Teaching Materials for Plant Transformation
by Andika Gunadi – [email protected]
American Society of Plant Biologists – Conviron Scholar
February 8, 2018
Plant DNA
New DNA
Plant DNA
Content
1. What is plant transformation
2. Why “transform” plants?
3. How are plants transformed
4. Big picture
5. Agrobacterium-mediated
6. Particle bombardment-mediated
7. Transformation to recovery
8. Agrobacterium vs. biolistics
9. Targeted genome editing
10. Future Challenges
11. Additional references
List of genetically modified crops taken from: http://www.isaaa.org/gmapprovaldatabase/cropslist/default.asp
Alfalfa RiceApple RoseArgentine Canola SoybeanCarnation SquashChicory Sugar BeetCommon Bean SugarcaneCotton Sweet pepperCreeping Bentgrass TobaccoEggplant TomatoEucalyptus WheatFlaxMaizeMelonPapayaPetuniaPlumPolish CanolaPoplarPotato
Many other transformable plants not listed…
What is plant transformation?• Process of inserting foreign DNA into plant tissue
• The foreign DNA is called “transgene”, the resulting plant tissue is called “transgenic”
• If foreign DNA is incorporated within the plant’s chromosome / mitochondrial / chloroplast DNA, it is called “DNA integration”, or “stable transformation”
• If foreign DNA is within plant nucleus / mitochondria / chloroplast but not incorporated within a chromosome/native DNA material, it is called “transient transformation”
Key terminologies
• DNA integration• Stable• Transient• Transgene • Transgenic
Image from:https://www.noble.org/research/areas/plant-transformation-genome-editing/
What is plant transformation?
Plant Cell
Key terminologies
• DNA integration• Stable• Transient• Transgene • Transgenic
Nucleus / mitochondria / chloroplast
Foreign DNACannot be
expressed or replicate
Foreign DNAcan be
expressed but cannot replicate
(Transient)
Plant DNA
Integration of foreign DNA, now it can be expressed and can replicate (Stable)
Why “transform” plants?• Valuable tool for understanding plant biology (ex. How genes
work, how genes are regulated)
• Can introduce new traits (ex. improved yield, disease resistance, tolerance to abiotic factors, bioremediation, enhanced nutritional content)
• Genetic engineering often can introduce desirable traits faster than conventional breeding
• Genome editing tools allow for targeted modifications
Examples:
For plant research - http://ptrc.ucr.edu/
For commercialization -https://sciencing.com/roundup-ready-corn-6762437.html
How are plants transformed?DNA has to be inserted into plant cells
Efficiencies differ between plant species and tissue-type
• DNA delivery by biotic agent- Agrobacterium (most widely used)- Other bacteria and viruses (tend to be less efficient)
• DNA delivery by abiotic agent- Particle bombardment / biolistics (also widely used)- Others… (tend to be less efficient)
References:Chung et al. (2005) Agrobacterium is not alone: gene transfer to plants by viruses and other bacteria. Trends in Plant Science.Darbani et al. (2008) DNA delivery methods to produce transgenic plants. BiotechnologyJoung et al. (2015) Plant transformation methods and applications. In Koh et al. (eds.) Current Technologies in Plant Molecular Breeding DOI 10.1007/978-94-017-9996-6_9
Key terminologies
• Agrobacterium• Particle
bombardment /biolistics
Big picture
Transgenic Plant
Plant growth
Plant physiology, tissue culture, plant propagation, plant pathology, ecology, microscopy,
robotics/engineering
DNA materials
Genetics, biochemistry,
molecular biology,
bioinformatics,computer science
Regulations
DNA Introduction
Microbiology,chemistry,
physics, mechanical engineering
Philosophy, law, ethics,economy, policy-making,
patenting, laboratory safety
Creating transgenic plants require multiple disciplines
Agrobacterium-mediated
• Agrobacterium: genus of gram-negative soil-borne bacteria with inherent plant-transforming capabilities
• Some isolates are considered plant pathogens• These bacteria (and viruses) transform plants long before we
could!
The sweet potato genome contains evidences of naturally-occurring plant transformation by Agrobacterium (https://www.npr.org/sections/goatsandsoda/2015/05/05/404198552/natural-gmo-sweet-potato-genetically-modified-8-000-years-ago)
Agrobacterium-mediatedPlant-transforming species commonly used:
• Agrobacterium tumefaciens – tumor inducing
• Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) – root inducing
These bacteria contain genes within their transfer DNA (T-DNA) that when transformed and expressed in plants, synthesizes plant hormones, resulting in unregulated plant tissue growth
Removal of their inherent transfer DNA (T-DNA) is a process called “disarming”
DNA sequence of interest can then be inserted to replace the T-DNA
Disarmed Agrobacterium still retain their virulence (Vir) genes, which are necessary for the bacterial machinery to perform plant transformation
“Binary vectors” are modified plasmids that are compatible for replicationin Agrobacterium, and have DNA of interest for plant transformation
Key terminologies
• T-DNA• Disarming• Vir-genes• Binary vector
Agrobacterium-mediated
Key terminologies
• T-DNA• Disarming• Vir-genes• Binary vector• Random
integration
Image borrowed from: https://passel.unl.edu/pages/informationmodule.php?idinformationmodule=1130447280&topicorder=6&maxto=10&minto=1
Foreign DNAintroduced byAgrobacteriumare integrated randomly into the plant genome
Agrobacterium-mediated
Additional references:
• http://www.apsnet.org/publications/apsnetfeatures/Pages/Agrobacterium.aspx
• Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev.
• http://archive.bio.ed.ac.uk/jdeacon/microbes/crown.htm
• Ron et al. (2014) Hairy root transformation usingAgrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiology.
Particle bombardment-mediated• Uses “gene gun”: Equipment that propels DNA into plant cells• Just like Agrobacterium transformation, foreign DNA is inserted
randomly, but often with higher copies• The number of copies of a transgene that gets integrated into the
plant genome is called “copy number”
Key terminologies
• Gene gun• Copy number
Examples of commercial devices: http://www.bio-rad.com/en-us/category/biolistic-particle-delivery-systems
Example of customized device:Particle Inflow Gun (PIG)http://u.osu.edu/plantranslab/pig/
Particle bombardment-mediated• Usually uses metal particles (positively charged) to attract
DNA (negatively charged), forming DNA-coated particles
• Tungsten and Gold particles are most commonly used
• DNA-coated particles are propelled in high velocity into plant cells
Key terminologies
• Gene gun• Copy number
Tomato fruit endocarp tissue bombarded with green fluorescent protein gene (GFP). Middle picture: several transformed cells express GFP. Right picture: Zoomed in view of several transformed cell. Middle cell likely has higher copy number than surrounding cells.
Transformation to plant recovery• Plant tissue that is cultured separately
from the whole plant is called “explant”
• Each stably transformed cell and tissue or plants that is regenerated from it is called a “transgenic event”
• Chemicals introduced during event recovery that favor the survival of transgenic events is termed “selection agent”. Using selection agent allows fasterdetection of transgenic events
Example: Using hygromycin in plant growth media to favor the survival of transgenic plants with resistance to hygromycin
Harrison et al. (2006) A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation. Plant Methods.
Image taken from: http://plantcellbiology.masters.grkraj.org/
Key terminologies
• Explant• Transgenic event• Selection agent
Transformation to plant recovery• Once an explant is stably transformed, it is called T0 Generation
• Progeny from T0 Generation is called T1 and so on…
• The presence of foreign DNA, the copy number, as well as the zigosity can be detected through molecular biology techniques
Key terminologies
• Generation• Zygosity
(homozygous or heterozygous)
Image from: Tizaoui and Kchouk (2012) http://dx.doi.org/10.1590/S1415-47572012000400015
Agrobacterium vs. BiolisticsAgrobacterium Biolistics
Copy numberGenerally less;
Less flexible for titrating transgene amounts
Generally more;More flexible for titrating
transgene amounts
Introduction speedAgrobacterium incubation
time takes longer time
Transient transformation DNA expression can be observed within hours
after bombardment
Damage to plant tissue when properly used
Generally less Generally more
Transgene DNA shearingLess likely (cleaner
introduction)More likely (mix of broken and intact transgene DNA)
Transient assaysAgroinfiltration, some plants not compatible
Mix of transient and stable events, most plants
compatible
Stable transgenic plant recovery
Variety and species dependent
Variety and species dependent
Targeted genome editing• Agrobacterium and biolistic transformation tools create random
DNA integration• Additional tools need to be added on top of plant transformation
methods to do “targeted DNA integration”• Targeted genome editing require the use of “nucleases”: Protein
enzymes that cut DNA at a pre-defined target within plant genome
• Types of targeted genome editing tools:- TALENS- Meganucleases- Zinc-finger nucleases- CRISPR
References:
Yin et al. (2017) Progress and prospects in plant genome editing. Nature Plants.
https://www.genome.gov/27569222/genome-editing/
Key terminologies
• Nuclease• TALENS• Meganuclease• Zinc-finger
nuclease• CRISPR
Targeted genome editing
Key terminologies
• HDR• NHEJ
Plant DNA Plant DNA
Nuclease recognizes and cuts DNA at
targeted site
Plant’s innate DNA repair machinery drives the next steps…Two key competing repair mechanisms:Homology Directed Repair (HDR) and Non-Homologous End-Joining (NHEJ)
Donor DNA absent:• HDR repairs broken DNA using plant’s
unbroken homologous DNA• NHEJ joins/ligates two strands together• NHEJ may introduce several base pairs
of insertion/deletions
Donor DNA added:• HDR can repair broken DNA while
inserting transgene in between• NHEJ can also repair broken DNA by
introducing transgene in between• HDR and NHEJ can also repair as if
donor DNA is absent
Transgene donor can be added for targeted insertion
Future challenges• Many plants are still difficult to transform
• Efficiencies for genome editing (Especially for targeted transgene insertion) are low
• Improving strategies for generating transgenic plants (more precise integrations, sustainable, better genetic components)
• Need more efficient methods to remove unneeded transgenes for final agricultural product
• Nucleases can be engineered for new functions (already in progress)
• Public perception of genetically engineered plants
A tool is only as good as the hands that wield it
In plant transformation, creativity is the limit
Additional references
Resources for plant transformation
• https://sips.cals.cornell.edu/research/plant-transformation-facility/what-plant-transformation
• http://u.osu.edu/plantranslab/
• http://parrottlab.uga.edu/parrottlab/
• https://web.uri.edu/pbl/plant-transformation/
• https://www.ndsu.edu/pubweb/~mcclean/plsc731/transgenic/transgenic1.htm
• http://plantcellbiology.masters.grkraj.org/html/Genetic_Engineering4D-Transformation-Plant_Cells.htm