Introduction:

The Orchid Family, Orchidaceae, is one of the two largest family of flowering plants in the world. These are truly remarkable plants that are coveted all over the world for their beauty, variety of pleasing fragrances, and economic importance; vanilla for example is an orchid. While the number of orchid species numbers well into the tens of thousands, finding a genus or species to propagate may seem intimidating. The orchid Dendrobium kingianum, is often a great starting point for new orchid growers as it is a hearty orchid when established, is quite beautiful, and has a pleasantly sweet fragrance. Kingianum is called the “Pink Rock Orchid”, because like most orchids it is an epiphyte which means it grows on other plants and sometimes rock surfaces rather than soil, and also because of its pink to white color.

This page will take you through the steps of propagation of orchids via tissue culture. This can be a challenging method of propagation as many things can go wrong, and the necessary resources (more to come later), but is rewarding if done properly as many new plants can be produced. Tissue culture is possible because of all plants ability of totipotency which allows a new plant to be created from undifferentiated cells from any part of the plant; basically this means regeneration. These cells are grown in a sterile medium, in a sterile invirto environment until they are ready to be placed in a permanent growing medium and location. Tissue culture is a preferred method of propagating orchids because other methods are simply too difficult and often just do not work. Many orchids are rare in nature due to difficulty in propagation and prefer the very humid regions of southeast Asia. More experienced growers can try the other various methods such as seed propagation, stem cuttings, and bulb division.

Step One:

Before any other steps can be taken in tissue culture propagation the media must be created. Tissue culture media can either be purchased or created in a laboratory. Depending on resources either could be beneficial. All media contain the same general ingredients. Inorganic salts that contain the micro and macro nutrients the plant needs and often added in the form of solutions made from stock solutions diluted with as pure of water that is available. Using pure water is necessary to help control the pH which should fall between 5.5 and 6. Organic compounds such as hormones, carbohydrates, and vitamins are also extremely important. The appropriate ratio of auxin : cytokinin varies among tissue cultures but for our purposes about 0.5ppm NAA auxin to 1.5ppm BAP cytokinin is an appropriate ratio of 3 : 1 and will promote adequate shoot formation and root formation in the tissue culture. Carbohydrates will provide food to the plant as it cannot photosynthesize yet. Sucrose is typically used in concentrations of 2 – 4%, but glucose can also be used if necessary. Thiamin and nicotinic acid are always added to the media as vitamins that the plant needs, both at 0.5ppm. Citric Acid can also be beneficial to the media because it helps prevent browning and cell death during the subculturing steps. The last ingredient is the gelling agent of the media. Agar, or an agar substitute called Gelrite can be used. Agar is a substance made from algae and can be a gel or liquid form depending on temperature (which is valuable in a tissue culture operation). This agar makes the media able to support the plant and should be added to a quantity of 0.5 – 0.6%. It is important to not add to much or too little agar as this will not provide a good osmotic potential and the plants will desiccate or the plants will sink into the media; also bad. When the media is made, it should be autoclaved to make it sterile at about 120 degrees Celsius and 15 psi for 20 minutes.

 

Step Two:

Now that the media is completed, it is time to select explant material. As with any asexual propagation method only the healthiest and ideal plant material should be used. Do not use any material that is diseased, damaged, or towards the end of the life cycle. This tissue culture method will make use of the meristem of the plant stem which is located in the center of the shoot tip. The small mass of cells can be as small as one mm, but is a good explant candidate because it is undifferentiated and is the youngest part of the plant. Before the meristem is excised, sterilize the plant part in soapy water for about 20 – 30 seconds, and then in a 10% bleach solution for 15 minutes.

 

Step Three:

From this point on, sterility and cleanliness are absolutely essential. Any contamination, even dust, breathing on the culture, or any unsterilized tools will completely ruin culture. If this happens you will not produce orchids, but rather a nasty looking bacteria or fungus culture. So in the lab, wash your hands, or if you wish to use latex gloves wash them with an ethanol solution. Use the same solution to wipe down and sterilize your working area.

Work will be done in a laminar flow hood which is used to flush clean air outwards to remove contaminated air from the work area. Take the explant out of the bleach and rinse it multiple times in clean water to remove any remaining bleach. Use a sterile scalpel or knife to remove outer layers of the plant to reach the tiny meristematic cell cluster. Carefully divide this into as many pieces as possible, ideally 20 pieces and places them on top of the tissue culture medium being careful not to press them into the medium. Use parafilm to seal the perti dish containing your culture and place it into a growth chamber at about 25 degrees Celsius and a light source.  This step is called “establishment” because this is where the cells will multiply and become established to their environment.

 

Step Four:

After a few weeks subculturing can be done if desired which is similar to what was done the first time in the lab. The masses of cells that have been growing will all be divided to exponentially increase the number of cultures you have. When these new cultures have grown, the shoot multiplication step will begin. This step is merely maintaining a stable culture while the number of shoots increases. When shoots have developed they can be excised and placed into tubes with the tissue culture medium. You may wish to make another medium with a slightly higher auxin concentration, of 2ppm to enhance the root initiation step. This is invitro root initiation rather than exvitro root initiation which is done for other species of plants; so the culture will remain in the growth chamber for this process.

Step Five:

The plants have successfully rooted and are growing quite nicely and have been for a year. It is time for the acclimation step. Plantlets are removed from their tubes and culture and are washed to remove any remaining culture medium material. The plants are placed in a medium, typically sphagnum moss and peat. Potting soil may be used for seedlings, but eventually will need to be replaced with a wood chips because orchids are epiphytic. A hardening off period is initiated so that the plants are not stressed, and shocked, or dead when they leave the facility. They should be kept under a mist to prevent desiccation and brought slowly to common greenhouse conditions (70% humidity, 21 – 24 degrees Celsius, and a 16 light to 8 hours dark photoperiod).

 

Conclusion:

Now you have orchids. These remarkable plants should have no problem being sold to a wide variety of customers. The tissue culture propagation method is a difficult process to master, but when done properly can be a very effective way to mass produce orchids. Dendrobiums will likely take about a year or more to fully develop into a flowering plant.

 

Literature Cited:

Hartmann, H., and Kester, D. Plant Propagation, Principles and Practices. Upper Saddle River, NJ: Prentice Hall, 2011. Print.

Murashige, T. “Plant Propagation Through Tissue Cultures.” Annual Review of Plant Physiology,  Issue 25 (1974): 135 -166. Print.

Pant, B., and Thapa D. “In vitro mass propagation of an epiphytic orchid, Dendrobium primulinum Lindl. through shoot tip culture.” African Journal of Biotechnology. Vol 11.42 (2012): 9970 – 9974. Web. 10 March. 2014. http://www.academia.edu/4977378/In_vitro_mass_propagation_of_an_epiphytic_orchid_Dendrobium_primulinum_Lindl._through_shoot_tip_culture

“Introduction to Orchids, orchid propagation”. Venamy Orchids.  2012. 10 March. 2014. http://www.orchidsusa.com/1Introduction.htm

 

 

By Liam Farrell