Teaching Materials for Plant Transformation

by Andika Gunadi – gunadi.9@buckeyemail.osu.edu

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