Countertop Tissue Culture Micro propagation
By Lynette Morgan 9 May 2018
Takeaway: Just as hydroponics was once the tool of scientists until growers modified the technology for their own use, tissue culture is now being carried out in home kitchens, on spare countertops, or in modified indoor growing areas.
Tissue culture, or micro-propagation, is a high-tech tool for the rapid multiplication of plant material. However, it is no longer primarily the domain of commercial labs with expensive equipment. Basic tissue culture is relatively simple and easily adapted to indoor gardens where artificial light and a clean, protected environment are readily available to facilitate the process. All growers need are steady hands and attention to aseptic procedures, and they’ll be rewarded with rapid production of clones or the multiplication of difficult or slow to propagate species.
The term “tissue culture” describes a wide range of procedures that all involve taking small parts of plants, tissues, or cells and growing them inside sterile containers in which the environment can be carefully controlled. Some methods are highly advanced and more suited to being carried out in a lab as they require specialized equipment. These include cell suspension culture and protoplast culture. However, basic plant multiplication is relatively easy on most plant species.
The medium most commonly used in tissue culture is agar gel, which holds the plant material in place at the base of the flask. This gel substrate provides all the nutritional requirements of the new tissue as well as directs growth and development of the new plantlets by containing various hormones. The most commonly used hormones in agar media are cytokinins to stimulate shoot development and auxins to stimulate new root development. An agar medium will also contain carbohydrates, such as sucrose or glucose; vitamins, such as thiamine (B1), nicotinic acid, and pyridoxine; and other compounds essential for the species being cultured.
Advantages of Tissue Culture
For indoor gardeners who have a few prized plants that require propagation, tissue culture is a method worth considering. Growers who use vegetative propagation usually select their best quality plants to multiply, but a single plant can only provide a handful of cuttings, making bulking up numbers slow. However, using basic tissue culture methods, even a small grower can rapidly produce high numbers of clones from a single plant, either for their own use or for sale. New plantlets, once they have come through the culture process, can be easily shipped to other growers and even around the world while still in their sterile tissue culture flasks. In fact, many orchid varieties can now be purchased as flasks of tissue cultured plantlets, which are ready to be shipped around the world to new growers, potted up, and grown on. Overall, it’s a cost-effective way of buying and selling newer plant material. Tissue culture flasks can also be used to store or hold plantlets maintained in a sterile, disease-free environment in-vitro as future propagation stock, taking up less space than mature specimens.
The most widely used tissue culture method is based on adventitious shoot formation, where a small piece of plant (root, leaf, stem, bulb scales, or similar) is taken and induced to produce many small shoots through the application of the correct plant growth hormone. Normally, such plant parts would not produce new shoots, let alone masses of them, but the conditions inside the tissue culture flask and the application of a plant growth regulator stimulates this growth to occur. These shoots, once sufficiently developed, are divided up into individual clumps and grown on in another flask where they are induced to form new tiny roots by application of another plant growth hormone. From there onwards, the young plant is grown on until it is large enough to leave the protected environment of the flask, be potted up, and grown on in a nursery situation.
The tissue culture medium inside sterile flasks can also be used to germinate very small seeds or spores. Orchid seeds are so small, they appear like a fine dust and have embryos that are not fully developed. In the wild, these seeds are dependant on a symbiotic relationship with certain microbes in the bark of trees, which provide nutrition for germination. However, orchid seeds can be raised on tissue culture media that supplies the inorganic salts and sugars required for germination. Fern spores can also be raised in this way and the use of tissue culture media and aseptic methods improves the rate of multiplication of ferns from spores.
The basic process of plant multiplication with tissue culture starts with healthy, well-grown stock plants from which the propagation material is taken from. In this example, we’ll use African violet, as it is an easy-to-work-with plant used by many to develop their culturing skills before venturing onto more valuable species. The first step is to cut a mature leaf from the parent plant and sterilize this material by immersing it in a solution of diluted bleach. This kills any surface microbes that would otherwise contaminate the plant material inside the culture flask.
The leaf is then rinsed with sterilized water and further prepared inside a sterile still air cabinet, or “clean box,” which is used whenever working with open flasks and transferring the plant material. In commercial tissue culture labs, a laminar flow hood carries out this role of preventing microbial contamination of the sterilized flasks and media by passing filtered air from the rear of the hood outward on a positive pressure gradient. In small or non-professional settings, a countertop cabinet can be made using a glass fish tank on its side, a large plastic container, or even a do-it-yourself cabinet made from plastic film stretched over PVC piping. These enclosed transfer boxes work well provided they are regularly sterilized by a misting spray of bleach solution or other disinfectant. The still air cabinet needs to have sufficient working space, and dimensions of 24x20x20 inches is usually adequate for small propagators.
Inside the clean box, the sterilized leaf is cut into one to two centimeter pieces and transferred to the agar medium inside a culture flask or jar (baby food jars are recommended for this). The gel-like agar needs to be purchased pre-made or prepared ahead of time. The agar-based media is prepared by mixing together the liquid medium, agar powder, and other ingredients; placing the mixture into the flasks or jars; and heat treated in either the microwave or a pressure cooker for a certain length of time. All tools and utensils such as scalpels, forceps, and knives must also be sterilized in this way. Isopropyl alcoholor ethyl alcohol is also used to flame cutting blades before they are used on plant material to ensure no microbes contaminate the plant tissue.
Once the pieces of leaf have been placed into the culture jars and sealed (the lid must be closed before the jar is removed from the clean box cabinet), they are left to generate new shoots. During this period, African violet leaves first form small bumps. Over two to four weeks, these develop into a mass of tiny shoots, a process that is promoted by the hormones in the tissue culture agar media. The jars are often kept on shelves with lights positioned nine to 12 inches above them. Cool-white fluorescent propagation lights are ideal, as they don’t create a heat buildup inside the flasks. An ideal lighting period of 16 hours per day and temperature control is also important for this process.
Once many new shoots have formed, these are transferred to a new flask with a fresh culture medium that does not contain shoot promoting growth regulators. (Instead, this second medium often contain auxins, which allow the shoots to elongate further and roots to rapidly form.) The mass of shoots can be extracted from the jar using sterilized forceps and a sharp knife. They can then be divided or pulled apart into many individual plantlets. These divisions are then transferred into the new culture medium to develop for a few weeks under lights. This process is similar for many plants that are propagated from leaves.
Once the young plantlets inside the flasks are sufficiently developed, they are taken from the culture flask and carefully hardened off. (The plantlets inside the jars or flasks have been protected with warmth and high humidity, so they are initially very tender.) This process applies both to growers who have purchased flasks of plantlets from a tissue culture lab and those who have carried out their own micro-propagation. The hardening off process is carried out using hydroponic methods; that is, using sterile soilless media under low-level natural or artificial fluorescent lighting in a protected environment. The plantlets are removed from the culture jar or flask, and any excess agar media is washed away. The young plant can then be potted into small containers or trays of sterile media. Fine-grade perlite or perlite/vermiculite mixtures work well for this process, as does stone wool and similar hydroponic media. The plantlets of most species can then be placed in a high-humidity tent, in a plastic bag for small pots, or under intermittent misting to retain humidity as the plant adjusts to normal growing conditions. Initially, only distilled or RO water should be given for the first few days, then a dilute quarter-strength seedling nutrient can be applied. Young plants typically require four to six weeks to harden off sufficiently. As they grow, light levels are increased, the humidity can be gradually lowered, and the nutrient can gradually increase in strength.
Materials and Supplies
For those new to tissue culture, a basic starter kit can be purchased from some suppliers and, increasingly, from hydroponic retailers. Kits are a good way to initially develop culturing skills, as all the hard work of sourcing agar medium ingredients, suitable jars or flasks, and other equipment has already been done. For those who are a little more DIY, many online retailers selling home tissue culture supplies can provide specific tissue culture agar mediums for different plants species and procedures, such as shoot multiplication, root development, and growing on plantlets. This removes the complicated step of formulating an agar medium for home propagation.
Tissue culture methods may seem a little complex and challenging at first, but once the basics of preparing plant material and a good understanding of aseptic methods have been developed, the process can be highly rewarding. Not only can favorite or carefully selected plants be cloned rapidly in high numbers, but more advanced methods can germinate difficult seeds or spores, produce virus-free planting stock, and play a vital role in ongoing plant breeding and improvement programs. Small-scale tissue culture is also well-suited to an indoor garden environment where artificial lighting and carefully controlled conditions can be provided to grow new plantlets from tiny pieces of excised tissue.