Want to work on creating cold resistant wheat

[John D]

Hello Fellow Biohackers,

I want to work on creating cold resistant forms of wheat. I don't have a lot of experience working with plants; mostly I have done ecology research. I have done some work in phylogenetics as well. If anyone has any resources, learning materials, etc, or know of any labs in the Boston area that I could work with that would be wonderful!

Best,

John

[Mega [Andreas Stuermer]]

Hi, it is a great idea, but do you know where the bottleneck is? Are all its proteins optimized for higher temperatures? Then it will be pretty difficult. Only one or few proteins - doable. Unless you add some exotic arctic ( was it shrimp?) chaperons that keep all the enzymes working at lower temperatures.

[Cathal Garvey]

I think the main issue with cold isn't that enzymes slow down, but that
frost causes structural damage to the plant. So, the fish proteins used
for other cold-resistant crops weren't chaperones exactly, they were
antifreeze proteins that prevent ice crystals from forming.

And, they work, although because they bind ice crystals directly I
imagine they work best if they are expressed in proportion to ice hazard?

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[Mega [Andreas Stuermer]]

If it is "just" about expressing antifreeze protein in wheat, that seems fairly doable. Probably you also find a cold induced promoter out of the literature.

I was told that monocots are different to regenerate from a gew transformed cells, though.
Agrobacterium may be easier, but then your organism is regulated by FDA and USDA.

Here is a small intro to plant trafos (transient). Might be worth a look.
http://diyspartanbiotech.files.wordpress.com/2014/03/genetic-engineering-and-synbio-for-beginners-v1.pdf

[David Weichselbaum]

This may prove interesting to you: cold resistant "fish tomato"

[Sebastian S Cocioba]

One issue with cereals is that they don't prove to be a good agrobacterium host even with additional stimulant (phenolic like molecules like vanillin or acetosyringone). You don't see crown gall disease on cereals for a reason. A gene gun may prove easier and there are designs online that are cheap. I made one similar to the one from the VIGGS paper here:

http://www.plantmethods.com/content/pdf/1746-4811-8-27.pdf

And it worked fairly well. I use tungsten so the buffer needs to be at a higher pH and you'll need to work cold. I get my particles from Usnano.com its the 800um tungsten. Last i checked it was $42 for 5g and 16mg gives you 7ish shots. The process is super low efficiency so the first thing I always recommend to newbies (and is seldom considered) is to start with getting tissue culture down packed. Its the hardest of the processes and will make or break your experiments. Plants From Test Tubes is a great book for beginners and a follow up is Introduction to Plant Biotechnology by Chawala...and hopefully I'll finish my book by winter (shameless plug).

You can take this article as a good basis for ICE pathway genes from Arabi:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC219064/

I work with plants and have been for a long time. If you need more info just ask. I'm an open book. Good luck!



Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC

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[John D]

Yes what Cathy says, frost causes damage to plants.

[John D]

Hi Everyone,

Here are some updates on some of the preliminary research I have done.

1. Identified preliminary targets for better crops (biofortification, resistance to fungus/viruses, resistance to sucking pests, drought tolerance, temperature tolerance) http://www.sciencemag.org/content/327/5967/812.full#T1 and https://royalsociety.org/~/media/Royal_Society_Content/policy/publications/2009/4294967719.pdf are some interesting reviews of what sort of technological properties are needed and possible to feed our growing population.

2. Chose biofortification over temperature tolerance. My guess is it is going to be easier to create agricultural cultivars that express a vitamin than it is to express an antifreeze protein(s).

3. Chose what to be expressed. I am going to start with a GFP and herbicide tolerance and go from there; I will decide on the vitamin later. If anyone has any recommendations please let me know.

4. Chose model organism (maize aka corn) Although rice and wheat are more widely consumed; the maize genome has been fully sequenced and there is more support for it.

5. Found protocols for raising maize http://cshprotocols.cshlp.org/content/2009/10/pdb.emo132.full

My next steps are:

1. Get some maize cultivars

2. Find a place to grow them

3. Grow maize cultivars

4. Chose protocol for transformation

[John D]

Probably want to include a reporter gene such as a gfp so you don't have to sequence all of the plants.

[John D]

You might want to have something that increases expression when it is cold http://www.nature.com/scitable/topicpage/environmental-influences-on-gene-expression-536 some genes in rabbits are activated by lower temperature. Although if the protein doesn't damage the plant/not a health concern it should be fine to have constant expression. The other issue is finding a protein that protects plant tissues from frost damage and getting those gene(s) into the plant.

On Sunday, August 9, 2015 at 8:42:40 PM UTC+9, Cathal Garvey wrote:

[Mega [Andreas Stuermer]]

If you need gene synthesis let me know. I might have something to synthesize too, but some companies do minimum order size to get a fair prize. GFP and herbizide tolerance -> that's all in pCambia 1302 http://www.markergene.com/pcambia1302-plant-expression-vector.html
http://www.snapgene.com/resources/plasmid_files/plant_vectors/pCAMBIA1302/

You can also tell the synthesis company to cut your synthesized DNA into a vector (such as pCambia) so you don't need subcloning work yourself. May accelerate your research, if your lab is not that well equiped

[Mega [Andreas Stuermer]]

The patent for Bt and roundup are expiring soon/have expired... Maybe you want some open source mainstream GMO?

On Saturday, August 8, 2015 at 7:43:51 PM UTC+2, John D wrote:

[Sebastian S Cocioba]

Sorry, with regards to my last message, there was a typo in the website address. Its us-nano.com. They supply graphene and nanotubes as well as other materials at the micron and nanometer scale. They also sell gold for microprojectile gene gun shots but I am morally opposed to shooting gold down the drain...although its a wonderfully inert and heavy metal. \m/

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC