Tissue Culture of Dendrobium Orchid

1. Preparation Eksplan
Parent eksplan sources used for seed production angggrek Dendrobium spp. in vitro is to clear type, species, and varieties and should be healthy and free from pests and diseases. Sugito (2007) adds that the plants will be eksplan source must have high economic value, the market liked the production, the plants healthy, grow well and normal. Also used indukan age to 1.5 years old at the time of flowering and placed in a green house.
To assist or facilitate the pollination of orchids, an attempt is made with the help of artificial pollination with the help of humans. At this pollination, cross pollination is often done. Cross pollination aims to reproduce the type or types of orchids, for example by crossing the color or the nature-siat other superior.
In general orchid orchids out odor when ripe for pollinated, but not all orchids out odor when ripe. Most appropriate one can be pollinated flowers is to smell it in the morning, afternoon and evening. We smelled the strong odor / smell and pistils mucus, that's the best time to do the pollination.
The smell and beautiful colors are attractive to insects and other animals that assist in the process of pollination. Purple is the color most favored by bees or other insects, which in general do pollination in the morning, before at 08.00-10.00 WIB. The flowers are inconspicuous and less attractive, such as light yellow white, etc., will be conducted by penyerbukanya the Active insects at night.
During the fruit ripening process, are encouraged to perform routine maintenance to include orchids indukan watering, fertilizing, and pest and disease control to improve the quality of seeds produced.
In general, fruit dark green orchids, and when cooked, turned into yellowish. Dendrobium orchids form of fruit is a small round. At one flower stalk can grow some fruit orchids. However, if the number of flower stalks more than two, then the seeds inside the fruit will experience cell death (there are only testa alone, without protocorm, so just like cotton). Orchid fruit shapes to form a six ribs, but really only three true ribs. When the cut across it will be collected at three angles true ribs. (Ir. Daisy 2000).

2. Making Media
Media is one of the most important thing in the culture in vitro. Medi cultures in vitro contain the various elements of nutrients that can meet the growth needs of the dendrobium orchids.
A very rapid development of nutrient medium for plant cell growth starting in the 1960s and 1970s. Basic nutrition for plant cell culture is basically similar to the nutrients needed by the plant itself. However, the variation depending on the nutritional composition of cells, tissue-nets, the organs and protoplasm and plant species will dikulturkan. Before making nutrient medium, one very important thing to note is knowing in advance which type of culture that will be used, for example: callus, cells, organs or protoplas that will be examined and the final goal of the study. A different type of culture will have one or more unique composition of the media.
Tissue culture medium formulation was first made based on the composition of the solution used for hydroponics, especially the composition makronya elements. Nutrient elements are given in the form of inorganic salts. Koposisis media and formulation development is based on the type of tissue, organs and plants used and the approach of each researcher. Some types are sensitive to high concentrations of macro compounds or substances require specific regulators for growth. Media for growth of roots that developed by White (Gunawan 1934 in 1988), the first White to use the media containing inorganic salts, yeast extract and sucrose, but then the yeast extract was replaced with 3 kinds of vitamins B, namely pyridoxine, thiamine and nicotinic acid (Chawla, 2002).
In general, an orchid seed plant organs that contain no food reserves. So that cell metabolism in orchid seeds for growth can not last. The lack of supply or additional nutrients, cells will die. Therefore, the provision of nutrients in the growth media, is absolutely necessary (Ir. Daisy 2000).
Orchid seeds have no food reserves. But in nature, capable of germination, although in a very small percentage. This is because the organic materials in the supply by the Mycorrhiza fungi that live in orchid seed. Therefore, on artificial media in the bottle, should be available December food reserves of carbon, which is necessary for orchid seed germination. (Ir. Daisy 2000).
It is almost certain that the success of in vitro culture activities will be very determined and depends on the choice of media used. Should be remembered kultu in vitro pressing environmental eksplan cook so can grow and develop. A suitable environment, some will be fulfilled if the selected media to consider what is needed by plants. In general, plants need the same nutrients, but in particular it is different. The similarity is a plant needs: elements of macro and micro nutrients, vitamins, carbohydrates (sugars), amino acids and organic materials, substances regulating growth, substance compactor (in order) (Untug Santoso 2004).
In making the media, takes accuracy when weighing the components of the media because of errors in the weighing could be fatal for the growth of culture. In general, the main composition of the planting medium in vitro culture, consisting of zpt (growth regulator substances) macro elements and micro elements, zpt, vitamins, amino acids, compactor material (so), in the form of the sugar glucose, sterile water. The addition of natural organic material in the media will also increase the growth and development of the embryo dendrobium orchids. Natural organic materials that are added to this medium is coconut milk, banana extract, corn extract, and fish emulsion.

Media used of Orchids are the media (VW). Composition of culture media each stage has a combination of different cultures according to the stages that will be developed, such as media tebar, sub culture, and the trans end.
Embryo culture techniques (sowing seeds) on solid media is a way of plant propagation is most often performed. Because, besides not require a lot of knowledge, as well as the growth rate of seeds into PLB, and so become planlet, will grow faster. Seeds taken from fruit orchids have the right cook. Tanman orchids will produce high value when the fruit comes from a cross between two types of orchids, each of which has a beautiful flower colors and high durability of terhadapa diseases and environmental stresses. Because the fruit of hard skin, the fruit of sterilization can be carried out by mechanical means which can be burned with spirtus fire. Implementation of the burning done in enkan space, so that when the fruit in sterile conditions, do not need to be moved to a distant room. This is done so that contamination can be avoided. (Ir. Daisy 2000)

There are three types of media to use, namely tebar phase media, subculture, and transplanting. Media foundation of the three media is the media VW (Vacin & Went). According to Hendaryono (2000), based on research that has repeatedly, it is best orchid in the media dikulturkan VW.
Media tebar, subculture, or transplanting in principle the same, but the difference is the composition of additional organic materials and the added hormones. This difference occurs because of the many nutrients that are needed by each PLB which dikulturkan on different media. Media tebar made simpler composition than the media subculture or transplanting. This is because according to research Handoyo Budi Orchids in mind that nutrition is given to the media has been able to meet the needs of PLB orchids to grow and thrive. PLB is planted in the media subculture requires fewer nutrients than planlet planted in the media transplanting (Satwika, 2003).

3. Sterilization
Effort to overcome a microorganisms is crucial in culture in vitro. All media, culture bottles, and tools used to handle this should be made to sterile as planting materials were used. To menecegah happening is the place where the activities carried out in vitro culture should be free from microorganisms and pathogens. The success of the place, neatness and accuracy of work needs to be maintained in order to reduce the risk of contamination.
Sterilization is one of the procedures used to kill microorganisms. Maintaining sterile and disease (keaseptikan) or sterile conditions are essential for success in tissue culture procedures. Aseptic conditions are required for all bottles to be used culture, culture media, equipment to be used in investment activities eksplan.
Life can be a contaminant fungi, bacteria, insects and their eggs, mites and spores. If these contaminants are not removed, then the media containing sugar, vitamins and minerals, contaminants, especially fungi and bacteria will grow rapidly. In a few days, contaminants will meet the entire bottle culture. Closed Eksplan contaminants eventually die. Example enkas. Enkas should be sterilized by using UV subs 30 minutes, but in enkas Formalin sterilized using tablets. Enkas in every corner of formaldehyde in Place 7 tablets. This is very efefktif as a way of sterilizing enkas useful ekspal As with the planting.
In addition the tools used such as spatulas, tweezers, etc. dielapi denganb alcohol 96% and then burned.
For fruit orchids as applied enough planlet alcohol 96%, and burned up all the microorganisms die and can reduce contamination.

4. Subculture
Subculture in principle aims to double the Protocorm Like Bodies (PLB) or propagated planting material and keep it in certain circumstances, so any time may be continued for the next stage.
Subculture on this dendrobium orchid is the transfer of dendrobium orchids are still very small from old media to new media aseptically performed in enkas. Which aims to get a supply plant nutrients for growth.
The ability to reproduce true from a multiplication of the in-vitro lies in whether or not a material easily re-planted for multiplication (Wetherell, 1976). Orchid sprouts or prtocorm like bodies (PLB) is in good condition and not contaminated from the stage or the stocking of disubkulturkan transferred to media containing cytokines. PLB produced in the number doubled disubkulturkan continue repeatedly until achieved the expected number of propagule. After that, micro shoots can be rooted and diaklimatisasikan.
Subcultures can be done over and over again until the number of shoots that we expect. But subculture that too much can reduce the quality of shoots produced, such as the occurrence of genetic aberration (aberration), causing a symptom abnormalities (vitrifikasi) and the frequency of off-type plants is very large.
Subcultures can be done with the view of some components of the media was thinned so that the element haranya is up, growth has filled the bottle and when needed according to the specified time. PLB after 3 months of tebar's time in subculture. PLB's green and many taken using the long spoon and then transferred to the subculture medium and PLB evenly attached to the media. Subcultures can be done repeatedly until achieved the expected number of PLB without sacrificing the quality buds. According to Yusita (2004) subculture that too much can reduce the quality of the shoot as vitrifikasi (a physiological disorder symptoms) and aberration (genetic drift). This situation occurs because of growing subculture that is more frequent in the media conditioned plants that contain cytokines, which increases power regeneration.

5. Transplanting
Transplanting an enlarged stage and rooting PLB subculture results. Transplanting done in the same room as the enkas tebar process seeds and subculture, seek a sterile culture results. Transplanting are activities that aim to achieve a good quality seed for sale and diaklimatisasi that the process requires precision and patience choose planlet of subculture results. PLB-PLB is not the time for the transplanting in another subculture subculture in the media.
Failure in transplanting caused the planters themselves. Contamination is one limiting factor in breeding success of tissue culture plants, it can happen any time in the culture. So must keep still so as not exposed to sterile miroorganisme (fungi, bacteria, or viruses) that enter and multiply rapidly which will attack the plants in culture bottles which consequently can inhibit plant growth or kill plants.
In addition to the failure in transplanting contamination can result from sterilization of transplanting a burning tool (tweezers) and the mouth of the bottle is still too hot to do. This mengakbatkan tanamn become dark brown / burnt so dead. But not all plants are not dead, there are still a living, then the revision.
Revision is the replacement of the dead planlet after the trans end. Revision usually results from steriliasi (burning mouth bottle) that is too long. Heating the usual cause is too long hangusnya plant tissue, because the network is still trlalu young plants. Revisions can also occur due to separation of the trans planlet at the end of inadvertent. Planlet separation must be done carefully, because the network is still young planlet very vulnerable to injuries causing friction. The wound tissue became bruised from the friction, it will become rotten and dead.
After transplanting the bottles are stored in the incubation room. This dal preparation bottle endeavored to avoid overcrowding. Thus, in addition to avoiding contamination, as well as facilitate clean room. It jams up the contamination so that the bottle was finished in transplanting the lid again given the plastic lid and tied with rubber.
At the time of incubation there will be quality control. Is transferring control Kuality bottle good orchid growth and uniforms to the level of the shelves in the incubation room. It is useful also to know the age and quality of dendrobium orchids that grow sediri.

6. Acclimatization
Acclimatization is the period of adaptation in plant breeding of the original tissue culture conditions are controlled and changed in the condition field conditions are not controlled anymore, besides that, the plant also must change the pattern of plant life heterotrop to tanama autotrop.
In doing grouping plantlet acclimatization selection results. Plantlet grouped by size to obtain uniform seedlings. Before planting plantlet should be selected first based on the completeness of the organ, color, hekeran growth, and size. Plantlet good is the full organ, has shoots and roots, the color green bud that is not stable and transparent good root growth.
According to Poster (2005) the characteristics of very good seed is planlet looked healthy and not moldy, planlet uniform size, fresh green leaves, and no yellowing. In addition to normal growth planlet, not stunted, leaf and root composition balanced, Pseudobulb or pseudo bulbs begin to appear and a small portion has issued new shoots, and has a number of fibrous roots from 3 to 4 roots with a length of 1.5 to 2.5 cm. Breeding procedure with a new in vitro culture could be said to succeed if planlet can diaklimatisasi to external conditions with high success. Acclimatization planlet aims to prepare to be ready planted in the field. Acclimatization stage is absolutely done on plants produced in vitro propagation because planlet will experience physiological changes caused by environmental factors. This can be understood because breeding in vitro (in bottles) all controlled environmental factors while in the field of environmental factors difficult to control (Herawan, 2006; Yusnita, 2004).
In the culture bottle, the humidity is almost always 100%. Acclimatization is critical stage because the micro-climate conditions in a greenhouse, plastic house, seed house, and the field is very much different. Conditions outside bottle berkelembapan much lower relative, not aseptic, and the light intensity level is much higher than the conditions in the bud botol.planlet or more micro heterotrofik since grown accustomed to the very high berkelembaban condition, aseptically, and the supply of mineral nutrients and abundance of energy resources.
In addition, these plants memperlihhatkan abnormal symptoms, such as is very succulent, thin cuticle layer, and the network is not fully developed vasikulernya, abnormal leaf morphology with no properly functioning stomata, mesophyll structure changed, and very low photosynthetic activity.
Acclimatization is done by conditioned media planlet the ex vitro rooting. Media that we use in the process of acclimatization to the ferns and orchids are charcoal / tiles. In addition, the moisture in the acclimatization remain high set in the first week, declining gradually in the following weeks until new roots grow from planlet. The light set of low intensity, gradually increasing. The acclimatization temperature should be maintained so as not to exceed 32oC.
After the acclimatization process of orchids were treated as follows:
a). Compotting
The size of the pot that is used to compote diameter of about 7 cm in this pot filled with about 30 seeds or seedlings depending on the size of orchid seedlings. First of all pots to be used sterofoam filled with about 1 / 3 parts, then chopped fern and orchid seedlings neatly arranged ..

b). Seedling (the Single Investment Pot)
Seedling is the process of moving seeds from compote into individual pots. Seedling performed at 5-month-old seedlings. If the plant can cause late diseedling compote seeds in the competition so that the absorption of nutrients and root obstructed at risk of being corrupted. Usually done seedling placed in a glass of mineral water used. Media used for each of orchids vary depending on the needs of the water. Media for Dendrobium is wrapped in sphagnum which plant roots, and plants grown in plastic cups that have been filled chopped sterofoam and ferns. Usually the media are also grown in the fern bars and tied with raffia rope. The characteristics of the seed is ready for the seedling roots was marked by growing more powerful and leaves the leaf was already out of the lip of the pot.

c). Overpot (Transfer of seeds)
Overpot done when the plants in a single pot are eligible to be moved, which indicated the number of tubers premises. Tanamn moved to a larger pot. Usually done after 2-3 months-old seedling. Medium used is a piece of fern bars arranged on a regular basis or one by one and tied with raffia rope premises.

d). Repotting
Repotting or re pengepotan is moving plants potted plant from the old to the new pot. Repotting orchids do if the pot has grown seedling and filled plastic popt. Pengepotan re-done on the grounds of the media in seedling pots have decayed and destroyed so that a low ph (acid) and susceptible to disease (Parnata, 2005). In addition, in anticipation of the media who have run out of nutrients. Media for repotting is also different for each type of orchid needs depends on the water.

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