The ultimate objective of modern plant breeding is to improve a top variety in one single additional character in a predictable and precise manner without disturbing the rest of the genome. Today this is being realised through examples of successful transfer of specific traits into higher plants by gene transfer.
Techniques that open up to the plant breeder the possibility of transferring in a planned manner characters from one organism to another have been developed in microbial genetics. It should be stressed right at the outset that the expression “gene” has different meanings in agriculture and in molecular biology.
Gene Transfer Methods:
The gene transfer techniques in plant genetic transformation are broadly grouped into two categories:
I. Vector-mediated gene transfer
II. Direct or vector less DNA transfer
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
Vector mediated gene transfer methods for transgenesis in Plants.Akshay More
Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.
☺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.
Agrobacterium mediated gene transfer, Ti-plasmid, cloning vectors based on Ti-plasmid, advantages disadvantages regarding cloning vectors based on Ti-plasmid are major areas covered in this Presentation.
This bacterium has a large plasmid that induces tumor, and for this reason, it was named tumor-inducing (Ti) plasmid.
This is process of altering the genetic makeup of an organism using Recombinant DNA Technology.
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
Vector mediated gene transfer methods for transgenesis in Plants.Akshay More
Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.
☺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.
Agrobacterium mediated gene transfer, Ti-plasmid, cloning vectors based on Ti-plasmid, advantages disadvantages regarding cloning vectors based on Ti-plasmid are major areas covered in this Presentation.
This bacterium has a large plasmid that induces tumor, and for this reason, it was named tumor-inducing (Ti) plasmid.
This is process of altering the genetic makeup of an organism using Recombinant DNA Technology.
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.
Agrobacterium and other methods of plant transformation including gene gun, i...PABOLU TEJASREE
The process of transfer, integration and expression of transgene in the host cells is known as genetic transformation. A foreign gene (transgene) encoding the trait must be incorporated into plant cells, along with a "cassette" of extra genetic material to add a desirable trait to a crop. The cassette includes a sequence of DNA called a "promoter", which determines where and when the foreign gene is expressed in the host, and a "marker gene" which allows breeders to determine by screening or selection which plants contain the inserted gene. For example, marker genes may make plants resistant to antibiotics not used routinely (e.g., agrimycin, kanamycin) or tolerant of some herbicides.
It is a rod-shaped, Gram-negative, Peritrichous flagella, Soil bacterium. Agrobacterium is well known for its ability to transfer DNA between itself become an important tool for genetic engineering.
Biotransformation by Dr. Jayarama Reddy, St. Joseph's College, Bengaluru-27Dr. Jayarama Reddy
The genus Agrobacterium has been divided into a number of species. However, this division has reflected, for the most part, disease symptomology and host range. Thus, A. radiobacter is an “avirulent” species, A. tumefaciens causes crown gall disease, A. rhizogenes causes hairy root disease, and A. rubi causes cane gall disease. More recently, a new species has been proposed, A. vitis, which causes galls on grape and a few other plant species. We now know that symptoms follow, for the most part, the type of tumorigenic plasmid contained within a particular strain. Curing a particular plasmid and replacing this plasmid with another type of tumorigenic plasmid can alter disease symptoms. For example, infection of plants with A. tumefaciens C58, containing the nopaline-type Ti plasmid pTiC58, results in the formation of crown gall teratomas. When this plasmid is cured, the strain becomes nonpathogenic. Introduction of Ri plasmids into the cured strain “converts” the bacterium into a rhizogenic strain. Regardless of the current confusion in species classification, for the purposes of plant genetic engineering, the most important aspect may be the host range of different Agrobacterium strains. As a genus, Agrobacterium can transfer DNA to a remarkably broad group of organisms including numerous dicot and monocot angiosperm species and gymnosperms. In addition, Agrobacterium can transform fungi, including yeasts, ascomycetes, and basidiomycetes. Recently, Agrobacterium was reported to transfer DNA to human cells. The molecular and genetic basis for the host range of a given Agrobacterium strain remains unclear. Early work indicated that the Ti plasmid, rather than chromosomal genes, was the major genetic determinant of host range. Several virulence (vir) loci on the Ti plasmid, including virC and virF, were shown to determine the range of plant species that could be transformed to yield crown gall tumors. The virH (formerly called pinF) locus appeared to be involved in the ability of Agrobacterium to transform maize, as established by an assay in which symptoms of maize streak virus infection were determined following agroinoculation of maize plants. Other vir genes, including virG, contribute to the “hypervirulence” of particular strains.
The problems attract worldwide attention K/a Global Environmental Problems.
The top three environmental problems are: (1) Greenhouse Effect and Global Warming (2) Depletion of Ozone and (3) Acid Rain.
Aim1: To study the method of genome identification through ENSEMBL browser.
Aim2: To study the method of genome identification through VISTA.
Aim3: To study the method of genome identification through UCSC Genome Browser.
Aim4: To study the method of genome and amino acid sequences through UCSC Genome Browser.
Intracellular Components
We will now begin our discussion of intracellular organelles. As we have mentioned, only eukaryotic cells have intracellular sub-divisions, so our discussion will exclude prokaryotic cells. We will also focus on animal cells, since plant cells have a number of further specialized structures. In this section we will discuss the importance of the cell nucleus, mitochondria, peroxisomes, endoplasmic reticulum, golgi apparatus, and lysosome.
Types of Receptors
Receptors are protein molecules in the target cell or on its surface that bind ligands. There are two types of receptors: internal receptors and cell-surface receptors.
Microbial biomass conversion processes take advantage of the ability of microorganisms to consume and digest biomass and release hydrogen. Depending on the pathway, this research could result in commercial-scale systems in the mid- to long-term timeframe that could be suitable for distributed, semi-central, or central hydrogen production scales, depending on the feedstock used.
The cells derived from root apical and shoot-apical meristems and cambium differentiate and mature to perform specific functions. This act leading to maturation is termed as differentiation. During differentiation, cells undergo few to major structural changes both in their cell walls and protoplasm. The living differentiated cells, that by now have lost the capacity to divide can regain the capacity of division under certain conditions. This phenomenon is termed as dedifferentiation. For example, formation of meristems – interfascicular cambium and cork cambium from fully differentiated parenchyma cells. While doing so, such meristems / tissues are able to divide and produce cells that once again lose the capacity to divide but mature to perform specific functions, i.e., get redifferentiated.
Meat and milk from farmed animals including livestock (cattle, goat and buffalo) and poultry are sources of high quality protein and essential amino acids, minerals, fats and fatty acids, readily available vitamins, small quantities of carbohydrates and other bioactive components.1 The Food and Agriculture Organization (FAO) 2008 estimate shows that meat consumption has grown with increase in population. The average global per capita meat consumption is 42.1 kg/year with 82.9 kg/year in developed and 31.1 kg/year in developing countries in a recommended daily animal-sourced protein per capita of 50 kg per year2. Milk on the other hand is consumed in various forms: liquid, cheese, powder, and cream at a global per capita consumption of 108 kg per person per year which is way below the FAO recommended daily consumption of 200 kg.
Antibodies, also known as immunoglobulins, are secreted by B cells (plasma cells) to neutralize antigens such as bacteria and viruses. The classical representation of an antibody is a Y-shaped molecule composed of four polypeptides-two heavy chains and two light chains. Each tip of the "Y" contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision. The ability of binding to an antigen has led to their ubiquitous use in a variety of life science and medical science. These antibodies can be classified into two primary types (monoclonal and polyclonal) by the means in which they are created from lymphocytes. Each of them has important role in the immune system, diagnostic exams, and treatments.
There are many characteristics of biological data. All these characteristics make the management of biological information a particularly challenging problem. Here mainly we will focus on characteristics of biological information and multidisciplinary field called bioinformatics. Bioinformatics, now a days has emerged with graduate degree programs in several universities.
Hormones, Proteins, etc. present in blood in minute concentration can be assayed by the recent advanced technique of “Enzyme Immuno Assay” without involving any disadvantage. The basic reaction is the interaction between an antibody and an antigen.
Meat and milk from farmed animals including livestock (cattle, goat and buffalo) and poultry are sources of high quality protein and essential amino acids, minerals, fats and fatty acids, readily available vitamins, small quantities of carbohydrates and other bioactive components.1 The Food and Agriculture Organization (FAO) 2008 estimate shows that meat consumption has grown with increase in population. The average global per capita meat consumption is 42.1 kg/year with 82.9 kg/year in developed and 31.1 kg/year in developing countries in a recommended daily animal-sourced protein per capita of 50 kg per year2. Milk on the other hand is consumed in various forms: liquid, cheese, powder, and cream at a global per capita consumption of 108 kg per person per year which is way below the FAO recommended daily consumption of 200 kg.
In shotgun sequencing the genome is broken randomly into short fragments (1 to 2 kbp long) suitable for sequencing. The fragments are ligated into a suitable vector and then partially sequenced. Around 400–500 bp of sequence can be generated from each fragment in a single sequencing run. In some cases, both ends of a fragment are sequenced. Computerized searching for overlaps between individual sequences then assembles the complete sequence.
Sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).
The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces. Besides the obvious difficulty of this task, there are some extra practical issues: the original may have many repeated paragraphs, and some shreds may be modified during shredding to have typos. Excerpts from another book may also be added in, and some shreds may be completely unrecognizable.
Vaccine (L. vacca = cow) is a preparation/suspension or extract of dead/attenuated (weakened) germs of a disease which on inoculation (injection) into a healthy person provides temporary/permanent active/passive immunity by inducing antibodies formation.
Thus antibody provoking agents are called vaccines.
Biological treatment is an important and integral part of any wastewater treatment plant that treats wastewater from either municipality or industry having soluble organic impurities or a mix of the two types of wastewater sources.
The four processes are: (1) Preliminary Treatment (2) Primary Treatment (3) Secondary or Biological Treatment and (4) Tertiary or Advanced Treatment
The genetic variations found in the in vitro cultured cells are collectively referred to as somaclonal variations.
The plants derived from such cells are referred to somaclones. Some authors use the terms calliclones and proto-clones to represent cultures obtained from callus and protoplasts respectively.
The growth of plant cells in vitro is an asexual process involving only mitotic division of cells. Thus, culturing of cells is the method to clone a particular genotype. It is therefore expected that plants arising from a given tissue culture should be the exact copies of the parental plant.
The occurrence of phenotypic variants among the regenerated plants (from tissue cultures) has been known for several years. These variations were earlier dismissed as tissue culture artefacts. The term somaclonal variations was first used by Larkin and Scowcraft (1981) for variations arising due to culture of cells, i.e., variability generated by a tissue culture. This term is now universally accepted.
As described elsewhere the explant used in tissue culture may come from any part of the plant organs or cells. These include leaves, roots, protoplasts, microspores and embryos. Somaclonal variations are reported in all types of plant tissue cultures.
In recent years, the term gametoclonal variations is used for the variations observed in the regenerated plants from gametic cells (e.g., anther cultures). For the plants obtained from protoplast cultures, proto-clonal variations is used.
Solid waste management is a polite term for garbage management. As long as humans have been living in settled communities, solid waste, or garbage, has been an issue, and modern societies generate far more solid waste than early humans ever did.
The chemical compounds produced by plants are collectively referred to as phytochemicals. Biotechnologists have special interest in plant tissue culture for the large scale production of commercially important compounds. These include pharmaceuticals, flavours, fragrances, cosmetics, food additives, feed stocks and antimicrobials.
Most of these products are secondary metabolites— chemical compounds that do not participate in metabolism of plants. Thus, secondary metabolites are not directly needed by plants as they do not perform any physiological function (as is the case with primary metabolites such as amino acids, nucleic acids etc.). Although the native plants are capable of producing the secondary metabolites of commercial interest, tissue culture systems are preferred.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
Embracing GenAI - A Strategic ImperativePeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Acetabularia Information For Class 9 .docxvaibhavrinwa19
Acetabularia acetabulum is a single-celled green alga that in its vegetative state is morphologically differentiated into a basal rhizoid and an axially elongated stalk, which bears whorls of branching hairs. The single diploid nucleus resides in the rhizoid.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
1.4 modern child centered education - mahatma gandhi-2.pptx
Plant transformation gene transfer methods in plants
1. Plant Transformation- Gene Transfer Methods in Plants
Dr. Naveen Gaurav
Associate Professor and Head
Department of Biotechnology
Shri Guru Ram Rai University
Dehradun
2. General Principles of Gene Transfer:
The ultimate objective of modern plant breeding is to improve a top variety in one single
additional character in a predictable and precise manner without disturbing the rest of the
genome. Today this is being realised through examples of successful transfer of specific traits
into higher plants by gene transfer.
Techniques that open up to the plant breeder the possibility of transferring in a planned
manner characters from one organism to another have been developed in microbial
genetics. It should be stressed right at the outset that the expression “gene” has different
meanings in agriculture and in molecular biology.
Gene Transfer Methods:
The gene transfer techniques in plant genetic transformation are broadly grouped into two
categories:
I. Vector-mediated gene transfer
II. Direct or vector less DNA transfer
The salient features of the commonly used gene (DNA) transfer methods are given in Table
49.1.
3. Method # I. Vector-Mediated Gene Transfer:
Vector-mediated gene transfer is carried out either by Agrobacterium-mediated
transformation or by use of plant viruses as vectors.
Agrobacterium-Mediated Gene Transfer:
Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium. It is rod shaped and
motile, and belongs to the bacterial family of Rhizobiaceae. A. tumefaciens is a
phytopathogen, and is treated as the nature’s most effective plant genetic engineer.
Some workers consider this bacterium as the natural expert of inter-kingdom gene transfer.
In fact, the major credit for the development of plant transformation techniques goes to the
natural unique capability of A. tumefaciens. Thus, this bacterium is the most beloved by
plant biotechnologists.
There are mainly two species of Agrobacterium:
i. A. tumefaciens that induces crown gall disease.
ii. A. rhizogenes that induces hairy root disease.
Crown Gall Disease and Ti Plasmid:
Almost 100 years ago (1907), Smith and Townsend postulated that a bacterium was the
causative agent of crown gall tumors, although its importance was recognized much later. As
A. tumefaciens infects wounded or damaged plant tissues, in induces the formation of a
plant tumor called crown gall (Fig. 49.1). The entry of the bacterium into the plant tissues is
facilitated by the release of certain phenolic compounds (acetosyringone,
hydroxyacetosyringone) by the wounded sites.
4.
5. Crown gall formation occurs when the bacterium releases its Ti plasmid (tumor- inducing
plasmid) into the plant cell cytoplasm. A fragment (segment) of Ti plasmid, referred to as T-
DNA, is actually transferred from the bacterium into the host where it gets integrated into
the plant cell chromosome (i.e. host genome). Thus, crown gall disease is a naturally
evolved genetic engineering process.
The T-DNA carries genes that code for proteins involved in the biosynthesis of growth
hormones (auxin and cytokinin) and novel plant metabolites namely opines — amino acid
derivatives and agropines — sugar derivatives (Fig. 49.2).
The growth hormones cause plant cells to proliferate and form the gall while opines and
agropines are utilized by A. tumefaciens as sources of carbon and energy. As such, opines
and agropines are not normally part of the plant metabolism (neither produced nor
metabolised). Thus, A. tumefaciens genetically transforms plant cells and creates a
biosynthetic machinery to produce nutrients for its own use.
6. As the bacteria multiply and continue infection, grown gall develops which is a visible mass
of the accumulated bacteria and plant material. Crown gall formation is the consequence of
the transfer, integration and expression of genes of T-DNA (or Ti plasmid) of A. tumefaciens
in the infected plant.
The genetic transformation leads to the formation of crown gall tumors, which interfere with
the normal growth of the plant. Several dicotyledonous plants (dicots) are affected by crown
gall disease e.g. grapes, roses, stone-fruit trees.
The Ti plasmids (approximate size 200 kb each) exist as independent replicating circular DNA
molecules within the Agrobacterium cells. The T-DNA (transferred DNA) is variable in length
in the range of 12 to 24 kb, which depends on the bacterial strain from which Ti plasmids
come. Nopaline strains of Ti plasmid have one T-DNA with length of 20 kb while octopine
strains have two T-DNA regions referred to as TL and TR that are respectively 14 kb and 7 kb in
length. A diagrammatic representation of a Ti plasmid is depicted in Fig. 49.3. The Ti plasmid
has three important regions.
7. 1. T-DNA region: This region has the genes for the biosynthesis of auxin (aux), cytokinin (cyt) and opine
(ocs), and is flanked by left and right borders. These three genes-aux, cyto and ocs are referred to as
oncogenes, as they are the determinants of the tumor phenotype.
T-DNA borders — A set of 24 kb sequences present on either side (right and left) of T-DNA are also
transferred to the plant cells. It is now clearly established that the right border is more critical for T-
DNA transfer and tumori-genesis.
2. Virulence region:
The genes responsible for the transfer of T-DNA into the host plant are located outside T-DNA and the
region is referred to as vir or virulence region. Vir region codes for proteins involved in T-DNA transfer.
At least nine vir-gene operons have been identified. These include vir A, vir G, vir B1, vir C1, vir D1,
D2 and D4, and vir E1, and E2.
3. Opine catabolism region:
This region codes for proteins involved in the uptake and metabolisms of opines. Besides the above
three, there is ori region that is responsible for the origin of DNA replication which permits the Ti
plasmid to be stably maintained in A. tumefaciens.
Plant Transformation Technique Using Agrobacterium:
Agrobacterium-mediated technique is the most widely used for the transformation of plants and
generation of transgenic plants. The important requirements for gene transfer in higher plants through
Agrobacterium mediation are listed.
i. The explants of the plant must produce phenolic compounds (e.g. autosyringone) for activation of
virulence genes.
ii. Transformed cells/tissues should be capable to regenerate into whole plants.
In general, most of the Agrobacterium-mediated plant transformations have the following basic
protocol (Fig. 49.7)
8.
9. 1. Development of Agrobacterium carrying the co-integrate or binary vector with
the desired gene.
2. Identification of a suitable explant e.g. cells, protoplasts, tissues, calluses,
organs.
3. Co-culture of explants with Agrobacterium.
4. Killing of Agrobacterium with a suitable antibiotic without harming the plant
tissue.
5. Selection of transformed plant cells.
6. Regeneration of whole plants.
Advantages of Agrobacterium- mediated transformation:
i. This is a natural method of gene transfer.
ii. Agrobacterium can conveniently infect any explant (cells/tissues/organs).
iii. Even large fragments of DNA can be efficiently transferred.
iv. Stability of transferred DNA is reasonably good.
v. Transformed plants can be regenerated effectively.
Limitations of Agrobacterium- mediated transformation:
i. There is a limitation of host plants for Agrobacterium, since many crop plants
(monocotyledons e.g. cereals) are not infected by it. In recent years, virulent
strains of Agrobacterium that can infect a wide range of plants have been
developed.
ii. The cells that regenerate more efficiently are often difficult to transform, e.g.
embryonic cells lie in deep layers which are not easy targets for Agrobacterium.
10. Method # II. Direct or Vector-less DNA Transfer:
The term direct or vector less transfer of DNA is used when the foreign DNA is directly
introduced into the plant genome. Direct DNA transfer methods rely on the delivery of
naked DNA into the plant cells. This is in contrast to the Agrobacterium or vector-mediated
DNA transfer which may be regarded as indirect methods. Majority of the direct DNA
transfer methods are simple and effective. And in fact, several transgenic plants have been
developed by this approach.
Limitations of direct DNA transfer:
The major disadvantage of direct gene transfer is that the frequency of transgene
rearrangements is high. This results in higher transgene copy number, and high frequencies
of gene silencing.
Types of direct DNA transfer:
The direct DNA transfer can be broadly divided into three categories.
1. Physical gene transfer methods—electro- portion, particle bombardment, microinjection,
liposome fusion, silicon carbide fibres.
2. Chemical gene transfer methods—Polyethylene glycol (PEG)-mediated, diethyl amino
ethyl (DEAE) dextran-mediated, calcium phosphate precipitation.
3. DNA imbibition by cells/tissues/organs.
The salient features of the different methods for direct DNA transfer are given in Table 49.1 .
(A) Physical Gene Transfer Methods:
An overview of the general scheme for the production of transgenic plants by employing
physical transfer methods is depicted in Fig. 49.9. Some details of the different techniques
are described.
11.
12. 1. Electroporation:
Electroporation basically involves the use of high field strength electrical impulses to
reversibly permeabilize the cell membranes for the uptake of DNA. This technique can be
used for the delivery of DNA into intact plant cells and protoplasts.
The plant material is incubated in a buffer solution containing the desired foreign/target
DNA, and subjected to high voltage electrical impulses. This results in the formation of
pores in the plasma membrane through which DNA enters and gets integrated into the
host cell genome.
In the early years, only protoplasts were used for gene transfer by electroporation. Now a
days, intact cells, callus cultures and immature embryos can be used with suitable pre- and
post-electroporation treatments. Electroporation has been successfully used for the
production of transgenic plants of many cereals e.g. rice, wheat, maize.
Advantages of electroporation:
i. This technique is simple, convenient and rapid, besides being cost-effective.
ii. The transformed cells are at the same physiological state after electroporation.
iii. Efficiency of transformation can be improved by optimising the electrical field strength,
and addition of spermidine.
Limitations of electroporation:
i. Under normal conditions, the amount of DNA delivered into plant cells is very low.
ii. Efficiency of electroporation is highly variable depending on the plant material and the
treatment conditions.
iii. Regeneration of plants is not very easy, particularly when protoplasts are used.
13. 2. Particle Bombardment (Biolistics):
Particle (or micro projectile) bombardment is the most effective method for gene transfer, and
creation of transgenic plants. This method is versatile due to the fact that it can be successfully
used for the DNA transfer in mammalian cells and microorganisms.
The micro projectile bombardment method was initially named as biolistics by its inventor
Sanford (1988). Biolistics is a combination of biological and ballistics. There are other names for
this technique- particle gun, gene gun, bio blaster. A diagrammatic representation of micro
projectile bombardment system for the transfer of genes in plants is depicted in Fig. 49.10, and
briefly described below.
14. Micro carriers (micro projectiles), the tungsten or gold particles coated with DNA, are carried by macro
carriers (macro projectiles). These macro-carriers are inserted into the apparatus and pushed
downward by rupturing the disc.
The stopping plate does not permit the movement of macro carrier while the micro carriers (with DNA)
are propelled at a high speed into the plant material. Here the DNA segments are released which enter
the plant cells and integrate with the genome.
Plant material used in bombardment:
Two types of plant tissue are commonly used for particle bombardment:
1. Primary explants which can be subjected to bombardment that are subsequently induced to become
embryo genic and regenerate.
2. Proliferating embryonic tissues that can be bombarded in cultures and then allowed to proliferate
and regenerate.
In order to protect plant tissues from being damaged by bombardment, cultures are maintained on high
osmoticum media or subjected to limited plasmolysis.
Transgene integration in bombardment:
It is believed (based on the gene transfer in rice by biolistics) that the gene transfer in particle
bombardment is a two stage process.
1. In the pre-integration phase, the vector DNA molecules are spliced together. This results in fragments
carrying multiple gene copies.
2. Integrative phase is characterized by the insertion of gene copies into the host plant genome.
The integrative phase facilitates further transgene integration which may occur at the same point or a
point close to it. The net result is that particle bombardment is frequently associated with high copy
number at a single locus. This type of single locus may be beneficial for regeneration of plants.
15. The success of bombardment:
The particle bombardment technique was first introduced in 1987. It has been successfully
used for the transformation of many cereals, e.g. rice, wheat, maize. In fact, the first
commercial genetically modified (CM) crops such as maize containing Bt-toxin gene were
developed by this approach.
A selected list of the transgenic plants (developed by biolistics) along with the sources of the
plant materials used is given in Table 49.2.
16. Factors affecting bombardment:
Several attempts are made to study the various factors, and optimize the system of particle bombardment for
its most efficient use. Some of the important parameters are described.
Nature of micro particles:Inert metals such as tungsten, gold and platinum are used as micro particles to
carry DNA. These particles with relatively higher mass will have a better chance to move fast when
bombarded and penetrate the tissues.
Nature of tissues/cells:The target cells that are capable of undergoing division are suitable for transformation.
Some more details on the choice of plant material used in bombardment are already given.
Amount of DNA:The transformation may be low when too little DNA is used. On the other hand, too much
DNA may result is high copy number and rearrangement of transgenes. Therefore, the quantity of DNA used
should be balanced. Recently, some workers have started using the chemical aminosiloxane to coat the micro
particles with low quantities of DNA adequate enough to achieve high efficiency of transformation.
Environmental parameters:Many environmental variables are known to influence particle bombardment.
These factors (temperature, humidity, photoperiod etc.) influence the physiology of the plant material, and
consequently the gene transfer. It is also observed that some explants, after bombardment may require
special regimes of light, humidity, temperature etc.
The technology of particle bombardment has been improved in recent years, particularly with regard to the
use of equipment. A commercially produced particle bombardment apparatus namely PDS-1000/HC is widely
used these days.
Advantages of particle bombardment:
i. Gene transfer can be efficiently done in organized tissues.
ii. Different species of plants can be used to develop transgenic plants.
Limitations of particle bombardment:
i. The major complication is the production of high transgene copy number. This may result in instability of
transgene expression due to gene silencing.
ii. The target tissue may often get damaged due to lack of control of bombardment velocity.
iii. Sometimes, undesirable chimeric plants may be regenerated
17. 3. Microinjection:
Microinjection is a direct physical method involving the mechanical insertion of the desirable DNA into a
target cell. The target cell may be the one identified from intact cells, protoplasts, callus, embryos,
meristems etc. Microinjection is used for the transfer of cellular organelles and for the manipulation of
chromosomes.
The technique of microinjection involves the transfer of the gene through a micropipette (0.5-10.0 pm
tip) into the cytoplasm/nucleus of a plant cell or protoplast. While the gene transfer is done, the
recipient cells are kept immobilized in agarose embedding, and held by a suction holding pipette (Fig.
49.11).
As the process of microinjection is complete, the transformed cell is cultured and grown to develop into
a transgenic plant. In fact, transgenic tobacco and Brassica napus have been developed by this approach.
The major limitations of microinjection are that it is slow, expensive, and has to be performed by trained
and skilled personnel.
18. 4. Liposome-Mediated Transformation:
Liposomes are artificially created lipid vesicles containing a phospholipid membrane. They are successfully
used in mammalian cells for the delivery of proteins, drugs etc. Liposomes carrying genes can be employed to
fuse with protoplasts and transfer the genes.
The efficiency of transformation increases when the process is carried out in conjunction with polyethylene
glycol (PEG). Liposome-mediated transformation involves adhesion of liposomes to the protoplast surface, its
fusion at the site of attachment and release of plasmids inside the cell (Fig. 49.12).
Advantages of liposome fusion:
i. Being present in an encapsulated form of liposomes, DNA is protected from environmental insults and
damage.
ii. DNA is stable and can be stored for some time in liposomes prior to transfer.
iii. Applicable to a wide range of plant cells.
iv. There is good reproducibility in the technique.
Limitations of liposome fusion:
The major problem with liposome-mediated transformation is the difficulty associated with the regeneration
of plants from transformed protoplasts.
19. 5. Silicon Carbide Fibre-Mediated Transformation:
The silicon carbide fibres (SCF) are about 0.3-0.6 pm in diameter and 10-100 pm
in length. These fibres are capable of penetrating the cell wall and plasma
membrane, and thus can deliver DNA into the cells. The DNA coated silicon
carbide fibres are vortexed with ‘plant material (suspension culture, calluses).
During the mixing, DNA adhering to the fibres enters the cells and gets stably
integrated with the host genome. The silicon carbide fibres with the trade name
Whiskers are available in the market.
Advantages of SCF-mediated transformation:
i. Direct delivery of DNA into intact walled cells. This avoids the protoplast
isolation.
ii. Procedure is simple and does not involve costly equipment.
Disadvantages of SCF-mediated transformation:
i. Silicon carbide fibres are carcinogenic and therefore have to be carefully
handled.
ii. The embryonic plant cells are hard and compact and are resistant to SCF
penetration.
In recent years, some improvements have been made in SCF-mediated
transformation. This has helped in the transformation of rice, wheat, maize and
barley by using this technique.
20. (B) Chemical Gene Transfer Methods:
1. Polyethylene glycol (PEG)-mediated transfer:
Polyethylene glycol (PEG), in the presence of divalent cations (using Ca2+), destabilizes the plasma membrane
of protoplasts and renders it permeable to naked DNA. In this way, the DNA enters nucleus of the protoplasts
and gets integrated with the genome.
The procedure involves the isolation of protoplasts and their suspension, addition of plasmid DNA, followed by
a slow addition of 40% PEG-4000 (w/v) dissolved in mannitol and calcium nitrate solution. As this mixture is
incubated, protoplasts get transformed.
Advantages of PEG-mediated transformation:
i. A large number of protoplasts can be simultaneously transformed.
ii. This technique can be successfully used for a wide range of plant species.
Limitations of PEG-mediated transformation:
i. The DNA is susceptible for degradation and rearrangement.
ii. Random integration of foreign DNA into genome may result in undesirable traits.
iii. Regeneration of plants from transformed protoplasts is a difficult task.
2. Deae Dextran-Mediated transfer:The desirable DNA can be complexed with a high molecular weight
polymer diethyl amino ethyl (DEAE) dextran and transferred. The major limitation of this approach is
that it does not yield stable trans-formants.
3. Calcium Phosphate Co- Precipitation-Mediated Transfer:The DNA is allowed to mix with calcium
chloride solution and isotonic phosphate buffer to form DNA-calcium phosphate precipitate. When the
actively dividing cells in culture are exposed to this precipitate for several hours, the cells get
transformed. The success of this method is dependent on the high concentration of DNA and the
protection of the complex precipitate. Addition of dimethyl sulfoxide (DMSO) increases the efficiency of
transformation.
DNA Imbibition By Cells/Tissues:Some workers have seriously tried to transform cells by incubating cell
suspensions, tissues, embryos and even seeds with DNA. The belief is that the DNA gets imbibed, and
the cells get transformed. DNA imbibition approach has met with little or no success.