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The Corroboree

Darklight

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Posts posted by Darklight


  1. Erg, sorry mate the previous sounds all rushed and condescending, didn't mean to talk down to you

     

    You know a whole lot more about the wider aspects of this field than I


  2. They look morelly and I haven't found anything local and non-morelly they could be confused with, but that wouldn't be enough for me:

     

    Atlas of Living Australia has a bunch of Morchella elata entries and they look similar enough and the northernmost range is currently described as Brisbane:

    Morchella elata at ALA

     

    Maybe add an ALA entry? If you send off the pic to Fungimap they will automatically include them in the ALA database, check the Fungimap page for submission guidelines.

     

    Most importantly- were they tasty?

     

    Please pass on my thanks  to the fairies for their eagle eyes and contribution to local knowledge- and kudos to you for keeping and documenting them.


  3. the feedback is fully appreciated and , actually needed, once in a while, thank you... 

     

    Not surprised, you have done some gorgeous work in this and I can imagine it would be hard to keep up the momentum sometimes

     

    sometimes I am wonder why the heck I keep posting in these threads

     

    This thread and the mangrove log are some of my favourites on the entire site. Threads like these keep all the valuable info in one place and follow the progress of your species and work over years. One of the important things ( at least for me ) is that I can see actual results, in succession, over time- there are lots of forums and posted information online but most of the time it is difficult to tell when a contributor's post is just an idea they had, whether they tried it, if it worked and how the processes/ experiments were adapted

     

    It also ( again, it could just be IMO ) marks you out as someone who is dedicated and thorough, not just mouthing off random thought bubbles and leaving the room. If I needed any Ephedra info in the future you would be one of the people I would ask first

     

    other times it feels like a calling for a collaborator or more on reviewing the idea of a monograph on ephedra 

     

    Yum! An Ephedra monograph? Bring it on! Sounds like a big task but it would be a lovely thing indeed

     

     

     Its only suitable that two of the oldest ephedra in the world, foeminea which is in fact the oldest existing sp. and fragilis , are climber and semi-climber respectively..  and are native to greece! fuck I didn't even know we have 4 fucking species of ephedra..  and along with a couple other climber species of middle east like aphylla (syn. E. alte) they seem to be by far the fastest growing ephedras. seeds large too..  So they they're not only the only extant group of species which is entomophilous, older, and faster growing, but that also means that the rest of species are all newer to them . ephedra genus shifted its manners from entomophilus to anemophilus in order to survive, which seems like a pretty funky adaptation move in a group of plants of gymnosperms that might have played a role or in appearance of angiosperms, the "newer" plants....  

     

    More lovely things I did not know, thank you

     

     

    • Like 5

  4.  

    Hello, I've been doing a lot of research and would love to get any feedback on my first attempt at a Mitragyna Speciosa tissue culture (which is legal plant/tree in my location). 

     

    I appreciate the time, energy, and resources that the members here have put into their practice, and would love to politely pick their brains; I understand that experience was hard-won, so I don't want to offend and ask for "info-freebies". My first hurdle is that I have limited mother plant material, and would really like to have at least one successful replication before I run out of plant. I've been working on this for a few months and have not had one yet; many were lost to contamination.

    A little about my method:

    -Working under plastic bin hood

    - WPM at label suggested strength

    - SIDNC (Milton Tablets) at 5000ppm for 15 minutes, but I wonder if my tissue samples are too large? Anyone tried 300-400ppm for 24 or so hours?

    -I also mix the SIDNC into the medium at 0.032%, but having a hard time adding it at the right time (ie. low enough temp to keep avail chlorine) without the medium ending up a little too soft. It's almost as if I need to heat/microwave the medium again, but that would destroy the SIDNC protection, right? For medium prep via microwave, pre SIDNC, how long do you let it boil?

    - 10% v/v coconut water

    - 4% Honey

    - 0.5 mg/l TDZ (this strength was at suggestion of fellow practitioner, open to suggestions on strength or using it at all?) Best way to dissolve TDZ powder?

     

    I've heard that petioles are best with this species (can anyone confirm that; goal is micropropagation) (I knocked off a leaf, so I decided to try that also, anyone had success with leaves of this plant?) What's the best way to position the sample on/in the medium? Size and cut of sample?

     

    Has anyone been able to micropropagate this with only two phases of medium (#1 medium=produce shoots AND roots, then #2 medium=hardening), OR does it need three phases of medium (#1=shoots, #2=roots, #3=hardening), OR four phases of medium (#1=callus, #2=shoots, #3=roots, #4=hardening) OR different combo...? Any thoughts on hormone use for these phases, I know 2,4 D is good for this particular plant to produce callus, but callus is not my end goal, Im not sure how to get callus to reorganize to shoots & roots. 

     

    Air supply (hole in lid), I've read both nays and yays as to whether or not it needs it, right now, no holes, too worried about dust mites, etc getting in.

     

    I saw a thread post on here from a few years ago saying there might be an article/paper/protocol put out or published on this by Darklight and a few others, did that ever happen? I searched and searched, but could not find anything; I wish there were a manual for this;)  

     

    After jumping in and doing a few trial runs, I can deeply appreciate how challenging and rewarding this can be. I wholeheartedly appreciate any and all feedback and tips! And I hope I've followed proper protocol for rules and etiquette on this post!  I normally keep to myself, but after lack of success I realized I needed to reach out for help on this one.

     

    I only have pics of my most recent batch. The last one (#5) must be contaminated, can anyone "Name That Contam"?

     

     

    Hey sorry, I only just saw this today. Congratulations on both starting and keeping going! Is hard yakka but I expect you will get it eventually and learn heaps in the meantime

     

    I never ended up publishing my media and methods but several other publications have come out on M. speciosa TC in the last few years- I think I linked to them in the thread where shonman and I talked it over.

     

    First critique ( please don't take this personally, this stuff happens to everyone especially this early in the learning curve )- you're throwing too much Milton at your tender babies. They only need sterilising for a few min, 1 tablet in 100ml water will do it, start with 10 minutes and work more/ less time into it depending on symptoms.

     

    Pretreatment of your parent plant is mission critical- make sure it only gets watered at soil level for a week or so beforehand. Harvest plant parts for TC on a warm sunny morning. This really does make a huge difference

     

    Now I have absolutely no experience with microwave sterilisation so can't speak to that. But next time you do a run keep a jar uninoculated and unopened to make sure it works for you and doesn't contaminate. Don't whack any Milton into it at all. Overkill, and some plants don't like it

     

    Have had no success with leaf culture, which by no means makes it impossible, keep trying.

     

    Ended up after a few generations with a single stage protocol- I could have produced more plants faster with hormones, but the market for them didn't warrant the haste and the added risk of producing off-types over multiple generations

     

    From the pics it looks like you have been overcutting your explants ( I could be wrong )- you do need more than just a stem. Two nodes minimum, and don't cut the petioles all the way back to the stem- leave 4-5mm on them, the extra lengths on them make them useful handles to avoid bruising the stem during transfers. Bah, I misread, sorry. Use the stem section. At least two nodes. If you don't have that spare on your plant, pinch out the main growing tip and make more stems. Always keep at least one active growing stem on the parent plant so you make more parent material if you ever need it

     

    Early stage your main priority is to get it clean, they will live without hormones for a while, and even grow. Once you have a few clean stems you can worry about hormones.

     

    Oi @shonman, how'd your cultured clones go? Any further hints?


  5.  

    If here wasnt the appropriate place to talk about tissue culture, than why continue to talk about it? 

     

    Sorry to be derailing your thread @Kierbob if my posts are annoying you ask a moderator to remove them. This will be my final post in the thread. 

     

     

    Youse are nuts. That was the point of my question. I took your comment and ran a gazillion miles with it and if the OP asks us to move this info to another thread I'm happy to do it.

     

    Looks like a nice flow hood, I'll repeat that for those of you at the back of the class. Nice flow hood, well stored. Those are good units, I have worked with one and was pleasantly surprised.

     

    Yes, it is facile to get callus. Good quality callus is another issue. I'm not calling you out on the quality of your callus, I assumed it was good or you wouldn't have bothered continuing with it

     

    For all I know there are people with much more experience than I on this and many many other topics on the forum and who are keeping quiet. Or laughing at you. Or me. Actually I pretty much assume this is the case :)

     

    You asked for collaboration, you got it. You took the whole thing the wrong way and now you have the bad hurtyfeels. I'm happy to continue collaborating with you but you might want to grow up a bit for groupwork.

     

    Failure is a constant in science. For you. For me. For everyone. Good on you for keeping going. We all do, because that's how science happens. Get a little sooky about it and move on. If something I say upsets or stops you, then you need another job

     

    There are pics of some of my cactus callus tissue in the gallery. From like the early 2000s. Callus quality was shit ( you really do need to stop taking this stuff personally, shit callus happens to all of us, it's a part of the experimental progress ). Regen was good, good enough to continue running that batch while working on other protocols. However deflasking caused the inner parts of the cactus to collapse in many individuals and it took someone else to handle the deflasking for contam reasons. By then the crop wasn't as financially viable as it was at the start. I still have callus stored for that species, and some whole plants in TC. The callus is still shit. It happens. If I wanted embryogenic callus I'd have to work and fail and keep working to get it- just like you do with your species.

     

    Nice flow hood. I hope someone buys it and gets lots of use out of it

    • Like 2

  6. Go to this. Absolutely go to this. Put it on your calenders, wend your way to Melbs and just go

     

    I have loved every single EGA I have been to . It's not just the presentations, it's the networking and the catching up with old friends you get to see all at once

     

    If you've never been and are worried about not knowing anyone yet, start thinking about that now, start a thread here or on FB, and people will often put themselves forward to make sure you don't feel isolated and have an excellent time

     

    Run by lovely, friendly, professional people

    • Like 7

  7. Ah, sweet. I used one of these units a coupla years ago at a mate's and was surprised how effective it was.

     

    Am used to a full flow hood setup, but you probably don't want a full flow hood if you're short on space or moving house ever. They're heavy and take up massive amounts of room

     

    Would recommend the units above, do keep the prefilters clean and do run a test with some open PDA plates when you get it just in case seals shift during transport ( simple to fix )- you need to do this with any flow hood regardless of size

     

    Sounds like OP has looked after it really well

     

     

    I cant teach you what to do with the callus cultures as im still learning, but it would be great to have another member to bounce idea off :)

     

     

    Is here the appropriate place? Do you have callus pics?

     

    Callus induction is pretty facile, it's the quality of callus you're looking for and if you can regenerate it back into whole plants you've finished the cycle and know your experiment is working. Do this first before you proceed further with the experiment

     

    Callus- especially embryonic callus, those tumescent, friable, egg shaped buggers and the subsequent torpedo shaped embryonic calli- can really speed up the replication process. Embryonic tissue is more 'plastic'- responsive to lower amounts of hormones. Callus requires less energy to reproduce itself, not needing to bother with massive cell and organ differentation.

     

    Trick is to regenerate it into whole plants early on in the piece to confirm concept, and not to run so many culture generations it forgets how to regenerate ( unless you're specifically after callus cultures for a specific purpose ).

     

    Many protocols regenerate whole plants from callus by reducing the sugars and the nutrients by half, and sometimes by adding an auxin, like IBA to the regeneration mix. If that doesn't work, there is a bunch of other stuff to try, but the above protocol is the standard to try when regenerating whole plants which lack a published protocol.

     

    Plant callus is also useful for mutation studies- throw enough hormones at it and you can get 'off types' which, if regenerated, can produce useful horticultural mutants. You can put callus under UV or throw actual mutagens at it ( be very careful with those ) to the same effect

     

    If you are using callus for mutation, be aware that while the process itself is a little tricky for noobs, the actual work and time comes from running proper controls and kill curves and maintaining them for the life of the experiment ( minimum a year, sometimes five or six ) monitoring *all* of them until the trait you are seeking is observed in at least your controls ( allow time for the others to catch up, mutations can cause lag in the first generation ). If your mutants propagate by seed only ( esp if you have thrown out all your callus/ sterile explants at that time- do not do this ) you will need to stabilise the mutation over at least one or more generations

     

    The logging and observations take time and space and a high degree of thoroughness. Minimum 100 plants incl controls, but serious statistical chance for success kicks in at about 1000 plants. Numbers will reduce over time as you remove unsuccessful candidates from the treated pool.

     

    The above is physically easy, but taxing long term as your personal situation will likely change over time. This is the bit anyone can do, but usually don't


  8.  

    @Darklight

    I can send you a few seeds when I next have a bunch. I'll message you when they are ready.

     

     

    Oh Rahli thank you, that's a lovely offer, but I can't promise I will do anything with them for a few years as the garden is quite full now and so is my brain.

     

    Sell them off to server costs? Or donate them to a new member? Something something... better ideas welcome

     

    I'd love to have a play with seed eventually, but right now they will sit there and go to waste, which would be a shame

    • Like 1

  9.  

    I agree with Darklight's comment "The exxy bit is getting the sequence data analysed by someone who knows what they're doing."

     

    This is ongoing for Lophophora right now so I can greatly appreciate what that means.


     

     

    OMG omg omg Trucha, and thanks for sharing this. So excited you're doing this from scratch

     

    What trucha is describing folks is the current gold standard for starting from scratch. Whole genome sequencing analysis. Then comes the development of marker sets. Two separate processes. There is a lot of overlap in common parlance, and even some scientists will take the linguistic shortcut.

     

    There is a surprising degree of capriciousness within this picture so a disturbing part of what has been published is really no more solid than a house of cards. The bar really needs to be raised a notch imho.

     

    Yep that blew me out. I was looking at whole genome sequencing of Ganoderma in 2014, talking to a couple of genetics post-docs and aside from the initial expense I was blown out that results can even differ depending on which brand machine you use. There is no one-size-fits-all standard, or wasn't at the time. And the blinkenlights machines are exxy and are superseded every few years. Whole genome sequencing is frequently outsourced even in mid-size facilities where the exxy toys of +5 years ago aren't up for replacement and yet almost obsolete

    • Like 2

  10.  

    A T section cutting is made by cutting a 4cm section of stem, around 2cm either side of a young shoot. The section could be layered using moist coir and course sand and wrapped with a stocking to allow easy watering. I'm not sure how long you would leave the layer cutting before checking if roots have formed. Once the roots have formed the cutting can be removed.

     

    Thank you! I had done a bit of googling and there were a number of teks suggested for the term so I got confused.

     

    Yours is a beautiful, clear answer and I'll be able to follow all that easily

     

    I don't want a million cuttings, just a couple of backups. Seymour is doing splendidly growing in the bathroom but I do like multiple copies just in case something happens to the original

    • Like 1

  11. Sent you the link to the sequencing place Change. It's not a state secret, but dealing with them does require a degree of technical knowledge

     

    CRISPR is for inserting and knocking out genes, not mutating them AFAIK. Happy to be corrected on this. Are you as technically up to speed on all this as you think you are?

     

    Mate, if you are in the Hunter and are playing with CRISPR you prolly want to be working out of a registered PC2 facility. Not sure about knockouts and OGTR, but unless you want to make ugly front page news I'd do a thorough OGTR legislation check and print out and keep the relevant documents next to you laminated in case of incursion.

     

    What you're proposing sounds like a PhD at the very least. More likely six PhDs.

     

    • Like 2

  12. Another example is drought tolerance within a species.
     

    It may be known, for example, that the presence of certain genetic markers in that species has a high correlation with drought tolerance.

     

    You can use those markers to screen a large population of that species, and select individuals to test for drought tolerance to breed from

     

    etc


  13.  

    The exxy bit is getting the sequence data analysed by someone who knows what they're doing. I know a few such people socially and once they started to explain the process to me my head assploded.

     

     

    I was wrong. There are places in .au which will extract your DNA and run all kinds of cool tests for about $100. They can give you results in spreadsheet form which are easily understood by people with minimal experience. They can provide more help for more cost.

     

    The problem is that you need  genetic *markers* for whatever it is you are seeking. These are similar to the controls you run in any experimental setup.

     

    You need specific markers for your specific test. And if these markers don't already exist on some database, you need to develop them, and that's the exxy part.

     

    For example, if you need to determine whether your Acacia is species x or species y, you will need the genetic markers for one or both of those species, so you can tell whether your sample fits the profile. If there are no markers on public record, you will need to track down the part of the genetic code which is consistently different in either species. Apparently this can be a PITA, time consuming and exxy.

     

    However ( a separate example here ) if you need to determine whether your sample is a clone of Acacia x, or a seed grown individual, you will need different markers to those outlined in the example above. Again, if these markers do not exist on public record you will need to develop them.

     

    Plant species such as tobacco, rice, wheat, and that bloody tedious Arabidopsis thaliana have plenty of markers on record, often available to the public. Is less likely that your obscure ethnobotanicals will have such records on them. There are places to check, but no idea where to find them. One place to start looking is formal published genetics studies on your species of interest. Another are the databases.

     

    Got some sequencing done recently for a common species, there were markers on public record for it. All up the bill was cheap, with supplied analytical spreadsheet.

     

     

    • Like 5

  14. Am a huge fan of commercial Trichoderma spp blends as a liquid fertiliser addition to my home garden.

     

    The commercial products are usually sold as dry powders- spore mixes treated for storage.

     

    I have run right out of these and need some soon. I have Trichoderma spp cultures in my library, and I can easily do liquid cultures based on my standard fertiliser blend.

     

    Would the shock of throwing a growing but otherwise sterile culture, even at log phase, into the wild to compete with other organisms potentially any advantage?

     

    What sort of optical density of the LC Trichoderma would I need as a fertiliser base, to add as 10x, 100x or 1000x. Can I wing it or do I need to switch the spectrophotometer on?

     

    Anyone had any experience trying this?


  15. On 06/10/2016 at 11:00 AM, obtuse said:

    Today marks 10 years since i joined SAB, and i just want to say a very big thank you to the SAB community.

     

     

    Love you Ob, know how you feel. Was a gamechanger for me too, and I suspect for a few others as well.

     

    When T first spoke about starting it I gave it six months til SAB was shut down. I love being wrong sometimes :D

     

    As a result of serious drug education and discussion I find I take a startlingly large amount of less actual drugs- like about 99.9%, and have furthered my education and professional development. Back in the early days not a few of us went on to study science or continue other studies inspired by what we learned here. A side effect I had not considered at all. Whoda thunk?

    • Like 4
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