The document discusses various methods for screening and selecting recombinant cells. Direct selection methods include antibiotic resistance screening and blue-white color screening. Indirect selection methods include screening by nucleic acid hybridization, colony hybridization, immunological assays, and detecting protein/enzyme activity. These screening methods allow identification of recombinant cells that contain the gene of interest from a mixture of transformed cells.
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
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.
This document discusses somaclonal variation, which refers to genetic variation that arises during tissue culture or plant regeneration from cell cultures. It provides definitions and history of the term as coined by Larkin and Scowcroft in 1981. The document outlines the various causes and types of somaclonal variation including physiological, genetic, and biochemical causes. It also describes methods for generating somaclonal variation both with and without in vitro selection. Finally, it discusses applications for detecting and isolating somaclonal variants, particularly for developing disease resistance in various crop species.
This document discusses various methods of genetic transfer, including natural genetic transfer between organisms as well as technological methods developed to manipulate genes. It describes how donor DNA can enter a recipient cell and recombine, producing genetically distinct offspring. Several gene transfer technologies are then outlined, including microinjection, biolistics, calcium phosphate precipitation, lipofection, and electroporation. The document explains the basic mechanisms and applications of each method while also noting their limitations for different purposes like gene therapy. In the conclusion, it emphasizes that gene transfer technologies now allow relatively easy and accurate introduction of genes into target cells to potentially cure diseases.
Microinjection is a gene transfer technique where DNA is directly injected into cells using a fine glass micropipette. It is highly efficient at the individual cell level and was originally used for transfecting hard-to-transfect cells. The procedure involves holding a cell using one pipette while another pipette is used to inject DNA into the cell's cytoplasm or nucleus. It allows for stable transfection efficiencies of around 20% and is used to generate transgenic animals by injecting DNA into oocytes, eggs or embryos. However, it is time-consuming and can only be done for a small number of cells.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
The document discusses various methods for screening and selecting recombinant cells. Direct selection methods include antibiotic resistance screening and blue-white color screening. Indirect selection methods include screening by nucleic acid hybridization, colony hybridization, immunological assays, and detecting protein/enzyme activity. These screening methods allow identification of recombinant cells that contain the gene of interest from a mixture of transformed cells.
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
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.
This document discusses somaclonal variation, which refers to genetic variation that arises during tissue culture or plant regeneration from cell cultures. It provides definitions and history of the term as coined by Larkin and Scowcroft in 1981. The document outlines the various causes and types of somaclonal variation including physiological, genetic, and biochemical causes. It also describes methods for generating somaclonal variation both with and without in vitro selection. Finally, it discusses applications for detecting and isolating somaclonal variants, particularly for developing disease resistance in various crop species.
This document discusses various methods of genetic transfer, including natural genetic transfer between organisms as well as technological methods developed to manipulate genes. It describes how donor DNA can enter a recipient cell and recombine, producing genetically distinct offspring. Several gene transfer technologies are then outlined, including microinjection, biolistics, calcium phosphate precipitation, lipofection, and electroporation. The document explains the basic mechanisms and applications of each method while also noting their limitations for different purposes like gene therapy. In the conclusion, it emphasizes that gene transfer technologies now allow relatively easy and accurate introduction of genes into target cells to potentially cure diseases.
Microinjection is a gene transfer technique where DNA is directly injected into cells using a fine glass micropipette. It is highly efficient at the individual cell level and was originally used for transfecting hard-to-transfect cells. The procedure involves holding a cell using one pipette while another pipette is used to inject DNA into the cell's cytoplasm or nucleus. It allows for stable transfection efficiencies of around 20% and is used to generate transgenic animals by injecting DNA into oocytes, eggs or embryos. However, it is time-consuming and can only be done for a small number of cells.
Site-directed mutagenesis is a technique used to introduce specific changes to the DNA sequence of a gene by altering the nucleotide sequence. It allows researchers to study the impact of mutations by changing individual bases, deleting bases, or inserting new bases. There are different methods of site-directed mutagenesis including oligonucleotide-based methods and PCR-based methods. Site-directed mutagenesis has applications in research, production of desired proteins, and development of engineered proteins for commercial uses like detergents.
Marker and reporter genes are used in plant genetic transformation. Selectable marker genes confer resistance to antibiotics or herbicides, allowing selection of transformed cells. Common selectable markers confer resistance to kanamycin, hygromycin, and glyphosate. Reporter genes like GFP, GUS, luciferase encode proteins that can be easily detected, allowing screening of transformation events. These genes help recover transformed cells and detect transgene expression without damaging cells.
A presentation covering the process of protoplast culture including protoplast isolation, protoplast fusion, culture of protoplast, its application, factors affecting protoplast culture and the future of protoplasts.
This document discusses metabolic engineering and summarizes key points about manipulating metabolic pathways in organisms. Metabolic engineering involves genetically modifying organisms to modulate their metabolism and produce desired products. It can be done by directly manipulating genes encoding enzymes in pathways or indirectly altering regulatory pathways. The document outlines several approaches to metabolic engineering, including overexpressing rate-limiting enzymes, inhibiting competing pathways, and expressing heterologous genes from other organisms. It also summarizes applications of metabolic engineering for producing amino acids, antibiotics, and manipulating plant metabolism to create foods with improved nutrients or biofuel properties.
PRODUCTION OF HAPLOID PLANTS AND HOMOZYGOUS DIPLOID LINESAmbika Prajapati
This document presents information on producing haploid plants and homozygous diploid lines through plant biotechnology. It discusses two approaches for producing haploid plants, in vivo and in vitro. It also describes techniques for identifying haploids, diploidizing haploids through colchicine treatment or endomitosis to produce homozygous plants, and the significance of haploid plants for plant breeding programs.
Genetically modified or transgenic plants are plants that have been modified using genetic engineering techniques to introduce new traits. This document discusses the history and process of creating transgenic plants. It describes how transgenic plants are generated by transferring genes from other species into the target plant using either indirect methods like Agrobacterium-mediated transformation or direct physical methods like biolistics. The document provides details on the various applications of transgenic plants including producing herbicide resistance, insect resistance, virus resistance, and improving nutritional quality.
The document discusses selectable marker genes that are commonly used in plant transformation systems. Selectable marker genes are included in transformation vectors along with the target gene of interest. They confer resistance to transformed cells when grown on media containing toxic substances like antibiotics, herbicides, or antimetabolites. This allows transformed cells to survive while non-transformed cells die. There are three main categories of selectable marker genes: antibiotic resistance genes, antimetabolite marker genes, and herbicide resistance genes. Common examples of genes used include nptII for kanamycin resistance, pat/bar for phosphinothricin/glufosinate resistance, and epsps/aroA for glyphosate resistance.
Introduction
Definition of an Insect Resistant Plant
What is the Bt gene?
History
The crystal ( cry)Proteins
Definition of cry protein
How does Bt work?
Mechanism of Bt toxicity
Mode of Action of Insecticidal Crystal Protein
Bt Technology
The Insect Resistance Problem
Advantages
Limitations
Conclusion
References
introduction
What is virus
What is virus resistance plant
History
Gene use for develop virus resistance plant
Coat protein gene
cDNA of satellite RNA
Defective viral genome
Antisense RNA approach and
Ribozyme – mediated protection
conclusion
References
The document summarizes a seminar on the Ti plasmid. It discusses that the Ti plasmid is found in Agrobacterium tumefaciens and is responsible for crown gall tumor formation in plants. It describes the organization and structure of the Ti plasmid, including the T-DNA region flanked by borders that is transferred to plant cells. Two common vector systems used for plant transformation, the cointegrate vector and binary vector, are explained. The cointegrate vector involves integration of an intermediate vector with the Ti plasmid, while the binary vector separates the plasmid and virulence genes. Finally, the general process of Agrobacterium-mediated plant transformation is outlined.
Chromosome walking is a method used to isolate and clone a particular gene or allele through positional cloning. It involves using overlapping clones that contain DNA fragments near the target gene to "walk" through the chromosome until reaching the gene. Each successive clone is tested to map its precise location until eventually reaching the target gene. Chromosome walking was developed in the early 1980s and can be used to analyze genetically transmitted diseases and find single nucleotide polymorphisms. However, it has limitations such as being a slow process and difficulty walking through repeated sequences.
1. Yeast plasmids like the 2 micron circle have been extensively studied and developed into yeast cloning vectors.
2. Shuttle vectors like YEp vectors contain selectable marker genes like LEU2 and bacterial plasmid origins of replication like pBR322, allowing them to replicate in both E. coli and yeast.
3. The 2 micron circle is a 6kb endogenous yeast plasmid that replicates autonomously through an ARS sequence and is maintained at 50-100 copies per cell.
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
This document summarizes terminator gene technology, which genetically modifies plants to produce sterile seeds. It was developed by the seed industry to prevent seed saving. There are two types: varietal GURT (V-GURT) renders all subsequent seeds sterile, while trait GURT (T-GURT) switches traits on/off using chemical treatments. While it provides benefits to industry, it is controversial due to concerns over loss of biodiversity and impact on small farmers who rely on seed saving. Most countries have imposed a moratorium on field testing and commercialization of terminator seeds.
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
The document discusses organogenesis, which is the development of adventitious organs or primordial from undifferentiated plant cell mass through differentiation. It describes the process, including dedifferentiation and redifferentiation stages. There are two types of organogenesis - direct organogenesis which does not involve callus formation, and indirect organogenesis which involves callus formation. Organogenesis is used in plant tissue culture to regenerate plants through shoot or root cultures and is influenced by factors like explant source and size, plant growth regulators, and culture conditions. It has commercial applications in micropropagation of plants.
Introduction
Transcriptome analysis
Goal of functional genomics
Why we need functional genomics
Technique
1. At DNA level
2.At RNA level
3. At protein level
4. loss of function
5. functional genomic and bioinformatics
Application
Latest research and reviews
Websites of functional genomics
Conclusions
Reference
Genetic variations can occur in plants produced through plant tissue culture and be detected as changes in genetic characteristics or phenotypes. Variations commonly include changes in chromosome number and structure. Regenerated plants with chromosomal changes often show alterations in traits like leaf shape and color, growth rate, and fertility. These heritable mutations can persist when plants are transplanted to fields. Somaclonal variations are caused by genetic factors like pre-existing variations in explant cells or mutations during tissue culture, and can result in changes in plant characteristics that are useful for crop improvement.
Embryo culture involves growing plant embryos artificially in order to enhance survival rates. It is commonly used to rescue weak or immature embryos that may not otherwise survive to become viable plants. The process involves excising embryos from seeds or ovaries and placing them onto sterile nutrient-rich media under suitable temperature, light, and humidity conditions. Embryo culture has various applications in plant breeding, including shortening breeding cycles, overcoming seed dormancy, producing hybrids, and conserving plant germplasm. It is an important technique in modern plant breeding and development of new crop varieties.
Sequence alig Sequence Alignment Pairwise alignment:-naveed ul mushtaq
Sequence Alignment Pairwise alignment:- Global Alignment and Local AlignmentTwo types of alignment Progressive Programs for multiple sequence alignment BLOSUM Point accepted mutation (PAM)PAM VS BLOSUM
Plant Disease Resistant And Genetic EngineeringShweta Jhakhar
Study the adverse effects of different viruses and other fungal diseases on the plants and their growth. Discuss the methods e.g. plant disease resistant and genetic engineering to protect the plants.
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.
Marker and reporter genes are used in plant genetic transformation. Selectable marker genes confer resistance to antibiotics or herbicides, allowing selection of transformed cells. Common selectable markers confer resistance to kanamycin, hygromycin, and glyphosate. Reporter genes like GFP, GUS, luciferase encode proteins that can be easily detected, allowing screening of transformation events. These genes help recover transformed cells and detect transgene expression without damaging cells.
A presentation covering the process of protoplast culture including protoplast isolation, protoplast fusion, culture of protoplast, its application, factors affecting protoplast culture and the future of protoplasts.
This document discusses metabolic engineering and summarizes key points about manipulating metabolic pathways in organisms. Metabolic engineering involves genetically modifying organisms to modulate their metabolism and produce desired products. It can be done by directly manipulating genes encoding enzymes in pathways or indirectly altering regulatory pathways. The document outlines several approaches to metabolic engineering, including overexpressing rate-limiting enzymes, inhibiting competing pathways, and expressing heterologous genes from other organisms. It also summarizes applications of metabolic engineering for producing amino acids, antibiotics, and manipulating plant metabolism to create foods with improved nutrients or biofuel properties.
PRODUCTION OF HAPLOID PLANTS AND HOMOZYGOUS DIPLOID LINESAmbika Prajapati
This document presents information on producing haploid plants and homozygous diploid lines through plant biotechnology. It discusses two approaches for producing haploid plants, in vivo and in vitro. It also describes techniques for identifying haploids, diploidizing haploids through colchicine treatment or endomitosis to produce homozygous plants, and the significance of haploid plants for plant breeding programs.
Genetically modified or transgenic plants are plants that have been modified using genetic engineering techniques to introduce new traits. This document discusses the history and process of creating transgenic plants. It describes how transgenic plants are generated by transferring genes from other species into the target plant using either indirect methods like Agrobacterium-mediated transformation or direct physical methods like biolistics. The document provides details on the various applications of transgenic plants including producing herbicide resistance, insect resistance, virus resistance, and improving nutritional quality.
The document discusses selectable marker genes that are commonly used in plant transformation systems. Selectable marker genes are included in transformation vectors along with the target gene of interest. They confer resistance to transformed cells when grown on media containing toxic substances like antibiotics, herbicides, or antimetabolites. This allows transformed cells to survive while non-transformed cells die. There are three main categories of selectable marker genes: antibiotic resistance genes, antimetabolite marker genes, and herbicide resistance genes. Common examples of genes used include nptII for kanamycin resistance, pat/bar for phosphinothricin/glufosinate resistance, and epsps/aroA for glyphosate resistance.
Introduction
Definition of an Insect Resistant Plant
What is the Bt gene?
History
The crystal ( cry)Proteins
Definition of cry protein
How does Bt work?
Mechanism of Bt toxicity
Mode of Action of Insecticidal Crystal Protein
Bt Technology
The Insect Resistance Problem
Advantages
Limitations
Conclusion
References
introduction
What is virus
What is virus resistance plant
History
Gene use for develop virus resistance plant
Coat protein gene
cDNA of satellite RNA
Defective viral genome
Antisense RNA approach and
Ribozyme – mediated protection
conclusion
References
The document summarizes a seminar on the Ti plasmid. It discusses that the Ti plasmid is found in Agrobacterium tumefaciens and is responsible for crown gall tumor formation in plants. It describes the organization and structure of the Ti plasmid, including the T-DNA region flanked by borders that is transferred to plant cells. Two common vector systems used for plant transformation, the cointegrate vector and binary vector, are explained. The cointegrate vector involves integration of an intermediate vector with the Ti plasmid, while the binary vector separates the plasmid and virulence genes. Finally, the general process of Agrobacterium-mediated plant transformation is outlined.
Chromosome walking is a method used to isolate and clone a particular gene or allele through positional cloning. It involves using overlapping clones that contain DNA fragments near the target gene to "walk" through the chromosome until reaching the gene. Each successive clone is tested to map its precise location until eventually reaching the target gene. Chromosome walking was developed in the early 1980s and can be used to analyze genetically transmitted diseases and find single nucleotide polymorphisms. However, it has limitations such as being a slow process and difficulty walking through repeated sequences.
1. Yeast plasmids like the 2 micron circle have been extensively studied and developed into yeast cloning vectors.
2. Shuttle vectors like YEp vectors contain selectable marker genes like LEU2 and bacterial plasmid origins of replication like pBR322, allowing them to replicate in both E. coli and yeast.
3. The 2 micron circle is a 6kb endogenous yeast plasmid that replicates autonomously through an ARS sequence and is maintained at 50-100 copies per cell.
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
This document summarizes terminator gene technology, which genetically modifies plants to produce sterile seeds. It was developed by the seed industry to prevent seed saving. There are two types: varietal GURT (V-GURT) renders all subsequent seeds sterile, while trait GURT (T-GURT) switches traits on/off using chemical treatments. While it provides benefits to industry, it is controversial due to concerns over loss of biodiversity and impact on small farmers who rely on seed saving. Most countries have imposed a moratorium on field testing and commercialization of terminator seeds.
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
The document discusses organogenesis, which is the development of adventitious organs or primordial from undifferentiated plant cell mass through differentiation. It describes the process, including dedifferentiation and redifferentiation stages. There are two types of organogenesis - direct organogenesis which does not involve callus formation, and indirect organogenesis which involves callus formation. Organogenesis is used in plant tissue culture to regenerate plants through shoot or root cultures and is influenced by factors like explant source and size, plant growth regulators, and culture conditions. It has commercial applications in micropropagation of plants.
Introduction
Transcriptome analysis
Goal of functional genomics
Why we need functional genomics
Technique
1. At DNA level
2.At RNA level
3. At protein level
4. loss of function
5. functional genomic and bioinformatics
Application
Latest research and reviews
Websites of functional genomics
Conclusions
Reference
Genetic variations can occur in plants produced through plant tissue culture and be detected as changes in genetic characteristics or phenotypes. Variations commonly include changes in chromosome number and structure. Regenerated plants with chromosomal changes often show alterations in traits like leaf shape and color, growth rate, and fertility. These heritable mutations can persist when plants are transplanted to fields. Somaclonal variations are caused by genetic factors like pre-existing variations in explant cells or mutations during tissue culture, and can result in changes in plant characteristics that are useful for crop improvement.
Embryo culture involves growing plant embryos artificially in order to enhance survival rates. It is commonly used to rescue weak or immature embryos that may not otherwise survive to become viable plants. The process involves excising embryos from seeds or ovaries and placing them onto sterile nutrient-rich media under suitable temperature, light, and humidity conditions. Embryo culture has various applications in plant breeding, including shortening breeding cycles, overcoming seed dormancy, producing hybrids, and conserving plant germplasm. It is an important technique in modern plant breeding and development of new crop varieties.
Sequence alig Sequence Alignment Pairwise alignment:-naveed ul mushtaq
Sequence Alignment Pairwise alignment:- Global Alignment and Local AlignmentTwo types of alignment Progressive Programs for multiple sequence alignment BLOSUM Point accepted mutation (PAM)PAM VS BLOSUM
Plant Disease Resistant And Genetic EngineeringShweta Jhakhar
Study the adverse effects of different viruses and other fungal diseases on the plants and their growth. Discuss the methods e.g. plant disease resistant and genetic engineering to protect the plants.
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.
Evolution of plant resistance to a fungal pathogenJason Price, PhD
Jason Price defended his Ph.D dissertation at Indiana University on September 17, 2002. The dissertation assessed the potential for evolution of quantitative resistance to leaf rust disease in the clonal plant species Euthamia graminifolia. Price found that (1) resistance levels varied between host genotypes, (2) resistance was heritable by vegetative offspring, and (3) disease negatively impacted vegetative reproduction by decreasing rhizome biomass. This met the three necessary conditions for evolution of resistance through natural selection via changes in genotype frequencies during vegetative reproduction within populations. However, seedling recruitment was very low in established populations, suggesting vegetative reproduction would be more important for changes in genetic makeup between populations over time.
Review Paper- Durable Resistance against Fungal pathogen 1Shanika Mihirani
This document discusses strategies for durable resistance against fungal plant pathogens. It defines durable resistance as resistance that can last for many years and defines several mechanisms that can provide durable resistance, including gene pyramiding, partial resistance, and genes like the wheat gene Lr34 that confers resistance through an ABC transporter protein. The document also discusses that durable resistance is influenced by factors like the farming system, pathogen characteristics, resistance gene mutation rates, and pathogen population size and interactions between host and pathogen genes.
Cisgenics as a next generation GMO crops. This concept is new and alternative to transgenic crops...can avoid fear of transgenics w.r.t health and environment problems.
it cover almost all content in cis/intragesis, right from introduction definition, explanation, production of marker free transgenic, intragenic vector construction, regulatory guide lines, current and future status, limitation, advantage over existing technique, swot analysis etc
its very useful for your seminar and presentations. it contain lot of picture, table, figure for your easy understanding
thank you
Mahesh
This document presents a seminar on cisgenesis and intragenesis as new tools in crop improvement. It begins with introductions to cisgenesis and intragenesis, noting they allow for the introduction of isolated genes from crossable species or the crop itself. It then discusses why cisgenesis/intragenesis are important alternatives to issues with transgenesis, traditional breeding, and translocation breeding. Methods for developing cisgenic/intragenic plants including vector design and transformation techniques are covered. Examples of crops modified with cisgenesis/intragenesis including late blight resistant potato and apple scab resistant apple are provided. A case study on stacking two late blight resistance genes in potato cisgenically is also summarized
Fungal Transformation in yeast and filamentous fungi
Introduction to Fungi
Background of fungal transformation
Transformation protocol
Transformation vectors
Integration into chromosomes
Biological applications of fungi
Conclusion
References
The use of the term cisgenesis is an attempt to distinguish GM plants or other organisms produced in this way from transgenics that is GM plants that contain DNA from unrelated organisms. Schouten et al. (2006) introduced the term cisgenesis and defined cisgenesis as the modification in the genetic background of a recipient plant by a naturally derived gene from a cross compatible species including its introns and its native promoter and terminator flanked in the normal sense orientation. Since cisgenes shared a common gene pool available for traditional breeding the final cisgenic plant should be devoid of any kind of foreign DNA viz., selection markers and vector- backbone sequences. Sometimes the word cisgenesis is also referred to as Agrobacterium-mediated gene transfer from a sexually compatible plant where only the T-DNA borders may be present in the recipient organism after transformation (EFSA, 2012). The cisgenesis precludes linkage drag, and hence, prevents hazards from unidentified hitch hiking genes (Schouten, and Jacobsen, 2008). Compared to transgenesis, one of the disadvantages shared by cisgenesis is that characters outside the sexually compatible gene pool cannot be introduced. Furthermore, development of cisgenic crops involves extraordinary proficiency and time compared to transgenic crops. Therefore, the required genes or fragments of genes may not be readily accessible but have to be isolated from the sexually compatible gene pool (Holme et al., 2013).
On 16 February 2012, European Food Safety Authority (EFSA, 2012) reported the detail study concerning the safety aspects of cisgenic plants and validated that cisgenic plants are secure to be used in terms of environment, food and feed, similar to the traditionally bred plants. However, the present GMO regulation keeps the cisgenic micro-organisms out from its supervision. The first scientific statement of bringing forth a true plant obtained by cisgenic approach was reported in apple through the insertion of the internal scab resistance gene HcrVf2 influenced by their own regulatory genes into the cultivar Gala, a scab susceptible cultivar (Vanblaere et al., 2011). Barley with improved phytase activity was produced successfully by Holme et al. 2011, through cisgenic approach. Late blight resistant potatoes have developed by cisgene stacking of R- gene (jo et al., 2014).
This document describes the Cre-loxP system for creating conditional gene knockouts in mice. The system involves introducing the Cre recombinase enzyme and loxP sites, which are not naturally present in mice. LoxP sites are inserted on either side of the gene to be knocked out in one "floxed" mouse line. A separate "Cre mouse" line is created that expresses Cre recombinase in a tissue-specific manner. Crossing the two mouse lines results in Cre recombinase recombining the loxP sites and deleting the gene of interest, but only in the tissues where Cre is expressed, allowing conditional rather than complete knockout of the gene.
This document discusses disseminated fungal infections, including candidiasis. It notes that fungal infections in hospitals have increased dramatically due to rising immunosuppressed populations. Candida species are the most common cause. Risk factors include ICU stay, immunosuppression, and use of broad-spectrum antibiotics and catheters. Diagnosis relies on blood and tissue cultures but has low sensitivity. Early antifungal treatment improves outcomes, and options include fluconazole, amphotericin B, and echinocandins. Prevention strategies focus on hand hygiene and reducing unnecessary fluconazole use.
Transgenesis is the process of introducing an exogenous gene into an organism to produce a new trait. It allows for more specific, faster, and flexible introduction of traits compared to selective breeding. Golden rice was developed using transgenesis to introduce beta-carotene genes into rice, providing vitamin A. While this could help address vitamin A deficiency, there are also risks like gene transfer and unintended effects that require careful evaluation.
mechanism of resistance in plant against insectsaylove
The document discusses different mechanisms of resistance in plants against insects, including antixenosis, antibiosis, and tolerance. It provides details on each mechanism:
Antixenosis deters or reduces insect colonization through physical and chemical traits that discourage feeding, oviposition, and orientation. Antibiosis impacts insects after colonization through toxic compounds or nutrient deficiencies that cause death, stunted growth, or other negative effects. Tolerance does not affect insect population growth directly but raises the damage threshold for the plant. The document then focuses on specific antixenotic and antibiotic plant traits and compounds.
role of NPK in plant fungal disease resistanceShilpa Malaghan
This document discusses the role of nutrients NPK in plant disease resistance. It states that high nitrogen can promote disease by weakening cell walls and improving conditions for pathogens, while moderate nitrogen helps resistance. Phosphorus and potassium also play important roles. Maintaining balanced nutrition helps physical and chemical resistance mechanisms, promoting healthier tissue structure less susceptible to infection.
Transgenic plants can be engineered for insect resistance by introducing genes from bacteria, plants or animals. This study generated transgenic broccoli expressing a Cry1Aa gene from Bacillus thuringiensis for resistance against diamondback moth. A second study transformed two cabbage varieties with a sporamin gene, conferring resistance against diamondback moth. Transgene insertion was confirmed, and resistant transgenic lines were selected and shown to inherit resistance stably to subsequent generations. Transgenic crops hold promise for reduced pesticide use and increased sustainable food production.
Tissue culture techniques in plant protectionJayantyadav94
Tissue culture is used to produce plants through biotechnology. Key points:
- Explant tissue is cultured on nutrient media and hormones induce callus growth.
- Plants can be regenerated from single cells through tissue culture techniques.
- Transgenic plants are produced by transferring foreign genes into plant cells using Agrobacterium or direct methods. Genetically engineered plants help with crop improvement traits.
- While tissue culture and genetic engineering offer benefits, there are also risks like unintended gene transfer and loss of crop diversity that require careful risk assessment.
Plant Genetic engineering ,Basic steps ,Advantages and disadvantagesTessaRaju
plant genetic engineering,first genetically engineered crop plant,first genetically engineered foods,genome editing,uses of GE,transgenic plants,basic process of plant genetic enginering,advantages and disadvantages of genetic engineering.
Bio saftey in transgenics & its productsVipin Shukla
Transgenic plants are those plants were we insert an foreign gene in an host genome to modify its characters such as Stress tolerance, Virus resistant, Biotic and Abiotic Tolerance etc.
This document discusses transgenic plants and provides several examples. It begins by defining a transgenic as a genotype developed through genetic engineering containing a foreign or modified gene. Several successful commercial transgenic crops are mentioned, including Bt cotton, golden rice, and Flavr Savr tomato. The document then covers the process of creating transgenic plants including identifying genes of interest, cloning genes, transforming plant cells, and backcross breeding. Methods of gene transfer like Agrobacterium-mediated transformation and particle bombardment are described. The document concludes by outlining the development and objectives of first and second generation transgenic crops.
Tarns-genesis and development of transgenic plantAhmad Ali khan
This document provides an overview of transgenesis and the development of transgenic plants. It defines key terms like transgene and transgenic plants. It describes traditional plant breeding techniques and compares them to transgenic technology. Transgenic technology allows genes to be transferred between any organisms, while traditional breeding is limited to the same genus. Reasons for developing transgenic plants include crop improvement, disease resistance, and stress tolerance. The document outlines the process of developing transgenic plants, including vector-mediated gene transfer using Agrobacterium and biolistic methods. It provides examples of transgenic plants created for insect resistance, herbicide tolerance, drought tolerance, and more. Both advantages and disadvantages of transgenic plants are discussed.
Biotechnology involves applying technology to modify organisms by adding genes from other species. Genetic engineering is the basic tool, involving isolating, modifying, and inserting genes into new species to develop transgenes. This allows developing organisms with novel traits like insect or herbicide resistance. Common methods are using Agrobacterium or gene guns to insert transformation cassettes containing the gene of interest, selectable markers, and insertion sequences. Pakistan uses Bt cotton containing genes for toxins killing bollworm pests. Researchers are also developing Bt rice containing genes for resistance to bacterial leaf blight diseases. Proponents argue GM foods are as safe or safer than conventional foods after regulatory testing and oversight.
The document discusses transgenic plants and their role in pest management. It covers the history of transgenic plant development, including the first genetically engineered crops in the 1980s. It also describes methods of producing transgenic plants and introducing genes for insect resistance from microorganisms like Bacillus thuringiensis and higher plants, such as Bt genes, protease inhibitors, and lectins. The document outlines the major insect resistant transgenic crops developed so far like Bt cotton, tomato, and maize and their commercial success in reducing pesticide use.
This document discusses genetically modified foods. It begins by defining genetically modified organisms and foods as organisms and foods produced through gene transfer between unrelated organisms. It then discusses the reasons for producing GM foods, including higher yields and improved resistance to pests and climate conditions. The history of genetic engineering and GM foods is reviewed from the 1960s to present. Current types of GM foods including herbicide resistant, insect resistant, nutritionally enhanced crops are described. Advantages and status of GM foods in India are also summarized.
The document discusses various modern techniques for increasing crop yield, including genetic engineering, tissue culture, mutagenesis, molecular breeding, RNA interference, and nanotechnology. Genetic engineering techniques like agrobacterium-mediated transfer and biolistic transformation are used to introduce genes that increase stress resistance or yield. Tissue culture, mutagenesis, and molecular breeding also introduce beneficial traits. RNA interference works at the post-transcriptional level to silence genes. Nanoparticles like carbon nanotubes and titanium dioxide have been shown to enhance seed germination and plant growth. The overall aim of these techniques is to develop crop varieties with higher yields.
Detection of Genetically modified plants and Organic Seed production.NSStudents
The Presentation is prepared by the N.S Institution of science, Markapur.
It consists of a basic introduction related to Detection of Genetically modified plants and Organic Seed production.
The document discusses several methods for producing virus-free plants, including meristem culture, heat treatment, chemotherapy, and electrotherapy. It provides details on experiments conducted on potato, black raspberry, sugarcane, and gladiolus plants infected with various viruses. For potato, meristem culture was used to produce virus-free plantlets of three varieties infected with potato virus Y. Heat treatment eliminated the black raspberry necrosis virus from black raspberry explants. In sugarcane, combining meristem culture and chemotherapy with ribavirin helped eliminate mosaic viruses. Experiments on gladiolus used thermo, electro, and chemotherapy methods to eliminate bean yellow mosaic virus from three cultivars. RT-PCR testing confirmed
Helping Farmers to Grow Healthier Crops: Use of qPCR to Monitor Plant Resista...Jonathan Clarke
1. Importance of crop diseases
2. Disease control measures
3. Use of qPCR in selected pathosystems:
• Downy mildew of opium poppy
• Verticillium wilt of olives
A high frequency microcloning protocol for subsequent cryopreservation in Kae...iosrphr_editor
This document describes a high frequency micropropagation protocol for Kaempferia galanga, an endangered medicinal plant. Key findings:
- Shoot cultures were established from rhizome explants on MS medium with BA and multiplied through subculturing. Maximum shoot multiplication (10.6 shoots/explant) was achieved on MS with 4 mg/L BA, 1 mg/L kinetin, and 1 mg/L NAA.
- Repeated subculturing every 4 weeks increased shoot multiplication 3-4x, allowing production of up to 30 shoots/explant within 12 weeks.
- Shoots elongated and rooted simultaneously without needing separate media, reducing production time versus previous protocols.
-
This document summarizes a research project studying the molecular response of tephritid fruit flies to sublethal stresses. The project aims to:
1) Identify the molecular pathways activated in fruit flies exposed to heat, cold, radiation, and parasitic infection stresses.
2) Construct a regulatory network of stress response genes in two key fruit fly pests.
3) Validate specific genes' roles in stress response through functional assays.
The goal is to develop more effective and environmentally friendly postharvest treatments for controlling fruit flies, providing benefits to the horticulture industry, government pest management strategies, and researchers.
This document discusses biotechnology and genetically modified crops. It provides examples of GM crops developed for traits like herbicide tolerance, insect resistance, and virus resistance. It also discusses the global area under cultivation of major GM crops and countries growing them. Both benefits and risks of biotechnology are outlined. The document emphasizes the importance of assessing ecological risks and managing risks through strategies like conducting laboratory, small-scale and large-scale field trials before commercial release of GM crops.
This document summarizes the process of plant genetic transformation using Agrobacterium tumefaciens. It describes how A. tumefaciens transfers T-DNA from its Ti plasmid into plant cells, integrating the T-DNA into the plant genome and expressing genes that cause crown gall disease. The document also outlines the key steps in the process, from gene transfer to the plant cell through regeneration of a transformed whole plant and methods to detect successful transformation events. Common genes inserted into transgenic crops are also listed, including genes for herbicide and insect resistance.
This document summarizes Agrobacterium-mediated gene transfer, which is a process where genes are transferred from Agrobacterium tumefaciens bacteria to plant cells. Key points include: A. tumefaciens transfers oncogenes from its Ti plasmid to plant genomes, integrating the T-DNA and causing crown gall disease; the process involves virulence genes and results in opine production in the plant; common methods are wounding, co-cultivation, and leaf disks; transformation allows foreign genes to be stably integrated and expressed in plants.
Similar to Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens (20)
To create a reporter knock-in mouse, a target vector construct containing a LacZ gene, neomycin gene, and HSV-tk is introduced into ES cells via electroporation. The ES cells are then screened and genotyped to confirm homologous recombination. Generated ES cells are injected into blastocysts that are implanted into a surrogate mother to generate chimeric mice. Analysis of reporter gene expression is carried out via X-gal staining of tissue from Foxk2+/LacZ, Foxk2LacZ/LacZ, and wild-type mice. Fluorescent immunohistochemistry can further validate results.
The document analyzes the monoamine oxidase A (MAO-A) gene through a bioinformatics study. It finds that amino acid sites 374 and 406 are conserved across 106 sequences and mutations there eliminate protein activity. Gene and protein expression of MAO-A vary between tissues and organisms. While gene expression is high in many tissues, protein levels are high in most tissues except the lateral ventricle, hippocampus, lymph node, and bone marrow.
1) The document discusses various methods for industrial biotechnology including strain engineering techniques like adaptive evolution, genome shuffling, and MAGE that aim to generate genetic diversity in strains.
2) It also covers the potential for algae-based biofuels but notes the current high production costs and need for further research and strain optimization over 10-15 years before being commercially viable.
3) The techniques discussed generate diversity in strains to increase metabolic capacity for industrial applications, though each method has strengths and weaknesses in terms of complexity, targeting abilities, and other factors.
Phytoremediation of Crude Oil Contaminated Marine Water with Halophytes endow...Kalyani Rajalingham
This document discusses using transgenic halophytic algae Macrocystis pyrifera for phytoremediation of naphthalene in marine oil spills. The objective is to insert genes for naphthalene degradation from Pseudomonas putida into M. pyrifera to create plants that can break down naphthalene. Experiments will transform M. pyrifera with these genes using agrobacterium, then test the transgenic plants' ability to degrade naphthalene from simulated oil spills in seawater tanks over 2 weeks, with or without additional P. putida on the leaves.
1) The document discusses using biotechnology to genetically modify crops to increase their freezing tolerance through the introduction of CBF genes from Arabidopsis thaliana.
2) While constitutive expression of CBF genes confers freezing resistance, it also leads to reduced growth and productivity.
3) The study by Pino et al. replaced the constitutive promoter of the introduced CBF gene with a stress-inducible promoter, allowing production of the transgene only under cold stress. This resulted in potato plants that were both highly productive and resistant to freezing.
Comparative mitochondrial zygomycetes: bacterial-like Rnase P RNAs, mobile el...Kalyani Rajalingham
Comparative mitochondrial zygomycetes: bacterial-like Rnase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperm,
By
Kalyani Rajalingham
Somayeh Haji Kazem Nili
1) The document discusses various methods for drug discovery screening, including combinatorial chemistry to generate drug samples and high-throughput screening using automation in multi-well plates.
2) It describes different detection systems for high-throughput screening such as radiometric detection, fluorescence assays including time-resolved fluorescence, fluorescence polarization, and fluorescence correlation spectroscopy.
3) The document also discusses cell-based assays, which are performed in vivo using immortalized cell lines. Various types of cell-based assays are described such as second messenger assays, reporter gene assays, and cell proliferation assays.
The binding of cosmological structures by massless topological defectsSérgio Sacani
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Unlocking the mysteries of reproduction: Exploring fecundity and gonadosomati...AbdullaAlAsif1
The pygmy halfbeak Dermogenys colletei, is known for its viviparous nature, this presents an intriguing case of relatively low fecundity, raising questions about potential compensatory reproductive strategies employed by this species. Our study delves into the examination of fecundity and the Gonadosomatic Index (GSI) in the Pygmy Halfbeak, D. colletei (Meisner, 2001), an intriguing viviparous fish indigenous to Sarawak, Borneo. We hypothesize that the Pygmy halfbeak, D. colletei, may exhibit unique reproductive adaptations to offset its low fecundity, thus enhancing its survival and fitness. To address this, we conducted a comprehensive study utilizing 28 mature female specimens of D. colletei, carefully measuring fecundity and GSI to shed light on the reproductive adaptations of this species. Our findings reveal that D. colletei indeed exhibits low fecundity, with a mean of 16.76 ± 2.01, and a mean GSI of 12.83 ± 1.27, providing crucial insights into the reproductive mechanisms at play in this species. These results underscore the existence of unique reproductive strategies in D. colletei, enabling its adaptation and persistence in Borneo's diverse aquatic ecosystems, and call for further ecological research to elucidate these mechanisms. This study lends to a better understanding of viviparous fish in Borneo and contributes to the broader field of aquatic ecology, enhancing our knowledge of species adaptations to unique ecological challenges.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
ESPP presentation to EU Waste Water Network, 4th June 2024 “EU policies driving nutrient removal and recycling
and the revised UWWTD (Urban Waste Water Treatment Directive)”
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...Leonel Morgado
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
5. Chitinase genes from Trichoderma harzianum used to
confer resistance to crop plants
6. Purpose
“The present study was undertaken to examine the
effectiveness of the 42 kDa endochitinase genes from
T. virens in protecting cotton from fungal diseases.
In addition, tobacco plants were initially
transformed to test the expression of various
endochitinase clones, and then these were evaluated
for their resistance to A. alternata. “
7. Materials and
Methods
Step 1 – Choose genes, and deliver it to plant
genome
Step 2 – Verify incorporation into genome.
Copy Number?
Step 3 – Protein levels – How much protein
does it make?
Step 4 – Treatments
13. Materials and
Methods
Step 3 – Protein levels using
Fluorometric gel-based endochitinase
assay
4-methylumbelliferyl B-D-
N-N,N-triacetylchitotrioside
14. Materials and
Methods Step 4 – Test effectiveness of insert in
COTTON using the fungus R. Solani
Measure disease symptoms
15. Materials and
Methods Step 4 – Test effectiveness of insert IN
TOBACCO using the fungus A. Alternata
% necrosis on leaf after 2 weeks was
A. Alternata
One ot two
agar plugs
16. Results
Step 1 – Check that the transgene was inserted.
In how many lines?
Step 2 – How many copies inserted?
Step 3 – Protein levels?
Step 4 – Present in subsequent generations?
Step 5 – Present in leaves, and roots?
Step 6 – Is transgenic plant resistant to R.
Solani, and A. Alternata?
31. Discussion
1 - only one (Tv-ech1) of the three
Trichoderma virens endochitinase cDNA
clones tested as determined by the enzyme
activity assays on leaf extracts
2 – no morphological abnormalities in
transgenic plants
3 - high expressing lines identified in T0
generation were not always found to
maintain chitinase activity in the T1
generation
32. Discussion
4 - T2 seeds from several high
endochitinase-expressing lines were
subjected to infection by planting them in
soil infested with R. Solani
5 - At moderate inoculum pressure (0.28 g
culture /L of mix), a majority of the
untransformed seedlings (98%) in infested
soil died due to post-emergence infections
or the seeds failing to germinate; however,
> 67% of the transgenic seedlings remained
healthy even after 2 weeks in the infested
mix
33. Discussion
6 - when the inoculum pressure was
doubled (0.56 g /L), 15 of the 28 transgenic
seedlings examined 9 days after planting
were free of disease symptoms, while none
of the control plants survived
34. Discussion
7 - A possible explanation for the high
levels of protection observed in our study
may therefore be that transgenic
Trichoderma endochitinase activity in plant
tissues may release compounds from the
cell wall of the invading fungi, that in turn
elicit in the plant a faster and more
comprehensive defensive response.