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.
@@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 😅
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.
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.
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.
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
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
@@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 😌🌱
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.
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!
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.
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.
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.
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".
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
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.
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?
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.
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.
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: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-fj9hgMeo50w.html
@@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.
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).
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.
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")
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.
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.. 🙏🙏
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).
@@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
@@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.
