Producing Orchid Seedlings and Its Acclimatitation

PRODUCING ORCHID SEEDLINGS AND ITS ACCLIMATITATION

BY: RINDANG DWIYANI

FACULTY AGRICULTURE, UDAYANA UNIVERSITY

rindangdwiyani@unud.ac.id

SPECIFICATION OF ORCHID FLOWERS

            Unlike other angiosperms, pollen (male sex cells) orchids clump together to form structures called polinia. This polinia is in the same position as the pistil or stigma (female genitals) in a structure called a gynostemium or column (some Indonesian literature refers to as "tugu"). The stigma in this column only appears to be a basin; meanwhile, the polinia is covered by an overcullum, which is easily opened by the pollinator. This clotted pollen structure makes it difficult for the orchid pollen to be blown off, so naturally pollination is highly dependent on the presence of pollinators such as insects or birds that carry polinia from one flower to the flower pistil. But this clotted pollen structure also causes easy pollination done by humans.

            Orchids are plants that are easy to be crossed with a high degree of compatibility, both either inter genera (inter generic) or inter species (inter specific). Compatible means that there is a match between the ovule (egg cell) as a female sex cell with pollen (in polinia) as a male sex cell to unite in the process of fertilization and seed formation.

ARTIFICIAL POLLINATION

            Artificial pollination of orchids is done by taking and removing pollen (polinia) from a flower to the flower's pistil of the same flower or  other flower. Pollen is male sex cells while the pistils are female sex cells. Pollination can be done by selfing  or crossing.  Selfing can be done between pollen and pistil originating from one flower or different flowers in one plant, or different plants but in the same variety. Whereas crossing can be done between plants with different varieties in one species, between species in one genus, or between one genus and another genus. For example, the cross between Phalaenopsis amabilis and Vanda tricolor is called crossing between different genera, between Dendrobium nobile and Dendrobium macrophyllum called between different species in one genus, whereas between V. tricolor var. Suavis with V.tricolor .var. tricolor is called between varieties within a species.

            The selection of plants to be crossed is very closely related to the hybrid character that you want to get.  But keep in mind that the closer the relationship among the orchid plant in the taxon, the higher the success rate in its crossing. Although many factors influence success, compatibility in artificial pollination is closely related to the relationship of plant taxon.

Steps in artificial pollination are described in the following:

1. Take pollen from the orchid by using a needle or toothpick and accommodating it in a container or tissue.
2. Place the pollen on the pistil which is located on the columna. Artificial pollination can be done by selfing, where the pollen is placed on the pistil of the same flower. But as mentioned earlier, pollination can also be done by crossing. Next,  the flowers must be labeled, containing information about male and female of the mother plants and the date of the pollination.
3. After 5-7 days, perianths of the flower will fall, ovaries swell and form fruit or capsules.
4. The capsule is harvested in the appropriate time. The harvest time varies depending on the genus of orchid used as the female parent. The Vanda genus takes 6-7 months after pollination, Dendrobium 3-4 months, while Phalaenopsis 7-8 months.  Harvesting must be done on time, the fruit should not be too young or too old. Capsules that are too young have seeds with low viability, making it difficult to germinate. While the seeds of broken capsule is difficult to be sterilized.
5. Harvest the capsules and prepare it for sowing the seeds in the laboratory. The orchids that are harvested are picked from the plants and the label is still recorded, because this label is also written on the bottle. The label is important as containing information about  the origin of the parents and the date of the artificial pollination done.

WHY DO THE ORCHID SEEDS MUST BE GROWN IN THE LABORATORY?

            One more specific thing about orchids is the seeds. Regarding the existence of food reserves (endosperm), orchid seeds, are different from seeds of  other angiospermsas they have very little or no food reserves at all. Naturally seed germination is assisted by a type of fungus attached to the trunk of trees, so we often find orchid seedlings attached to the  large tree in nature. However, only a small portion (about 1%) of the seeds in a capsule can be germinated naturally. The endosperm in plant seeds is required by the embryo to grow and emerge to be a small plants. This endosperm is required as food reserve by a process of cell respiration . Respiration requires a substrate in the form of food reserves (carbohydrates) to  produce energy (Adenosine Tri Phosphate or ATP) for embryo growth. 

            This lack of food reserves makes orchid seeds must be germinated in-vitro in the laboratory. The bottle containing orchid seedlings that we see on the market, are actually the results of sowing the orchid seeds on the artificial media in a bottle in the laboratory.

            However, orchid seedlings in a bottle can actually be produced from plant vegetative organs such as apical shoots, leaves, stems or roots. Broadly speaking, these organs are planted in vitro in artificial media containing growth hormones for induction and multiplication of propagules.  Propagules can be in the form of callus first, then subsequently induced to shoot. These mini shoots are then planted on the rooting media to become plantlets. This regeneration system in the in vitro culture is called indirect organogenesis because the formation of organs (in this case shoots) through the formation of callus first. If the multiplication stage does not go through the callus phase, it is called direct organogenesis. Propagation methods that use planting material in the form of plant parts that contain somatic cells are mainly carried out to obtain progenies that are genetically the same as the parent, for example multiplying hybrids with superior traits. Because if the superior hybrids are propagated through seeds (selfing), it will get a variety of progenies in which the characteristics of the parent will reappear in the progenies.

MAKING OF CULTURE MEDIA

            The sowing of seeds begins with the making of culture media. The media for seed sowing can be in the form of basic media sold in the market, such as Vacin & Went (VW), Murashige & Skoog (MS), New Phalaenopsis (NP).   However, media can also made from foliar fertilizer for an economically purpose as it is cheaper. Media of orchid seedlings in bottle is usually added by natural organic ingredients such as coconut water, tomato extract, banana juice, bean sprout extract, coconut milk, and so on. The amount used per liter of media ranges from 50-150 ml. The culture media for orchid seeds does not require plant growth regulator as needed in organ culture.  Beside that, 20-30 g of sucrose (sugar) is added. Bioagar (7 g per liter) or gellan gum (2 g per liter) is added to solidify the media. The steps of making media (example for making 1 liter of media)is described in the following:

1. Weigh the media to be used. The amount of needs that must be weighed for making one liter of media is usually written in the label of media, for example for MS media requires 4.3 g per liter of media. If using media from foliar fertilizer, we use 2 g of foliar fertilizer added with 2 mL of fish emultion, and 1 mL of  Atonik.
2. Place the media on a measuring cup
3. Add 200 cc of water
4. While continuing to stir, add natural organic ingredients to be used (for example 100 mL of tomato juice)
5. Add 30 g of sugar
6. Add more water until the volume becomes one liter
7. Measure the pH of the media solution, and adjust  it to pH 5.8-6.0 by adding a NaOH solution or HCl solution.  
8. Transfer the media solution from the measuring cup to a stainless / aluminum pan
9. Add 7 g of bio agar or 2 g of gellan gum a solidifier
10. Cook on the stove until boiling, while keep stirring constantly.
11. Pour the media in the culture bottle provided.
12. Cover the bottle with aluminum foil / plastic.
13. Sterilize the media in the bottle using an autoclave for 30 minutes. As a substitute for an autoclave, a pressure cooker can also be used to place the media.
14. Turn off the stove after 30 minutes, allow the presto / autoclave pan to cool, then remove the media from the pan

SOWING THE SEED

            The first step, the capsule that is ready to be sown is washed thoroughly with detergent while being brushed repeatedly, rinsed thoroughly. The fruit is then dipped in spiritus and burned in a bunsen fire, this step  is done up to three times. Then the orchid capsule is placed on a sterile petridish inside the laminar. The sterile work table can be enkas or laminar.  Enkas is much cheaper and can be homemade, whereas laminar is relatively expensive. Basically, the principle of its use is the same, which is maintained in sterile conditions. Enkas is sterilized by spraying alcohol before use, while laminar is sprayed with alcohol and sterilized by U.V. lamp.

            Within 7 days, orchid seeds germinate and are called protocorms. The protocorms eventually grow into small orchid plant with the emergence of roots and shoots. These orchid seedling are called plantlets in the tissue culture technology.

ACCLIMATIZATION

            The acclimatization of orchid seedlings is removing orchid seeds from the bottle so that they adapt to the new environment (the environment outside the bottle).   In the next,  the seedlings will grow into vigorous seedlings and then grow into flowering orchids that are ready for sale.

The acclimatization steps are as follows:

1. Orchid seedlings are removed from the bottle
2. Cleaned it from agar which is still attached and soaked in a fungicide solution (2 g / L Dithane or Benlate)
3. Then they are air-dried
4. They are Planted on the media of in a  container , referred to as compot (community pot).  The container can be a plastic tray or a pot.
5. Seedlings in the community pot that are sufficiently high (5 cm) then can to be transferred to individual pots.

REFERENCES

Dwiyani, R. (2014).  Anggrek Vanda tricolor Lindl. var suavis. Udayana University Press, Denpasar. ISBN: 978-602-7776-92-0 (In Indonesian)

Dwiyani, R.  (2015).  Kultur Jaringan Tanaman.  Pelawasari, Denpasar.  ISBN: 978-602-8409-44-5 (In Indonesian)