Tissue Culture

When mentioning the term tissue culture, some may conger up images of replacement organs being cultured in dishes, or racks upon racks of genetically identical test tube encapsulated plantlets growing in a laboratory setting. Maybe or maybe not, but greenhouse growers have been taking advantage of micro-propagation techniques such as tissue culture for years. Many home growers may also take advantage of these techniques by initiating changes in cell division in plant tissue following sterile cultural practices. Some commercially grown plants are too difficult to propagate by more traditional methods, so tissue culture can be an alternative. Saskatoon berry plants and orchids are an example. Seeds can be germinated in sterile culture conditions to ensure that they are in virus free, reducing the risk of infecting apparently healthy stock. Once desirable and uncontaminated cultures are developed, the plants can be propagated infinitely and even held in a sort of suspended animation for gene preservation in a very limited space. Some viruses, if present, can be eliminated from the plant tissue, so future divisions are virus free. Growers who have been growing a cloned variety for years only to discover some virus has begun pissing in their favorite gene pool can attempt eradication.

Only the very smallest stem tips (aprrox. ¼") from actively growing stem tissue are incubated at very high temperatures (32 to 40 degrees C) in an attempt to rid the tissue of the virus. The theory is that the plant material can survive a higher temperature than the virus or other infection. This has tremendous possibilities for preserving pedigree breeding stock.

One of the most common applications for tissue culture is the rapid production of many identical, disease free individuals from a single donor. This greatly increases the grower's chances of producing successful crops with fresh, new stock each time they plant. If you are considering some experimentation with tissue culture, it is important to have a basic understanding of the change in plant cell development and how it can be influenced to the growers' advantage. A good example is the formation of roots from a stem cutting, or commonly, "cloning".

In basic theory the following changes occur: The cutting is taken from an actively growing shoot portion of the plant, the cut portion is treated with a root stimulating auxin, and fed the appropriate nutrient diet under appropriate environmental conditions and eventually, roots are formed.

On more of a "micro" level, you may look at the same scenario this way:

Plant tips, or apical meristems, have specific cell growth and division. Specialized, actively dividing cells produce the vegetative growth that we have programmed the pant to produce through the manipulation of environmental factors. By the addition of auxins to stem cells and changing the environmental conditions, we promote root development. In doing so, we have triggered the plant to produce hormones to regulate that specific type of cell development. Tissue culture helps to take some of the focus from the environmental triggers we provide, but initiates the hormonal changes directly. Cytokinins are produced by the plant in the root tips, and regulate the development of stem tissue in the plant. An increase in cytokinins will typically increase the rate of branching and growth in a plant. Auxin concentrations, produced in the upper growing tips (apical meristems) influence the rate of root development. Note that apical meristematic tissue is the actively dividing cell region in both root and shoot tips. So, if we take a section of actively dividing, specialized plant cells, and dedifferentiate their growth (they no longer "know" what type of cells they should become, ie. root or shoot), we can then chemically influence what type of cell they are to become by additons of hormones such as cytokinin and auxin. This means that green shoots can be developed from tiny portions of root tips or vice versa. One of the technique using these principles is holding an unrooted growing tip for many years, producing thousands of identical offspring for production, without ever even initaiating roots on the parent material. Thousands of future production plants can be stored in the same space that used to only house space for hundreds. Growth regulating substances can be derived from naturally occurring sources or by the addition of synthetics. This article is only a glimpse into tissue culture, and does not deal with the potentially carcinogenic and mutanagenic synthetics which most home users are not equipped for.

All materials, equipment, and handling must be sterile. Each time you touch anything, the instrument must be sterilized over an ethyl alcohol burner.

The plant material is suspended in an agar (fortified gel) medium in a sterile test tube, or for the home user, a sterile baby jar, new pharmacy containers, or transluscent film canisters. The containers are kept in room temperature with 18hrs on / 6 hrs off, of gentle, fluorescent lighting. Cooler temperatures can suspend cell development for storage and preservation of genetic material without ever having to put the plants into production. All areas used in tissue culture must be sterile. It cannot be over emphasized the level of diligence that is required to produce uncontaminated cultures. There are a lot of books on detailed tissue culture practices. Many websites are an excellent resource.

The first step is to have the agar medium prepared, the recipe is from Dr. Acram Taji's website. (jars must be sterilized in a pressure cooker, and must be kept sterile throughout all handling):
2-cups rainwater
¼ cup of sugar
½ tablespoon complete, general application hydroponic nutrient in 1 Litre of water -use 250ml of this stock for this recipe.
½ a 500mg tablet of Inositiol (human vitamin supplement)
½ Thiamine tablet (human vitamin supplement)
4 tablespoons (60ml) agar flakes. This is the geling agent. It can be found in health food stores and Asian grocery stores. Make sure you get unflavoured and uncoloured.
Add ½ cup of coconut milk for the multiplication media (for dividing stem pieces). For rooting (after you have divided the plant the desired amount of times) use ½ cup coconut milk and ½ cup malt.

Mix the above in a sterile, stainless steel saucepan and gently boil until all of the agar has dissolved, stirring often. Check and adjust the pH as required. Put into a bay food jar to about 1-1/2" depth. Poke a small hole in the lid of the babyfood jar. Fill the hole with cotton soaked in alcohol, then loosely cover the lid with aluminium foil. This will act as a sterile "breathing passage". Put the jars with agar mix in a pressure cooker for about 15 minutes. A small piece of cotton soaked with alcohol should also be in the release valve, so when the pressure is released air that is drawn in will not contaminate the culture jars.

Meanwhile, you will have collected parent plant tissue along the following lines:

Spray the parent plant with distilled water and a wetting agent to rinse off any particles which may contain microorganisms, etc, which can contaminate the culture. Only the growing tips are required. Now you may quickly dip the tiny nodes into 70% isopropyl alcohol or 1 part bleach in 10 parts water. This will require some experimentation. If soaked for too long, the shoots will die. If soaked too quickly, the culture may become contaminated and must be discarded. Rinse with fresh distilled water and place the tissue into the culture dish and seal.

The above sounds easy, but carrying out these procedures without contamination is the real trick. Even the slightest movement of air in your area of operation sends thousand of spores into the air. A laminar air flow cabinet with a HEPA filter or glove box apparatus can be constructed at home. It can be as simple as sideways fishtank with a cardboard lid and rubber kitchen gloves inserted into the work area. Mushroom growers are very familiar with this concept. The quality and construction of your design will influence your success rate.

Your hands, clothing, and equipment must also remain as sterile as possible. Remember that you are dealing with open tissue, much like surgery. Your scalpel, tweezers, etc, should also be sterilized in the pressure cooker, as per the medium sterilization. Each time any equipment comes into contact it must be resterilized from a pass over an alcohol burning flame, and/ or dipped in bleach. Remember, that you are attempting laboratory procedures in a home setting, so methods that work best for you will need to be discovered by trial and error unless you intend to construct a specialized area in your growing facility.

Seeds can be treated much the same as plant tissue for ensuring clean stock. The seeds can also be soaked in bleach and follow the rest of the guidelines. Within hours or a few days you should spot contaminated and uncontaminated cultures. White or dark fuzz is a good indicator of contamination. After a few weeks in the tubes, the shoots should be ready for multiplication. Remember all you need is one little node. Your original may now have stretched to three or four nodes, giving you four new potential plants in a short period of time. Following sterile procedure, on a sterile surface, the growing points can be "surgically divided" and treated as before (soaking, new medium, etc).

Once you have about 1.5X the number of plants you will require, repeat the procedure but transfer into an agar medium with ½ cup coconut milk and ½ cup malt, as this will encourage rooting. After satisfactory root growth you will now need to acclimate the laboratory plants to the real world. Remember these plants may require a month or more to be ready to be put into production. Carefully rinse the agar medium away. Soaking the plantlets in a mild B-1 solution during transplant will help the tender sprouts from drying out during handling. Plant into a medium such as very fine peat or coconut coir. The plants will require very gentle light and very little air movement for the first 2 to 3 weeks before gradually increasing. Minimal nutrients and water will be required. An occasional foiliar spray should help perk things along. There, now through trial and error you have learned to create an army of identical superplants. There are many applications for micro-propagation. Cultures can be developed from plant fluids, genes can be mixed… the possibilities are virtually limitless.