by Joe Coleman
Plants available from tissue culture developed rapidly from the 1970’s and 1980’s as techniques were refined and protocols established. Begun primarily in university and college scientific labs, the practice has expanded into the commercial and industrial realm. The first commercial use in the 1950's was developed for the mass propagation of orchids. Since that time, specialized media have been developed for most other plants. As demand for larger numbers of new hybrids has grown, the fastest method of production remains tissue culture.
While the term "tissue culture" applies to several related methodologies, most frequently it is a means to propagate plants by growing them as shoot cultures derived from the parent plant. The term ‘tissue culture’ is actually a misnomer because plant micropropagation is basically concerned with the whole plantlet and not just isolated tissues, even though the tissue may come from a particular plant part. In an environment free of microorganisms and provided with a diet of balanced chemicals, a portion of a plant called an explant can produce plantlets that can in turn multiply indefinitely, given proper conditions. The growing medium, which may be a liquid or gel, is a mixture of chemical compounds that form a nutrient rich base for growing cultures whether cells alone, organs, or plantlets. There are four stages of growth:
- Initiation or explant establishment
- Hardening off into the environment.
These stages can vary widely from plant to plant and often overlap. When material is started in vitro or in glass (test tube or baby food jar or similar container), all of the offspring from a single plant is classified as a clone. All cutting grown plants are also clones. These have the same genetic material as the mother plant.
Plants raised from seed will not be identical to the seed parent but may have some characteristics of the pollen parent and some characteristics of the seed parent, even if the characteristics are dormant in the parents. Even in clones, a mutation can cause a change in characteristics. A mutation is a sudden abnormal change in the genetic order that will change some characteristic or even the basic number of chromosomes.
Reasons for Tissue Culture
Tissue culture is often the only practical way to produce the large numbers of plants required. If propagating by seed, an ample supply of viable seed is absolutely necessary. If using cuttings, a large source of quality material is necessary. Both require labor and expense to produce. The tipping point may well be the number of plants needed. Once you pass the thousand mark, tissue culture becomes the more attractive method. Laboratory conditions are ideal for growth and development, and can be scheduled for release at the best growing time for a nursery. One explant can in theory produce an infinite number of plants, thus fewer stock plants need to be maintained. Most woody plants take several months to start multiplying, but as they double the number every month, a single explant can provide 1024 plants in 10 months and 2048 in 11 months. Initially, tissue culture avoids extensive nursery care at first while under glass, but as they are grown along, they will have the same basic requirements as all growing woody plants ̶ watering, up-potting, fertilization, weeding, and supervision. There are also a number of plants that are extremely difficult to propagate by usual cutting procedures. Kalmia or mountain laurels would never be really marketable except by tissue culture production. Seed propagation is quite slow and lacks the advantage of being able to produce identical plants. Cutting propagation of Kalmia is very difficult.
Commercial establishments are compelled to make tissue culture a profitable venture. Whether taking plant material from a test tube all the way to a three gallon plant or merely establishing plantlets that can be sold to nurseries for growing on, the prime need is a market for the plants. Most tissue cultured plants are true to type, more vigorous, more disease resistant, and disease free. The end product, produced in quantity, costs less than the conventionally propagated plants.
The absolute minimum to maintain the clean conditions is a laboratory, the size of which is governed by the expected use of the facility. Whereas very simple work can be done by a hobbyist even in a kitchen as one basic course advertizes, in working with woody plants you must be prepared to create a clean laboratory space free of dust, smoke, molds, spores, and chemicals. Air quality and direction of air flow are a primary concern. A transfer chamber or hood is necessary to provide a positive air flow of filtered air, free of contaminates. All work in vitro should be done under the most sterile conditions possible, otherwise you will be growing a variety of beautiful fungi. A clean area for preparing medium with necessary chemicals, a quality scale for weighing minute amounts, pH meter, sink with hot and cold water, a refrigerator, stove or heating source or a hot plate with stirrer, storage area for glassware, and a clean, warm well lit growing area are necessary. Ideally, the growing area or transfer room should be separate from heavy traffic areas to help prevent contamination. The air in these areas should be filtered to reduce the particle count. Provision needs to be made for using sterile water in all applications.
An autoclave or large pressure cooker is important in medium preparation. A hot bead sterilizer is a useful addition to the work area under a hood, as well as alcohol spray bottles, good lighting, and when needed, magnifying glasses or scopes for close work. Also quite necessary is a labeling system that can follow the material through every stage of development. If you think that this operation is cheap and easy, now is the time to disabuse yourself of that concept. This takes money! And having invested in every necessity, you must still be prepared for total disappointment!
So You Don't Like Chemistry
The difference between success and failure is pure chemistry, like it or not! Fortunately, the research has been done so that that the formulas for most media are available, and the chemicals can be obtained from reputable sources. What is necessary is a basic knowledge of weights and measures in order to follow formulas in media preparation. Required is an understanding of pH values and how to adjust it using sodium hydroxide (NaOH) and hydrochloric acid (HCl) solutions or similar basic and acidic solutions. A number of the inorganic chemicals are found in most fertilizers and are essential for plant growth: calcium, iron, magnesium, nitrogen, phosphorus, potassium, and sulfur. All must be present for if one is missing, the presence of the others is useless. Minor elements need to be available in minute amounts. These micronutrients or trace elements are essential for good growth and are present in water, soil, even dust, but can be quite toxic if found in excessive amounts. Such elements as boron, chlorine, cobalt, copper, iodine, manganese, even zinc promote healthy growth when in tiny amounts. The most important chemicals are the organics: carbohydrates, hormones, proteins, and enzymes. All contain carbon, are mostly insoluble in water, do not conduct electricity in water, can burn and are slow to react. Plants normally make their own organic compounds so it is rare to feed them to outdoor plants, but in vitro tissues need to have these substances added to give a little oomph to the tissue growth! Vitamins, growth regulators, plant hormones like auxins and cytokinins, gibberellins, amino acids, and antibiotics just add to the complexity, some to be used initially and others to be added later to promote a steady growth and maturation. For an established lab, the list of preferred chemicals is extensive, and the problem can arise of keeping them fresh and up to date. Just order what is absolutely necessary, follow the medium recipes, and leave the experimenting to researchers.
Keep It Clean
After establishing a clean lab and having prepared sterile media, the most difficult problem remains before you. Obtaining clean explants for culture requires great diligence. The explant may be meristems, shoot tips, macerated stem pieces, peduncles (flower stalk pieces), anthers, petals, seeds, stolons, root pieces, leaf or flower bud tissue, etc. Only disease free plants can be used in tissue culture.
In preparation, stock plants should be moved into a greenhouse or other shelter where they can develop in a controlled environment, free of dust and disease. Their cultural needs must be monitored and the plants fed nutrition at the highest level. Taking samples from plants outdoors may be the only option in obtaining material from the wild, but be prepared to perform extensive cleaning procedures. Such plant material is very dirty, particularly under trees with rain, wind, fog, etc. Yet, sometimes forced dormant material (i.e., buds grown on wild-collected plant samples) can yield clean new growth which can be used for explants.
Basic rules to follow include: the smaller the sample, the less contamination; new shoots are cleaner than old wood; plant material at the base of a plant near the ground can be younger than that at the top; explants are cleaner in the morning; material should be refrigerated until use; and root material is always cleaner in an artificial medium. The most common disinfectant is bleach, from 5% to 12%, with the addition of Tween 20 as a surfactant shaken for several minutes. For dirty samples, such as those collected in the wild, several sessions of cleansing are necessary followed by distilled water rinses to remove contaminates before putting the explant material into culture. Success or failure depends on the cleanliness protocol in place.
The complexity of tissue culture can scare off the hobbyist from making use of this method of propagation. Most home practitioners do not have a need for thousands of the same plant or to maintain a repository of cultures, but it might be possible to propagate a plant from the wild that might not respond to cuttings or yield seeds for propagation of siblings. With the complexities, it would be much easier to have a commercial lab start the process and leave the growing on to you. A number of labs have already started doing this and have side operations for distributing tiny, tender plantlets at a fair price. Perhaps it is the challenge that inspires the hobbyist to continue trying tissue culture.
Kyte, Lydiane and John Kleyn. (1996). Plants From Test Tubes. Portland, OR: Timber Press.