic levels. For plant biochemists,
study of plant genes, their functions and regulation, is resulting in a quantum leap
in the understanding of familiar biochemical pathways, and the elucidation of less
familiar ones.
28. REFERENCE
❖ Gene expression in plant https://www.sciencedirect.com/topics/biochemistry-genetics-
and-molecular-biology/gene-expression-in-plant
❖ Plant genes for abiotic stress https://www.intechopen.com/books/abiotic-stress-in-plants-
mechanisms-and-adaptations/plant-genes-for-abiotic-stress
❖ RNAhttps://en.wikipedia.org/wiki/RNA
❖ Complementary DNA
https://www.sciencedirect.com/topics/neuroscience/complementary-dna
❖ Plant stress. What causes it –how to reduce it https://www.coolplanet.com/blog/plant-
stress-what-causes-plant-stress-and-how-to-reduce-it/
❖ Introduction to plant stress https://link.springer.com/chapter/10.1007/978-3-319-59379-
1_1
❖ Water Stress in Plants: Causes, Effects and Responses
https://www.researchgate.net/publication/221921924_Water_Stress_in_Plants_Causes
_Effects_and_Responses
❖ Response of plants to water stress
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3952189/
❖ NucleoSpin®
RNA Plant https://www.mn-net.com/tabid/1327/default.aspx
❖ Manual http://www.genetixbiotech.com/manual.php