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Role of amino acids in phlebophylla metabolism

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Ok there's not been a great deal of joy from the silica/ low chloride media- yet. Growth is *much* slower than normal, to say the least, and we can continue on that line for a while- morphology is otherwise fine.

The next small batch of seed were allowed to emerge from the seed coat naturally ( longer soaking period resulted in increased contam as expected )but as the last lot were excised from their seed coat and much of the surrounding tissue ( a partial embryo rescue ) before fully germinated, I wanted to compare results.

To nobody's surprise but my own the seeds germinated entire with roots are growing faster and look much healthier than their predecessors. This doesn't usually apply in TC where all nutrient requirements are theoretically met within the media and root formation is uneccessary in most spp until prior to deflasking. Embryo rescue has a lower contam rate but the advantage of keeping the roots on can't be dismissed in this species atm. Which gives birth to the latest theory we're trialling- the inclusion of l-tryptophan in the media

Apparantly l-trypto is usually metabolised into IAA, as Auxin would know, a compound which is responsible for internode length, cell size, and I think chlorophyll development and gawd knows what else, and this happens in the roots ( please somebody correct me if I'm wrong, it's early and I can't see far enough ahead to locate my notes ).

Given that l-tryptophan is a precursor for the phlebo major constituent DMT, it seems like a good idea ( and not much more than a hunch ) to kick some into the media and see how it flies. I hate wasting seed but, so if anyone has any comment I'd love to hear it.

I'm gunna keep one entire and grow it out to 150mm in a larger tube to compare and contrast in progress

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Darklight,

this is something that comes up in relation to the phlebs...silica hydride.

http://www.phisciences.com/megah.html

I have not encountered anything of this sort of benefit for the human organism, ever as this kind of supplement. It's the PERFECT comedown, energy boost supplement and the body assimilates it very well...the effect really is of a fresh mountain streaming cleaning out one's blood.

Maybe the phlebs need some negative ion/antioxidant action?...I'll do some tests soon with this. Wouldn't do any harm I'd say!

Julian.

[ 06. February 2004, 09:58: Message edited by: folias ]

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Thanks for that Julian, I'll look into it :)

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It doesn't seem very likely that a source of sufficient tryptophan would be present in the natural environment unless it it couples with a symbiont immediately on germination.

Ammonia is a good candidate for poisoning things in extreme conditions but that is all I can say before I see the experimental conditions.

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theobromos:

It doesn't seem very likely that a source of sufficient tryptophan would be present in the natural environment unless it it couples with a symbiont immediately on germination.

Spot on. But I am working on what is known as Today's Hypothesis ( where I jump round and shout Eureka until I find out I'm wrong ) which is- several plants grown in media with their roots on are showing a better response than those with their roots removed. This isn't common in successful TC at all- the most popular form of bulk proliferation is either regeneration from callus- or via axillary bud mulitplication from parent plants with root systems removed

Today's Hypothesis involves having two seedlings maintained intact in a modified B5 media, which produces the best growth I've seen, and going for ax bud prolif from other explants with the roots removed- in the same media, plus 50mg/L l-tryptophan. Plants are little factories, and if the l-trypto is metabolised into IAA then it might just sort some of the problems I've been experiencing in culture- internode shortening and leaf shrinkage and browning over successive subcultures

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Specifically for Acacias:

http://aob.oupjournals.org/cgi/reprint/80/3/257.pdf

This paper on parasitism by Quandong gives some analysis of Acacia root xylem sap contents for A. pulchella and A. rostellifera, including that asparagine is the major N transport and majority by mole. Only Acacias of all the plants studied and at only one of the two sites had pipecolic acid in their xylem at 30% of the amino acids by mole. The other site had much less pipecolic acid but about the same asparagine and much more aspartic acid.

Inorganic salts were much the same in the sap of all host species.

Organic acids were dominated by malate at 60%+ by mole in all species of hosts, though citric acid was only present in the two Acacia species. They don't give the details so bug the responding author, in fact you could send some of our bigger forum members round to see him (John S. Pate) at the Department of Botany, The University of Western Australia, Nedlands, WA 6907.

Perhaps the plant is very sensitive to the particular gibberellin that it makes for itself, getting back to general botany:

"The idea that they are produced in the roots seemed logical though indications exist that they are only converted in the roots, i.e. that one gibberellin is transported from the shoot into the root where it is converted another one. The new product is then transported back via the xylem into the shoot. Both in xylem- and phloem exuded matter have gibberellins been detected showing how the distribution mechanism for hormones works in this type of plant."

http://www.biologie.uni-hamburg.de/b-onlin...ine/e31/31d.htm

Also xylem transport from root to shoot appears to be true of cytokinins and ethylene, though not for auxins, according to http://www.uoguelph.ca/~mgoss/two/Sr_ratio.html They also give jasmonic and abscisic acids as "phloem mobile" and the latter also in the xylem.

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Some data on xylem sap for Acacia trachycarpa and A. ampliceps from http://aob.oupjournals.org/cgi/reprint/82/5/675.pdf Again arginine but with subtantial amounts of threonine and djenkolic acid in A.trachycarpa. With djenkolic acid being "a regular feature of Acacia xylem sap" according to three references.

While malate dominated in A. ampliceps citrate was the majority of the organic acid fraction in A. trachycarpa.

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I am not sure what to make of the fact that the gums like gum arabic from members of the Phyllodineae are usually associated with the xylem cells. Or that the oils from the seeds have a range of non-uses in food. I assume that last was a transcription error.

http://science.calm.wa.gov.au/cswajournal/...4-3/109-116.pdf

And one of the top of my head, if Acacia phlebophylla likes methylating things give it some SAMe to play with.

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theobromos:

asparagine is the major N transport and majority by mole.

Thanks theo, yer a god :) Most of this is babble to me as my chemistry is lousy, thank heavens TC is empirical observation at this level, if one has an idea, they add or subtract it to a generic media and note its results before moving out into bat country and adding vegemite or orange juice.

There are two other amino acid candidates I was looking at- trying to find them at this hour is diffucult cos I can't open my eyes wide enough to see the small print til my second coffee. But I found a new ref to l-tryptophan in David Seigler: Plant Secondary Metabolism Kluwer Academic Publications and I don't have the edition cos someone removed those pages. Anyhow it gives the pathway for IAA as coming via l-tryptophan.

Wish I could find the other reference, it listed two other amino acids as contenders for the next round of experiments, and its somewhere in the 2nd edition of " Medicinal Natural Products", Paul M Derwick, John Wiley & Sons, 2001. And I'll def keep the asparagine thing on the plate for the next round of trials

They don't give the details so bug the responding author, in fact you could send some of our bigger forum members round to see him

Crackup- I can see the headlines now... Fractal what are you doing this weekend?

"The idea that they are produced in the roots seemed logical though indications exist that they are only converted in the roots,

I did try both GA3 and coconut milk several years ago, but to no avail. Prolly not surprising- how many gibberellins are available commercially- and how many more might be identified?

I think I'll have a clever phytobotanist sit down with me next week and go over this post of yours theo, it seems like a bit of a goldmine, and me standing here without a shovel or a clue. Thanks tho, it adds weight to the amino acid argument!

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How may I be of service? So what's my question exactly? Sorry I've been meaning to have a munch on this thread for a few days. It'll require a trip to uni. They are big on plant nitrogen metabolism at my uni. What do you want to know? Not sure what details of the parasitism experiment you are after.

Is this the general question?:

What role could amino acid supplements (in the medium) play in helping the little fellas:

a) take up / transport nitrogen

OR

B) synthesise growth hormones

I'll be in there full time starting monday, so I'd love to help out.

Now I will begin to waffle on about semi-relevant trains of thought. I haven't seriously looked into this yet so I don't know if there is any evidence to suggest it is true but...

I've been wondering about (and i'm sure others have) the symbiont issues. Apart from the supply of nutritional molecules, there may signalling/elicitor molecules of symbiont origin normally needed to induce proper regulation of plant genes that are required for the metabolism of the incoming goodies by the plant. Maybe some chitin or something?

[ 11. February 2004, 10:12: Message edited by: Fractalhead ]

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Fractalhead:

How may I be of service?

I think theo was suggesting we send someone around to stand over a particular researcher for more details on their paper. I nominated you

What role could amino acid supplements (in the medium) play in helping the little fellas: (snip)

That's the one. Can you keep it to answers of one syllable or less for those up the back whose chemistry hasn't progressed beyond making instant coffee? Like me?

Goddamn it I wish I had time to get the phlebo web page together, yours is just the sort of occasion it would come in really handy for, as then they'd know what I've tried and what the results were.

Currently trialling 50mg/L l-tryptophan to a modified B5 media with low chlorine and phosphorous and some silica- won't have results in for two or three weeks

Planning to add a couple of other amino acids to the modified media ( both full and 1/2 str versions )if l-trypto produces no discernable response- asparagine, arginine, can't remember the other one and can't find the bloody reference. If they have any other suggestions, I'm happy to hear them

Also going to send any discarded plant material left over from each unsuccessful TC run away for elemental analysis, separated by batch of course, to the mob who did the soil analysis. Maybe that will identify some kinda trend...

I've been wondering about (and i'm sure others have) the symbiont issues.

I kinda stopped, the obvious mechanical challenge of microbial co-cultivation aside. When I picked up the soil sample, I asked the nice bloke who runs the environmental analysislab whether any symbiont would come into play. He looked at the soil profile and shook his head a lot.

Still, given the high & low levels of odd stuff in the soil, if there is a relationship, the cultivation of the responsible organism as a crop innoculant could have serious benefits for agriculture ( and if any of youse get out there and make a zillion bucks from this idea you owe me at *least* a dinner, OK? And a pat on the head. And a hefty donation to a worthwhile community conservation project such as Landcare. OK? )

molecules, there may signalling/elicitor molecules of symbiont origin normally needed to induce proper regulation of plant genes that are required for the metabolism of the incoming goodies by the plant.

I almost understood that :)But I have neither the facilities or the knowledge base to consider it as an experiment for here. Someone else might though.

Maybe some chitin or something?

I'll squash some cockroaches into the next batch, promise

Theo I'm still chomping through those URLs, I almost understand them too :) Taa again

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I'm not sure how one could draw any conclusions about the role of a symbiont from an elemental analysis of a soil sample. Will think about this some more.

I can speak with the fella next week.

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Thanks fractal, I appreciate that :)

Ah yeah, the other two amino acids I was thinking of adding ( found my notes ) were l-alanine and glycine. On the spurious grounds that they're the building blocks for all other amino acids, and since plants want to live, most of the time all you have to do is give them a hint of what they like.

You should see some of the bizarre justifications I've givn myself for picking and choosing TC formula, I suspect I'm not the only researcher who operates 50% by vibe tho -sometimes it even works

Sheesh I gotta keep outta the grow room, I'm positively pouring over my babies. One of the real dangers is I'll handle them so often I'll drop the little darlings or knock them over and nullify the whole experiment. Trouble is its also the only cool room in the house and is otherwise irresistable

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Blind swag time, probably not helpful- but...

I've never heard of brassinosteroids being mentioned in relation to A. phlebophylla- maby 1 ppm of a cheap brassinolide alternative would help :confused:

Some compounds fortify plant defences- like 10^-4 M salicylic acid in the growth media, or a few ppm methyl salicylate in the air. Maby in the field a decomposing leaf gives it salicylic acid or a nearby flower gives off methyl salicylate- telling the phleb to boost its defences.

 

quote:


It doesn't seem very likely that a source of sufficient tryptophan would be present in the natural environment unless it it couples with a symbiont immediately on germination.

BUT- In the field the babies are near the mother plant, right? The mother sheds its leaves and with it DMT- bacteria, fungi, insects, animals, etc. convert DMT to IAA.

Same thing.

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Quite good considering I am working blind... :cool:

If the plant is lacking glycine and alanine it is going to have a hard life, if it gets that far. Isn't basic amino acid production in the leaf with modifications (especially to the non-protein ones like pipecolic and djenkolic acids) perhaps in other places like the root? Can't expect vegemite to work on this one.

Zero vanadium in your culture media, I presume?

"Auxin conjugates with aspartic acid have been discovered in rhizobia and may participate in nodulation" http://www.ppws.vt.edu/~bacteria/WILT&TUM99.pdf

Sorry DL but that chemical will probably cost a million per milligram unless you make it yourself. It is easy to purify chitin, though.

Would IAA really hang around after being produced by the bacteria, Auxin? Endophytic bacteria use IAA to control their plant host. However, if there were no benefit to the bacteria I am sure it would be metabolised to a range of other things.

Fractalhead, the question is:

What do the roots supply to the growing plant that Darklight hasn't?

For this we need to know as many analyses of root xylem contents of Acacias of this section of the genus as possible. This species would be nice but unlikely.

Oh, and what do the plants smell like?

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theobromos:

Would IAA really hang around after being produced by the bacteria, Auxin?

IAA doesnt last long outside the lab, but it could last long enough... like I said, just makin blind swags

-Auxin

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Well I know its been a while, but a few weeks ago I went to go and talk to Prof Pate only to find out he has retired. I might be able to talk to some other TC gurus around the place for advice, but as for Acacia root xylum analysis data, we might be stuck with what's in the literature.

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Thanks for trying tho fractal.

No discernable improvement in morphology or growth rate as a result of adding l-tryptophan- if anything its negligible and could be attributed to randomness or small sample size.

The growth rate is so slow atm ( for which I suspect a lowering of 4C in the lab environment is at least a contributing factor but so may increased substrate rigidity due to the addition of silica in the media- we'll test those next ) that it is likewise impossible to tell if we're getting a repitition of the previous problem- a decrease in leaf size over subsequent subcultures.

I'll keep going on all current formula with the present ones and add some new tricks to the next germination media to see if anything improves

Coculturning with the contaminant outlined in another thread is also underway- again, no results yet, could be another month

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Hey DL,

sorry this isn't on topic, but...

whats the seed stock situation of A. phlebophylla like?

Just curious, because a few months back, i came across a seed company in france (i think) which had phleb. on it's list. I forgot to ask them if they do actually have any(i will). But if they do, it's probably worth trying to get alot of it back in oz for better chances of regeneration, or at least more research into it. What do you think?

here is the page, waiting on email now.

http://www.b-and-t-world-seeds.com/39098.htm

[ 20. March 2004, 12:39: Message edited by: gerbil ]

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re: folias' link- so hydride ion is good for you now because it's an "antioxidant"! I must get some LiAlH or HI/P and flog it as a health supplement.

H- + H20 ----> OH- + H2+ heat ---> a long and happy life?

It's a con I'm afraid Julian.

[ 22. March 2004, 14:23: Message edited by: Tryptameanie ]

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I've been giving people the Silica Hydride capsules for months now and everyone gives them huge big thumbs up!

I love them...I have found nothing more powerful to wake me up, feel refreshed and uplifted...if its all psychosomatic/placebo, long live the placebo! And I wish I'd found a better placebo alternative earlier ;-)

Look at the site, I think there's more to it than meets the eyes. Patrick Flanagan is a champ...the neurophone is an incredible invention, I've never used one, but have friends who have owned them...you can listen to music through your nervous system...it took the dude decades to even get a patent for it, because they couldn't believe it was real!

a big btw, the phlebophyllas are coming up in droves!

I will have photos up in a few weeks when I have made a large report on many different species I am testing.

Julian.

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I know this might sound dumb, and Im no plant tech guy myself, definitely not my area of specialty, but I had a "dream" that the reason these phlebs were not doing so well was because the CO2 levels were too LOW.

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inpsyght:

heard today about a melbourne guy who is supposed to be the bee's knees when it comes to tissue culturing obscure and rare plants.

I've heard of Mike too, and his reputation is excellent :)

perhaps good for some advice or even better to take it on as well.

We're still running experiments as devised by jasper and myself, and discussions with other TC ppl have indicated we're running the sort of stuff they'd try themselves anyhow.

Once I've collated all the data on *all* experiments we've run and put it on the website ( hmmm... now to find time to build it... ) it will be easier to invite comment from colleagues- at the moment it would take literally hours to explain what has happened so far.

At the moment the project is still without funding, and runs in spare moments in the TC lab here. If someone else wants to go for funding and set up their own tc project, they are welcome to use our data as a starting point- frankly I view the time wasted applying for public grants as mostly lost productive time. But to pay a commercial lab to take this on as a job would be extremely prohibitive. Like seriously extremely prohibitive: mine is the second lab to have experienced problems with this plant in culture- the first is run by a dear & respected colleague who is my former TC teacher.

We've run about 30 starts and hundreds of subcultures- we've gotten past the chlorosis finally but seem to have encountered a nitrogen problem which we're working on. L-tryptophan didn't solve it so we're looking further back down that pathway to l-glutamine, and to activated charcoal to adsorb anything the plant doesn't like. Oh, and some cold weather experiments. That's enough variables to keep us in dirty test tubes for a year or so

Fingers crossed :-)

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