Agar Substitutes

Agar is one of those staples of the tissue culture industry we generally can not do without. Agar is a gelatin like substance which is derived from common seaweed. You can find it used in many old fashion and modern desserts such as those found in Japan and are known as anmitsu.

Agar has traditionally been employed universally throughout the world as a medium by which cultures of bacteria or fungus could be readily grown. Usually this product is sold in bulk quantities as a powder and mixed with water for use as it is required. Although agar is the product of choice there are always active amateur scientists that are looking for alternate items to use. I personally have used common grocery store purchased unflavored gelatin as a suitable substitute for agar. It worked very well and I was able to culture quite a variety of plants from it.

Several years back I heard of an individual who did some work on the use of sand in place of actual agar. Naturally I had to try this method out and also found that it too worked as expected.

The procedure for using the sand is to start with the type of sand that is generally employed for a child’s sand box. It is frequently referred to as play sand. Prior to using the sand in any sort of tissue culture protocol you should wash it well to remove any visible contaminates. Next place approximately one ounce of the sand into each of your culture flasks or baby food jars if you are using them for your cultures. Seal the top of the jars with a strip of aluminum foil and place it on a cookie sheet. Your oven should be set for 350 degrees and you should let the jars remain in it for at least one hour.

The jars should be cooled and approximately 30 ml of sterilized medium should be added at this time. The key is to add enough of the medium to saturate the sand but not so much that you cause your explants to float around. Finally you will want to aseptically close your jar with a sterilized lid. I boiled mine and then place them in the oven for a period of time in order to kill off any contaminates.

During the course of your tissue culture experiments if the culture starts to dry out you should then add some additional sterile medium to the container. Naturally you will want to add other items such as nutrients, etc to help with the explants growth. You should at this stage of the game use the sand as if it were the actual agar. If you would normally add a specific ingredient to your agar then add it to the sand.

You should have no problems using the sand in place of the agar and I personally have seen some very interesting results while using it.

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Fern cultures

Several people have written to me for information relating to fern spore cultures. I am not an expert at this phase of micropropagation and have only a vague familiarity with this type of cultured plants but here is what I do know. Hopefully it is of use to you.

Although there are currently at least 20 different species of ferns which are found here in America, I would venture to say that the same process could easily work with any of them. So without any further wait here is the information.

The fern spores are usually collected in the late autumn from mature fern plants. We will use the domestic “Platycerium superbum” as an example. The spores would naturally have to be sterilized and this is usually accomplished by way of a centrifuging technique. The spores are quickly shaken in a test tube using sterile water with a couple of drops of Tweeen 20 added. If you have access to a centrifuge you could place the tube in it and rotate it at the rate of 2800 RPM for 5 minutes. This will settle the spores to the bottom of the test tube. Next, discard the liquid in the tube and replace it with clean sterile water. Once again shake and let the tube sit at room temperature for a 24 hour period.

The following day re-centrifuge the tube and discard the water again. Now add 10ml of common household bleach. Let the bleach remain in the test tube for approximately 5 minutes. Finally, centrifuge again and discard the solution and place the aseptically clean plant spores into a media as described below.

These spores you will note were planted into the media without a final wash with sterile water. The cultures were then incubated for 16 hours. The temperature should be kept at about 25 degrees C.

This is a slow growth method and your spores will start to germinate and produce prothallus in about three month’s time. You will likely need to sub-divide these early cultures as they start to form. In a future article I shall cover construction of a home made centrifuge as well as an incubator.

This media should be successful for just about any fern cultures that you wish to try it on.

300 mg MS salts
500 mg NaH2PO4
5 mg Inositol
20000 mg Sucrose
8000 mg agar

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Using the Microwave on your Media

Experimenters are always on the look out for unique and unusual methods by which they can sterilize the particular media that they are employing for their tissue culture work. The time honored home lab process is by the use of a home pressure cooker. Although there is one easier and simpler method that is often overlooked – the use of the family microwave oven.

I have previously covered the use of a microwave oven to sterilize the plastic containers that are used for the process but in this article I am going to provide you with a new insight on using it to sterilize the media itself.

The procedure is essentially very simple. You merely place the container of your selected liquid media into your microwave oven and heat it to just below boiling which should be approximately 4 minutes. What could be easier? Keep firmly in mind that should you forget and allow the media to boil for more then several seconds you will have one heck of a mess to clean up in your microwave.

Several major variables that will affect your procedure are the amount of the media that you will be sterilizing and the maximum power output from your particular microwave. It will more then likely be necessary for you to do a bit of experimenting at first by using different power settings and time for your particular containers. You could fill your vessels with water during your experimental stages in place of actual media. It is usually a lot cheaper using a mixture of water, a little agar, 2 percent sucrose and some tomato juice when doing your tests. The tests if accomplished properly can teach you a lot of valuable information that relates to the problem of contamination and how you best can eliminate it.

You will want to be especially careful if your microwave oven has what is known as “hot spots”. In this case you may need to move your media round slightly to ensure that it sterilizes all of the intended media. You may actually end up increasing your heating time but however you look at it you are still saving a lot of time over the conventional pressure cooker method. A very useful item that you may decide to purchase for use in your microwave is the microwave pressure cooker called the "Micro-Go-Round". It is a microwave turntable made by Nordic. This does an excellent job of microwave tissue culture sterilization. If you are using baby food jars it can sterilize the media right in the jars.

Although this will work well for the home lab you would not want to use it if you eventually go professional as it is simply too small. The unit is 7 inches in diameter and 4 inches high. That’s about 87 cubic inches of useable space. Unfortunately as I mention you could not place too many Petri dishes or tissue flashes in this little marvel. You could place a small plastic rack of some sort in the bottom of it and include a bit of water in order to generate some steam which would exceed 100 degrees C.

Do not place a cap on your containers nor use any sort of aluminum foil as a cover. You can easily use some plastic caps on your culture tubes or a single hole stoppers on culture flasks.

You may be interested in knowing that the process has a tendency to work very well. Media that has been sterilized in this manner has not become cloudy as a result of bacteria and it shows no signs of developing mold even after several weeks of sitting.

The truth of the matter is that you may perhaps contaminate your project as a result of handling your explants as opposed to the media itself being contaminated.

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Don’t be a failure statistic

Failing in tissue culture experiments is very easy if you are slack in your procedures and careless when you do your initial explants setups. Some of the major reasons that have been cited for unproductive cultures are as follows:

1. Don’t select a difficult plant to clone initially. Leave that “Hemerocallis” alone and select one of the simpler plants to duplicate. You have plenty of time to work your way up to the more complex plant cloning procedures so take your time.

2. Don’t try and rush to get your explants out of their sterile home and into the open. Give them plenty of time to get accustomed to a new environment. It usually will take several weeks to condition your new explants to the open air life so properly pre-treat them in an enclosed system. Failure to heed this advice could cause contamination of the plant that can not at all be eliminated by the mere surface sterilization and ultimately lead to the destruction of your plant.

3. Make certain that you follow the correct procedures to ensure that you do not induce any sort of contamination. Following all advised procedures that pertain to sterilizing instruments and cleaning the various cutting surfaces properly. Make sure that you use your glove box as it was designed to prevent contamination while you are working. I have actually when first becoming involved in micropropagation used my glove box to grow several of the explants in.

4. Don’t try and rush the procedures. If a specific time is stated for leaving your explants in something such as detergent then by all means make sure that you adhere to that time specified. These are not arbitrary time frames but have been established by many trial and error experiments.

Growing tissue cultured plants can be a lot of fun and is an excellent way to spend your spare time so don’t become one of the failure statistics and make the usual mistakes that most newbie’s tend to make. When in doubt ask someone with a bit more knowledge as it will save you a lot of heartache in the long run.

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Sterilization of TC Containers by Microwave

I generally purchase my tissue culture containers by the case and it goes without saying that they can get rather expensive. The particular ones that I like to use are made out of plastic which tends to make them slightly more affordable then those created from pure glass.

I am especially fond of recycling whenever it is feasibly possible and it seems like such a waste to toss these perfectly good containers into the trash when I am finished with them. In the past the major disadvantage of the plastic containers has been the inability to properly decontaminate them and to be able to reuse it for additional cultures. By being able to do so greatly cuts the cost of the tissue culture containers down to a minimum.

With this thought in mind I decided it was time to reuse these vessels once again. Naturally, they would need to be in a sterile condition in order to prevent contamination of the next series of explants that would be developing within them.

It obviously seemed like a very good idea to use the microwave oven for this exact purpose. The microwave can effectively kill any type of bacteria present and successfully decontaminate the plastic containers for further tissue culture work. Tests have shown that our common home microwave is well capable of decontaminating these containers with only a 3 minute cycle.

Initially cleaning the plastic tissue culture containers is fairly easy. One merely uses their usual dish detergent and washes them well followed by a rinse in fresh water. Generally, this will take most of the residue out of the containers and provide you with a clean container for reuse. It is the sterilization of the containers that used to pose you the greatest difficulty however with the use of the small microwave ovens as described above you are well on your way to an effective means of sterilizing these containers quickly and inexpensively.

So as a home lab user it is now possible to save a lot of money with not having to repurchase your cloning vessels after each and every batch.

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Homemade Alcohol Lamp

Build your own alcohol lamp from easily available materials.

Items you will need:

1. A small jar with lid. Baby food jars work well but any jar will do.
2. A tool to make a small hole
3. Cotton Ball
4. 95 % Ethyl alcohol (ethanol)

Steps

1. Make a small hole in the lid of the Jar.

2. Pull apart a cotton ball.

3. Roll the cotton in your hands to make the wick. The wick needs to fit snug in the hole and reach to the bottom of the jar.

4. Now thread your wick through the small hole in the lid.

5. Now fill the jar with alcohol and put the lid on.

Done!

The wick should be sticking out about 1/4 in. The more wick that sticks out the bigger the flame will be.

When you don't use the lamp, store in a tightly closed plastic bag to prevent the alcohol from evaporating.

Original at: http://www.sporetradingpost.com/lamp.htm

Mixer for Test Tubes

In this video I will show you how to make a mixer for test tubes. It is pretty easy and you can build it in less than 10 min.

Mixer For Test Tubes Tutorial - The funniest home videos are here

Step 1 - Get an e-Motor and Magnets

Almost any type of e-motor will work but I prefer the tiny ones which come out of a CD/DVD driver. They work at 1.5 Volts and the rotation speed is not too fast. The magnet I use on the shaft of the Motor is also from a CD/DVD-driver (you can find 2 of them close to the lens).

Step 2 - Attach the parts

First superglue one magnet to the shaft of the motor. Do not glue shaft to the housing or the unit will not work any longer.

Secondly, mount the motor to the bottom side of the rack. I used hot glue. The magnet should be close to a test tube hole. If you have a different looking rack then you have to find a place near a tube and attach it there.

Step 3 - Connect the battery

This is an easy step. Just connect the wires to a 1.5 Volts battery. The polarity does not matter. You can also put a switch in the circuit to turn the unit on and off but it is your choice.

Step 4 - Put a magnet into the tube

Put a magnet in the test tube you are about to use. I used a, 1cm (~0.4inch) in diameter and 1mm (0.04 inch) thick, neodym magnet. But you can also use one of the CD/DVD driver ones as well.

Do not use bigger and stronger magnets or the glass will crack.

Common Sense Laboratory Equipment

As I previously had stated in another article I would start to let you know how to build or fabricate much of your own equipment. Quality equipment for a laboratory can be extremely costly and any time that you can construct your own you are likely to save a considerable amount of hard earned dollars.

In just about every bit of work that you will be doing in cloning plants involves measurements of some sort. It could be measures for bleach or for liquid dish detergent but the measurements should end up being accurate and honest. As such we will begin our lab supplies by creating some simple measuring devices.

Usually there will be no special equipment required in order to learn about cloning plants or micropropagation of plant tissue. A quick glance at your kitchen is likely to reveal a considerable amount of capabilities for your new hobby lab. Before you seriously begin any laboratory work what so ever you should learn how to make a few very useful items from your kitchen such as creating empty dispenses into storage bottles for your various ingredients that are usually needed in your cloning projects. Make sure that these bottles are always properly labeled and out of reach of small children. These various bottles can range from empty salad dressing jars to ketchup containers.

Being able to accurately perform measurement conversions is important. The following points may help you in this respect after all with a home lab you may not have access to graduates or pipets of any sort in order to measure volumes. Usually the metric system of measurement is employed in any sort of laboratory work so we will naturally follow suit and use the same thing here. I have included a list of approximations that you may wish to hang near your work area.

A set of kitchen measuring spoons can provide you with a vast number of measurements suitable for laboratory work. You can use a straight edge of your knife to slide off the excess material so that you end up with an exactly level spoonful.

* 1/4 teaspoon is equal to 1.10 grams (Packed)
* 1 teaspoon is equal to 4.35 grams (Packed)
* 1 teaspoon is equal to 4.25 grams (unpacked)
* 1 tablespoon is 13.0 grams (Packed)
* 1 tablespoon is 12.6 grams (Unpacked)

To create some quick and simple volumetric tools you should take two 500 ml soda bottles. Now fill the first one up to the neck with water. Pour the water into a second identical bottle until you show both at the same level. Mark both bottles at that point as 250 ml. Discard the water within one of the bottles and repeat the procedure again. This time mark the bottles as 125 ml on each bottle.

You can take a 1 liter soda bottle and using the kitchen measuring cup filled to 100 ml of water proceed to pour this water into the one liter bottle and mark it as 100 ml. Add another 100 ml and mark it as 200 ml. You can continue in this manner until you have completely filled the one liter bottle with water. You can perform a similar procedure on just about any bottle and mark them with 5, 10, 15, 20 and 30 ml.

As you can readily see you don’t need to spend a lot of money on equipment when you can use your imagination and come up with products around the home to use. Our next article will expand upon this idea a bit more

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Tissue Culture Part III

Dr. Sandy Wilson, Associate Professor of Environmental Horticulture at the University of Florida lectures on the topics of Tissue Culture and Micropropagation.

Tissue Culture Part II

Dr. Sandy Wilson, Associate Professor of Horticulture at the University of Florida lectures on the topics of Tissue Culture and Micropropagation.

Tissue Culture Part I

Dr. Sandy Wilson, Associate Professor of Horticulture at the University of Florida lectures on the topics of Tissue Culture and Micropropagation.

The Perfect Explants

The Perfect Explants By Joseph Parish

We have briefly discussed cloning principles in the past so now we are hoping to get into some of the finer details concerning plant cloning. Let’s start with the original plant that we wish to clone and learn a bit about how to select the perfect cloning material.

Cloning is one of the most effective means at our disposal for duplicating plants. It allows us to keep the best plants that we have and make thousands of the same exact species. In the words of a botanist we could say that clones are nothing more then the end results of asexual propagation as compared to seeds which are considered to be sexual propagation.

When we take a cutting from a plant to clone, we usually use a branch or fresh growing section of the plant. We then cut and place a small portion into a media of some sort and watch it take root. I can not express the feeling that one gets as they watch this piece of plant become an actual living thing right before our eyes.

Seeds are an unpredictable media for outcome. We never know what the end result will be when we plant seeds however with cloning we always know exactly what we will have for a final product after all just take a look at the parent plant! Naturally the first step of this process involves selecting the proper parent to begin with.

Select the parent plant by what ever characteristics you are looking for. Try and find the healthiest and the best sample for the species that you are selecting. You should select a plant for the parent that is strong and at least several months old.

I like to try and clean out my plant as much as I can prior to starting the cloning process although this next step is not completely necessary. I use it in order to clean the plant’s insides prior to cloning it. After I have selected my chosen plant I leach the soil with one gallon of water for every five gallons of dirt that the plant is currently growing in. I will do this for five days in a row before I start the actual cloning process.

You may have heard me mention the word explants several times in my articles and perhaps you are not sure exactly what it is. Explants are that portion of the plant cuttings which will be used to produce the clone. Select a young plant and look for the location of the bud that might appear on it. Find one that has not opened up yet. Cut the plant off about one inch from the end. If you have to transport the plant any distances from its current location to your laboratory area make sure that you use a plastic bag that contains a damp paper towel and wrap the plant cutting gently in it.

Keep in mind that these procedures explained below will be accomplished within the confines of your glove box and not in the open where your plant sample can become contaminated. Here is a list of the equipment and supplies you will need for this process.

Glove box
A supply of sterile water
Dish washing detergent or Tween 20
Baby food jars
70 percent alcohol
Tweezers
Household bleach

You should start with a pint jar ½ full of sterile water. I prefer to boil the water or to use a pressure cooker. As a last resort you can purchase sterile, distilled water from your local pharmacy.

To this pint of water you will want to add several drops of liquid dish washing detergent. In an actual laboratory they would use a product called “Tween 20”. This is nothing more then a wetting agent and you can achieve the same thing with the dish detergent.

Place the plant cutting in this solution and put the lid on the container. Baby food jars are excellent for use in a home micropropagation lab. Vigorously shake your container for approximately one minute and then pour liquid out. Rinse the plant several times with fresh water and repeat the procedure once again. Now dip the stem in a 70 percent alcohol solution for several seconds and once again rinse it well.

Take an additional container and place 30 ml of your household bleach diluted with 270 ml of sterile water. Add two drops of detergent to this combination. Now place the explants into this jar and once again shake it intermittently for a 10 minute period. After the ten minutes is up drain the explants and rinse in sterile water. Cover them again and shake well. Your explants are now ready for use in cloning.

It may seem like a lot of work but the key here is cleanliness and sanitation. The cleaner you can get your explants to be the less chances you will have of any sort of contamination destroying your cloning attempts.

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Cloning Plants

Cloning Plants By Joseph Parish

There may perhaps come a day when cloning a plant would be the only way that you and I can obtain one of our favorite tomato or pepper plants for our garden. As survivalists this can be very important to us in order to ensure our continued existence. In that case I would like to impart his article on plant cloning.

When we say we are cloning a plant we are not talking about physically going into the nucleus of the cell and modifying the DNA or any such procedures as that. What we are talking about is scraping the tissues in this case of a plant and effectively growing it in a sterile environment. Our sterile environment in this case is a cultured media.

Plants are in fact composed of live cells and when we have transferred a minute piece into a culture media we are cultivating these cells which ultimately will continue to divide until such time as it provides us with a perfectly executed genetic copy of our original tissue sample.

This is the technique which can be used on any plant which grows on the face of the earth. When a plant is brought in from nature to our laboratory setting we must provide as much of a sterile environment as we possibly can. This sterile status should include every item that will touch the explants as they are called from the Plant itself to the media.

About ten years ago, I wrote a technical paper on sterile techniques involving a micropropagation laboratory and the principles that I brought forth at that time are just as effective now as then. The object is to isolate the pure plant tissue from its natural contaminants which normally would coexist with it in the natural outdoor situation.

Your laboratory should consist of the following items of equipment and supplies:

A Glove Box for doing the culturing in
A fresh plant
A hobby X-Acto knife
Tweezers
Several Sterile Agar filled Petri dishes or baby food containers
A solution of at least 91percent Alcohol contained in a small dish
A paper towel soaked in alcohol
A cigarette lighter or alcohol lamp

The procedure is as follows:

Cloning is a very easy procedure and can be accomplished in your own kitchen if necessary. A glove box is an enclosed container where you can insert you hands and not infect the items inside. All the following procedures that I am going to explain to you should be completed in the sterile confines of the Glove Box. I can not stress enough that you maintain a completely sterile environment at all times.

Turn the glove box on and place all your equipment and supplies inside. Inside you will place the dishes of prepared agar which has previously been microwave in order to kill any possible pollutants. Also put your sample plant in the glove box as well along with your alcohol soaked paper towel and the remaining supplies. After completing this phase of the process wash your hands thoroughly with antibacterial soap.

Dip your x-acto knife blade into the alcohol and proceed to flame it with your cigarette lighter or alcohol lamp after which you will place it on the alcohol towel to keep it sterile.

Slice the stem of the plant in half lengthwise in order to open up the virgin tissue inside that has never previously been exposed to the open air. With tweezers carefully select a small piece of the plant tissue from within the inside of the stem. Place this small section of tissue in the container of alcohol. Let it sit for several minutes and then transfer it to the center of the agar that you have placed in the baby jar or Petri dish.

Place the x-acto knife into the alcohol once again and flame it as before. Repeat the above process three times for a total of three samples in order to guarantee you will have at least one successful growth.

Set the dishes in a safe place where they will not be distributed and check them everyday. Your explants should remain clean and you will eventually see what appears to be cells multiplying and forming a new plant. This plant is exactly the same as the sample plant that it came from.

There are a number of other variables which come into play as you clone various plants. These variables range from the nutrients that should be placed into the agar all the way to the growth process itself. If you happen to really be interested in this process please by all means ask me for further information, as this has been one of my major interests from years back.

Copyright @ Joseph Parish

Welcome to “Cloning for the Common Man”

Welcome to “Cloning for the Common Man”
By Joseph Parish

I have been involved with plants and “micropropagation” for many years now. To this day I am still awed at the prospect that I can take a single cell from a plant and create thousands of miniature duplicates, each and every one the exact image of the original.

No I am not a mad scientist nor am I some sort of crazed doctor. What I am is a botanist who enjoys the abilities that are bestowed upon us by the generosity of science.

In case you are not familiar with the term “micropropagation” then I will briefly explain it to you. The term refers to a method by which plants can economically be reproduced in large quantities. This system of plant reproduction permits a vast number of plants to be quickly propagated in a short period of time from a single high quality parent plant.

In theory, under laboratory conditions the growing portion of a plant or as we refer to it the meristem is sliced and artificially cultivated in a very sterile environment. With attention to detail this single sample can become hundred and thousands of similar plantlets. Naturally the amount of time that it takes depends upon the type of plant that the explants are taken from.

Although the actual techniques work best under laboratory conditions I intend to explain everything that you will need to know in this blog over a period of time. I will provide you with all the fine details on creating much of the required equipment that you will need as well as to provide you with formulas and other useful information.

No longer should mankind worry about a plant becoming extinct for we have at our disposal the means by which we can prevent this from happening. By following this blog you can successfully create clones of your favorite plants all in your kitchen if necessary. So I leave you with these thoughts in mind to consider until I place the next blog entry online. That entry will be a short how to on actually cloning the plants. I will not get into a lot of detail on it at his time but you will get the idea.

Copyright @ 2009 Joseph Parish