|Tissue culture of Orchids|
Orchids are beautiful, mysterious and slow groiwng pernnial plants which can be easily cultured in your home (lab). Their cultivation is simple as compared to other plants since their requirements are minimum for optimum growth. It is generally believed out of experience that orchids are destroyed due to over care rather than by neglect. However cultivation of all types of orchids calls for proper care and conditions for good growth and regular yield of flowers.
Sterile water will be needed to rinse plant pieces after they are soaked in bleach solution. Fill pint jar 1/2 full with tap water or distilled water. Place cap on loosely. Set aside until media preparation is finished.
Medium: Everything a living plant needs as a heterotrophic organism (that is, when growing in vitro) must be available to it through the nutrient medium. The nutrition is more specific and sensitive than for an adult pot-plant and for plants living in situ . The composition of nutrients differ somewhat between media, especially between media for epiphytes and media for terrestrials. Terrestrials generally grow in a soil with higher pH than pH-values that epiphytes are used to (adult plants). In culture, the pH has to be somewhat acidic, since the up-take of some salts is more or less restricted at higher pH-values. Tropical epiphytes are usually cultured in media with higher nutrient values than media suitable for terrestrials.
The purpose of the clean area: It is to limit the number of particles that fall into your tissue culture jar. These airborne particles carry bacteria and fungi, and can kill your plant tissues because they grow faster than the plants. A clean area can be made from a plastic-lined cardboard box or a plastic storage box with a "window" cut out.
The inside of the clean box and the surface of the clean area should be wiped down, or sprayed, with 70% alcohol or a 10% bleach solution. All items that are put into the clean area (media jars, bleach container, sterile water jar, "dipping" alcohol) need to be wiped down, or sprayed, to get rid of possible contaminants. Hands should be washed in soap and water for at least 20 seconds, and then wiped with 70% alcohol. Dip or soak instruments in 70% alcohol.Gelling agents and support: Orchids are grown on some kind of gel or other solid substrate when cultured in vitro . Agar is used to concentrations of 5-7g/L (sometimes up to [14g/L]) to produce a solid gel. Other gelling agents (gelrite, agaros, gelatine) and supporting inert substrates (such as sand or cotton) can of course be used.
A gel that is re-heated (i.e. melted) may not always solidify again. Higher concentrations of salts may require higher concentrations of agar.
Inorganic salts of macronutrients, e.g. N, P, K, Ca, Mg, (of which the plant has a large need of) and micronutrients (such as Fe, Mn, Cu etc.) make up the base of the medium (basal salts). Tryptone is sometimes added. It's a mix of amino acids to increase the nitrogen levels. There are some evidence for better growth in cultures partly enriched with organic nitrogen.
The micronutrients are especially important for the function of various enzymes and many processes going on in the cells. Iron (Fe) is an important 'nutrient' for all green plants, mainly because of its importance for the synthesis of chlorophyll. (Chealated iron is used as the iron salts are difficult to dissolve). In the old days, various salts of iron were used in the media, which meant that the most of the iron pretty much was useless for the plant). (Duchefa, 2004)
Energy for a heterotroph: A medium probably contains some source of carbon (for energy). Cells that has the ability to photosynthesise, suddenly find themselves in a container where the carbon dioxide levels decrease and where a lot of free sugars are available, naturally they go for a heterotrophic life style. There have been various studies on which carbon source that is the best for culturing orchids. It seems like sugars can influence the growth in a positive and in a negative way and that sucrose is one of the better sugars as energy source for the cells. Sucrose is added to a concentration of 20g/L (that's 2%). myo-inositol is often added to [100mg/L] and is a cyclic alcohol that has proven to be a good additive, promoting the growth; especially for monocotyledons (such as orchids).
Vitamins: Vitamins are important in small amounts. Niacin (nicotinic acid) [1,0mg/L] is probably the most important one (Arditti, 1993). Thiamine (B1) [10mg/L] and pyridoxine (B6) [1,0mg/L] are two related vitamins which seem to have effects on the growth as well. Other often used additives are: hormones, amino acids, and certain complex organic additives (see further).
pH: The pH-value of a medium should be somewhat acidic (5,25-6,25) when culturing epiphytes. It's possible to change the pH with a diluted acid (HCl, HNO3) or a diluted base (KOH, NaOH, Ca(OH)2). Buffers can also be used, e.g. MES [1g/L].
At a 'wrong' pH-value the uptake of nutrients are less efficient. Some salts are insoluble at certain pH-values.
Complex substances are sometimes added to the media. E.g. potato extract, beech sap, banana pulp; [80g/L], coconut water [10-15cl/L], tomato juice, orange juice (citrus cultures), fish semen (contains adenine from the nucleic acids), and pineapple juice.
Extracts and juices may give extra sugars, vitamins, or other growth stimulating factors (e.g. hormone-like substances etc.).
Antibiotics: Antibiotics are used by some professionals to inhibit growth of contaminants. There are many substances that can be used and most of them are only sold to certified laboratories. PPM is however a product which has won the trust of many growers. It's usually used at concentrations of 1,0ml/L. PPM has its own website for further product information.
Phenolic substances: A common problem in cultures is the exudation of phenols. (This is in most cases due to an iron deficiency. (Duchefa, 2004)) Phenols can kill the cells or explant or inhibit the growth. A sign of an exudation is a spreading black or greyish contamination of the media, localy around the explant. The larger size of the explant, the higher is the chance of an exudation.
Some substances absorb phenols, activated carbon is one good example (Carbo medicinalis) which easily can be added (concentrations of ca. [0,5-2g/L]). Activated carbon is likely to interfere with other additives as well (Arditti et. al, 1993).
Ascorbic acid (vitamin C) has an inhibiting effect on the exudation of phenols. The explant can either be pre-treated with an ascorbic acid solution (before inoculation) or one may add ascorbic acid to the medium (Arditti et. al, 1993)
Ventilation: Smaller plants growing in sealed containers must be able to exchange gas with the atmosphere. Plants can be choked to death! Cotton can be used as stoppers if no bacteria-safe filter is available. However, cotton doesn't stop bacteria (which basically means that something protecting the cotton might be good to use, e.g. aluminum foil). It's important that the cotton plug is dry (bacteria or fungi are more easily spread through wet cotton).
Micropore sticking plaster (e.g. 3M's Micropore) can be used if no other filter-material is available. Remember though that it isn't bacteria and spore safe (it's 'non-woven' and doesn't protect good enough). A few layers on a small hole would probably be enough.
A small hole is sufficient. An opening that is too large would let much moisture out of the sealed space and make the medium dry (eventually kill the plant).