People who are engaged in propagating new and sought-after plant clones meant for the commercial market find the tissue culture laboratory an excellent means to achieve their target. Tissue culture helps to increase a clone at a very rapid pace. For tissue culture, all that one needs is a solitary selected seedling and it can yield up to 2 million bulbs in just two years! A specific form of tissue culture is called embryo culture, which is undertaken with the particular objective of avoiding any premature death of hybrids obtained from any atypical, mismatched cross.
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Tissue culture is defined as a technique wherein plants are propagated in laboratories. It involves placing an extremely small amount of plant tissue in a disinfected and nutrient medium. Subsequently, various plant growth hormones are included in the medium with a view to stimulate the cells to proliferate as well as differentiate till the time they develop minute bulblets. Incidentally, this method of propagation has transformed the horticultural industry by allowing growers to produce vast number of progenies from a particular plant. It allows the growers to choose a specific seedling and produce hundreds of thousands of bulblets in just two years. These bulblets can be grown to a satisfactory size and marketed in another couple of years.
Generally it is said that tissue culture helps to produce plants that are free from viruses. However, the process involved in tissue culture is very complicated compared to what people usually think. When you select a plant tissue for culture, first you need to examine it using an enzyme-linked test called immunosorbent assay to detect whether it has any of the viruses common to lilies. In case you find the presence of any virus, you need to get rid of it by undertaking a process called meristemming, wherein minute pieces of quickly developing meristem tissues are eliminated, cultured and tested again. This method may take several repeated procedures and over a year to obtain genuinely virus-free tissues. However, when the plants grown by this method are out of the laboratory, they can be infected quickly. Therefore, the top priority is to breed lilies that are resilient to infections by viruses.
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It is worth mentioning here that usually the material that is used to commence a lily tissue culture is obtained from the bulb scales. In addition, stem segments having flower buds (internodes) can also be used for lily tissue culture. It is important to exercise utmost care while digging out a bulb if you wish to prevent forfeiting a valuable plant. While digging out the bulb, care should be taken to leave the stem unharmed in the ground, as it usually continues to produce stem bulblets there. On several occasions, unopened flower buds have been used to undertake tissue cultures of wild lily species already in the danger of extinction.
Ideally, the donor plant ought to be one that has possibly not come in contact with viruses. In addition, the material that would be used for tissue culture ought to be kept fresh and also stored in a dry condition in order to avoid any fungal growth.
After the material that is to be used for tissue culture arrives in the laboratory, its surface is sterilized by dipping it in a 10 percent solution of domestically used bleach for anything between 20 minutes and 30 minutes. Subsequently, the material is washed and severed into little square-shaped segments, which are known as "explants", measuring about 5 mm (0.25 inch). The lower half of a scale is employed for this purpose and it has been found that the part of the material that is nearest to the basal plate of the bulb develops bulblets most enthusiastically. Then the explants are placed in a medium in their respective tissue culture vessels, covered with plastic and stored at a temperature of about 21°C (70°F). Usually, a significant portion of the new cultures is unsuccessful owing to contamination by organisms present in the soil. On the other hand, it takes anything between four weeks to six weeks for the cultures that survive or are successful to generate visible bulblets.
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The medium that is used for growing the explants is basically a solution comprising basic fertilizer nutrients and some compounds similar to complex vitamins. Usually, a number of man-made compounds are also included. In fact, majority of the media used to grow lilies enclose 0.03 mg of a synthetic auxin called naphthalene acetic acid (NAA) per litre. Even table sugar (sucrose) is added to the medium with a view to provide the explants with vigour. The medium is usually made into a gel by adding agar or any patented agent like Gelrite. The concentration of sugar is considered to be the most vital aspect of the medium. The sugar concentration is responsible for supplying the entire energy that any average growing plant would obtain from photosynthesis. It is worth noting that tissue culture always takes place in complete absence of light. Compared to several other plants developed by tissue culture, lilies require more sugar. Therefore, sugar is added to the medium in the ratio of anything between 45 grams and 60 grams per litre. In the later stages of growth, when the bulblets are forming, the amount of sugar content in the medium can be raised up to 90 grams per litre.
As discussed earlier, tissue cultures are developed in total darkness and at temperatures ranging between 20°C and 24°C (68°F and 75°F). During this period, hardly any leaves are produced, as most of the energy is diverted to leaf production and the growth of bulblets suffer. Therefore, you need to ensure that there are very few leaves, if any, as it will help produce maximum bulblets. Following the initiation of the culture, it is tested over and over again with a view to ascertain that there are no viruses. Such testing continues till the technicians are somewhat sure that the culture is completely free from virus. The multiplication phase begins after this. During the multiplication period, the cultures are maintained in a manner that helps them to achieve very active growth. When the conditions are optimal, "sub-cultures" can be made from these cultures. In other words, the cultures can be divided into numerous new cultures once in every 8 weeks to 10 weeks.
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Precisely speaking, sub-culturing occurs when the cultures have already produced small clusters of bulbs, each measuring anything between 0.5 cm and 1 cm (0.25 inch and 0.5 inch) across. Eventually, the cluster of bulbs is separated and replanted in a fresh medium. The process of separating the bulb clumps is called "singulation". Each of these separated bulbs forms new clumps after about 8 weeks to 10 weeks.
On the other hand, you may also break down the small bulbs to obtain their individual scales. While this process will facilitate production of more bulbs, it actually needs a longer cycle. It is also possible to sub-culture a number of varieties by slicing the small clusters of bulbs into square pieces, each measuring roughly 1 mm.
The rooting and bulbing stage comprises the final phase of tissue culture, also called micro propagation of lilies. During this phase, technicians work to make the sub-culture produce the largest possible single bulblet. Unlike the medium used for explants, the medium for bulbing does not contain any growth hormone. This is primarily because multiplication is not desirable during this process. On the other hand, the bulbing medium contains augmented sucrose concentration - about 60 grams to 90 grams every litre. It takes anything between 10 weeks and 16 weeks to produce a superior quality bulblet.
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It is easiest to grow large single bulbs, especially after they have been taken out from tissue culture. A number of laboratories produce small clusters of lily bulbs in the final stage of tissue culture. In such cases, the grower will have to spend a lot of time to divide these clumps prior to planting. As a result, the losses during the first year are generally higher. It is vital to carefully protect the bulblets derived from the sterilized environment of the tissue culture laboratories from various infections, particularly those caused by fungi, during the initial stages of their growth. In addition, the timing is also very vital. For instance, lily varieties that require a vernalization period ahead of spring planting should never be bought very late.
After the bulblets are taken away from the tissue culture vessels, they should be rinsed meticulously in running water to ensure that all traces of agar are removed. Subsequently, the bulblets are immersed in a suitable fungicide with a view to protect them from fungal diseases borne by the soil, for instance Cylindrocarpon and Fusarium. It has been found that using a blend of captan and TBZ (Mertect) is extremely effective for this purpose.
Subsequently, the bulblets originated in tissue culture laboratories are packed using plastic bags in low moisture, well-ventilated medium. Ideally, you should use a blend of dry sphagnum peat and damp vermiculite, as it has been proven to be very useful. While packing it is important to ensure that the bulblets as well as their roots are kept under best possible condition - not very wet or very dry. Only pack a limited number of bulblets in one bag and also make sure the aeration is good. The plastic bags are stored for a minimum period of 8 weeks to 12 weeks prior to planting. While Asiatic lilies are stored for 8 weeks, Oriental lilies can be stored for 12 weeks before planting. The bulblets are planted in spring in sterilized beds inside a greenhouse. Alternatively, they can also be planted in trays containing pasteurized soil. These trays are also kept in a sterilized greenhouse.
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