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
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
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.
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.
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
1.What is plant tissue culture?
2.Production of virus free plants.
3.History.
4.Virus elimination by heat treatment.
5.Virus elimination by Meristem Tip culture.
6.Factor affecting virus eradication by Meristem Tip culture.
7.Chemotherapy.
8.Virus elimination through in vitro shoot-tip Grafting.
9.Virus Indexing.
10.Conclusion .
11.References .
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.
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.
Agrobacterium mediated gene transfer in plants.ICHHA PURAK
This power point presentation consist of 41 slides. Attempts have been made to illustrate how Agrobacterium behaves us natural genetic engineer. How it can infect a plant through wound and a part of DNA present on Ti plasmid is Tranferred and causes disease as crown gall in the infected plant. In second part of the presentation attempts have been made to describe how Agrobacterium can be utilized for iinsertion of desired gene into the plant,what manipulation are to be made with Agrobacterium.How infection and transfer of desired gene can be made possible.What is the role of plant tissue culture etc.
This presentation is all about biotechnology. It is about the basic aspects of Biotechnology and covers a lot of topics under biotechnology, recombinant DNA technology. This is specifically for the HSC students of Mumbai. I hope that it helps.
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
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.
LF Energy Webinar: Electrical Grid Modelling and Simulation Through PowSyBl -...DanBrown980551
Do you want to learn how to model and simulate an electrical network from scratch in under an hour?
Then welcome to this PowSyBl workshop, hosted by Rte, the French Transmission System Operator (TSO)!
During the webinar, you will discover the PowSyBl ecosystem as well as handle and study an electrical network through an interactive Python notebook.
PowSyBl is an open source project hosted by LF Energy, which offers a comprehensive set of features for electrical grid modelling and simulation. Among other advanced features, PowSyBl provides:
- A fully editable and extendable library for grid component modelling;
- Visualization tools to display your network;
- Grid simulation tools, such as power flows, security analyses (with or without remedial actions) and sensitivity analyses;
The framework is mostly written in Java, with a Python binding so that Python developers can access PowSyBl functionalities as well.
What you will learn during the webinar:
- For beginners: discover PowSyBl's functionalities through a quick general presentation and the notebook, without needing any expert coding skills;
- For advanced developers: master the skills to efficiently apply PowSyBl functionalities to your real-world scenarios.
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
Slack (or Teams) Automation for Bonterra Impact Management (fka Social Soluti...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on the notifications, alerts, and approval requests using Slack for Bonterra Impact Management. The solutions covered in this webinar can also be deployed for Microsoft Teams.
Interested in deploying notification automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Connector Corner: Automate dynamic content and events by pushing a buttonDianaGray10
Here is something new! In our next Connector Corner webinar, we will demonstrate how you can use a single workflow to:
Create a campaign using Mailchimp with merge tags/fields
Send an interactive Slack channel message (using buttons)
Have the message received by managers and peers along with a test email for review
But there’s more:
In a second workflow supporting the same use case, you’ll see:
Your campaign sent to target colleagues for approval
If the “Approve” button is clicked, a Jira/Zendesk ticket is created for the marketing design team
But—if the “Reject” button is pushed, colleagues will be alerted via Slack message
Join us to learn more about this new, human-in-the-loop capability, brought to you by Integration Service connectors.
And...
Speakers:
Akshay Agnihotri, Product Manager
Charlie Greenberg, Host
Encryption in Microsoft 365 - ExpertsLive Netherlands 2024Albert Hoitingh
In this session I delve into the encryption technology used in Microsoft 365 and Microsoft Purview. Including the concepts of Customer Key and Double Key Encryption.
Dev Dives: Train smarter, not harder – active learning and UiPath LLMs for do...UiPathCommunity
💥 Speed, accuracy, and scaling – discover the superpowers of GenAI in action with UiPath Document Understanding and Communications Mining™:
See how to accelerate model training and optimize model performance with active learning
Learn about the latest enhancements to out-of-the-box document processing – with little to no training required
Get an exclusive demo of the new family of UiPath LLMs – GenAI models specialized for processing different types of documents and messages
This is a hands-on session specifically designed for automation developers and AI enthusiasts seeking to enhance their knowledge in leveraging the latest intelligent document processing capabilities offered by UiPath.
Speakers:
👨🏫 Andras Palfi, Senior Product Manager, UiPath
👩🏫 Lenka Dulovicova, Product Program Manager, UiPath
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
In this webinar, we will review the benefits of leveraging InfluxDB and Grafana when executing load tests and demonstrate how these tools are used to visualize performance metrics.
Length: 30 minutes
Session Overview
-------------------------------------------
During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Essentials of Automations: Optimizing FME Workflows with ParametersSafe Software
Are you looking to streamline your workflows and boost your projects’ efficiency? Do you find yourself searching for ways to add flexibility and control over your FME workflows? If so, you’re in the right place.
Join us for an insightful dive into the world of FME parameters, a critical element in optimizing workflow efficiency. This webinar marks the beginning of our three-part “Essentials of Automation” series. This first webinar is designed to equip you with the knowledge and skills to utilize parameters effectively: enhancing the flexibility, maintainability, and user control of your FME projects.
Here’s what you’ll gain:
- Essentials of FME Parameters: Understand the pivotal role of parameters, including Reader/Writer, Transformer, User, and FME Flow categories. Discover how they are the key to unlocking automation and optimization within your workflows.
- Practical Applications in FME Form: Delve into key user parameter types including choice, connections, and file URLs. Allow users to control how a workflow runs, making your workflows more reusable. Learn to import values and deliver the best user experience for your workflows while enhancing accuracy.
- Optimization Strategies in FME Flow: Explore the creation and strategic deployment of parameters in FME Flow, including the use of deployment and geometry parameters, to maximize workflow efficiency.
- Pro Tips for Success: Gain insights on parameterizing connections and leveraging new features like Conditional Visibility for clarity and simplicity.
We’ll wrap up with a glimpse into future webinars, followed by a Q&A session to address your specific questions surrounding this topic.
Don’t miss this opportunity to elevate your FME expertise and drive your projects to new heights of efficiency.
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Ramesh Iyer
In today's fast-changing business world, Companies that adapt and embrace new ideas often need help to keep up with the competition. However, fostering a culture of innovation takes much work. It takes vision, leadership and willingness to take risks in the right proportion. Sachin Dev Duggal, co-founder of Builder.ai, has perfected the art of this balance, creating a company culture where creativity and growth are nurtured at each stage.
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
GraphRAG is All You need? LLM & Knowledge GraphGuy Korland
Guy Korland, CEO and Co-founder of FalkorDB, will review two articles on the integration of language models with knowledge graphs.
1. Unifying Large Language Models and Knowledge Graphs: A Roadmap.
https://arxiv.org/abs/2306.08302
2. Microsoft Research's GraphRAG paper and a review paper on various uses of knowledge graphs:
https://www.microsoft.com/en-us/research/blog/graphrag-unlocking-llm-discovery-on-narrative-private-data/
2. INTRODUCTION
Plant genetic engineering has become one of the most
important molecular tools in the modern molecular
breeding of crops.
Over the last decade, significant progress has been made in
the development of new and efficient transformation
methods in plants.
Despite a variety of available DNA delivery methods,
Agrobacterium- and biolistic-mediated transformation
remain the two predominantly employed approaches.
3. In particular, progress in Agrobacterium-mediated
transformation of cereals and other recalcitrant dicot
species has been quite remarkable.
In the meantime, other transgenic-enabling technologies
have emerged, including generation of marker-free
transgenics, gene targeting, and chromosomal engineering.
4. Although transformation of some plant species or elite
germplasm remains a challenge, further advancement in
transformation technology is expected because the
mechanisms of governing the regeneration and
transformation processes are now better understood and
are being creatively applied to designing improved
transformation methods or to developing new enabling
technologies.
5. The genetic constitution of plants can be altered in the
laboratory by a process called transformation, whereby a
segment of DNA (deoxyribonucleic acid) is introduced
that becomes inserted in one of the plant chromosomes.
Several methods to accomplish plant transformation have
been devised. In all these methods, single cells are
transformed and thereafter regenerated into complete,
fertile plants by tissue culture procedures.
Some of the transformation methods can only be applied
to protoplasts.
6. Nowadays, particle bombardment and the natural vector
Agrobacterium tumefaciens are preferred because they can
also cope with whole plant tissues such as roots and leaves,
which are easier to handle, more stable and require less of
the lengthy steps that are required for plant regeneration.
Transgenes are integrated at random positions in the plant
genome, but procedures for targeted integration become
more and more efficient.
7. Production of transgenic plants
Isolate and clone gene of interest
Add DNA segments to initiate or
enhance gene expression
Add selectable markers
Introduce gene construct into plant
cells (transformation)
Select transformed cells or tissues
Regenerate whole plants
9. Techniques for plant genetic
transformation
Indirect method- Agrobacterium mediated gene
transfer
Direct methods-
Particle bombardment (biolistics)
Microprojectile gun method
Electroporation
Silicon carbide fibres
Polyethylene glycol (PEG)/protoplast fusion
Liposome mediated gene transfer
10. Properties of good host
1. Easy to transform
2. Supports the replications of recombinant DNA
3. Is free from elements that interfere with replication of
recombinant DNA
4. Lacks active restriction enzymes ex: E.coli k12 substrain
HB 101
5. Does not have methylases
6. Is deficient in normal recombination function : so that
it is not altered by recombination events.
11. Transformation vector requirements
Origin of replication
Bacterial selectable marker
Gene constructs of interest
T-DNA borders and other Agrobacterium genes if using
Agrobacterium
Compatible with helper plasmid if using Agrobacterium
12. Agrobacterium mediated gene transfer
Agrobacterium-
Soil borne, gram negative, rod shaped, motile found in
rhizosphere
Causative agents of “Crown gall” disease of dicoltyledones
Have ability transfer bacterial genes to plant genome
Attracted to wound site via chemotaxis in response to chemicals
(sugar and Phenolic molecules: acetosyringone) released from
damaged plant cells
Contains Ti plasmid which can transfer its T-DNA region into
genome of host plants
13. Ti-plasmid features
Two strains of Ti-plasmid:
-Octopine strains- contains two T-DNA region: TL (14
kb) and TR ( 7 kb)
-Nopaline strains- contain one T-DNA region(20 kb)
Size is about 200 kb
Has a central role in Crown-gall formation
Contains one or more T-DNA region that is integrated
into the genome of host plants
Contain a vir region ~ 40 kb at least 8~11 vir genes
Has origin of replication
Contains a region enabling conjugative transfer
Has genes for the catabolism of opines
16. Forms of T-DNA that are found in Agrobacterium
ds circles - found only in induced bacteria, not
(apparently) in plant cells.
ds linear T-DNA - found only in induced bacteria,
not (apparently) in plant cells.
ss linear T-DNA - found in bacteria and plant
cells.
what is not found - Ti plasmids with evidence that
T-DNA has been precisely deleted.
17. Process of T-DNA transfer and integration
1. Signal recognition by Agrobacterium:
-Agrobacterium perceive signals such as sugar and phenolic compounds
which are released from plants.
2. Attachment to plants cells:
Two step processes: i) initial attachment via polysaccharide ii) mesh of
cellulose fiber is produced by bacteria.
Virulence genes (chv genes) are involved in the attachment of bacterial
cells to the plants cells.
3. Vir gene induction:
VirA senses phenolics ans subsequently phosphorylating and thereby
activating VirG. VirG then induces expression of all the vir genes.
4. T-strand production:VirD1/virD2 complex recognises the LB and
RB. virD2 produces single-stranded nicks in DNA. Then virD2 attached
to ssDNA. virC may assist this process.
18. Process of T-DNA transfer and integration (continu)
5. Transfer of T-DNA out of bacterial cells: T-DNA/VirD2 is
exported from the bacterial cell by “T-pilus” composed of
proteins encoded by virB operon and VirD2. VirE2 and VirF are
also exported from bacterial cells.
6. Transfer of the T-DNA and Vir proteins into the plant
nuclear localization: T-DNA/VirD2 complex and other Vir
proteins cross the plasma membrane through channels formed
from VirE2. VirE2 protect T-DNA from nucleases, facilitate
nuclear localization and confer the correct conformation to the
T-DNA/virD2 complex for passage through the nuclear pore
complex (NPC). The T-DNA/VirD2/VirF2 /plant protein
complex the nucleus through nuclear pore complex. And
integrated into host chromosome.
19. Agrobacterium tumefaciens mediated
transforamtion in plants
Explant
{ decapitated seedlings, cells, protoplast, leaf discs}
Addition of wounding
Acetosyringone Agrobacteria
co cultivation to allow infection
antibiotics to kill bacteria
transformed and non transformed tissue
selective media to kill the non transformed tissues
i.e, addition of kanamycin and hygromycin etc
transformed tissue / callus
transformed shoots
rooted shoots
adult plants
SEEDS
20. Practical application of Agrobacterium-mediated
plant transformation
1. Agrobacterium mediated transformation
methods are thought to induce less
rearrangement of the transgene.
Lower transgene copy number that direct DNA
delivery methods.
Successful production of transgenic plants
depends on the suitable transformation
protocols.
21.
22.
23. The basic protocol used for any Agrobacteruim
mediated transformation experiments
1. Identify a suitable explants: Suitable plant tissue is
removed and sterilized. Leaf is used for Tobacco.
2. Co-cultivate with the Agrobacterium: Leaf tissue is cut
into small pieces and placed into a culture of Agrobacterium
for about 30 mins. The explants are subsequently removed
from the bacterial culture and placed on to the MS medium
that contain no selective agent. The incubation of explants
with Agrobacterium is allowed to continue for 2 days to allow
transfer of the T-DNA transfer to the plant cells.
3. Kill the Agrobacterium with a suitable antibiotic: The
explants are removed from the medium and washed in
antibiotic (cefotaxime) solution that kill Agrobacterium cells.
24. The basic protocol used for any Agrobacteruim
mediated transformation experiments (Conti-)
4. Select for transformed plant cells: The explant
are transferred to fresh solid medium supplemented
with a selective agent (kanamycin). It also contains
cefotaxime. Auxin, Cytokinin are used to encourage the
regeneration of by organogenesis. High cytokinin to
auxin ratio promotes shoot formation from the
explants.
5. Regeneration of whole plant :The shoot can be
rooted by placing them on solid medium containing a
high auxin to cytokinin ratio.
25.
26. Advantages of A.bacterium mediated transformation
It is a natural mean of transfer and is perceived as more acceptable
technique to those who feel natural is best.
It is capable of infecting intact plant cells, tissues and organs. As result
of tissue culture limitations are much less of a problem. Transformed
plants can be regenerated more rapidly.
It is having a capable of transferring large fragments of DNA very
efficiently without substantial rearrangements.
Integration of T-DNA is relatively precise process, it serves as an ideal
insertional mutagenesis vehicle as it introduces one to several copies
of the transferred DNA into the intact genome at one or a few loci.
The stability of gene transferred is excellent. Integrated T- DNA gives
consistent maps and appropriate segregation ratios. Introduces traits
have been found to be stable over many generations.
This at ability is critical to commercialization of transgenic plants.
27. Disadvantages
It has limitation of host range,
Some of the important food crops can not be infected by
this although, lately much progress has been achieved to
over come this limitation by the development of highly
virulent strains of Agrobacterium.
Sometimes, the cells in a tissue that are able to regenarate,
are difficult to transform. it might be that these are in
layers too deep to be reached by Agrobacterium, or
simply are not targets for T-DNA transfer.
28. Virus mediated
Vectors based on viruses are desirable because of high efficiency
of gene transfer that can be obtained by infection and because of
the amplification of transferred gene that occurs via viral genome
replication.
Vectors for transferring gene into plants are based on DNA or
RNA molecules, that naturally express their genetic information
in the plant cells.
The replicating genomes of plant viruses are non-integrative
vectors, as compared to vectors based on the T-DNA of A.
Tumefaciens which are integrative gene vectors.
29. The plant virus vectors do not integrate into the
plant cells or host genome , rather they spread
systematically within a plant and accumulate to
high copy number in their respective target cells.
In most of the cases , the viral genome has modified
to accommodate the insertion of foreign sequences,
which are transferred, multiplied and expressed in
plant as part of recombinant virus genome.
30. Plant viruses as vectors
Caulimoviruses – ds DNA – CaMV
Geminiviruses - 2ss DNA – maize streak virus
RNA plant viruses - TMV
31. Vectorless or Direct DNA transfer
The trem “Direct gene transfer” is used to discriminate
between the methods of plant transformation that rely on
Agrobacterium (indirect method) and those that do not (direct
methods). Direct gene transfer methods all rely on the delivery
of large amount of naked DNA whilst plant is transiently
permeabilised.
Direct methods-
Particle bombardment (biolistics)
Microprojectile gun method
Electroporation
Silicon carbide fibres
Polyethylene glycol (PEG)/protoplast
fusion
Liposome mediated gene transfer
32. Physical methods
DMGT: DNA Mediated Gene Transfer
A general scheme for production of transgenic plants using
various direct DNA delivery methods have been
commercialized.
Electroporation
Particle bombardment / Micro projectile / Biolistics
Electro injection
Micro injection
macro injection
Liposome mediated
Silicon carbide fibre mediated
ultra sound mediated
DNA transfer via pollen
33. Electroporation technique.
Is the process where by electrical impulses of high
field strength are used to reversibly permeabalize cell
membrane to facilitates uptake of large molecules,
including DNA.
It has been used for long time for transient and
integrative transformation of protoplasts.
1 to 1.5 k V. so uses low capacitance hence short
decay time .
34. Electroporation
It can be used to deliver DNA into plant cells and protoplasts.
The genes of interest require plant regulatory sequence.
Plant materials is incubated in a buffer solution containing
DNA and subjected to high-voltage electric pulse.
The DNA then migrates through high-voltage-induced pores in
the plasma membrane and integrates into the genome.
It can be used to transform all the major cereals particularly
rice, wheat, maize.
Advantages and disadvantages:
Both intact cells and tissue can be transformed.
The efficiency of transformation depends upon the plant
materials, electroporation and tissue treatment conditions used
for transformation.
~40 to 50% incubated cells receive DNA
~50% of the transformed cells can survive
35. Duracell
DNA containing
the gene of interest
Plant cell
Protoplast
Electroporation Technique
Power supply
DNA inside the
plant cell
The plant cell with
the new gene
36. Biolistic/Particle bombardment
High velocity micro projectile were utilize to
deliver nucleic acids into loving cells.
Advantages :
Transformation of organized tissue
Universal delivery system
Transformation of recalcitrant spp
Study of basic plant development processes.
37. Particle bombardment
Why Biolistics or Biolistic bombardment?
Is the most powerful method for introducing nucleic acids
into plants, because the helium pressure can drive
microcarriers through cell walls
Is much easier and less time consuming than microinjecting
nucleic acids into plant cells or embryos
Allows transformation of animal cells that have unique
growth requirements and that are not amenable to gene
transfer using any other method
Requires less DNA and fewer cells than other methods, and
can be used for either transient or stable transformation
38. Principle
The gold or tungsten particles are coated with the DNA that
is used to be transform the plant tissue.
The particles are propelled at high speed into the target
plant material where the DNA is released within then cell
and can integrate into the genome.
Two types of plant tissues are used for particle
bombardment:
a) Primary explants that are bombarded and then induced
to become embryogenic
b) Proliferating embryonic cultures that are bombarded
and then allowed to proliferate further and subsequently
regenerate.
39. PDS-1000/He bombardment System
Fig: Schematic representation of the PDS-1000/He
system upon activation. The arrows indicate the
direction of helium flow
Fig: The PDS-1000/He system, shown here
with magnified view of the Hepta adaptor.
40. How the PDS-1000/He System Works
The sample to be transformed is placed in the bombardment chamber,
which is evacuated to subatmospheric pressure
The instrument is fired; helium flows into the gas acceleration tube and is
held until the specific pressure of the rupture disk is reached
The disk bursts, and the ensuing helium shock wave drives the
macrocarrier disk (which carries the coated microparticles) a short
distance toward the stopping screen
The stopping screen retains the macrocarrier, while the microparticles
pass through the screen into the bombardment chamber and penetrate
the target cells
The launch velocity of microcarriers depends on a number of adjustable
parameters: the helium pressure (rupture disk selection, 450–2,200 psi),
the amount of vacuum, the distance from the rupture disk to the
macrocarrier, the distance from the microcarrier launch assembly to the
stopping screen, and the distance between the stopping screen and target
cells. Adjusting these parameters allows you to produce a range of
velocities to optimally transform many different cell types.
42. Microinjection
Under a microscope, a cell is manipulated to a blunt
capillary. Gentle suction holds the cell in place.
With a micromanipulator, a very fine tipped pipet is
inserted into the cytoplasm or nucleus.
DNA or RNA is injected directly into the nucleus or
cytoplasm.
Microinjection has been successfully used with large frog
eggs, cultured mammalian cells, mammalian embryos,
and plant protoplasts and tissues
43. Microinjection
Microinjection techniques for plant protoplasts utilize a holding
pipette for immobilizing the protoplast while an injection pipette is
utilized to inject the macromolecule.
In order to manipulate the protoplasts without damage, the
protoplasts are cultured for from about 1 to 5 days before the
injection is performed to allow for partial regeneration of the cell
wall.
It was found that injection through the partially regenerated cell
wall could still be accomplished and particular compartments of the
cell could be targeted.
The methods are particularly useful for transformation of plant
protoplasts with exogenous genes.
46. Silicon carbide fibres-Whiskers
Plant materials (Cells in suspension culture, embryos and
embryo-derived callus) is introduced into a buffer containing
DNA and the silicon fibers which is then vortexed.
The fibers (0.3-0.6 μm in diameter and 10-100μm long)
penetrate the cell wall and plasma membrane, allowing the
DNA to gain access to the inside of the cells.
Disadvantages and advantages
The drawbacks of this technique relate to the availability of
suitable plant material and the inherent dangers of the fibers,
which require careful handing.
Many cereals, produce embryonic callus that is hard and
compact and not easily transformed with this technique.
Despite the some disadvantages, this method is recently used
for successful transformation of wheat, baerly, and maize
without the need to cell suspension.
47. Silicon carbide fibre mediated
0.6 micro m dia, 10 – 80 micro m lth.
vortexing in an eppendorf tube a mixture of plasmid DNA encoding
a selectable or screen able marker gene, si fibre and the explants
tissue.
DNA delivery in his system is presumably due to cell wall penetration
by DNA coated si C F.
cellular penetration is likely enhanced by frequent and forceful
intercellular collisions encountered in the votrexed millue.
DNA bound to the fibre surface is carried into the penetrated cell and
becomes integrated into the nuclear genome and stably transform
plant cells with plant regeneration capacity will unable to the
application of Si c f mediated.
49. Ultrasound mediated
Immersion of explant in sonication buffer containing plasmid
DNA and is then sonicated with an ultra sonic pulse
regenerater at 0.5 w/cm2 aucostic intensity for 30 mins.
Then the samples are rinsed in buffer soln, and then cultured
for Gth and deferentiation.
Advantage:
Being simple method
Inexpensive and multi functional equipment.
There is no need of tissue culturists.
50. DNA transfer via pollen
Y pollen as v?
Fertilization of zygotic embryogenesis, and ovules are
difficult to isolate and injection of DNA into embryo sac in
insitu seemed to be too tedious and unpredictable..
Ex: DNA treated pollen as vector for pollinating fertile
plants of Maize by ohta.
52. Chemical gene transfer method
PEG mediated
Calcium phosphate Co-precipitation
The polycation DMSO technique
DEAE dextran procedure
53. Calcium phosphate Co-precipitation
DNA is mixed with CaCl2 soln & isotonic buffer to form
DNA CaPO4 ppt. this ppt is allowed to react with actively
dividing cells for several hours, washed and then incubated
in fresh culture medium.
The efficiency of transformation though low can be increased
by giving them a physiological shock with DMSO.
relative success depends upon high DNA concentration and
its apparent protection in the ppt.
54. The polycation DMSO technique
Involves polycation, polybrene to increase the
adsorption of DNA to the surface followed by a
brief treatment with 25-30% of DMSO to increase
the membrane permeability and enhance uptake.
Advantage :
Less toxic than other polycations,
High transformation efficiency and allows very
small quantities of plasmid DNA to be used.
55. DEAE dextran procedure
Transformation of cells with DNA complexed to the high molecular
weight polymer Di Ethyl Amino Ethyl dextran is used obtain
efficient transient expression. The efficiency increases to 80% when
DMSO shock id given, but it wont produce stable transformants.
Advantage:
Direct gene transfer method
Ex: cereals
Disadvantage
After transformation the further improvement of plant tissue
culture is not good so..
It show unpredictable pattern of foreign DNA integration
During their passage into the nucleus , the DNA are subjected to
nucleolytic cleavage resulting in truncation, recombination,
rearrangement or silencing of DNA occurs.
56. Tomatoes comes in many
varieties, colors and shapes
Transgenic tomatoes -
expressing different malarial
antigens
57. Normal and mutant tomato fruit
high-pigment 1 (hp1/hp1), high-pigment
2 (hp2/hp2), Never-ripe (Nr/Nr),
Green-ripe (Gr/Gr), Colorless non-
ripening (Cnr/Cnr) &
ripening-inhibitor (rin/rin) mutations
58. Delivery of a corn-based edible
vaccine
Transgenic corn kernels (a)
Corn snack (b) or
Embryo or germ cells (c)
59. Dr Eady
Crop & Food Research in New Zealand and
his collaborators in Japan
Tearless Onion