Here's my take on plant breeding vs. Genetic Engineering. It ain't a GMO unless a technique beyond breeding and hybridizing was used.
1. Breeding Historical
2. "Gene Gun"
3. Agrobacterium plasmids (late 1970s?)
4. New, 2013 "second generation GE techniques: CRISPR & Talens
1. Plant Breeding
Traditional plant breeding involves finding varieties with traits they want to add to some other variety. Sometimes they search for wild 'ancestral' varieties or just wild cousins. They control which plants pollinate other plants, then select for descendants that are closer to what they want. Every cross that gave them one gene they DID want brought in 50% of genes that you probably do NOT want. So they had to back-cross and select for years to get a possible improvement.
Today they may use DNA sequencing to make better guesses about which wild genes may be helpful, but that isn't called genetic engineering as long as the genes are combined only through one plant pollinating another, and saving seed.
2. Biolistics, a.k.a. bioballistics, a.k.a. the "gene gun"
http://en.wikipedia.org/wiki/G...
For decades, plant biologists wished they had a better way to manipulate plant genomes. They knew how to "transform" bacteria, but not plants. Eventually they stumbled onto ballistics, which is VERY inefficient and clumsy. But better than nothing.
The earliest "gene guns" were modified Crossman air pistols! I've read that researchers had been trying for years and found no good way to "transform" plant cells. Then (I speculate), some lab assistant probably brought in his pellet gun and they had a good laugh but tried it anyway. Then they found that it worked better than the other methods.
They have fancier gene guns now - involving powdered tungsten. I heard that is still sometimes used to blast genes into 4-cell embryos in animal GE research.
One downside is inefficiency - even if a fragment of the DNA you want happens to be blasted into a nucleus, that DNA usually just sat there until it degraded. There was no mechanism to encourage it to cross over into the plants' chromosomes.
Another downside is randomness. Even in the rare case where the DNA winds up near a chromosome AND crosses into it, the location of insertion is random. Existing mechanisms that would normally control the expression of the gene (initiators, promoters and what-not) won't exist, so you have to try to inject an entire DNA sequence with the gene(s) you want plus the regions for initiating and promoting or regulating transcription.
Another downside is crudeness: since you're just shotgunning DNA into random locations in random chromosomes, you need to try to inject entire gene complexes. Biolistics (and the next method) are so crude that the only practical approach is to take DNA from any species, genus or kingdom that might have a desired effect. This "foreign" DNA is called "transgenic" if it came from a species that could not naturally have crossed with the target plant in nature (not the same species).
3. "Agrobacterium plasmid-mediated transformation of plant cells with DNA"
Up until the last few years, this "plasmid" method is what was meant by
"genetic engineering" of crops.
It is as random as "biolistics" but more efficient. You can infect a Petri dish full of plant cells, and get lots of transformed cells, whereas shooting it with a pellet gun or accelerated tungsten powder works about as often as you might expect. Researchers using Agrobacterium plasmids still seem to use a lot of transgenic DNA to get results.
Agrobacterium species have some efficient mechanism for inserting DNA INTO plant cell DNA. In nature, this causes small galls or warts or tumors.
Molecular biologists identified the plasmids that do that (like a zipper that can unzip itself and then re-zip so that it is merged INTO the plant chromosome). The genetic engineering trick is to add the foreign or transgenic DNA
to a plasmid and then let the plasmid infect plant cells (it is good at that).
Typically the plasmid, initiator and promoter regions of DNA are selected from digital libraries of DNA sequences other researchers have found helpful. There are also libraries of plant genes and gene sequences, but people still look at wild relatives, distant relatives, different species, genuses (genera) and Kingdoms to find DNA worth all the time and expense of plasmid transformation and then selection. It's faster and more efficient than "biolistics", but still random and clunky.
Sadly, the magic-zipper plasmid is carried along with the transgenes and other wanted sequences are present in every cell of every GM crop. So their presence in the environment has been multiplied by something like a billion-fold or trillion-fold.
(Also, each plasmid and transgenic DNA region in every cell in every GE plant is likely to have some "leftover" or "unwanted" DNA sequences that happened to come "along for the ride" when someone extracted a trans-gene from any source whatsoever (plant, animal, fish, fowl, bacteria, fungus, virus or purely synthetic). Researchers spend time and money to get functional DNA sequences, but how much time and money do they "waste" to trim off DNA that has no known function before adding them to a digital library? And when a team trying to alter a crop takes "Bacterial initiator XYZ" from a library, does he spend time and money "tidying it up" even BEFORE using it to try to improve a crop plant?)
I think this is what bothers people who consider GE an ecological hazard: Agrobacterium plasmids and other transgenic DNA are jammed into plants that never evolved them. Then we multiply that invented genome billions of times in fields of monocrops. "Horizontal transfer" of transgenic DNA is encouraged.
Chronologically, many DNA techniques were perfected in bacteria: power tools including sequencing, cutting, recombinant DNA methods and polymerase chain reaction. Those tools let researchers whittle, insert and copy-paste DNA, then save the result in a digital library from which the sequences can be re-created or edited further. All these genetic engineering techniques are used on DNA before injecting it into plant cells using the "shotgun" methods "biolistics" and Agrobacterium plasmids.
4. CRISPR and Talens
Technology Review Talens & CRISPR "Second Generation" GE Techniques
March-April 2014
http://www.technologyreview.co...
http://en.wikipedia.org/wiki/C...
The newer techniques (Talens and CRISPR, say 2013) EDIT the DNA in the plant in a precise way in a predetermined spot. That is their advantage. Like editing one word in a book instead of trying to use a shotgun to blast a corrected page or chapter "somewhere" into the book.
If you see a version of a gene that you want to test, from a related variety or wild cousin, you can
specify the exact DNA nucleotides that you want to change (by sequencing the plant, which is now easy and pretty cheap). Then you build your CRISPR sequence to identify THAT EXACT sequence of DNA within THAT SPECIFIC gene, and then edit only the nucleotides that you WANT to edit.
No need to add foreign initiators and promoters or regulators - you just let the plant continue using the regulatory regions that the plant already had wrapped around that gene sequence.
No need to import entire foreign gene SEQUENCES: the plant already has that sequence in place and you can just edit it.
The old ways (biolistics AND the Agrobacterium plasmid) were like shotguns: they
added huge chunks of new DNA randomly, wherever it happened to attach to a chromosome. And usually parts of those new gene sequences were transgenic, which seems to be the main thing that many people consider ecologically risky.
Technology Review : Why we will need GMOs to feed the world:
Jan-Feb 2014
http://www.technologyreview.co...
