Some Progress...

It's musical season. Because I have been getting home at 9:30 every day and have had very little time to do some reading. I've talked with a few biologists, though, and have been collecting materials and refined the procedure. For this project I need a very specific kind of fertilizer to feed the tissue. They come in ratios of Nitrogen-Phosphorous-Potassium. I need 10-10-10 fertilizer. The only kind I found of this at Lowe's were sticks of slow-release fertilizer, which I learned have all kinds of other things that won't help my project. I found the proper fertilizer and ordered it on Amazon. Ya, it was a really boring weekend.

So Sunday night I decided to break out some power tools. I got my dad's tin snips (basically these super powerful pliers that shred plastic tubs like scissors through paper) and went to work making my sterile area. 10 minutes later (not counting the five it took to convince my dog that a fallen piece of plastic was not a potato chip) I had this...

This is just my work area. I'll have to spray it down with bleach every time I decide to use it, and clean everything that goes inside. These two square feet will be the most important place in this whole process.

The plant I'm going to multiply is looking AWESOME. I'm really excited to start the cloning.

I ordered some agar online. Once I get it and the fertilizer, I can start mixing the nutrient gel. This weekend I'll also have bought my Walmart pressure cooker. The Biology department at my school is lending me some long tweezers. I'll finally have all the materials for my base formula. Then comes the experimenting.

In the horticultural world, a common way to make more plants is by taking a cutting, planting it in soil, and making it grow roots. There is a root hormone I bought at Sears for a few bucks that is supposed to help with this process. It's readily available, cheap, and incredibly dangerous. So I bought a filter mask, too. Basically, I intend to make another mixture with this root hormone, which I will hopefully be able to attempt a transfer from the original flask to a new gel that will help the clones root and get ready for real soil. I have no idea how much I will need of the hormone, but I probably shouldn't use a ton.

Another thing I found in my search for cheap tissue culture is corn starch. Agar is normally used to make the nutrient liquid gel, which allows for the samples to rest on top and bury when it begins growth. Cornstarch is a nonnewtonian fluid that can create a viscous liquid. Though it would make a cloudy gel that would make it difficult to identify contamination, it would be a very cheap alternative. Cornstarch goes for about $5 a pound, the same price for 20oz of agar. I have agar ordered, but I will also be trying cornstarch. Here is some clear cornstarch I may try later.

This weekend I intend to make the nutrient liquid and finish collecting materials. Until next week...

Getting Started

Hello. My name is Drew, and people say I'm obsessed with carnivorous plants. I currently grow about 65 different species, with a total collection of well over 100 plants. I have been growing them for the past five years, and, with the encouragement of my parents (I honestly don't think they knew what they were getting themselves into), have grown my collection to nearly every room of my house. To me carnivorous plants are so cool because they are a living example of evolution and give me the opportunity to experiment and try new things. This blog will detail my biggest experiment to date.

About five months ago, I received seed of my favorite carnivorous plant variety, the sundew. These seeds were from the a rare 'Giant Red' form of the species Drosera burmannii. However, as I have raised one of these plants from seed, a unique trait arose. It was brown. The past few weeks I have been showing my "coffee-flavored" plant to other carnivorous plant enthusiasts, and no one has seen this before. Naturally, one questions come to mind:

What do I do with this plant?

Sundews are typically very easy to propagate (or get to regenerate to make identical plants). Typically perennial plants will grow back from cuttings of leaves or roots and become copies of the parent. However, Drosera burmannii is one of those few plants that are annual, or live for one season. It has not developed this ability. There is the possibility of collecting seeds from flowers I have self-pollinated. However, this will by no means make exact clones. I may lose this trace forever, and I have about eight months before this plant reaches the end of its growing season and dies.

Once I realized that my plant was going to die soon, I delved back into some research I had seen this December during winter break. There is a very effective process for creating hundreds of clones of difficult to propagate plants. It's called tissue culture.

Tissue culture is a pretty complex process, and is still gaining widespread acceptance. The key to how it works is a sterile environment, and a whole mess of plant nutrients, sugar, and growth hormones. First, a sterile environment is prepared, often something as simple as a glass baby food jar. This is filled with a sterile cocktail of the above ingredients, which should solidify into a soft gel. Some kind of plant tissue is selected from a plant and added to this culture. The tissue can either be the "flesh" of the plant, like a leaf or a stem, or it can be a seed. In this nutrient-rich, sterile environment with no other competition, the plant cells exert all of their energy into multiplying. If done correctly, tissue culture can make thousands of plants from a single sample.

So, tissue culture is a pretty remarkable process, and I have chosen it as the subject for my Genius Project. For any readers not in my class, this is an opportunity where for seven weeks I get to pursue any passion of mine. I will be blogging about my progress, and will eventually give a presentation on what I have learned. I will also produce some kind of product as the culmination of the process.

My goal for this project is to end with one uncontaminated flask of growing tissue. Maintaining a perfectly sterile environment will include establishing a sterile place where I can work, as well as actively sterilizing every surface in this space, and everything that enters it. It will be meticulous and extremely hard. Tissue culture is typically done in $15,000 university labs with hardcore chemicals and equipment. I'm going to try to do it in my kitchen. Because most of the written work explaining tissue culture and how to go about it is directed at those who have access to a formal lab, this process will be based mostly upon the experience of other home "TCers," as well as anything I can come up with. If you're here looking for information on how to do this formally, I apologize, this is not the blog for you. I'm in high school, so I'm going to have to work with cheap materials and make do. This is going to be one huge experiment, and I don't know if I will be successful. But I'm going to try very hard to be, so I can get it right the second time around.

Here is the timeline I have created for these next few weeks.

1. Collect materials (2/24-3/10)
2. Mix solution (2/24-3/2)
3. Add agar and heat (3/9)
4. Add to baby food jars (3/9)
5. Autoclave baby food jars (3/10)
6. Collect explants (3/16-3/17)
7. Sterilize explants (3/16-3/17)
8. Wash explants (3/16-3/17)
9. Add explants to jars (3/16-3/17)
10. Seal jars (3/16-3/17)
11. Wait (3/17-4/1)

Well there's all the introduction. I've done a lot of research with resources like the Home Tissue Culture Group and Phytotech Laboratories. Honestly, all this information has blended into my single understanding, so I am not able to cite where every idea comes from. I'll begin the description of the process I am going to follow with a list of materials. The bolded items are those I have already acquired.

General Materials:

• A plastic box as a pseudo-sterile hood (see picture below)
• 91% isopropyl alcohol
• 70% isopropyl alcohol
• Hydrogen peroxide (3% I believe)
• 8.5% bleach
• Spray bottle to sterilize surfaces
• Coffee filters to hold samples during sterilization
• Saran wrap
• Baby food jars
• Long (10”) forceps (tweezers)
• Pressure cooker (by using the 15psi and high temperature conditions in a pressure cooker, I can sterilize things that would be hard to clean with chemicals)

Nutrient Gel (as detailed to me by a very experienced California biologist):

• Table sugar (sucrose): 1/8 cup
• Distilled water: 1 cup
• 10:10:10 fertilizer (1/4 teaspoon dissolved in one gallon of water). Use 1/2 cup of that mix.
• Inositol tablet (250 mg) -- use 1/2 of tablet
• Vitamin tablet with thiamine -- use 1/4 tablet
• Agar flakes (amount varies with manufacturer)

In terms of procedure, I will have to measure this out in an old pan on my stove. I'll have to dilute it by 1/2 or 1/3, because carnivorous plants do not like as many nutrients as other plants. Once I have everything together, I will heat and stir until it is one homogenous mixture. I will deposite about 20mL of this into each baby food jar, and then place it into the pressure cooker for 25 minutes. This will completely sterilize them and the media.

Once I have the gel prepared inside the baby food jars, I will have to construct my sterile area. This can consist of a plastic bin with a small rectangle cut out of the side. If the bin's open side faces down, then and I sterilize the inside with the bleach solution listed before, I can safely work inside without fear of bacteria falling into the jars to contaminate the tissue. It should look like this:

Once I have sterilized this area, I will sterilize the cuttings of my sundew, and add them to the jars. Then I will wrap the jars in Saran wrap, place them under lights in my basement and hope for growth. I have made a very rough procedure to sort out the basic steps of what I have to do and where I have to do it, either in the sterile box or outside of it.

OUTSIDE STERIL WORK AREA

Making the Nutrient Gel

1. Mix together ingredients for the nutrient gel.
2. Add an extra cup or two of water.
3. Add agar.
4. Heat and stir until every ingredient is dissolved.
5. Use syringe and transport 20mL of warm solution to each baby food jar.
6. Close jars and put them in pressure cooker.
7. Autoclave for 25 minutes.

Collecting Tissue

1. Cut apart explants or find seeds.
2. Place seeds in coffee filter.
3. Wash samples in soap and water for 10 minutes. Rinse thoroughly.

INSIDE STERIL WORK AREA

Sterilize Samples and Add Them to Jars

1. Dunk cuttings in 91% alcohol extremely quickly. Skip this for seeds.
2. Dunk cuttings/seeds in 10% bleach solution for 5 minutes. Put seeds in here for 4 minutes.
3. Place in sterile jar of water (sterilized in pressure cooker) and shake for 2 minutes.
4. Pull samples out of jar with sterilized forceps.
5. Slightly open TC jar.
6. Place explants in jar extremely quickly.
7. Tightly close jar and wrap lid in Saran wrap.

STERILIZATION TECHNIQUE

1. Using tiny spray bottle, spray 10% bleach solution on every surface inside the work area.
2. Wipe down jars with paper towel of 10% bleach solution.
3. Wash hands with soap and water for 2 minutes, then wipe with 70% alcohol.

Well that's about all of the information I have at the moment. This week I intend to gather all the remaining materials, construct the sterile work area, and begin mixing the gel solution. Hopefully I have some interesting experiences for next week. Until then...