Disclaimer: This thread is very half assed and I have been working on it for a while but due to an extreme lack of focus I have decided to just release it as is and let it grow from there. If I have misplaced or mislabled references which im sure I have im sorry. There is much more material I have to add to this thread but if I don't put it out there now I may never do so. I chose this option because there appears to be a HUGE amount of voodoo associated with Genetic modification when viewed by those who are outside of the circle so to speak. I have done relatively little research into it and my views on the difficulty of GM have changed dramaticly.
The idea of Geneticly modifing plants always seemed like a pipe dream to me until a short time ago when I realized the potential of a new form of modification that can be performed using a common soil bacteria and the roots of various plants to produce large quantaties of secondary metabolites from common starting materials. This thread over at Blacklight peaked my intrest in the subject sometime back and there are a couple good references to check out pointing at the possibilitys of Genetic modification on an amature level. I am by no means an expert in this field but I will try my best to explain in in a fashion that people can understand to the best of my understanding.
Hairy Root Cultures(HRCs) are the result of an infection of a typical root by the invasion of an Ri Plasmid containing bacteria. These bacteria contain a circular form of DNA known as plasmids with the ability to splice itself into the normal structure of plant cells leading to something that resembles a tumor in structure and growth in many instances. Once the correct form of infectant is identified and isolated it is use to infect the host root in an attempt to produce a culture of hairy roots. These can then be cultured in a seperate Bioreactor simular to aireated brewing equipment shown by the text SECONDARY METABOLISMOFHAIRYROOTCULTURES IN BIOREACTORS[1] to produce a large amount of high cell density rootmass in a relativly small area. Once cultured the reactors have been known to keep the DNA stable for over a period of 4 years as evident in Link showing how a root was made to glow by inducing the DNA of a jellyfish to fuse thru the HRC process for over 15 years in some cases(Floresetal.,1999)
What is needed is to isolate a viable strain of bacteria that contains the correct root inducing(RI) plasmid which is primarly contained in the Agrobacterium rhizogenes soil bacteria. If you look at Complementation of Agrobacterium tumefaciens[2] you will see the obvious infection site in the root is disinquished by the large nodual on the root. If anyone has seen roots in the wild they will know that this sort of infection appears fairly common and first order of business would be isolation of the correct Agrobacteria to induce the formation of these Hairy root cultures. The lumps on wild infected plants seems like the best place to take a swab from to start isolation of the bacteria.
Lets jump ahead and assume we have isolated and cultured the root we wanted. Its at this point that a simple areated nutrient solution simular to how hydroponics works with the addition of some bioprecursor to the mix and these will convert large amounts of starting material to a finish product increasing concentrations well beyond the normal for a plant. These HRCs do not seem to suffer from the same metabolyte toxicity that plants do and as long as the precursor is feed in the reactor it will continue to produce the secondary metabolytes without death to the plant as is normal in nature. The most obvious use of this is the conversion of Eugenol to Safrole quickly and cleanly
You can see the simplicity of my micro "Bioreactor" as nothing more then an aireated tube filled with water and at the moment a Rather crude but if I didn't start somewhere I would never start. My main goal is to keep this little clump of grass alive for a long enough time without infection to prove its ability to run for extended period of time. This at the moment is nothing more then a form of hydroponic growing equipment and I think all I will feed it is a few ML of Suger solution every couple days since I don't think for these test it needs much more. In all honesty im trying to feed any bacteria present more then im trying to feed the grass because I want to see what kind of hold Fungi or bacteria might take in such an experiment. The initial weight of the grass is 2.3 grams and I will measure it after im done as well to see if it grows any at all thruout the course of the experiment. I do not have much faith in the experiment at the time but my main goal is to proceed to the stabalization of a culture of Rootbark from a sassafrass trees. I don't think a Hairy root culture is fully needed to test what I had in mind since the reaction need only a single enzyme in the conversion of Eugenol and not a multistep process that many chemicals needs involving the entire root.The lump of grass seen in the picture was kept alive under water for over 2 weeks and it was not until I stupidly refilled it with tap water when the solution started to evaporate did the grass die. Keeping the roots alive after getting it started I think would be fairly simple in a home setup.
Lets for a second take a step in another direction. HRCs are just one form of tissue culture and with simple techniques explain in reference [] you can produce things like Codeine, morphine ect...
Odds are this does also have the potential to make a large amount of cacti from a very small second of the source cacti.
This is where I began to lose focus and intend to finish reporting the subject and slowly cleaning up this thread as time permits. If you noticed missing links or misplaced references tell me and I will try to find them since I have more but am insure if I have linked to all of hem.
Further reading:
http://openpdf.com/ebook/hairy-root-pdf.html
Recieved references:
mutants by genes from the TR-region of the Ri plasmid of
Agrobacterium rhizogenes.
External references
The idea of Geneticly modifing plants always seemed like a pipe dream to me until a short time ago when I realized the potential of a new form of modification that can be performed using a common soil bacteria and the roots of various plants to produce large quantaties of secondary metabolites from common starting materials. This thread over at Blacklight peaked my intrest in the subject sometime back and there are a couple good references to check out pointing at the possibilitys of Genetic modification on an amature level. I am by no means an expert in this field but I will try my best to explain in in a fashion that people can understand to the best of my understanding.
Hairy Root Cultures(HRCs) are the result of an infection of a typical root by the invasion of an Ri Plasmid containing bacteria. These bacteria contain a circular form of DNA known as plasmids with the ability to splice itself into the normal structure of plant cells leading to something that resembles a tumor in structure and growth in many instances. Once the correct form of infectant is identified and isolated it is use to infect the host root in an attempt to produce a culture of hairy roots. These can then be cultured in a seperate Bioreactor simular to aireated brewing equipment shown by the text SECONDARY METABOLISMOFHAIRYROOTCULTURES IN BIOREACTORS[1] to produce a large amount of high cell density rootmass in a relativly small area. Once cultured the reactors have been known to keep the DNA stable for over a period of 4 years as evident in Link showing how a root was made to glow by inducing the DNA of a jellyfish to fuse thru the HRC process for over 15 years in some cases(Floresetal.,1999)
What is needed is to isolate a viable strain of bacteria that contains the correct root inducing(RI) plasmid which is primarly contained in the Agrobacterium rhizogenes soil bacteria. If you look at Complementation of Agrobacterium tumefaciens[2] you will see the obvious infection site in the root is disinquished by the large nodual on the root. If anyone has seen roots in the wild they will know that this sort of infection appears fairly common and first order of business would be isolation of the correct Agrobacteria to induce the formation of these Hairy root cultures. The lumps on wild infected plants seems like the best place to take a swab from to start isolation of the bacteria.
Lets jump ahead and assume we have isolated and cultured the root we wanted. Its at this point that a simple areated nutrient solution simular to how hydroponics works with the addition of some bioprecursor to the mix and these will convert large amounts of starting material to a finish product increasing concentrations well beyond the normal for a plant. These HRCs do not seem to suffer from the same metabolyte toxicity that plants do and as long as the precursor is feed in the reactor it will continue to produce the secondary metabolytes without death to the plant as is normal in nature. The most obvious use of this is the conversion of Eugenol to Safrole quickly and cleanly
You can see the simplicity of my micro "Bioreactor" as nothing more then an aireated tube filled with water and at the moment a Rather crude but if I didn't start somewhere I would never start. My main goal is to keep this little clump of grass alive for a long enough time without infection to prove its ability to run for extended period of time. This at the moment is nothing more then a form of hydroponic growing equipment and I think all I will feed it is a few ML of Suger solution every couple days since I don't think for these test it needs much more. In all honesty im trying to feed any bacteria present more then im trying to feed the grass because I want to see what kind of hold Fungi or bacteria might take in such an experiment. The initial weight of the grass is 2.3 grams and I will measure it after im done as well to see if it grows any at all thruout the course of the experiment. I do not have much faith in the experiment at the time but my main goal is to proceed to the stabalization of a culture of Rootbark from a sassafrass trees. I don't think a Hairy root culture is fully needed to test what I had in mind since the reaction need only a single enzyme in the conversion of Eugenol and not a multistep process that many chemicals needs involving the entire root.The lump of grass seen in the picture was kept alive under water for over 2 weeks and it was not until I stupidly refilled it with tap water when the solution started to evaporate did the grass die. Keeping the roots alive after getting it started I think would be fairly simple in a home setup.
Lets for a second take a step in another direction. HRCs are just one form of tissue culture and with simple techniques explain in reference [] you can produce things like Codeine, morphine ect...
Quote
Production of fine chemicals like Codeine, Diosgenin, Sitostero, Quinine, Vincristine, Atropine, Pyrethrin, Saffoon and Methanol which are presently produced from plant sources can be produced from cell culture. However, the commercial success has been obtained in production of `Shikonin' from cells of plant lithospermusm erythorhizon.Image in your head the potential of a tissue culture from the Morphine producing areas of the Poppy plant. A steady stream of waste liquor containing the substances desired could be saved and concentrated on an unlimited scale. This is all not to mention the potential that a tissue culture from the budding female pot plant could have.
Odds are this does also have the potential to make a large amount of cacti from a very small second of the source cacti.
This is where I began to lose focus and intend to finish reporting the subject and slowly cleaning up this thread as time permits. If you noticed missing links or misplaced references tell me and I will try to find them since I have more but am insure if I have linked to all of hem.
Further reading:
http://openpdf.com/ebook/hairy-root-pdf.html
Recieved references:
- [1]SECONDARY METABOLISM OF HAIRY ROOT CULTURES IN BIOREACTORS
- [2] Complementation of Agrobacterium tumefaciens tumor-inducing aux
mutants by genes from the TR-region of the Ri plasmid of
Agrobacterium rhizogenes.
External references
- Plants chemicals of instrest.
http://www.rain-tree.com/plantdrugs.htm - Genetic modification thread from Blacklight
http://forums.blacklight.me/viewtopic.php?f=31&t=811&p=13678&hilit=hairy+root#p13678 - Gene Extraction from Science Madness
http://www.sciencemadness.org/talk/viewthread.php?tid=1496&page=3 - Tissue Culture
http://www.indiaagronet.com/indiaagronet/seeds/CONTENTS/tissue_culture.htm