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The Corroboree

Darklight

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Posts posted by Darklight


  1. One question I have which I did not put very well earlier is this:

    Say I have induced shoot proliferation at the axial buds at an increased level with a hormone.

    Now, will I get a finished plant quicker, if I let these shoots grow longer attached to the main stem, with the rest of the shoots and the top

    and then separate them later, allow them to recover, grow and root.....

    Or, would I get them further along/faster by separating these newly proliferated shoots from the main plant when they have a few nodes,

    so that they grow into a separate plant on their own, starting as soon as possible, like when they have three nodes perhaps??

    I understand there is a recovery time after they are divided, but they would be divided eventually anyway...

    do I produce plants faster, by leaving the new branching out plants on the main stem,

    or, dividing them sooner, allowing them to recover and grow separately?

    Took me a while to work out what you meant, thanks for clarification

    This is a *suck it and see* question, individual to each species, media, growing conditions etc. These are the questions rarely specifically answered in scientific publications and need to be tailored by each lab for their own requirements.

    You could do a side-by-side experiment to see what works for you, but it would only be an important consideration if you had, say, a commercial contract for replication with a definitive and punitive time line that was, say, six- eight months away ( to give you time to run the experiment and change replication protocols )

    One other thing I have been wondering about...

    Is there some way to contaminate plants send out to market, in agar

    so they will be fine if 'deflasked' but cannot be used to produce functionally sterile cultures from/ would be contaminated 'in vitro'?

    Hee hee :) There was a lot of speculation on this in the late 90s- ways to maintain IP, mask TC media so plants were still saleable but the media wasn't suitable for analysis ( if it ever was )- addition of food colouring etc. Not sure where it got to, didn't keep up with it

    Not sure about your functional contam to maintain IP and commercial advantage. Short answer- I wouldn't try it. If there is a fart in an elevator's chance that whatever contam was in there could harm a worker ( spores, people with lower immune systems ) or an ecosystem ( including agriculture ) or a business I wouldn't touch it with a pole with a condom on the end. Nice theory tho

    If you have developed a distinct variety you want to maintain you could patent it and protect that patent by having the plant fully sequenced ( sequencing costs about $1k last I checked but prices dropping all the time ). Patenting is a few $k too, and last I checked a patent needed to be applied for in every country the plant is sold.Then you have to enforce the patent by maintaining intel on whatever it is coming out on the market, a full time job for someone

    I am in favour of patents on plant varieties which are very distinct and have been developed by the patent owner- particularly ornamental species. Some scummy companies were trying it on for older strains of food plants as a kind of astroturfing, which was a bastard act of the lowest kind. Patenting is an ethical issue so YMMV

    Thanks mate for keeping us up to date on your progress, you have raised many many good scientific and ethical questions which are relevant for both beginners and experienced people alike, and you're also helping my professional development as well :D

    I did check the TC email list too, on your advice, and ended up signing on again, the volume there has definitely shrunk since I was a member last time, it's down from about 40 messages a day to 4-5 a week. Most manageable, and the search function on the archives is still pretty OK


  2. Thanks again!

    I will post more/ask more soon, but wanted to reply to a question asked earler.

    Here are some bad photos of the rooting of micropropagated M. Speciosa plants obtained by adding honey and coconut water to the medium.

    Honey was from the grocery store, said to be pure raw honey but who knows?

    Sry for delay, I had to do a bit of background checking to confirm my memory was correct

    How much honey/ how much coconut water did you use per litre of media?

    Raw honey would be interesting, tho I suppose the autoclaving would knock the enzyme activity out as much as it would processed honey.

    All the reading I have done has suggested coconut water has a stronger cytokinin activity than auxin.

    Now the important factor with hormone interactions is not just how much of what hormone in which species, but also the ratio between the hormones you add in combination. So maybe the interplay is important with the coconut water. Or maybe it's some other aspect of coconut water which is beneficial with rooting. About to give it a go in some recalcitrant species, may have more info in a month or so

    Those biological indeterminates can be lovely but so elusive to pin down, but shonman your experience shows that addition may have some benefits for rooting as well

    Inspired me, so I'll try it anyhow :)

    Don't worry about the quality of your pics, they look pretty bloody good to me. Getting publication quality TC pics is super hard, carn, we all know that. Yours are fine!


  3. Mate you totally have the touch when it comes to easy teks and getting good fruit out of this place

    This tek has never worked for me at my place, I have too many fungus gnats, but I'm glad it works for you

    But you put a lot of work into it too and you deserve every success


  4. Goddamn it, I can still see your posts when I log out

    But you are on very, very shaky ground and I suggest you get help soon. Don't look to it from here, here is the place you are being a fuckwit and all you are going to get is laughed at. Whatever trauma your situation- real or imagined- has generated for you, you need to get help. If you are acting out like this IRL you are going to find it hard to get support if you don't make an appointment to see a professional about it before it gets worse

    ReShroomEd is a friend of mine. A very, very dear friend of mine. And he has been significantly unwell for some time, and I miss him like crazy and hope he is getting better or is as well as possible. Whatever it is you have accused him of, given that I don't care about you enough to check, you probably deserved it. ReShroomEd is a very old fashioned gentleman with no time for spruikers, fools, the pissweak or the whiny. If you are still traumatised about what he said to you after all those years you need to get yourself fixed and not look for sympathy among strangers while being a tool

    ReShroomed can be brusque too. But he is also one of the finest, smartest, most honourable and capable and generous of my friends.

    I count the time he took me and a bunch of motorcycling buddies out to the Otways for a few days camping as one of the happiest in my life. It was th first time I had met him IRL after years of correspondence, we hit it off immediately, he was exactly the person he had come across in his correspondence with me and we all had a fantastic time. He had no reason to offer his generous hosting to a stranger and a bunch of her mates, her family and our random Seppo companions who would sink piss and ride like demons, but he thought it would be a good idea, and it so was

    He was a significant contributor to the place back in the early days when it was a researcher's fun park and really helped a lot of people out. I hope he can still muster the energy to lurk. If you can see this, Reshroomed mate, sending you love from my heart

    Ed's hosted a few such camps off his own back in the early days pre-2010. Usually footing the expenses/ providing lifts/ food/ putting up with the occasional fuckwit. you name it.

    And one of the first high yield ID cactus clones in Australia came from him. His clone 'Eileen' is now safely world wide in many, many collections. Often given for free, or postage. If you have an Eileen cactus in your collection, they all originated from his yard

    Kid, I don't care what happens to you. You have no idea how little I care. If anything did happen to you tho, there are prolly a few soft or sweet people who would feel bad, and that's why I'm reminding them to play nice. I don't want them to feel bad. Go get help. Oh, and fuck off outta here like you said you would. You're the hairball in our free flowing sink of iniquity here

    But personally I do not give a fuck what happens to you. Except I hope you move to Northern NSW. I live in Northern NSW.

    ( Edited to say even more nice things about ReShroomEd )

    • Like 10

  5. The difference between fresh Damiana and dried commercial Damiana is like the difference between chamomile you harvest yourself and the chamomile you get in tea bags. The latter tastes like carpet sweepings in comparison to the former. Much of the beauty is lost

    • Like 2

  6. Do caapi take well to pruning, will she grow back bushier and stronger than before?

    Yes they do, and I reckon it will. Put it this way- if it doesn't, I shall be surprised.

    At least she didn't do it when it is any cooler here. Now is about as late as you can do it to really get Spring growth happening

    • Like 5

  7. Gelrite is cheap as! Its clear and soft and you can see infection with ease

    It also is .3% not 3% so you need very little

    http://www.plantculture.com.au/GELRITE_50_Grams/p3017392_13618904.aspx Only $20 for 50grams enough for 150 odd liters of media

    Lol gelrite works out at 4x cost the amount for agar. I guess that's a matter of scale. I don't like it's capacity to cause hyperhydricity, but it does have it's uses

    Just a quick note- this is *not* a criticism, but where you are typing in decimal amounts for scientific ( or any other ) publication it greatly enhances clarity and translation if you put a 0 in front of the decimal point.

    0.3% is clearer visually than .3%, and while they mean the same things, the ease of visual identification the former gives is polite and makes for fewer 3am critical errors- critical where ingredients are toxic when they are increased by that order of magnitude

    Am mentioning this because it caught me out once too. I used to think it was trivial. Then a colleague ( a PhD, who should have known better- they ram that ritual into your head early in most science degrees ) scribbled me a media list on a post-it. The decimal point required for one critical ingredient was not visible ( it was listed as 1% ). I did question the note but she said it would be OK. I ended up killing a batch of rare explants

    Am a convert to scientific notation. It's a good habit

    • Like 1

  8. also, speaking of weird ingredients and wondering about just how they were obtained, thought up etc,,,

    .....has anyone tried female stuff, like salmon eggs in the medium, 'Juice' or estrogen, etc?

    Nup. That's not to say it doesn't happen, or I haven't thought the question through enough to link it to some of the commonly used additives. And don't forget, commercial micropropagation people rarely publish their unique findings- so it's entirely possible

    Closest would be coconut water, the liquid endosperm of coconut seed

    Ha! Knew there would be something on a rethink. Google Foetal Calf Serum and Foetal Horse serum ( hint- it's ugly ). Not used in plant TC too often, but used in TC regularly. There will be others

    Edit: Also add: banana powder, tomato powder, V8 juice. Fruity goodness.

    • Like 1

  9. Has anyone propagated Mitragyna Speciosa from callus?

    I also found some info saying that tryptophan as a precursor, added to the plant, increases alkaloid content...

    in this case it was a cell suspension culture, and showed increased alkaloid content after six days

    Nup, never did it back when it was legal and I don't know of anyone who has tried it since

    Ax node proliferation was sufficient for the purpose of replicating the numbers of whole sale plants I needed- mucking round finding a suitable media ( there were no other protocols around back then that I could find ) would have been a waste of effort unless there had been an urgent need for more plants

    Plus suspension culture, like any liquid culture, is very prone to contamination and can be hard to keep clean, especially for large volume systems where you need to add sterile air

    • Like 1

  10. Has there been success with TC/MP with genetics that fruit with age rather than being photoperiodic? Is it possible even, or do the clones carry the same hormone that induces fruiting with age?

    Or can it be successfully done taking clones while the plant is still in its juvenile growth stage and propagated without the plants fruiting in vitro?

    It took me a while to work out what you were talking about sry

    Bamboo would be a prime example, I have a few colleagues who have worked on it extensively in TC, one said something about flowering at some point but I wasn't paying attention. I'll ask her again next time she is in touch

    ( Non-photoperiod sensitive pot would be another candidate, but there isn't the same amount of data on it/ I don't know if the same mechanism is involved re. flowering )

    • Like 2

  11. Thanks!

    Dang it, did my Mitragyna Speciosa thread disappear from the forum, or is it just buried?

    Is buried, there are like 3 or 4 good micropropagation threads on here now

    What is it with sperm and micropropagation? Adding Endosprem, herring sperm.....who thinks up this stuff?

    "I know. lets add some HERRING SPERM to the medium, that ought to do it !!"

    Next thing you know, I might as well experiment with ancient witches formulas, you know, eye of newt, toad, etc

    maybe a few bats boiled in cows milk at the full moon, then bury the medium in a graveyard at Midnight for one lunar cycle, and stuff like that.

    lol yeah I know, I've often wondered stuff like that myself. Some scientific publications read like film scripts once you get used to the flow ( all it takes is practice and experience, you do not need a tertiary education to get to that point )

    I remember seeing warfarin added to one media fairly early on, and vegemite to another, and thinking ' poor bastards got desperate, threw in their lunch or the lab rat bait or what was in front of them and hey it worked '- but there is actually some good science behind the reasoning for it. Once something is published that works, no matter how weird, people start trying it themselves and the repetition finesses the known effects of that ingredient and it enters general use.

    But who in their right mind was the first person who thought ... yeah, salmon sperm, we need salmon sperm. We have salmon, let's go and wank one until it comes and store it in a jar in the fridge '

    Still, formal science publishing etiquette means they take out the bits where you can hear everyone screaming in rage and frustration and fear. Only sometimes will the echoes remain...

    Will love to hear of the honey experiments. Was it raw honey or commercial? Honey would certainly be a biological indeterminate, so variable

    • Like 1

  12. ...and coconut milk (from a live green coconut is said to be best)

    Hehe, it is so so easy, given the infinite numbers of possible media formulations and hormone combinations, to forget that sometimes simple changes offer equally good chances of success

    I'd forgotten about coconut water as a possible hormone substitute/ elicitor for rooting, but I have some and I'm going to throw it into my next batch of media for trial rooting mix

    Ta for reminder mate

    • Like 1

  13. You really know your stuff!

    Please elaborate on gelrite?

    I wouldn't bother with gelrite for micropropagation unless you have very particular research needs. It's expensive, not really necessary for most commercial replication, and prone to causing hyperhydricity in many species ( cells swell, appear almost transluscent, and replication becomes unreliable )

    If you required absolute control over a media ( gelrite is more chemically consistent between batches, wheras agar is almost a biological indeterminate between agar batches of different origin ) then yeah, go the gelrite if you have a bulletproof protocol and enough material to keep back so hyperhydricity isn't an issue, then yeah, gelrite.

    Dammitt, I broke my Hannah instruments PH electrode taking it back and forth between hot and cold fluid, wont do that again!!

    I didn't know about that but in hindsight it's logical- ta for the warning!

    Do you think a plant like the coffee tree, or M. Speciosa, where allowed, could be poropagated from leaf/petiole?

    Theoretically yes, but I've never tried it

    ...sometimes I get overwhelmed, and leave cultures in medium too long.

    Me too. Just make a note of it. And keep enough material back so you don't lose the lot and can start again if needed

    1) How long can these plants stay in medium, before they will stop growing and or suffer in quality in some way?

    Until they do. There are few hard and fast rules. Most advice is informal from other TC people. I ran generations of Mitragyna til they ran out after about 5 years. Another media formulation could have made them last longer, or a different light and temp regime, or a different starting material. Running regen from generations of callus could have reduced the time or introduced variation and unreliability too

    2) How long does medium made in advance, keep in the refridgerator?

    The SDICN medium, says it is good for six weeks storage, and then use.

    If you've sterilised media in the fridge, the other problem is going to be risking contamination via temp changes, especially if your lids and containers are made from different material and you open the fridge door a lot. Domestic fridges don't have the same temp control as lab grade ones too. My advice, don't store autoclaved media in the fridge and expect it to be reliable

    Hormones in media, autoclaved or not, are another storage issue. Some like IAA degrade quickly. Others not so much. IDK the death curve, I have a mate with chemical knowledge I ask about this stuff when in doubt, and try not to store made up media too long at all

    3) also, if there is a phase where the plant adjusts itself after being divided,

    am I further ahead to let it grow while connected, then divide, or just divide right away?

    Eh? I don't understand

    4) what is the best way to make these grow quickly after deflasking?

    If they are fully acclimatised post deflask, then you need to talk to a nursery person. Sorry, no idea

    5) what are some profitable plants to produce via micropropagation?

    You keep asking this, you've already got a bunch of species you're working on which look good :D Go and talk to some local specialist nurseries to you.

    I would also add, that combining honey as a sugar source, and coconut milk (from a live green coconut is said to be best)

    into the medium seem to increase growth and rooting a bit

    I did not know that- thank you! Got any data to share for it? Have added coconut water occasionally, but not as a regular ingredient. Never tried honey, have some raw. How much do you add?

    • Like 1

  14. I have been experimenting with micropropagation for a while now...it is quite interesting!!

    Much thanks again to those who have lit the path before me, helping guide my steps...

    (...Darklight, Carol @ Kitchen Culture Kits, Shaman Australis Forum, and so, Torstein & friends...and the book, 'Plants from Test Tubes.)

    Dividing and multiplying the plants is very effective.

    They seem to put out roots, in some cases, in 1`.2 strength medium...oir, medium after the nutrients run out.

    They can be slowed down bv temperature, I have one batch that has not been transferred for five month!! (at 60F)

    Now, I am thinking about growing plants to a certain size, so they can be deflasked by others.

    The bigger the better up to a certain point.

    What might be the best way to do this part quickly?

    How big, is big enough?

    Here is what I am thinking:

    Divide explants until a certain number is reached.

    Root.,...in agar...

    Then, transfer to final sales container (any suggestions on these containers would be helpful).

    The medium in this final container in which the plant will be sold, would be full strength.

    They would have roots when placed in there.

    I would keep temperatures maybe at 70-75, and increase light.

    Theory being, that the plant would grow faster with light and full strength nutrients.

    Any thoughts on this are appreciated!!

    Thanks!

    Bigger is not necessarily better with direct sales TC plants.

    What you want is a saleable plant which will survive deflasking. A plant with a large surface area is going to be at a disadvantage after a certain size, because it will need to feed so much more of itself and learn to survive contam risks.It is also more likely to damage during shipping

    Other points against raising larger plants in TC include- time on the lab shelf which could be occupied by faster growing products, giving you more sales per square foot of grow shelf space. If you leave plants for six months prior to sale and they are six inches tall, you are probably not going to recover that against sale price when compared to the fact that the same space could be used to produce six smallersale plants every two months

    Finally, there is a tradeoff between plant size, and the increased risk of contamination when transferring it. More surface area on the plant, more potential for contamination to fall on it, or it to be injured during handling, and longer times in containers which may not be 100% contam proof

    I'm a fan on 30ml tubes for individual plant sales. Max 120ml. Polycarbonate, for ease and cost of shipping.

    Ta on yr update about cooling plant growth to 60F to maintain longer shelf life. I've tried it in a few species too

    • Like 1

  15. I would be very interested in 'at home' tissue culture but I don't know where to begin.

    Do a search here for starters. Heaps of information, especially on threads Shonman has contributed to

    There are also home TC links listed on here which use readily available ingredients

    Would there be a database of what hormone/media works best for specific plants/genus? I cant find anything substantial. Or is it basically trial and error? and years of study:)

    There's a TC wiki, but it is small and not well populated.

    There is no database, sorry. Yes it's years of trial and error

    Try checking scientific publications on Google first by species name, then genus for closely related species

    • Like 1

  16. Ta for replies everyone. It's going to be a process.

    I just got a coupla quotes from ppl to remove a very large Euc that is going to continue to block out most of the solar access year round for at least another 100 years ( planted pre-internet on the advice of a mate who planted 2 and had them die in 10 years, I thought this one would too )

    Quote is gunna be about 2k minimum, so... um... by the time that happens technology may have advanced significantly.

    Tradeoff could be tho if I spend 2k removing tree, I will save 2k on power bills. Leave it with me a bit :)


    Some concluding advice - look very closely at your power usage (in Kwh/day) - see whether you can shift your energy intensive usage to the daytime when you are generating maximum output this is likely to make the investment in solar an economically sound one.... Whittle down your consumption as much as you can (e.g. energy efficient fridge, solar hotwater, LED lights)... and look toward lithium batteries in a few years, this way you escape being gouged for the goldplating charges of the massive ($37 billion) investment in poles and wires across NSW (and seemingly other eastern states)...

    Fucking gold plating bastards. My aircon for the lab was approved by energy provider and overloaded the transformer to the point that living here was like working in a strobe light factory. Power fluctuated between 90-240v by the millisecond and chewed out a lot of circuit boards. Then they stuck another 2 houses on the transformer. Def didn't get my money's worth on that deal, my power prices rose while my gear burned out

    If I reinstalled a more efficient lab aircon system it would run 24hr/ day but that would mean I could offset autoclave and flow hood use to middle of the day. Unsure how that would pan out over time re. billing

    A mate told me to get in the roof and install some led wiring and drop a few in the roof of each room of the house, then hijack the original power points with the wiring to turn them on and off. You'd have a little spot in the linen cupboard to swap over the cheap twelve volt batteries that you'd charge on good days for free from your solar panels.

    If I did this if want a few 12v power points etc to charge mobiles and laptops at night too.

    Yup. Solar charged LED lighting for regular power outages we get here is a priority

    Your last statement says it all. If you want to be able to divorce your self from the grid, you have to purchase a system outright & forget about leasing one from GEM Energy Australia.

    The website is unclear whether it is a lease/ buy arrangement or a straight out loan. I haven't followed it up due to large tree described at post beginning.

    • Like 1

  17. Flowering like a bastard at my place this year in nnsw. I haven't seen it flower like this since 2007

    How many seed I get remains to be seen

    Not sure if it is because of a seasonal confluence, or the fact that I started fertilising it regularly with a foliar spray about eight months ago

    • Like 1

  18. Can anyone recommend a good, reliable, quality supplier of fruit trees in Darwin?

    I get Bunnings, with limited stock and range, and a couple of places which sell arids, and a bunch of empty links. Have contacted one or two places to see if I can find a pdf catalogue specifically for fruit

    Interested to hear of other's experiences buying fruit trees up there


  19. bro, sorry. I just checked the shed, and ants have been into the seed and they don't look viable. I planted about 6 weeks ago, and gave the extra seedlings away yesterday... I'll have a look locally for you and get back to you. But if there's anyone else out there with seed....

    Hey it happens, don't worry about it, I appreciate the trouble you have already gone to.

    Would love it if you find more fresh seed tho :)

    Anyone else? I seem to have just missed the season

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