Acacia phlebophylla TC weirdness

[Darklight]

Not sure what to make of this, so I'll just go BLAH, make a few observations and maybe see what everyone else thinks.

24 Acacia phlebophylla seed excised at various stages of germination and placed in one of three media- all based on modified, low chlorine & phosphorous Gamnborg's B5- one full strength, one half strength, and one full strength with charcoal. 100mg/L l-glutamine added to each media to try to get more auxins happening- for increased leaf size over subcultures. No hormones added.

OK so far we got the uniform nice light green healthy colour you'd aim for in TC. If we can keep that going over several subcultures I'd be a happy camper indeed.

Remember the closer something is to embryonic stage, the more responsive it is to variations in media- and all these plantlets are one subculture away from being a germinated seed. Some of them ( evenly distributed over each media ) had in fact just emerged and had remnant dicots, but no root tissue, still attached. It's hard to say which ones, cos now they all have a bit of callus around the base. Which previously has not happened in abundance and when it has happened usually indicates that those plants are going to be around to subculture off a bit longer

Several of them- in fact most- are achieving some kinda lengthening, which has not happened in such numbers before. 25% of samples, most in the 1/2str media, have developed extensive and really healthy root systems over the last few days. So healthy that if I'd done this on purpose I'd be pretty happy. Likewise this has never happened before- at all- even when I'd used auxins in much earlier experiments I'd never managed to initiate root formation.

So I'm kinda thinking that l-glutamine has been transformed down the pathways to some kinda auxin, which was the whole point. And wondering what to do next ( it's always stage fright when dealing with rare and small numbers of plants in experiments )...

Growth is still abysmally slow. Like twice or three times as slow as you'd hope for in a commercial situation. So something's still not right...

If I run another set of experiments- one without hormones and one with a cytokinin in it to initiate axillary formation ( which is the growth we want to get numbers up ) it means I am running six experiments of only four plants each- even less a statistically viable sample size IMO. And a pain in the arse to monitor.

Theoretically I could keep going- try to use the formula as though I am taking cuttings. It would be good to see if we're still getting the maintenance in leaf size as that would indicate success.

I still have experiments to run with the basic media on some emergent seeds- humic acid, casein hydrolysate for starters... I don't want to get too confused by running too many variables at once. Should I try next for uniform leaf size over several subcultures? Axillary bud replication? Constant root regeneration from each cutting? Faster growth?

Gawd I think I've just talked myself out of it. I might save the cytokinin experiments for later. Blah blah blah... midnight lab notes are always so vague but are good to get back to later and drag diamonds from the dross..If anyone has any ideas based on the above data, lemme know here?

[ 11. June 2004, 00:31: Message edited by: Darklight ]

[Stonehenge]

Very interesting. I hope you come up with some tecs for this plant. Most likely, other acacias would work well with whatever procedure worked for this one.

How much light are you using? In the batches with root formation, are there distinct individual plants or is it roots coming out of a mass? Do you find that sprouting seeds works better than leaf or stem tissue?

Stoney

[Darklight]

Stonehenge:

Most likely, other acacias would work well with whatever procedure worked for this one.

Not in my experience. I worked successfully with 4 species, two of which had no protocol, before starting on phlebs

How much light are you using?

Coolwhite fluros in banks of 2 x 56W, tubes changed every 3 months

In the batches with root formation, are there distinct individual plants or is it roots coming out of a mass?

As each of these is the first subculture from a germinating seed and most growth is upward rather than basal, they are still distinct individuals

Do you find that sprouting seeds works better than leaf or stem tissue?

In cases where you want to preserve the species rather than a desirable individual strain, seed is preferable IMO. Easier to disinfest and as I said above, more plastic and ready to respond to variations in media. And you don't have whole plants sitting round being prepped in a possibly unsuitable environment for them, waiting for more ax buds to become available.

Mature material is often hell to prep, with much higher contamination rates from the field. And often you have to bombard it with just that little bit more of everything. Mature material was a definite failure from the phlebs- which doesn't mean we can't try again once we have a protocol from seed which will give us a starting point.

Mature material is best when you have a protocol someone else has gone to all the trouble of inventing, and you want to preserve some particular characteristic posessed by very few within a population

You always ask good questions eh stoney

[Jojobafruit]

Yes this is very interesting. THough for someone who cant acquire seeds to try the tc method with leaf tissue would have to suffice. maybe slices of bark material. ? down to the xylem layer?

I would love to work with some acacia's in tc. I do have lots of different acacia seeds. sadly no

phlebophylla. I think ill start with maidenii. I took your advice darklight and im going to work with other More abundant plants for now. till i get the hang of it, then move onto the more scarce plants.

Do you think this could be done with protoplasts with enzymatic treatment ? I was also reading about the "Ti" "plasmid" vector, but i cant seem to find much info on the bacterium known as Agrobacterium tumefaciens that is used to infect the plant. Have you done anything like this?

Seems a little advanced but its nice to learn lots of techniques used for cloning..

Peace. jojoba

[Darklight]

Jojobafruit:

THough for someone who cant acquire seeds to try the tc method with leaf tissue would have to suffice. maybe slices of bark material. ? down to the xylem layer?

Theoretically replication into a whole organism from any single cell is possible- some cell types are more difficult than others and a few damn near impossible. But *theoretically* any cell can be regenerated to its entire organism cos it carries all the genetic information.

Bark material would additionally be very difficult to disinfest from contamination, and would probably require regeneration via callus- to put it simply, you initiate a particular type of undifferentiated cell clump, then get that cell clump to form embryo-type tissue in a different formula, then turn those embryo style cells into whole plants in a third media. Protocols are available for some species via callus regeneration/ somatic embryogenesis but are unlikely in your Acacia example to be available because of the problems of contamination from bark tissue and the relative ease of other methods

Leaf tissue works in some instances, but I've never seen one done for acacias. What you will probably find works if you can't get seed- and you will still have high rates of contamination, but don't let that stop you- is axillary bud proliferation. It's the most common, and usually the default type, of commercial TC propagation. Just take the growing tips as you would for a normal cutting, pretreat and sterilise.

I think ill start with maidenii.

Good choice- seed is plentiful and there are heaps of plants to take your tc clones from. I started with maidenii and did pretty well out of it results wise.

Funnily enough I tried again last year and the results weren't as good- this time round I used filtered tank water instead of normal tank water, and I may retry with unfiltered water ( lots more variables to choose from so I don't use it anymore )- which goes to show you how sensitive variable control in experimental TC processes really is

Do you think this could be done with protoplasts with enzymatic treatment ?

Might be going overboard for a beginner and you could achieve far more once protoplasts were isolated and combined, then regenerated than simple replication culture Ploidy, chimeras and hybrids for a start

I was also reading about the "Ti" "plasmid" vector, but i cant seem to find much info on the bacterium known as Agrobacterium tumefaciens that is used to infect the plant. Have you done anything like this?

That sounds like GM stuff, AFAIK plasmid insertion via agrobacterium is used to introduce new genetic material into sterile callus cultures. We don't do that here and I have no experience with the procedure. It goes way beyond simple axillary proliferation.

Check yer PMs jojobafruit, I'll have some info on basic teks over to you soon.

[Darklight]

Darklight:

I still have experiments to run with the basic media on some emergent seeds- humic acid, casein hydrolysate for starters...

Casein doesn't work- with or without the charcoal. Similar effects to previous media- leaf shrinkage over several subcultures etc.

Glutamine works and works better WITH charcoal- it's way back down the IAA pathway ( which I think is the same as the DMT pathway? )

And yes, I had tried both IAA and IBA in media with no success

Now all I need is to keep the axillary buds forming multiples at the base of each explant, rather than relying on nodal sections- without loss in leaf size or ability to re-form roots down the track...

...cross my fingers....

...and write the bloody thing up from scratch if it works I think I'll let someone else work out the cultivation problems.

The reddening at the margins of some older leaves still occurs, though it doesn't much seem to indicate any loss of growth potential anymore. Doesn't happen in all plantlets either. Wish I knew what it was, it might be the final fix

[Darklight]

And at least in TC, cold doesn't help. We put 25 plantlets under coolwhite on a 16/8 cycle at 4C for six weeks- and they stopped being happy. Most died when we placed them back at 25C

[Ed Dunkel]

Just a side topic:

Is it possible to graft A. phlebophylla onto a range of acacia root stocks as a way of propagating the plant for the masses (i.e. A. alpina)

I have no idea if acacias even graft well but maybe this could be a way of getting plants out to the massses.

[Darklight]

Ed Dunkel:

Is it possible to graft A. phlebophylla onto a range of acacia root stocks as a way of propagating the plant for the masses (i.e. A. alpina )

I understand this was unsuccessful

[Rev]

If that was the attempts at Wandjina then the results would be less than conclusive

Im sure the time taken to transport and also the new climate may have influenced success in ways that could be avoided if done again

So if you are serious about it dont lose hope

[Darklight]

Here is my latest stupid theory: if anyone wants to propose it as a major study topic you can pat me on the head OK?

Actually if this is someone else's theory it wouldn't surprise me... metabolic pathways ain't my field of expertise. And to prove that I just spent an hour looking for metabolic pathways for the auxin NAA ( which is synthetic, and explains why I couldn't find it anywhere in my metabolic pathways book )

Plants sometimes form secondary compounds as a byproduct or intermediary step in the process of root formation. Seems to work for phlebs anyhow- about 80% of those ( including some near death from prev experiments ) in the glutamine media have formed roots and I'm inclined to wait another two weeks before culturing them again to see if we have 100% and subsequent good leaf sizing for all individuals.

I have yet to confirm this in phleb leaf tissue, and for other species with different types of compounds. And I am totally not a chemist, so any chem boffos out there are welcome to comment. More when I get more results.