Plastid transformation allows for high levels of protein expression and improved biosafety compared to nuclear transformation. Plastids are organelles that perform important functions in plant cells, including photosynthesis, and contain their own circular genome. Transformation involves delivering foreign DNA to plastids using biolistics or PEG-mediated transformation, then selecting for integration into the plastid genome by homologous recombination. Successful plastid transformation has been achieved in many plant species and used to confer valuable agronomic traits and produce human therapeutic proteins.
This presentation is about chloroplast transformation, the importance of chloroplast transformation on nucleus transformation and strategies for making marker-free transplastomic plant
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
A transplastomic plant is a genetically modified plant in which the new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts.
This presentation is about chloroplast transformation, the importance of chloroplast transformation on nucleus transformation and strategies for making marker-free transplastomic plant
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
A transplastomic plant is a genetically modified plant in which the new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts.
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
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
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
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
☺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.
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
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
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
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
The presentation describes the advantages of plastid transformation over 'conventional' nuclear transformation, hurdles to plastid transformation, its advantages. The presentation also covers some successful plastid engineering and its potential.
Agrobacterium tumefaciensppt............it is a slide presentation on interki...Anwesha Banerjee
agrobacterium gene transfer is very important for all students learning biotechnology and microbiology , i have prepaed it for my college presentation class hope all my friends like it
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 lecture covers some nice stories about the origins of the words "genome" and the derived word "genomics". the lecture also introduces viral, bacterial, and eukaryotic genomes.
chloroplast being the second semi-autonomous organelle of the plant cell also harbours its genome. the presentation includes various characteristic features of this organelle genome along with its functional pecularities and significance
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
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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.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
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Biological screening of herbal drugs: Introduction and Need for
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2. Plastid
• Major organelle of plant and algal cells
• Site of manufacture and storage of important chemical
compounds
• Has circular, dsDNA copies
• Replicates autonomously of the cell
• Thought to have been originated from endosymbiotic
bacteria
• Plastid genes show maternal inheritance
4. Have diverse functions
• Chloroplasts – green plastids – for photosynthesis
• Chromoplasts – coloured plastids – for pigment synthesis and storage
• Gerontoplasts – control dismantling of photosynthetic apparatus
during senescence
• Leucoplasts – colourless plastids – monoterpene synthesis
• Leucoplasts include amyloplasts (starch), elaioplasts (fats),
proteinoplasts (proteins) and tannosomes (tannins)
5. Nuclear genome Plastid genome
Chromosomes Two copies of each of ~60 copies of a single circular
many chromosomes; chromosome per plastid
the number of ~50–60 chloroplasts per cell
chromosomes per
diploid cell is species
-specific
Genes per chromosome Could be thousands ~120–150
Arrangement and Each gene is separate Many genes are in
operons transcription of genes (individually transcribed )(transcribed together)
Comparison of the nuclear and plastid genomes of
angiosperm
6. Why plastid transformation?
• High protein expression levels
• Absence of epigenetic effects
• Uniparental inheritance is commercially favored
• Easy transgene stacking in operons
• Increased biosafety – Since plastids are maternally
inherited, they aren’t transmitted by pollen
7. Hurdles to ‘transplastomic’ plants
• Difficulty in delivering foreign DNA through double
membrane of the plastid
• The enormous copy number (polyploidy) of the plastid
genome
• The desired genetic modification must be in each copy of
plastid genome in each cell
• Failure to achieve homoplasmy results in rapid somatic
segregation and genetic instability
• Repeated rounds of selection and regeneration are required
8. Chloroplast transformation requires:
1. A chloroplast specific expression vector.
2. A method for DNA delivery through a double
membrane of the chloroplast.
3. An efficient selection for the transplastome.
9. DNA delivery into plastids
• 2 successful methods include biolistics and polyethylene
glycol-mediated transfer
• Biolistics is preferred as it is less time-consuming and
demanding
• Integration of foreign DNA into plastid genome occurs via
homologous recombination
• Homologous recombination operates in plastids at a high
efficiency
10. Transformation of the chloroplast genome by bombarding
tobacco leaves with microprojectiles coated with DNA.
Following bombardment, leaf discs are placed onto antibiotic-
containing medium (panel A). Transgenic plants are
regenerated from the transformed tissue that is able to develop
green chloroplasts (panel B)
11. Molecular biology of Chloroplast
Transformation
• Stable transformation system depends on integration of the
transforming DNA into the plastid genome by homologous
recombination.
• Sequence to be introduced into the plastid genome must flanked on
both side by region of homology with the chloroplast genome.
• Primary transplastomic event results hetroplasmic cells.
• Hetroplasmy is unstable so it will resolve into homoplasmy .
11
15. Marker removal
• Recombination between directly repeated sequences excises
the intervening DNA sequence and one copy of the direct
repeat.
• The breakage and joining of DNA strands involved in
recombination can be mediated by the native homologous
recombination machinery present in plastids
18. Protoplast isolation
• Lettuce seeds were sterilized and sown on MS
medium with 2% sucrose
• Shoot tips from leaves obtained were transferred to
MS medium with 3% sucrose
• The leaves were cut into pieces and incubated in PG
solution, followed by enzyme solution consisting of
1% cellulase and .25% macerozyme
• Protoplast suspension was filtered through nylon
mesh
• Protoplasts were collected at surface after
centrifugation at 70g for 8min
19. Transformation and culture
• 10µl transforming DNA and 0.6ml PEG solution was added
to protoplast suspension and incubated at 25ºC for 10min
• Protoplasts were mixed with 1:1 solution of B5 and 2%
agarose to a density of 3.6 X 104 protoplasts per ml
• The suspension was plated onto Petri dishes and cultured at
25ºC in the dark
• Selection was initiated on the 7th day by fresh medium
containing spectinomycin dihydrochloride
20. • 100% of spectinomycin-resistant lettuce cell
lines were true plastid transformants
• A limitation was the high frequency of
polyploid cell lines
21. Analyses
• PCR – specific primers were used to assess the
presence of aadA gene in resistant cell lines
• Immunoblot analysis – using HRP-conjugated
secondary antibodies
• Southern and Northern blots were performed to look
for target genes and their transcripts
22. Production of human therapeutic proteins
Why lettuce is favoured over tobacco?
• Most of the plant is leaf tissue and this tissue
contains the greatest number of plastids per cell
• Unlike tobacco, lettuce has no toxic alkaloids
that need to be removed - low purification and
downstream processing costs
• Lettuce is a relevant human foodstuff that can be
consumed without cooking
23. Milestone of chloroplast transformation
Year Milestone DNA
delivery
Approach Selection Reference
1988 Chlamydomonas reinhardtii
1st stable plastid transformation
Biolistic Homologous
targeting
Photosynthetic
competence
Boynton & Gillham
(Science, 240)
1990 Nicotiana tabacum
1st stable plastid transformation
Biolistic Homologous
targeting
Spectinomycin
(rrn16)
Svab et al (PNAS,
87)
1993 Nicotiana tabacum
1st high level foreign protein
(2.5% GUS)
PEG Homologous
targeting
Spectinomycin
Kanamycin
Golds et al (Biotech.
11)
O’Neill et al (Plant J.
3)
1995 Nicotiana tabacum
New agronomic trait: B.
thruingiensis
Marker gene elimination: co-
transformation
Biolistic Homologous
targeting
Spectinomycin McBride et al
(Biotech. 13)
Carrer and Maliga
(Biotech. 13)
1998 Arabidopsis thaliana
1st stable plastid transformation
Biolistic Homologous
targeting
Spectinomycin Sikdar et al (Plant
Cell Rep. 18)
1999 Solanum tuberosum (potato)
1st stable plastid transformation
Oryza sativa (rice)
1st stable plastid transformation
Biolistic Homologous
targeting
Spectinomycin Sidorov et al (Plant
J. 19)
Khan and Maliga
(Nat. Biotech. 17)
24. Year Milestone DNA
delivery
Approach Selection Reference
2000 Nicotiano tabacum
1st human protein expression
Biolistic Homologous
targeting
Spectinomycin Staub et al (Nat.
Biotech. 18)
2001 Lycopersicon esculentum (tomato)
1st foreign protein in fruit
Marker gene elimination: CRE-lox
New agronomic traits: glyphosate
tolerance and PPT resistance
Biolistic Homologous
targeting
Spectinomycin Ruf et al(Nat.
Biotech. 19)
Corneille et al (Plant
J. 19)
Ye et al (Plant J. 25)
Lutz et al (Plant
Physiol. 125)
2002 Porphyridium sp.
1st stable plastid transformation
Biolistic Homologous
targeting
Spectinomycin Lapidot et al (Plant
Physiol. 129)
2003 Chlamydomonas reinhardtii : Foot-
and-mouth disease virus VP1
protein expression
Brassicacea (oil seeds)
1st stable plastid transformation
Phytoremediation: Mercury
Biolistic Homologous
targeting
Spectinomycin Sun et al (Biotechnol
Lett. 25)
Skarjinskaia et al
(Transgenic Res. 12)
Ruiz et al (Plant
Physiol. 132)
2004 Gossypium hirsutum (cotton)
1st stable plastid transformation
Glycin max (soybean)
1st stable plastid transformation
Linum usitatissimum L. (flax):
PHB polymer expression
Biolistic Homologous
targeting
aph A-6
npt II
Spectinomycin
Kumar et al (PMB. 56)
Dufourmantel et al
(PMB. 55)
Wrobel et al (J.
Biotech. 107)
25. References
o Scotti, Manuela Rigano, Cardi. 2012 Production of
foreign proteins using plastid transformation,
Biotechnology Advances 30 : 387–397
o Day, Goldschmidt-Clermont .2011, The chloroplast
transformation toolbox: selectable markers and
marker removal. Plant Biotechnology Journal 9:
540–553