Here is an idea that came to me recently, please bear with me for an introduction and if in mood check the paper i have attached.
Introduction
Agrobacterium tumefaciens is a gram negative bacterium that can infect dicotyledons and is responsible for the "crown gall disease" in plants.The crown gall appears as a mass of undifferentiated cells that grow on the plant resembling what would look like a tumor.
The "mode of action" of the agrobacterium is fascinating. It usually lives in the soil and when a plant is wounded it is attracted to the wound ,due to chemotaxis being able to locate and move towards sugars and compounds like acetosyringone. The bacterium carries a plasmid ,called Ti Plasmid, that its expression enables it to perform something akin to bacterial conjucation but with the recipient to be the plant cell. It forms a "bridge" with the plant cell and utilising the vir genes, cuts a fraction of the plasmid called "TDNA" ,transfers it in the plant cell and incorporates it to its genome. The TDNA portion contains genes coding for AUXIN production, CYTOKININ production and OPINE production. The first two ,known plant growth regulators, de-differentiate the cells leading to the tumor formation. The opine genes are rather peculiar : they force the cells to make opines which are aminoacids reacted with keto-acids or sugars. So what the bacterium gets out of it? Simply, it feeds off the opines that while the now "genetically modified" plant cannot use ,the bacterium is perfectly capable of utilising!
Given the mechanism of action agrobacterium has received much attention from biotechnology , because if one substitutes the TDNA part with a gene of interest ,it will get carried in the plant cell and incorporated on its DNA, effectively modifying the plant. Here though ,the main interest is NOT genetic modification but the effects of the the "wild type" agrobacterium on the plant and secondary metabolism. It seems ,as far as i have looked around, that such an infection might raise secondary metabolism products by much and some scientists are considering a close relative of tumefaciens, Agrobacterium rhizogenes an option of increasing production of secondary metabolites in hairy root cultures in the lab.
A small collection of articles displaying the effect of the agrobacterium on secondary metabolism (alkaloids, terpenes?) is this :
Functions of rol genes in plant secondary metabolism
Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only
Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don
It seems promising, given that such remarks are made within the abstracts :
"The last ones accumulated between 4 (1.1 mg g-1 DW) and 27 (8 mg g-1 DW times more alkaloids than the intact roots (0.3 mg g 1 DW).This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures. "
"Dimeric alkaloid vincristine in the transformed cultures was present at a concentration of 0.011 that was tenfold higher compared to untransformed control cultures. "
Some increase eh?
Searching on literature for cacti i have not found much on tumefaciens, but i found an article on rhizogenes with the title "In vitro analysis of susceptibility to Agrobacterium rhizogenes in 65 species of Mexican cacti" which i am attaching at the end. It seems that the Agrobacterium species CAN infect cacti although at it was thought that it was very rare, at least in nature.Apart from infection ,the paper also examines secondary metabolites using Thin Layer Chromatography and UV light ,Dragendorff reagent and Marquis reagent to visualise secondary metabolism products.So here comes the interesting part :
"Transformed roots induced by A. rhizogenes are
widely used for the study and production of secondary
metabolites from many plant species. With the goal of
verifying if the cacti transformed roots conserve the
biosynthetic capacities found in the normal roots,
alkaloid-like compound production in some of the studied
cacti species was investigated using thin layer
chromatography. In all five species analyzed, and with
the three detection systems used, it was observed that the
patterns obtained in transformed and non-transformed
roots were qualitatively equal (Fig. 3). Also, some of the
spots appear more intense in the extracts obtained from
transformed roots. These results should be confirmed
using more sensitive techniques."
It seems that the secondary metabolism remained the same (same alkaloids) but it seems that in some cases (ones detected as more intense spots) they observed MORE alkaloids. So ,the "increase alkaloidal production" in cacti theory with Agrobacterium starts seeming more plausible!Keep in mind though that this may not be a systematic effect ,but limit itself in the crown gall. If it grows to an appreciable size though ,things could get interesting both biochemically and morphologicaly.
Proposal
In the light of the above ,i wonder if one would want to experiment by infecting cacti samples of "more or less known" potency with Agrobacterium tumefaciens and seeing how this would affect the plant, and propably alkaloidal production.The protocol for such a procedure can be simple ,if one locates crown galls in plants : They could be taken, propably blenderised in some distil water and "subcutaneously injected" in the cacti ,underneath the epidermis or applied in cacti wounds.One could perform a series of such treatments ,given that it might have very low infection rate. My personal recomentation would be to a) have a mixture of different crown galls from different plants, maybe this will ensure more strains in the inocculation liquid , b) using lots of them with little water so the inocculation liquid propably would have high load of bacterium, c)multiple sites of injection : the bacterium attaches to wounded tissue, d) if possible retain the liquid at the injecting site hence the "hypodermic" approach: a "bubble" of liquid could be formed causing also tissue damage if the needle was to be put just underneath the waxy/hard layer of cacti epidermis with orientation almost paralel to the to the axis of cactus growth and the plunger pushed so as the injected liquid to forcefully enter.
If one has access to commercial Agrobacterium strains or somehow can get his/her hands on them ,this would also be a nice idea .The first method though is low tech, requires one to purchace almost nothing just looking around for crown galls which can be easily spotted. A google image search on them will show you how they look.
Tell me what you think of the above please.
Introduction
Agrobacterium tumefaciens is a gram negative bacterium that can infect dicotyledons and is responsible for the "crown gall disease" in plants.The crown gall appears as a mass of undifferentiated cells that grow on the plant resembling what would look like a tumor.
The "mode of action" of the agrobacterium is fascinating. It usually lives in the soil and when a plant is wounded it is attracted to the wound ,due to chemotaxis being able to locate and move towards sugars and compounds like acetosyringone. The bacterium carries a plasmid ,called Ti Plasmid, that its expression enables it to perform something akin to bacterial conjucation but with the recipient to be the plant cell. It forms a "bridge" with the plant cell and utilising the vir genes, cuts a fraction of the plasmid called "TDNA" ,transfers it in the plant cell and incorporates it to its genome. The TDNA portion contains genes coding for AUXIN production, CYTOKININ production and OPINE production. The first two ,known plant growth regulators, de-differentiate the cells leading to the tumor formation. The opine genes are rather peculiar : they force the cells to make opines which are aminoacids reacted with keto-acids or sugars. So what the bacterium gets out of it? Simply, it feeds off the opines that while the now "genetically modified" plant cannot use ,the bacterium is perfectly capable of utilising!
Given the mechanism of action agrobacterium has received much attention from biotechnology , because if one substitutes the TDNA part with a gene of interest ,it will get carried in the plant cell and incorporated on its DNA, effectively modifying the plant. Here though ,the main interest is NOT genetic modification but the effects of the the "wild type" agrobacterium on the plant and secondary metabolism. It seems ,as far as i have looked around, that such an infection might raise secondary metabolism products by much and some scientists are considering a close relative of tumefaciens, Agrobacterium rhizogenes an option of increasing production of secondary metabolites in hairy root cultures in the lab.
A small collection of articles displaying the effect of the agrobacterium on secondary metabolism (alkaloids, terpenes?) is this :
Functions of rol genes in plant secondary metabolism
Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only
Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don
It seems promising, given that such remarks are made within the abstracts :
"The last ones accumulated between 4 (1.1 mg g-1 DW) and 27 (8 mg g-1 DW times more alkaloids than the intact roots (0.3 mg g 1 DW).This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures. "
"Dimeric alkaloid vincristine in the transformed cultures was present at a concentration of 0.011 that was tenfold higher compared to untransformed control cultures. "
Some increase eh?
Searching on literature for cacti i have not found much on tumefaciens, but i found an article on rhizogenes with the title "In vitro analysis of susceptibility to Agrobacterium rhizogenes in 65 species of Mexican cacti" which i am attaching at the end. It seems that the Agrobacterium species CAN infect cacti although at it was thought that it was very rare, at least in nature.Apart from infection ,the paper also examines secondary metabolites using Thin Layer Chromatography and UV light ,Dragendorff reagent and Marquis reagent to visualise secondary metabolism products.So here comes the interesting part :
"Transformed roots induced by A. rhizogenes are
widely used for the study and production of secondary
metabolites from many plant species. With the goal of
verifying if the cacti transformed roots conserve the
biosynthetic capacities found in the normal roots,
alkaloid-like compound production in some of the studied
cacti species was investigated using thin layer
chromatography. In all five species analyzed, and with
the three detection systems used, it was observed that the
patterns obtained in transformed and non-transformed
roots were qualitatively equal (Fig. 3). Also, some of the
spots appear more intense in the extracts obtained from
transformed roots. These results should be confirmed
using more sensitive techniques."
It seems that the secondary metabolism remained the same (same alkaloids) but it seems that in some cases (ones detected as more intense spots) they observed MORE alkaloids. So ,the "increase alkaloidal production" in cacti theory with Agrobacterium starts seeming more plausible!Keep in mind though that this may not be a systematic effect ,but limit itself in the crown gall. If it grows to an appreciable size though ,things could get interesting both biochemically and morphologicaly.
Proposal
In the light of the above ,i wonder if one would want to experiment by infecting cacti samples of "more or less known" potency with Agrobacterium tumefaciens and seeing how this would affect the plant, and propably alkaloidal production.The protocol for such a procedure can be simple ,if one locates crown galls in plants : They could be taken, propably blenderised in some distil water and "subcutaneously injected" in the cacti ,underneath the epidermis or applied in cacti wounds.One could perform a series of such treatments ,given that it might have very low infection rate. My personal recomentation would be to a) have a mixture of different crown galls from different plants, maybe this will ensure more strains in the inocculation liquid , b) using lots of them with little water so the inocculation liquid propably would have high load of bacterium, c)multiple sites of injection : the bacterium attaches to wounded tissue, d) if possible retain the liquid at the injecting site hence the "hypodermic" approach: a "bubble" of liquid could be formed causing also tissue damage if the needle was to be put just underneath the waxy/hard layer of cacti epidermis with orientation almost paralel to the to the axis of cactus growth and the plunger pushed so as the injected liquid to forcefully enter.
If one has access to commercial Agrobacterium strains or somehow can get his/her hands on them ,this would also be a nice idea .The first method though is low tech, requires one to purchace almost nothing just looking around for crown galls which can be easily spotted. A google image search on them will show you how they look.
Tell me what you think of the above please.
