Lecture 3 – Plant Biotechnology II: GMOs

Genetic Modification (Modern) vs. Molecular Breeding (Traditional)

MB: requires desired to trait be already present in the population, genetic resources must be available, and requires sexually reproducing plants to propagate

GM: acquires genes from any source, genetic resources aren’t required, plants can be propagated vegetatively (no need for crossing)

GMO Crops

Crops that have undergone genetic modification to acquire foreign DNA within its genome. Have been around since the 1980s, becoming more prevalent, and have gone through extenstive testing to be safe for human and animal consumption.

• Only large-acreage commercial producers can buy and plant GMO seeds

Steps of Transforming Plants

Modified T-DNA from Agrobacterium tumefaciens that has the gene/s of interest present

1. Pieces of a plant are infected, then undergo root & shoot growth

   1. Initial explant placed within a medium

   2. Infected to become a callus (cancerous)

   3. Auxin is used to stimulate root growth

   4. Cytokinin is used to stimulate shoot growth

2. The resistant plants that have grown are the ones that have been successfully transformed

How Does Agrobacterium tumefaciens Work?

Intiitally discovered in 1907 when Erwin Smith isoalted a bacterium from crown galls and inoculated them in other plants which then produced more galls (establishes link).

Agrobacterium-induced galls do not require bacterial persistence (for the bacteria to actually be there) to continue growing

• Even without the bacteria, they can persist in culture (with nutrients, resources, etc)

• This is different to other pathogen-induced growths

They also don’t require the presence of phytohormones auxins and cytokinins

• The gall tissues already express high levels of these phytohormones

The bacteirum is able to do this by:

• Having a Ti plasmid, a tumour inducing plasmid, that contains T-DNA, transfer DNA, which is naturally integrated into the plant genome

• The T-DNA contains genes for auxin, cytokinin, and octopine (it’s own resource) synthesis. All of which will support in cancerous growth to house more bacterium that is supported by octopine.

• Only the T-DNA is integrated

Applying T-DNA for GMOs

The T-DNA has a left and right border in which everything in-between is integrated and can be modified. We’ve taken this to our advantage by:

• Replacing and modifying the genes present with target genes of interest (removing octopine gene too)

• Multiple genes can be inserted (35-50 max)

• This also includes a selectable marker (typically antiobiotic resistance) to allow us to know which plants have been successfully transformed

• All other oncogenes are removed

As long as the necessary parts are present (promoter, coding region, stop & start codons, terminator, and so on) and the left & right borders are in-tact, expression of the interest gene/s should be successful (as long as transformation is successful

• Promoter that initiates gene expression (can specify where it occurs to, 35s promoter most common cuz highest expression

• Terminator to add PolyA tails

• Coding sequences (including the stop & start codons)

Where the T-DNA is inserted is random and expression can vary

• The insertion sites are random and when replicated, will result in different sites. We’d typically select for the plant that was transformed in a way in which the insertion site doesn’t interfere with other genes

Eukaryotic Gene Structure: Promoter

Region of DNA where proteins & transcription factors bind to help initiate transcription by facilitating the binding of RNA polymerase

Eukaryotic Gene Structure: Coding Region

Portion of a gene’s DNA/RNA that codes for proteins, with a start and stop codon. There are introns within the DNA’s coding region that are excised out and not included in the final mRNA

• Start Codon = AUG (RNA) or ATG (DNA)

• Stop Codon = UAG, UAA, UGA (RNA) or TAG, TAA, TGA (DNA)

Eukaryotic Gene Structure: Initiation & Termination Regions

Not the same as the start and stop codons. They are regions where transcription intiates and is terminated.

Bt Peanut Plants

Peanut plants are susceptible to insects. The Bt gene from Bacillus thuringiensis expresses insecticidal proteins known as Bt Cry toxins. These toxins are highly specific, only affecting insects with a specific receptor protein in their inestinal lining where it then forms a pore and kills the insect.

We have historically sprayed this Bt Cry toxin onto the plant itself, but with GM and T-DNA, we can make the plant express the toxin too

Purpose of Transgenes in GMOs

Herbicide resistance, which reduces total amount of herbicides sprayed and reduced the need to plough fields, reducing erosion

Insect resistance, making the crop toxic to some insects while still being safe for human consumption (and beneficial insects), reduces needs of insecticides

Virus resistance, allowing crops to withstand viruses that tend to spread fast and can threaten the viability, yields, and growth of crops

Changing traits, allows us to change the phenotypes and how crops look

• Seen in genes that prevent browning in arctic apples & white russet potatoes as well as genes that change flesh colour like the pinkglow pineapple

Other examples:

• Golden rice, carrying two genes for beta-carotene (from daffodils and soil bacteria) to be enriched with vitamin A to reliminate vitamin A deficiency

• Purple tomatoes, carrying purple pigment anthocyanin genes which are antioxidants

• Phytase corn, carrying phytase genes that breakdown antinutrient phytic acid to make phosphorus more bioavailable. This improves the health of animals that eat it and reduced phosphorus content in manure for less water pollution

Other Applications of Transgenics w/ Agrobacterium

Can be used for gene knockout studies to help us infer a gene’s function

• Done by overexpressing a gene to figure out function & pattern or by selecting for different phenotypes

Used for transient expression studies which is the short-term express of T-DNA genes to produce faster results

• Done mostly as controls and tests to ensure that something works before going through with transgenic plants

Genome Editing

Making targeted modifications in the genome without permanently introducing foreign DNA. A much more precise and faster method due to not needing to rely on random mutations (as in the case of traditional domestication & breeding)

The current technologies that utilise present are: meganucleases, zinc finger nucleases, TALE nucleases (TALENs), and CRISPR-Cas9 (Cas9 protein cuts, sgRNA guides Cas9)

Genome-edited crops are not the same as GMO crops because they don’t have any foreign DNA (as long as transgenic constructs are removed). The only different is the few base pairs/genes that are edited

• This means that it is hard to tell if a crop is genome-edited, and therefore aren’t as regulated as harshly as GMOs. Some more regulations being put in place (eg. number of base pairs allowed to be changed, etc)

• GE crops are currently on the rise and scientists are exploring using GE in other organisms too

How Does Genome Editing Work

A nuclease (that is produced from foreign DNA/organism) is introduced into a host cell. It will then cut DNA in a targeted manner in a specific site with the help of guide RNA

The resulting DNA break will trigger DNA repair where are a few outcomes are possible: an accidental insertion/deletion that results with a mutation, or a template DNA is introduced to control the repair process

Applications of GE

There are many applications:

• Gene knockout studies

• Polyploid/chromosome transformation improvements

• Multi-trait improvements

• De novo domestication (editing known genes of wild relatives to resemble modern crops)

• Large-scale gene screenings

• Gene stacking (to exphasise a gene)

• Fine-tuning gene expression (how often a gene is expressed, controlling expression)