Preparation of Leaf Disks for Callus Induction

[@agnelomascarenhas8990]

Thank you for the video about callus generation. Thank you for taking the time to reply to all the comments.

[@ItsLearnable]

You bet!

[@robertasilveira1600]

Thank you for the video! Really useful!

[@ItsLearnable]

Glad it was helpful!

[@ArtyMars]

Those massive commercial laminar flow hoods are so beautiful they even have a sneeze guard omg 😳 😍

[@ItsLearnable]

Hahaha! The Biosafety Cabinet I'm using is meant more for animal cell culture - it's a bit of an "overkill" for plant tissue culture - but it's what I have available in our teaching lab.

[@ArtyMars]

@@ItsLearnable it’s so cool! Yeah you can get away with less, My DIY hood is a HEPA filter blowing air inside garbage bags sticky taped together and it works 😅

[@HappyDayRoots]

❤

[@matchmedicines8209]

fantastic video, special thanks 🙏

[@ItsLearnable]

Glad you enjoyed it!

[@edmit2001]

Thank you for upload this :-)

[@ItsLearnable]

I'm glad you found it helpful.

[@rabbeyaharoon8118]

So satisfying

[@ItsLearnable]

Glad you enjoyed

[@davidnewton3064]

Absolutely fascinating. Makes me want to quit my career, beg borrow and steal enough money to go back to school, and get whatever job would allow me to do this science all day.

[@ItsLearnable]

Some people seem to make a career out of it at home. I do enjoy some of the videos here: kzbin.info

[@hardyshibiscusheaven977]

Great video thank you, just what I needed. I couldn’t find the next one. Is there one? I am going to try callus on hibiscus, any suggestions?

[@ItsLearnable]

I haven't had time to prepare too many videos for this specific course, but I do plan to add more in the future. I haven't worked with hibiscus, so I can't really speak with any authority on this.

[@ArtyMars]

Oh :O I thought you had to use a node from the stem I didn’t realise you could make Callus from the leaves 🍃 I have so many leaf scraps i can use from my fish tank hahha

[@ItsLearnable]

A node from a stem is a great way to propagate plants and doesn't require the production of a callus. (it already has a meristem with stem cells - no need to try to generate any). Using leaves can be a bit more tricky.

[@Drifting_Leaf]

Thanks for the video. Do they have to be circular, can we cut rectangular pieces with a knife instead. I use a Bunsen burner to sterilise my tools which are limited to forceps and a knife.

[@ItsLearnable]

Yes, definitely. You can use any shape really. I went with disks for the class because I had tools that would do it quickly and easily. When you're teaching a class, you want to use a protocol that will minimize the chances of contaminating things, and thus you go with the fewest steps required. Using a hole-punch just allows my students to make a bunch of small explants quickly and fairly easily. Do keep in mind that you shouldn't make the explants too small - try to aim for ~1cmx1cm.

[@Drifting_Leaf]

@@ItsLearnable Thanks.

[@AquaMayne]

did you use cytokinins and auxins to initiate callus or is the callus just initiated because the leaf portions are damaged?

[@ItsLearnable]

We did have a very small amount of auxins added to the media, but yes the damage to the leaf tissue was the main factor.

[@ArtyMars]

Do you have a video on what auxin to cytokinin ratio is best for callus formation? I think I read BAP 0.5mg/L + IBA 1mg/L for banana plants in a pdf some time ago

[@ItsLearnable]

Sorry no. In my experience, it can be a bit variable depending on the plant you're using. In general, it tends to be even, or almost even. In our case, we had some success with just 1mg/L NAA

[@ArtyMars]

@@ItsLearnable fascinating thanks, yeah I’ve noticed it’s almost even ratios in most plants, whereas the multiplication and transfer media is like 10 to 1 BAP to IBA 😌🌱

[@NagashiChidorii]

First of all, Thank you for sharing your knowledge and techniques! What is being sprayed in the spray bottle for disinfecting? Based on context from your other comment replies, I am guessing it must be ethanol? Why use ethanol instead of 70% iso alcohol or some bleach solution? Is it pure ethanol? Thank you!

[@maxmitchell70]

Pretty sure its 70% alcohol mixed from either pure, methylated or isopropyl. 70% percent has been shown to be more a effective sterilizing agent than pure.

[@jinty1232]

70% ethanol is used for spraying the items going into a sterile hood.

[@ItsLearnable]

it's 70% ethanol (as the others have already metioned) Here is a video covering this: kzbin.info/www/bejne/qnWun62GbcSso6c

[@rigobertorincon4578]

May sound a dumb question but... How deep should I press down disks on medium? the outline of leaves should be all cover?

[@ItsLearnable]

It's a bit hard to describe. You're not trying to "dig the disks into" the media, so don't press too hard. You just want to make sure that as much of the cut surface as possible is in contact with the hormones and nutrients in the media. This will maximize the likelihood that at least some cells in the cut area will start to dedifferentiate and form a callus. Keep in mind that calluses form where there is tissue damage, so if only the undamaged leaf surface is in contact with the media, then you probably won't get much callus growth.

[@ItsLearnable]

Also, in my experience, there are very few "dumb questions". If you're smart enough to realize that a relevant piece of information is not covered in sufficient detail, then it's unlikely that a question seeking to clarify that point could be considered "dumb".

[@karenmoyle4800]

I used the plant begonia for leak disks, I am trying to savage it because of contamination. Do you think its the surface area of the leaf being rough?

[@ItsLearnable]

Rough/"fuzzy" surfaces are definitely more difficult to sterilize. The addition of a little bit of detergent like Tween (or even dish soap if working at home) into the bleach solution can help with this. But always keep in mind that everything we do in the procedure it there to *minimize the risk* of contamination, we aren't always going to be successful.

[@bigdawg3002]

Sounds as if you said 50% bleach. Seems a little concentrated but it may very well be required. Just wanted to ask if in fact you said 50%?

[@ItsLearnable]

Noooooo. Sorry. I should probably be a bit more careful with how I say things. 😳 Were using 15% clorox.

[@ernestdenney2223]

how do you sterilize the paper towels you wipe everything with

[@Phyto.]

In an autoclave

[@ItsLearnable]

It's a good question. If you need sterile paper towels or filter paper for work inside the biosafety cabinet (BSC) or laminar flow hood, you can wrap it in tin foil and autoclave it. In my case, since I'm only using the paper towels to spread and wipe off excess ethanol from items before placing them in the BSC, I'm not using autoclaved paper - it's just a regular roll of paper towels (the paper towel will be disinfected by the ethanol as it's being used).

[@jinty1232]

Where can you buy the punch for making leaf discs? There's no information in the description of the video. Does the punch have a holder for the discs or have you removed it so they fall into the petri dish?

[@ItsLearnable]

It's just a regular hole punch (I just made sure it was all metal so I could autoclave it). Yes, I did remove the holder for the disks.

[@ArtyMars]

I found a circular punch at my local hobby store / sewing crafts store they have loads of shapes

[@ItsLearnable]

Just make sure it's something you can sterilize easily. In my case, I had to stay away from ones with plastic parts - they would be unlikely to survive multiple autoclaving cycles. Also, look for something that can generate larger disks.

[@dinocageman2311]

Hello Can I use a normal hole puncher or is this a specific one ?

[@ItsLearnable]

The one we used is just a regular single hole punch (if just had to be something we could autoclave). Try to find one that will generate relatively large holes/disks.

[@debwilliams3286]

How long would you expect these to take to make a callus?

[@ItsLearnable]

About 3-4 weeks should be enough to generate a callus. There are some awesome time-lapse videos of callus growth out there. Here's an example: kzbin.info/www/bejne/nJucmZqDmtRoZtk

[@drdirt505]

@@ItsLearnable My students and I really like Dr Cahoon's videos. We've been using them as a starting point for some of our projects. And yours, of course! Thank you for all of this good information.

[@ItsLearnable]

@@drdirt505 I'm glad you're finding it helpful.

[@matchmedicines8209]

and just a question, what is the media name inside of the Petri dish?

[@ItsLearnable]

Murashige-Skoog en.wikipedia.org/wiki/Murashige_and_Skoog_medium

[@rosemendonis8487]

Did you Try it to Patchouli plants?

[@ItsLearnable]

No.

[@jackixxiscariot1003]

Hiii, is there a citeable publication that goes with this demonstration for use in a in a formal application?

[@ItsLearnable]

no

[@dustinjohnson6033]

Curious what is in the media? Specifically? such as nutrients, hormones,? Aside from the common things such as sugars, agars?

[@ItsLearnable]

The media is Murashige-Skoog media (en.wikipedia.org/wiki/Murashige_and_Skoog_medium), with a small amount of 2,4-D (en.wikipedia.org/wiki/2,4-Dichlorophenoxyacetic_acid). I don't remember the exact amount at the moment.

[@joecorcoran9]

@@ItsLearnable Is this type of media plant specific, or will will it be effective for most plant species?

[@ItsLearnable]

MS Media is a fairly general media used in plant tissue culture, so it would probably work well with most plants. That said, some plants will have requirements that will not be well met by this media, so your mileage will vary. Sorry for the late reply - I just came across your comment. (it didn't seem to show up in my list of "Unanswered comments")

[@damalungorchid5347]

is this method can be use for phaleonopsis culture?

[@ItsLearnable]

I have no experience with orchids. Please scroll down to some earliest comments to this video - I linked some resources there.

[@ahlamabdulaziz6513]

hello , what is the medium and growth regulators used for callus induction

[@ItsLearnable]

The media is Murashige-Skoog media (en.wikipedia.org/wiki/Murashige_and_Skoog_medium). The growth regulator in this case was 1mg/L NAA, but it can vary depending on the species. In many cases, the ratio of auxin to cytokinin is close to 1:1.

[@kalpanarautela320]

Hello sir, I have tried an experiment for callus induction using pseudobulb as my explant, I have followed careful sterilization processes, hormonal balance and other necessary things all are in good condition...still I'm not getting any kind of callus induction in my culture tubes. (Cultures are 1.5 months old now) .... Could you please help me with this ... And suggest some modifications... Thanks in advance.. 🙏🙏

[@ItsLearnable]

Are you trying to culture orchids? I'm afraid I don't have much experience with those, so I can't give you any specific advice. I'm assuming you did your research and have the right media and hormones for your specific plant - different plant species can have very different requirements. Also, how do you *know* that the "hormonal balance and other necessary things are all in good condition"? Do you have a positive control of some kind? How many explants did you generate? Not every explant may generate a callus, so starting with many explants is a good idea (definitely more than 2). Also, be careful with your explant sterilization. If you overexpose the explant to hypochlorite, you could end up killing the cells that would be most likely to yield a callus (ie. meristematic tissues).

[@kalpanarautela320]

@@ItsLearnable thank you so muchh for your speedy response. Yes I'm trying for an orchid species, I have used auxin cytokinin ratio (1:1, 2:1, 1:2) following some research paper. It's been 2 months and the Culture tubes are showing nothing... I can only use pseudobulb parts as this is not the fruiting period of the species. Could you please suggest something which can be modified in order to get the callus ... If possible please reply.. Thank you

[@ItsLearnable]

@@kalpanarautela320 As I've already stated, I don't have any experience with culturing orchids, so I really can't provide any advice that isn't based on reading of online resources (something I assume you've done as well). In a quick search, I came across a few articles (none specifically dealing with starting a tissue culture from pseudobulbs): www.researchgate.net/publication/349140920_Prospects_of_Plant_Tissue_Culture_in_Orchid_Propagation_A_Review and www.jstor.org/stable/2474002?seq=1#metadata_info_tab_contents . It seems that you may have to dissect your pseudobulb to obtain the meristematic tissue. I also came across some presentation slides on orchid propagation (www.igdir.edu.tr/Addons/Resmi/uploads/files/orkide-mikroc%CC%A7og%CC%86altimi-meleks%CC%A7en-akin.pdf) by a university researcher. You might be able to get more helpful advice by contacting her directly.