Horticulture 202        Plant Propagation

Introduction:

 

Orchids are one of the prettiest and most

desired household flowers.  Their bright,

colorful, and unique flowers lighten up any

room.  Belonging to the family Orchidaceae,

orchids are one of the most diverse family

of flowering plants known to mankind. 

Orchid flowers grow all over the world,

specifically tropical rainforests.  Instead of

growing in the soil, most usually grow on

other plants due to their epiphytic nature.

However, they have been documented to

grow in many different areas and climates,

and anchor themselves to many different

objects.  Humans have been interacting with

orchids for over 2,000 years.  The earliest

records date back to 700 B.C.  At this time,

orchids were mainly used as herbs for

medicinal purposes.  However, they soon became collector items and the rich began to build orchid collections throughout colonial times in Europe.  Many other people across the world have used orchids for other purposes as well.  For example the Aztecs used an orchid for culinary purposes, giving us the wonderful flavor, vanilla.  Regardless of their past use, most orchids today are used as indoor houseplants.  To satisfy the demand for orchids, one used to go out and remove them from their natural habitats.  This is not sustainable, and it became evident that new forms of propagation would be needed.

 

 

 

Propagation:

 

Methods:

 

There are several methods currently used to propagate orchids.  Some cultivars and hybrids can be propagated vegetatively, others via seeds.  These methods are not normally practiced commercially.  Vegetative methods take too much time and are generally not successful.  Seed propagation is usually not plausible due to the fact that most orchid seeds are too small to store their own food (Chugh; et al., 580).  In nature, these seeds must form a mutualistic relationship with a certain fungi to allow them to acquire the food they need.  This complex relationship for these species of orchids makes seed propagation unfriendly towards commercial production.  However, various tissue culture techniques have been developed in the last few decades allowing growers to quickly and efficiently produce thousands upon thousands of new orchids in a quicker amount of time.  These techniques include the culturing of orchid shoots, meristem, flower buds, leaves, and rhizomes (Chugh; et al., 509-515).  Almost all of the produced plants are genetically identical to their parents and by using meristem culture the orchids produced will also be completely free of any systemic viruses (Deyoung; et al., 488).  Reproducing orchids through tissue culture can be very complicated and can require advanced equipment, therefore, it is difficult for the standard homeowner to propagate any orchids he or she may possess.  These tissue methods are mostly for the commercial grower.  Each type of culturing done has its own set of specifications, however, regardless of the type, they each follow these general steps and guidelines.

 

 

Step by Step Procedure for Tissue Culture Propagation:

 

Media Preparation:

 

1. There are specific growing medias for specific orchids, however, there are a few mixtures that can generally be used for most orchids.  One such example is Murashige and Skoog Basal Medium.  This comes in a powder mix that can include various vitamins, plant growth hormones, and gelling agents.

2. Take the selected media and mix the powder with distilled water.  Once dissolved, add any other supplements to the mixture.

3. Heat the mixture to ensure that the powder and supplements fully dissolve.

4. Add more distilled water to achieve the proper volume needed.  Adjust various amounts of media powder and supplements as needed so that the mixture does not become too diluted.

5. Check the pH of the media.  A pH value between 5.2 and 5.4 is ideal for orchid media.

6. If the pH is correct, skip this step.  If the pH is too low, add sodium hydroxide (NaOH) to the mixture.  If it is too high, add hydrochloric acid (HCl) to the mixture.

7. Sterilize the media by autoclaving it to 121˚C and 15 psi for at least 15 minutes.  The greater the volume of media being used, the longer it must sit in the autoclave (see table below).

8. Once sterilized, poor the media into the containers to be used and allow it to cool and solidify.

9. Please note that these directions are guidelines and that different medias may have more precise or different directions depending on their compositions.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Explant Preparation:

 

1. When choosing the tissue source (explant), only take from orchid plants that are free of diseases and are generally healthy. 

2. Also consider which type of culturing you would like to do, and know which kind of explant to take for different orchid genera.  Shoot tips are usually a safe bet if the specific explant type is not known (Murashige, 148).  Choose shoot tips from plants with active growth (Hartmann; et al, 173).

3. Remove the explant from the parent plant using clean snippers or a clean knife.  If the explant to be cultured is quite small (like in meristem culture), more advanced gear and techniques may be required.

4. Sterilize the explant by first rinsing it with soap and water.  Then place the explant in a 10% bleach solution for precisely 15 minutes to kill all microorganisms present.

 

 

Station Preparation:

 

1. Before transferring the explant to the media, one must clear and disinfect a flat surface to allow themselves room to work.

2. Remove all items from the flat surface and place them somewhere else in the room

3. Wash your hands and arms before proceeding.

4. Sterilize the area by wiping it down with a 10% bleach solution, allowing it to dry by itself.

5. Set up the various tools needed, such as forceps, cutting knives, rinse baths, and a heat sterilization machine.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Plating the Explant:

 

1. Before touching any tools or removing the explant from the bleach solution, wash your hands and arms thoroughly.  Once clean do not touch anything, not even your own skin.

2. Sit down at the station and sterilize the forceps and cutting knives before grabbing your explant.  Do this by letting each sit in the heat sterilization machine for about 20-30 seconds.

3. Rinse the tools and remove the explant from the bleach solution.  Rinse the explant as well, but leave it in the rinse bath.

4. Take a container, holding the media, and remove the parafilm from the edge.  Do not open it.

5. Remove the explant and place it onto a sterilized cutting plate.  Take the cutting knife and forceps and cut the explant as desired.  Take each fragment and place it in the container holding the media.  This is the only time to take the lid off of the container, and when doing this, only open it part way away from you.

6. After all explant pieces are placed in the container, take a new piece of parafilm and wrap it around the container’s edge, thus sealing it.

7. Repeat these steps as necessary.  When finished place the containers in a clean growth chamber that has been properly adjusted for the orchids being cultured.

8. Please note that one should sit up straight and keep their body back away from the plant and containers to avoid accidental contamination.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Subculturing:

 

1. Once microshoots form from the tissues, they can be removed and placed in new media containers.  This is called subculturing.

2. First, prepare a sterile area like before and use the same techniques outlined above.

3. Cut the microshoots that are 2-4 nodes in length and place them firmly in the new media containers.  Do not submerge any nodes within the media (Hartmann; et al., 716).

4. Seal the container and place it in a growth chamber.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Root Intiation and Greenhouse Acclimination:

 

 

1. The microcuttings must form adventitious roots so that they can survive in greenhouse conditions.  To allow this, the orchid microcuttings must be transferred to another media.

2. This media should have a higher concentration of auxin in it.  It should also be lower in inorganic salts and cytokinins than the previous medias (Hartmann; et al., 718).  Prepare this media as mentioned above.

3. Once roots are formed and well established, pull the plantlets from the rooting media and wish any media off of the plantlets.

4. Plant the plantlets in a sterilized potting soil mixture.

5. Once planted, place plantlets in the shade, and mist frequently to prevent dessication.  Keep humidity high.  Keep the temperature warm.  Do this for several days.

6. As the plants become more established, lower the humidity and allow more light to make contact with the plants.

7. Once plants have been established care for them like any other plants.

8. Congratulations!  You have successfully propagated orchids!

 

 

 

Conclusion:

 

No matter which type of tissue culture used for the orchids, each have to go through those specific steps.  Orchids are very beautiful flowers that many desire for their homes.  This has led to an explosion in the industry throughout the last few decades.  With the recent advancements in tissue culture techniques, it is now possible and relatively easy to propagate massive amounts of orchid clones from a single parent plant.  With over 30,000 cultivars and over 100,000 hybrids of orchids available, the true challenge is deciding which ones to propagate.  For more information on orchid propagation, please feel free to look at the references listed below:

 

 

 

References:

 

 

Chugh, S., Satyakam, G., I Usha Rao. “Micropropagation of orchids: A review on the potential of different explants.” Department of Botany. University of Delhi.  Published in Scientia Horticulturae. Vol. 122, Issue 4. Nov. 3, 2009. Print. Page 507-520. Retrieved From: http://ac.els-cdn.com/S0304423809003641/1-s2.0-S0304423809003641-main.pdf?_tid=85c622fc-ef13-11e5-b67f-00000aab0f6c&acdnat=1458530463_560785d3e69317a3886133903e240a86.  Accessed on March 21, 2016.

 

Deyound, G., Rowe, B., Erik Runkle. “Propagating Orchids.” 2011. Michigan State University. Retrieved From http://www.flor.hrt.msu.edu/assets/Uploads/Propagatingorchids.pdf. Accessed on March 20, 2016.

 

Hartmann, Hudson T., Dale E. Kester, Fred T. Davies, and Rovert L. Geneve. Hartmann & Kester’s Plant Propagation: Principles and Practices. 8th ed. Boston: Prentice Hall, 2011. Page 820. Print.

 

Murashige, T. “Plant Propagation Through Tissue Culture”. 1974. Department of Plant Sciences. University of California.  Published in Annual Review Plant Physiological. 1974. Retrieved From http://www.annualreviews.org/doi/pdf/10.1146/annurev.pp.25.060174.001031.  Accessed on March 20, 2016.

 

“Murashige and Skoog Basal Medium.” 2016. Sigma-Aldrich. Retrieved From: http://www.sigmaaldrich.com/catalog/product/sigma/m9274?lang=en&region=US. Accessed on March 20, 2016.

 

“Media Recommendation Guide.” 2011. PhytoTechnology Laboratiories.  Retrieved From: http://communities.naae.org/servlet/JiveServlet/previewBody/4620-102-1-5215/Orchid%20Tissue%20Culture.pdf. Accessed on March 20, 2016.

 

The North of England Orchid Society. N.d. “The Story of Orchids.”  Royal Horticultural Society. Retrieved From http://www.orchid.org.uk/orchidstories.htm.  Accessed on March 20, 2016.