1. Teaching Materials for Plant Transformation
New DNA
Plant DNA
Plant DNA
by Andika Gunadi –
American Society of Plant Biologists – Conviron Scholar
February 8, 2018

2. Content What is plant transformation Why “transform” plants?
Alfalfa Rice
Apple Rose
Argentine Canola Soybean
Carnation Squash
Chicory Sugar Beet
Common Bean Sugarcane
Cotton Sweet pepper
Creeping Bentgrass Tobacco
Eggplant Tomato
Eucalyptus Wheat
Flax
Maize
Melon
Papaya
Petunia
Plum
Polish Canola
Poplar
Potato
What is plant transformation
Why “transform” plants?
How are plants transformed
Big picture
Agrobacterium-mediated
Particle bombardment-mediated
Transformation to recovery
Agrobacterium vs. biolistics
Targeted genome editing
Future Challenges
Additional references
List of genetically modified crops taken from:
Many other transformable plants not listed…

3. 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:

4. What is plant transformation?
Plant Cell
Foreign DNA
Cannot be expressed or replicate
Nucleus / mitochondria / chloroplast
Key terminologies
DNA integration
Stable
Transient
Transgene
Transgenic
Foreign DNA
can be expressed but cannot replicate
(Transient)
Plant DNA
Integration of foreign DNA, now it can be expressed and can replicate (Stable)

5. 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 -
For commercialization -

6. 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. Biotechnology
Joung et al. (2015) Plant transformation methods and applications. In Koh et al. (eds.) Current Technologies in Plant Molecular Breeding DOI / _9
Key terminologies
Agrobacterium
Particle bombardment / biolistics
General workflow

7. Big picture Creating transgenic plants require multiple disciplines
Plant physiology, tissue culture, plant propagation, plant pathology, ecology, microscopy, robotics/engineering
Plant growth
Genetics, biochemistry,
molecular biology,
bioinformatics,
computer science
Microbiology,
chemistry, physics, mechanical engineering
Transgenic Plant
DNA materials
DNA Introduction
Regulations
Philosophy, law, ethics, economy, policy-making, patenting, laboratory safety

8. 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 (

9. Agrobacterium-mediated
Plant-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 replication in Agrobacterium, and have DNA of interest for plant transformation
Key terminologies
T-DNA
Disarming
Vir-genes
Binary vector

10. Agrobacterium-mediated
Foreign DNA introduced by Agrobacterium are integrated randomly into the plant genome
Key terminologies
T-DNA
Disarming
Vir-genes
Binary vector
Random integration
Image borrowed from:

11. Agrobacterium-mediated
Additional references:
robacterium.aspx
Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev.
Ron et al. (2014) Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type- specific gene expression and function using tomato as a model. Plant Physiology.

12. 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:
Example of customized device:
Particle Inflow Gun (PIG)

13. 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.

14. 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 faster detection 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.
Key terminologies
Explant
Transgenic event
Selection agent
Image taken from:

15. 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)

16. Agrobacterium vs. Biolistics
Copy number
Generally less;
Less flexible for titrating transgene amounts
Generally more;
More flexible for titrating transgene amounts
Introduction speed
Agrobacterium 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 shearing
Less likely (cleaner introduction)
More likely (mix of broken and intact transgene DNA)
Transient assays
Agroinfiltration, some plants not compatible
Mix of transient and stable events, most plants compatible
Stable transgenic plant recovery
Variety and species dependent

17. 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.
Key terminologies
Nuclease
TALENS
Meganuclease
Zinc-finger nuclease
CRISPR

18. Targeted genome editing
Nuclease recognizes and cuts DNA at targeted site
Plant DNA
Plant DNA
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)
Key terminologies
HDR
NHEJ
Transgene donor can be added for targeted insertion
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

19. Future challenges A tool is only as good as the hands that wield it
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

20. Additional references
Resources for plant transformation
transformation-facility/what-plant-transformation
enic/transgenic1.htm
ngineering4D-Transformation-Plant_Cells.htm