A masterclass in democratizing biotechnology that turns complex tissue culture into an accessible reality for the modern hobbyist. It proves that with enough bleach and precision, professional-grade science can thrive far beyond the ivory tower.
Deep Dive
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Deep Dive
I tried cloning strawberries in my lab ๐Added:
Bad news, guys. Strawberry season is over. A few days ago, I Amazon primed a strawberry plant to my house, thinking that it was still strawberry season and that I'd be able to plant it. But it turns out strawberry season is much earlier in Florida than the rest of the country. The good news is that I found this paper from 25 years ago called Invitro Preservation of Temperate Fruit Crops, and it got me feeling very inspired. I think what I'm going to do is I'm going to take the strawberry plant that I Amazon primed to my house earlier this week, cut it up, and clone it using tissue culture to make a whole bunch of strawberry plants. And then, because they're going to be ready way too early for the next strawberry season, I'm going to put them in my refrigerator to store them. There's a few different methods you can use to store plants in cold storage. The coolest and like most sci-fi method would be cryopreservation, which is basically storing plants at ultra low temperatures using liquid nitrogen. If you're like me and you don't have equipment to cryopreserve things, you can also do a much more DIY cold storage method just by putting tissue culture plants in the fridge. The colder temperatures slow down the plant's growth, but it doesn't stop it completely. So, it's better for shorter term storage like one to two years. The first thing that we have to do today is find a protocol to work with. I've already done that for us. This may be an unpopular statement, but I like using AI to find tissue culture protocols. If you just prompt chat GPT to write a protocol for you, it'll spit out some really hot stinky garbage. So, don't do that. But if you ask it to link you protocols, it actually finds some pretty good stuff that I'm not able to find on Research Gate or Google Scholar. Today I'm essentially going to be using two different protocols, a protocol mashup.
The first protocol is from 2004, and I'm going to be using the sterilization method from this protocol. This method is very simple because it only uses bleach and tween 20, both of which I have in the laboratory where we are.
funnily enough, this protocol also has some information about cold storage as well. So, thank you for that. The second protocol that I'm referencing is micropagation of strawberry through runner culture. And in this protocol, they basically just tested different concentrations of BAP to see which was the best for shoot proliferation. If BAP sounds familiar, that's because it's one of the products that I sell on my website, plantsandjars.shop.
We can see from the results that it looks like 0.5 milligrams per liter of BAP resulted in the highest average number of shoots per culture and also the longest average length of those shoots. They also included some photos of the resulting plants which I always appreciate because a lot of protocols don't have photos. This protocol also includes information for rooting.
Obviously, we need to wait for the plants to grow before we can root them in tissue culture. So, useful information, but not needed today. Okay, so now we're ready to make the media.
Let me show you our setup. This is everything basically that I'm going to need to make the media. The products that I'm using are MS, AAR, and BAP. As I mentioned before, all of these products are available on my website if you wanted to follow along and also test out the strawberry protocol.
So, I'm going to be making 2 lers because I need a decent amount of media.
The first thing I'm going to do is fill this up to around 1,800 milliliters.
There we go. Just going to turn on the magnet stir bar.
I'm actually going to get a smaller stir bar so it's not making so much noise.
This one is just a little bit big. Do not tell anyone I did this.
That's better. With the MS, the amount that you use per liter is on the bottle, 4.43 g. Since I'm making 2 L instead of one liter, I'm just going to double that. So, it'll be 8.86. 86 g.
Perfect is next. I'm going to add 50 g of sugar.
The protocol only used 20 g per liter, but that just seems kind of low. So, I'm going to be using 25 g per liter * 2 equals 50.
Now, I just need one single milligram of BAP.
We've already got it set. Perfect.
Before we can adjust the pH, we just need to fill up our container to the 2 L mark.
There we go. And I like to adjust the pH prior to adding the AAR just so that I don't damage the really sensitive probe tip on this thing. It looks like it is around four or slightly lower than four.
So, I want to raise it up to about 5.7.
I'm going to do that using one molar sodium hydroxide, but you can totally also just use the hydroponic up and down. There we go. Just a few drops at a time. So, right now I'm about 5.2. I just want to increase it just a little bit more.
We've made it, boys. Okay, I went a little too far. It's more like 5.8, but that's good enough. The last thing we need to do is add our AAR. This is our jelling agent. In the laboratory, I have just been adding the agar directly to the media bottle that the media is going to get autoclaved in instead of adding it to our mixture here. The way that I make media in the laboratory is a little bit different to how I make it at home.
So, I'm going to link a media making tutorial so that you guys can see how I make it at home if you want to copy what I'm doing. But at home, you would pour it into containers first and then autoclave it. In the laboratory, we autoclave it in a big media bottle like this. I want to have kind of soft media.
So, I'm only going to do 11 g of agar, which sounds like a lot until you remember we're making 2 L, so it's 5.5 g per liter.
I need another bottle.
Bonap petite. I'm also off camera going to prep some rose media. I want to repeat my rose bouquet experiment a third time. Um, if you remember my last vlog, I was cloning roses from bouquets of roses. So, I want to show you how they're doing. These are all that we have left from the first day. These three are contaminated, but there is hope. We can see there are shoots coming off some of them. This one has probably the largest chute. Uh, it's obviously contaminated here. Uh, but my experiment's working. So, if I can just get them cleaned up better, I think that this will be a great success. These two, you can tell they're contaminated because of the discoloration in the media. These two I don't think are contaminated.
And then all of the ones in the back were from the second round of my experiment. The only thing I did different that time is I sterilized the expplants for a few minutes longer. So, I did have a lot less contamination that way. I'm pretty sure I sterilized all of these for about 25 minutes. But you can see we actually have some shoots forming off of these nodes as well.
Also, people always ask me the difference between shoots and roots.
This is a Monsa Bulbasaur. And if we flip it over and look under the hood, we can see one root here. And then this one actually has two shoots, one there and one over here.
I also need to autoclave some containers for both the strawberries and the roses.
So, I'm going to be using these 5.5 oz portion cups. I have these linked on my Amazon if you're curious. They're polyropylene, so they can go in the autoclave. They can also go in a pressure cooker if you're doing this at home. And how I do this is I wrap them in a slow cooker bag.
These are the ones that I buy. And then I just seal it shut with autoclave tape.
Doing this is a bit of an art form.
And yes, I have mastered it.
I just put them in the bag like that.
Then I roll it shut.
I do the same thing for the lids, but the lids are a little bit more finicky.
This is everything that is going to be going into the autoclave. We have our rose media, our strawberry media. These I'm going to fill with water so that I have sterile water for washing expplants when we get to that step. And then I have the lids, the containers that we just wrapped up. I just put them between the bottles so that they don't all smash together. And then I also have a bunch of tools that need to be sterilized as well. And those are my gauntlet gloves.
They're very stylish. Let's ride.
This crazy thing is where I get the deionized water.
This is an autoclave. It's basically a giant pressure cooker. If you're at home and you're making tissue culture media, you can just use a regular pressure cooker or an Insta Pot.
I think it's the 29th.
26 minutes left until I've got to go get the stuff out of the autoclave. In the meantime, look at this poke bowl.
Absolutely beautiful. Over the weekend, I had a party at my house and we had a sushi boat and there was like a bunch of sushi left over afterwards. So, I was eating so much sushi all weekend. And then I guess I woke up this morning and I was like, didn't eat enough raw fish this weekend. Does this look like the face of someone with mercury poisoning?
Be honest. Also, I'm drinking water out of a Fisher brand media bottle because I forgot my water bottle. I went to go see Project Hail Mary last weekend. Very good. I would recommend it. I've also read the book and I did enjoy the book as well. People are saying it's like the best sci-fi movie of the decade, but also Dune 2 came out this decade, so I disagree with that statement. I don't want to spoil anything, so I don't know.
Fast forward if you don't want spoilers, but there is an alien encounter and they used visual effects, not CGI is what I'm trying to say. They did not CGI the alien. He's like a puppet, which I thought was really cool. The only thing I didn't like was the music. I guess Han Zimmer was booked and busy for this one because I didn't really like the soundtrack. They also kept spamming the same space music over and over and over.
And they would play it over any scene.
Like the guy is just eating lunch. Space music. Uh but overall, I would recommend it. I think I give it a 7.5 out of 10. I know what everyone's thinking.
Q1 is over. Did I complete my Q1 reading list? Almost. This quarter I read The Poppy War by RF Kuang. I won't talk about that one because I didn't really like it. I haven't liked her other books, Yellowface or Babel. So, I think I'm done reading her books going forward. I also read Snake Eater by Tek Kingisher. I love Tek Kingisher. I didn't like that book, so I'm not going to talk about that one either. The book I did really like was Dungeon Crawler Carl. And the reason that I didn't finish my Q1 reading list is because I'm now on book five of Dungeon Crawler Carl instead. I've been listening to the audio books and the narrator is the best narrator of any audio book I have ever listened to. What's his name? Cuz I got to give him a shout out. Jeff Hayes.
Jeff Hayes, you are a superstar. The premise of the book is that aliens invade Earth and they are harvesting it for its resources. All of the buildings collapse flat to the ground. So if you are outside, you live and you have the opportunity to walk into a dungeon, which is basically a game show broadcast to the entire universe, which is huge.
Quadrillions of aliens are watching this show. And it's a lit RPG.
If you don't know what an RPG is, you probably do if you're watching this channel, but it's a role playing game.
World of Warcraft and League of Legends are examples of RPGs. The characters are fighting different monsters to level up.
And as the story goes on, it becomes less about fighting monsters and more about the game itself and exploiting the mechanics of the game. The main characters are getting called away from the dungeon all of the time to do interviews on alien talk shows and reality shows and things like that. I would say it's probably my new favorite science fiction book of all time, but listen to the audio books. I did not read the physical version. I sometimes feel with different forms of media, whether it's books or movies or anime, the same tropes kind of get recycled over and over again. And Dungeon Crawler Carl like genuinely feels like something very new and different. Like I said, I'm on book five. I think there's eight out or the eighth one's coming out soon. I'm not sure how many books are planned total, but I hope a lot because it's so good. onto my Q2 reading list. First of all, I'm shelving Lonesome Dove. I do not feel like reading it. Uh, a lot of people say it's the best book I've ever read. I believe you, but I don't feel like reading it. Instead, I'm adding a different 1,000page thick book called, what is it? Pillars of the Earth, which is another one that a lot of people are like, that's the best book I ever read.
All I know is that it takes place in 12th century medieval England, I think.
and it's about a monk building a massive Gothic cathedral. Like I said, it's a really long book. So, that's the only book I'm putting on my reading list for Q2. Plus, I'll probably keep going with Dungeon Crawler Carl. It's time on time.
Oh my god. So now I'm just going to get the flow hood ready and all the stuff I'm going to need in the flow hood in order to pour the media. And then tomorrow I will be putting the plant into tissue culture.
So hopefully it arrived to my house today.
We will not be needing this. So I just push it to the side.
And that's it. This is obviously very hot still.
This is what 2 L of media looks like if you ever need to visualize it. This is our strawberry media. I'm just going to wait until the media actually solidifies before I go ahead and put the lids on.
Hello everyone. It is the next day and I brought in today's victim, our I almost said banana, our strawberry plant. I had a call with a Tanzanian banana lab this morning, so my mind's on banana. It was baking in the sun outside my house all of yesterday, so hopefully it still looks good. on Amazon. It has one review, so that's always a good sign.
Just as a reminder, here's what we're going to be doing to sterilize the expplants from this beautiful strawberry plant that we have right here. It says that the expplants are taken from recently formed runners of greenhouse and grown plants. I don't know how this plant is grown, but that's okay.
Plantlets are surface sterilized. I think they meant to say expplants there because plantlets doesn't make a lot of sense. Um, but they said plantlets are surface sterilized by placing them in a 10% bleach solution. The bleach is 5.25% sodium hypocchlorite with 0.1 ml per liter of 20 and then they put it on the rotary shaker for 10 minutes. It's always important to look at the bleach.
It says 5.25% sodium hypocchlorite. My bleach is actually uh 7.5%. So, I'm going to have to do some stoeometry.
Some of the runner tips are gone, unfortunately. So, I won't be able to use tissue that looks like this. But, I am going to use the nodes. So, that's an example of a node.
Oh, there's a strawberry. What I'm doing is I'm just taking mostly the nodes. To be honest, the shootute tips for the most part don't look that great on this plant. Well, this one actually looks pretty good. So, I'm taking both chute tips and nodes node. So, these were all the nodes and the runner tips that I was able to get from our strawberry plant here. The node spacing on the strawberry runners is really far apart. So, they're Oh, as I'm speaking, I found another node. I'm going to go wash these under the sink really quick and then they're going to go into some water with just a drop of Tween 20 prior to the bleached sterilization solution. Hello, voiceover lore here. One thing that I think gets overlooked in tissue culture is the value of cleaning your expplants really well before they even go into the bleach sterilizing solution. Dirt is packed with microbes and contaminants and organic debris that physically block the bleach from reaching the surface of the expplant. Bleach can only disinfect what it can actually touch. So, if there's a layer of dirt on your expplants, even if you can't see it with your naked eye, you end up with these little protected pockets where bacteria and fungi can survive the sterilization step completely untouched. No contact equals no sterilization. A really thorough pre-clean with running water at the sink and placing the expplants in water with a drop of tween 20 on either the orbital shaker or a magnetic stirer does two things. First, it physically removes a large percentage of the dirt and contaminants before you even start sterilizing. And second, it lets your bleach solution actually contact the plant tissue evenly and do its job. I did make a mistake here, which is once again using too much Tween 20. The smallest drop of that stuff goes the longest way. It was difficult to rinse the expplants and after three rinses in the sterile water, uh maybe more than that actually. I think I did like four or five rinses in the sterile water, but there were still a ton of bubbles from the Tween 20. You want zero residue left at this stage if it's still foaming.
You're not done rinsing. Tween 20 is basically lab soap. It acts as a surfactant so that the bleach can clean the chute tips and the nodes better or whatever type of expplant you're using.
I ended up getting a sterile container like the ones that I was drinking out of earlier, but not that exact one obviously because it needed to be sterile. And I rinsed them in that to get the rest of the tween 20 off as much as I could. There were still a few bubbles, but for the shoot tip expplants, I cut off the end that had been previously cut and exposed to the bleach. For the node expplants, I cut off both ends since both ends have been exposed to the bleach. It's important to remove the tissue that was directly exposed via a cut. If you don't, then the ends of the nodes become necrotic and that can cause contamination in your cultures. I'm not going to show every single expplant that I put into media because it's very repetitive. Uh, but this is the exact process that I did for all of them.
Here's the aftermath of the situation. I have all the strawberry expplants here.
Unfortunately, I made way too much media. So, I'm thinking about buying another strawberry plant and possibly repeating this experiment. Also, I ended up just using some of the stems that didn't contain nodes or a chute tip just to see if anything happens. I'm guessing probably not. There's not enough mermatic tissue in these types. Not me dropping it in these types of expplants.
But just because I had so much media, I was like, may as well.
I didn't film an outro, but I just finished editing the video and I wanted to say thank you guys so much for watching. If you're interested in learning how to do tissue culture, it's a really fun hobby and you can also do it from home. Even though in this video I was doing it in the laboratory, I'm going to link some beginner home tutorials below. They're designed for complete beginners. So, even if you have no experience doing tissue culture at all, you should be able to follow along and figure out what you need to get for the setup and how to get started with it. Let me know what other plants you guys want me to tissue culture. I'm testing a lot of different protocols this year. I kind of hinted at it in the video, but I will be doing bananas pretty soon. Uh, so yeah. Bye guys.
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