Seeds...

Well. That was terrible. Yesterday I collected seeds from the Drosera burmannii I have been trying to clone. Let me just say these seeds are extremely valuable to me, as they are my last tie to this plant before it dies. I had one jar of the tissue culture gel left. Showtime.

So everything started out well. I modified my sterilization process a little, because I was working with seeds and didn't want to use as many chemicals. I hauled the box upstairs and sprayed everything down with bleach. I put the seeds in a piece of coffee filter and paperclipped it shut. I placed this in a baby jar filled with 10% bleach for 13 minutes. I added a little dish soap so that the bleach would have a lower surface area and better cover the seeds. As I shook the jar to make sure the seeds were thoroughly cleaned, I noticed about ten seeds slipped out of the packet. This was ok, I had sort of planned for this. Not that big a deal.

So then I transfer the packet to a vial of hydrogen peroxide for about 4 minutes. After that I swirled it around in two different jars of steril water for 10 minutes each.

For the whole sterilization process, you aren't that worried about the seeds because they are about to get cleaned again and again. However, at the last stage where you have to transfer the seeds, you have to work FAST to make sure you don't compromise the sample and the jar. I pulled the filter paper out of the final jar quickly and started to unfold it. Unfortunately, it ripped. Stupidly, I just decided to keep ripping the filter paper apart to get at the seeds as quickly as possible. Emphasis on stupid. I had folded the paper enveloped multiple times to try and seal the seeds inside. So when I forcefully tried to rip it apart, all the layers compacted into a solid pulpy mass, trapping my seeds inside. I pulled and pulled and pried, but I could never get the seeds out from inside the filter paper.

I managed to get about four seeds of the forty seeds I has started with onto my finger (in hindsight I realized I had forgotten to wipe myself down with alcohol, so they were definitely compromised). These I transferred onto wet tweezers and tried to flick into the jar of nutrient gel. However, they stuck to the tweezers. After about four unsure bangs on the rim of the open jar (meanwhile millions of bacterium, algae, and mold spores are falling inside) I moved onto the other seeds and repeated the process. At the end of this miserable attempt, I found one black speck (which still might not be a seed) lodged in the nutrient gel.

Ya.

Well, I refuse to let this hinder me. While I do not have time to redo the entire project (plus I'm out of baby food jars), I would like to retry the process further down the road. The important thing is to learn from yesterday. Here is what I could come up with.

1. Always unfold the filter paper.
2. Practice flicking wet seeds into a jar, or sterilize a thin wire, get them onto that, and then carefully lower them down onto the gel (lab scientists use a thing called an inoculating loop).
3. Practice with seeds you don't care about.

Hopefully, I can get this right the next time around. While I still considered yesterday fun, I really hope the third time's the charm.


I almost forgot the good news! If you watch my vlog, you can see my previous plant grow area. Over spring break, I bought and built a GROWSHELF, complete with reflective bubble wrap I rigged up around the plants.

YEA BUDDY

That was fast...

Alas, the most likely outcome in this experiment has arrive. Contamination. I checked last night and every single leaf had some form of white/grey fuzzy mold on it. But hey! At least I could recognize it. And the problem was on the leaves, not the gel, which means my project isn't a total failure. I just need to reconsider. I will be redoing the experiment tomorrow with some new jars, and seeds instead of cuttings. These sundew leaves are particularly hard because of the hundreds of nooks and crannies created by the tentacles.

This was the best photo I could get. See the white smudge on the green leaf? That's the mold.

Seaweed Chutney

Now that I'm done tissue culture, I felt the need to do a little more reading to improve on my process. Remember a little while ago how I mentioned the need for plant growth hormones? Well I did a couple hours worth of reading on these substances, and it is really, really cool. Basically, these chemicals come in two forms, auxins and cytokinins. Both are responsible for cell enlargement and increased division, which makes for more plant in a short amount of time. Which is perfect for tissue culture.

I do not have a PhD in Chemistry and so was not able to decipher all of the jargon, but basically what I learned is that these chemicals are manufactured by the plant to help growth. They start at the roots and work there way up the stem to the apical growth (or middle stem). If this apical growth is removed or conditions are very nice, these plant hormones will trigger growth in the dormant nodes surrounding a plant (those funky little nubblets that surround a plant). This is why your prune plants- by severing the top you get those surrounding nodes to grow, making for a larger, bushier plant.

So where are these found? Well they are made by the plant. There are four auxins created by the plants themselves, though the most notable is called IAA (this is just an acronym, but because I don't want to type out the name and don't understand it at all, I'll leave it at that). However, this chemical is extremely unstable and frequently breaks down. In tissue culture, scientists have manufactured synthetic auxins and cytokinins that are cheap and way more powerful. Good for us!

However, I don't like to buy things from labs. It's too easy and still too expensive. So what if I make some chutney? As I was doing my reading, I came across "seaweed extract" as containing a potent mix of natural plant growth hormones. I tried to figure out why this is. It turns out that seaweed (or kelp) is one of the largest and fastest growing organisms on the planet. Some can grow two feet per day and top out at 260 feet long. Ya.

Now how does one "extract" these chemicals from seaweed. Well some further research showed that IAA is somewhat soluble in water, and one paper showed that these hormones can either be easily freed from the plant or are seriously locked inside proteins and won't leave. An extract is created when you mash the seaweed up and steep it in water and other solvents, and basically let it break down into a concentrated seaweed tea. Here's a video I found hysterical about a guy outlining his process, including the infamous "seaweed chutney."

So then I thought to myself, is there anything better than seaweed. Well as it turns out a lot of these extracts rich in plant growth hormones are also marketed as rejuvenating skin lotions. This is one of the primary selling points for coconut water, which I talked about earlier. But there is one plant on this earth that grows at an even more ungodly pace than seaweed.

Bamboo.

Bamboo is the rat of plants. With a growth rate of nearly three or four feet per day in some species, bamboo tops the charts of growth. So, what if I extracted the wicked cocktail of growth hormones in bamboo and applied it to samples in tissue culture. I couldn't find studies that had done this, though coconut water and seaweed had both been tried. I need to do a little more research into exactly what makes bamboo so hopped up on crazy dust and how one goes about extracting it. If this is possible, then here is a natural, flip floppin' unending source of growth hormone. Or I could always buy the laboratory stuff. WHOA. What if I could replace the growth hormone in bamboo with the ones we've made? Would it grow even faster? Or is bamboo nature's perfectly mutant result of 'roid rage?

I wanted to add one more note that just occurred to me. Seeds are very rich in growth hormones, which means that the seedlings do not produce very much, if at all. So whatever is substituted in the media (nutrient gel) of tissue culture is its main source of "inspiration" for growing.

I wanted to add another note. In an experiment based on the above logic, I could compare the effects of bamboo extract hormones vs. synthetic hormones on bamboo seeds. If the extract came from the same species, it would be a proper comparison.

DONE DONE DONE DONE DONE

I doubt this will be the last time I say it, but here it is anyway:

WOOHOOOOOOOOO!!!!

Yesterday I finished the first four jars of tissue culture. It all went pretty well, I cut off the first four developing leaves of my D. burmannii. Then I filled some jars/tubes with the following chemicals and started sterilization.

I was going to try and sterilize some tweezers with a lighter, and then realized I had these covering every surface. An oxygen source, two potent fuels, and a very reactive source of a toxic vapor.

Here's the procedure I followed, modified from when it was listed in the first post.

INSIDE HOOD

1. Dunk explants in 91% alcohol for 10 seconds. Skip this for seeds.
2. Dunk in 3% H2O2 for 4 minutes.
3. Dunk in 10% bleach solution for 7 minutes. Put seeds in here for 4 minutes.
4. Place in sterile jar of water and rinse for 5 minutes. Repeat.
5. Pull samples out of jar with sterilized forceps.
6. Loosen cap on TC jar.
7. Place explants in jar extremely quickly.

8. Wrap containers in Saran wrap.


STERILIZATION:

1. Using tiny spray bottle, spray 10% bleach solution on every surface.
2. Generously spray jars with 10% bleach solution.
3. Wash hands with soap and water, then wipe with 70% alcohol.


To prepare the steril water, I filled loose jars with distilled water and pressure cooked them for 25 minutes. For the bleach solution, I used 2/3 cup of extra-concentrated bleach in 9 1/3 cups of tap water. I figured any impurities in the tap water would be washed off in the final rinse. 

If I were to do this again, I would use flat forceps (tweezers) the whole time, for this I used surgical forceps with teeth that I am sure damaged the cuttings. I am really happy with how everything went. The only danger is that by using those toothy forceps some of the chemicals killed off the entire leaf. We'll see how it goes over this coming week, where contamination is the new hurdle.

I still have five jars left and some agar, so I may do the entire process over again later in the week. I wanted to get this done so that I have a solid two weeks before the presentation for something to grow. If I do it again, it will be with agar and not cornstarch.

Pictures!

The aftermath. My parents are lovely people.

The river of bleach that ran down my kitchen counter. I cannot describe the smell of bleach, alcohol, and peroxide mixed together.

The little green thing is the cutting. It sank into the cornstarch perfectly.

The cutting with terrible aim. I hope it grows.

Another cutting in its new home.

VIDEO BLOG

Here I talk about the process and detail my inspiration. It's all explained in the video, but with this project my main goal was to bring a complicated science to my home. Though it may seem like it, I've really simplified the process by synthesizing a lot of research.

 

Tissue Culture Vlog from Drew E on Vimeo.

Halfway There!

This week has been full of lots of fun research and concluded in me finishing my first five jars. But I'll start at the beginning. I spent the week learning about these chemicals called Plant Growth Hormones, or PGRs. They're pretty self-explanatory, they are natural chemicals found in plants that make them grow. Certain combinations either make plants grow new shoots or roots. In real tissue culture, you can buy ones made in a lab. However, I'm doing cheap tissue culture! So what I found was that coconut water contains a number of these PGRs, but is extremely unreliable. Here's a study I found explaining coconut water's various chemicals. Here's one showing how it applies to tissue culture. It was some really interesting reading. If you want to learn about PGRs, I found this, this, and this to be very helpful. In the end, I determined that seaweed extract would actually be the most consistent supply of PGRs, but I ended up not finding any. Maybe I'll make a new media over spring break that contains some seaweed. It'll help if I transplant some of the samples and make stimulate some real growth.

So now onto the real news. I continued to play around with corn starch, and cooked three jars with different amounts of it in my pressure cooker for about 10 minutes. I determined that 6 teaspoons per 1/2 cup of water worked the best.

4 teaspoons per 1/2 cup water. Notice the clear spot of liquid.

5 teaspoons per 1/2 cup water. Notice the small dots of liquid.

6 teaspoons per 1/2 cup water. Notice the white jelly goodness.

A quick note about pressure cookers. They are very thick metal pots that have a very tight lock on the top. They have one valve to let just enough steam out to maintain 15psi. They are HORRIFYING. The steam screams out of the valve, and I refused to stay in the same room as it. Pressurized shrapnel from glass jars would not feel good ripping through my face if it blew up. I dropped a baby food jar this weekend, and when they shatter, they are sharp.

Pressure Cooker from Drew E on Vimeo.

After these tests, it was time for the real deal. I mixed my nutrient liquid with some corn starch (see video blog), and added it to the pressure cooker. I cooked it on high for about 2 minutes and then let is simmer to make a total of 30 minutes. I pulled the jars out, and the liquid had gelled perfectly! One jar opened as I pulled it out, which is a big no-no because any bacteria that entered the jar can quickly spoil it. But I closed it up again and am going to leave it, just so I know what contamination looks like. The other jars I covered with Saran-wrap to further seal them and keep out any nasties. I plan to sterilize the tissue samples and add them to the jars on Tuesday. I still have five more jars to prepare for Spring Break with the high-quality gel, agar.

WOOHOOOOO!!!

A Sticky Situation

This weekend I played with corn. Cornstarch to be exact. I picked up two $1 packages at Walmart and was playing around with mixing different concentrations. I know cornstarch as a fun camp activity I did with kids in Honduras. When you add enough cornstarch to water, it appears to be a liquid, but when it undergoes stress compacts into a rigid solid. I know that gravy recipes call for cornstarch to thicken them. So, I added about 10 teaspoons of cornstarch to 1/2 cup of water, and microwaved it. Lo and behold, it thickened into a lovely gel. Well, I can't say lovely. It was a disgusting pale white color with a layer of moist cornstarch that had not gelled on top. I played around with it some more, and found that 5 teaspoons in 1/2 cup of water made a nice gel. One problem I could see was that as I microwaved it, the gel heated and rose up considerably. When it cooled it continued to stick on the sides of the glass jar, while the center collapsed. It was pretty gross, and a very uneven surface. I'm hoping the pressure cooker heats it evenly so that I don't have this problem, and have to smooth it out after I sterilize it. Also, the white color makes it look like it is already contaminated with bacteria. It will be really hard to spot infection if it occurs, unless I dye it some ridiculous neon color. Which I will likely end up doing.

If corn sneezed, this would come out.

Speaking of the pressure cooker, I finally assembled it! It looks very nice. I have to rinse all the parts before I can safely use it, but from what I can see it seals very well.

I'm collecting some jars, and may have to buy some cheap baby food. I currently have 5 jars, and would like 10-15.

I was told that this project is confusing, and I use a lot of botanical jargon. So here I will attempt to do a basic explanation of tissue culture. It is a scientific concept though, so I can not avoid a few terms.

Tissue culture is used to clone plants. This is not done the DNA-splicing techniques that are often associated with cloning labs. Cells are able to rapidly divide and preserve their DNA and traits in a process called asexual (without sex) reproduction. When this happens, exact copies are made of the original cells. In a normal environment, plant cells, or tissue, cannot grow very fast because of disease, contaminants, being eaten, etc. Additionally, plant tissue needs a lot of food to grow and divide at a fast pace.

What tissue culture aims to do is provide the perfect environment for cell division and plant growth. It does this by placing a tissue sample in a sterile environment, typically a glass jar. This jar has a sterile gel in it that is firm enough to allow the tissue to anchor itself and grow, while also supplying all of the energy and nutrients necessary for growth. When these two factors are present, the cells can grow and divide uninhibited.

As a tissue sample (a leaf for instance) divides, it forms a clump of cells called a callus. New growth points (the center of a plant where leaves and roots come from) emerge, and generate dozens or even hundreds of new plants. These are then divided and places in their own jars, where they will again divide. Different chemicals can be added (like the root hormone I mentioned earlier) that will stimulate certain growth in the plants to further establish them. When the plants are large enough they are removed from the jars and gradually introduced to real growing environments.

Now here is a description of the materials I am using:

A plastic box as a pseudo-sterile hood: By cutting a hole in the side and spraying it down with bleach, this place becomes a safe place to work free of contamination. Bacteria and mold spores cannot "fall" into the jars because the top is covered.

91% isopropyl alcohol, 70% isopropyl alcohol, 3% Hydrogen peroxide, 8.5% bleach: All of these are used to sterilize the tissue and cultures. Different combinations are used to effectively kill everything bad.

Pressure cooker: Pressure cookers do not let steam evaporating inside to escape, creating an environment with 15 pounds per square inch of force and high temperatures. These conditions sterilize things that would be hard to clean with chemicals.

20-20-20 fertilizer: 20-20-20 is a ratio of Nitrogen-Phosphorous-Potassium, which are three major nutrients plants need for good growth. It also has a lot of other good things.

Multivitamin: Another great nutrient source that covers all the bases.

Inositol: I honestly don't know exactly what this does. Claims say that it is a natural chemical that aids in nutrient absorption by cells. It may help the tissue samples to absorb the food I'm providing and help them grow.

Agar/cornstarch: These are natural ingredients that, when heated in a mixture of water, will turn into gels. This provides a surface that the tissue samples can anchor and grow into, as well as easily passing on the nutrients to the growing cells.

I hope this helps!

Sources

I realized that I haven't really cited my research. I did most of my research during winter break and then revisited a few of the sites for specifics. Additionally, I have received a lot of advice in procedure through the intangible sources of real biologists. Anywhere, here are all the sources that I have found:

A description of cheap home tissue culture.

A fantastic FAQ on a carnivorous plant forum with an incredible glossary.

A nice walkthrough of procedure with helpful pictures.

Here's a set of documents prepared by Phytotech Labs. The Technical Literature section explained all the chemicals and procedures involved in great detail.

Here's a forum thread explaining some people's attempt at cheap tissue culture. It was my inspiration.

Here's another forum thread created by one of the biologists who has helped me. It's a great explanation of procedure and materials.

The tissue culture thread of the other biologist who has helped me. It isn't as outright, but I found great information as I scrolled through.

CULTIVAR NEWS!!!

WOOHOOOOOO!!!

So I emailed the International Carnivorous Plant Society, and received a reply from the person in charge of cultivar registration. It went like this:

Dear Drew,

Stability of cultivar characteristics is one of the main issues in registering cultivar names (NB: we are registering names, not plants) but this stability refers to the phenotype only and not to lineage nor to procedures of generation or maintenance. The means of propagation may be a useful recommendation in the context of the description but it is not mandatory that a plant was generated using a specific method in order to apply a specific cultivar name to this plant because the definition and meaning of any cultivar name is governed exclusively by the phenotype described in the original publication. In your case this means you must be careful to mention everything you consider essential for making your new cultivar a special plant in the description of said cultivar. Irrespective of how other plants may have been generated, if they meet these criteria of your description, they may be given the name you propose in the description.

If the progeny of self-pollinated plants show variation in the characteristics that define the cultivar, only the plants that match the original description do belong to the cultivar. The remaining plants that deviate from the original description do not belong to the cultivar although they share the same lineage from a technical perspective. This may generate problems if seeds and not mature plants are traded because the defining characteristics may not be detectable in the seeds. One way to deal with this situation is to declare the seed batch in question as "seeds from selfing [Your Cultivar Name]", or similar.

Do you know what this means??? It basically means that when you register a plant as a cultivar, you are registering the appearance of a plant, not a specific individual and its genes. Therefore, any plant in the world that looks like the description is now that cultivar. By that logic also, seeds from the original that may not be exactly identical are still the cultivar as long as they look the same.

Do you know what this means??? It means that tissue culture is not my only option for registering this plant. I can grow it from seeds. Which also means I get to have a lot more fun with this project. I'm not rushed to do things by the book! I can experiment with less valuable plants with less of a risk, because I don't need to clone this exact plant. I will of course try to, but it is not my only sample (which also means I can make more cultures of many different plants).

I learned this week that cheap aluminum pressure cookers from Walmart may not result in the best sterilization. However, I will just "cook" everything a little longer than I had said before to counteract this. I'm trying to keep my budget under $100, and an $80 pressure cooker would really affect that. So, I will make due for this project. This way everything I'm using is cheap and readily available.

Here's the current tally of things I had to buy that I am not borrowing from school or weren't already in my house:

Fertilizer: $6
Inositol: $11
Agar powder: $5
Distilled water: $2
Sterilization chemicals, spray bottle, and plastic box: $25
Pressure cooker: $35

Not bad. This weekend I'm going to try and dispense the liquid into whatever jars I have and sterilize them. The week after I will sterilize and add the samples. Let's hope it works!

A quick cell phone picture of the D. burmannii I'm hoping to register. As it has aged it's gotten this beautiful red color. It's huge for its species, too, at two inches across. They are normally around one inch in diameter.

Nutrients? Check!

Just finished the liquid nutrients!!! It looks absolutely disgusting, but I am pretty convinced it should work.

Wouldn't you love to eat nothing but this for three weeks?

So I used the recipe I listed in my first post. I had found a 20-20-20 fertilizer (a ratio of Nitrogen-Phosphorous-Potassium, the major nutrients needed by plants), so I diluted it by 1/2. Otherwise everything was the same. I improvised with measuring, because I didn't have 1/8th cups, so it was a bit of a guessing game. Otherwise though, it looks good to go!

The materials I used: (Front row, left to right) Jack's Classic 20-20-20 Fertilizer, measuring cups, One A Day Women's Multivitamin, Inositol, measuring cup, measuring spoons. (Back row, left to right) 1 gallon distilled water with 1/16 teaspoon of fertilizer, pure distilled water, Domino cane sugar.

In other news, I finally bought my pressure cooker. It's a six-quart model from Walmart. It was steeper than I imagined at $35, but well worth it. Just look at that quality aluminum! I will be using this to sterilize the nutrient liquid once I add it to the jars and thicken it.

When I was at Walmart, the guy behind me in line told me that he hated aluminum pressure cookers because they always leak. Not exactly the best encouragement.

How will I thicken it? Good question! I also bought some corn starch today, and intend to play around with it. I don't know how much will make a good gel, just that I should add it to the mix, heat it so it dissolves, and then distribute to the jars.

I kind of feel like getting away from procedure now. I am really excited to list my plant as a cultivar (by definition: a race or variety of plants that has been selected intentionally and maintained in cultivation) if I can reproduce it. I emailed the International Carnivorous Plant Society for some input on how to go about registering it. I'm not sure if I made this process very clear in my first blog post, but I've done some more reading to understand propagation.

Basically, there are two ways a plant reproduces, either by sexual (with another plant) or asexual (without another plant) means. Sexual reproduction will never result in very similar plants, because two plants are exchanging traits. Asexual reproduction can occur either by the regeneration of some plant tissue (This is what tissue culture aims to do. By cutting off a part of a plant and forcing it to grow back, you get an exact clone of the original.) or by self pollination of a flower. But self pollination is tricky. Apparently, some genes that may be hidden in the parent can be revealed again when a plant pollinates itself. Essentially, the seeds aren't exact copies, even though they were formed by one plant. Because my unique sundew can't be cloned by regrowing leaves (except under the controlled conditions of tissue culture) the process of maintaining its traits is really difficult. If the ICPS does not allow me to multiply my plant with self pollinated seeds, than this tissue culture attempt is really my only hope at registering my own plant.

So no pressure.

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