Mericloning

Mericloning is the vegetative culture of a plant through the tissue culture of apical meristems that began in the 1960s. Plants produced through this process are relatively identical. Meristemic cells are undifferentiated cells that occur in areas of new growth. They are analogous to human stem cells. Shoot apical meristems are usually used but root apical meristem can also be used. When orchids are cultured through mericloning, they produce a mass of protocorms which can develop into plants.

Materials

1. Sterile environment: An environment without fungal spores such as a glove box or over steaming water.
2. Flask can be any glass bottle or petri dish
3. Cap (if used tape the sides of the container with water proof bandages or aluminum foil) to flask or rubber stopper (must have a hole on the top which is stuffed with cotton)
4. Stainless steel rod bent into a hoop at the end
5. Medium (What seeds are planted on)
6. Sterilizing agent: Bleach, Sodium hypochlorite, or ethanol/isopropanol alcohol can be used.
7. Sterilizing chemicals: 70% Ethanol, 0.5% Sodium hypoclorate solution, 1% Sodium hypoclorate solution
8. Forceps: must be sterilized.
9. small blade must be sterilized.

Procedure

Selecting meristem

1. Shoots are chosen from a mother plant. Plant should be cleaned to prevent microbial contaminations
2. Shoots are collected prior to period of most rapid growth.

Medium preparation

Typically meristem culture uses a modified version of the Murashige and Skoog, or the Knudson C formula medium. Hormones such as cytokinins and auxin are used as growth regulators.

Preparing flask

For flasking, all material must be sterilized before they can be used. This is done by using a pressure cooker.

1. First prepare the medium inside a flask. Make sure it is at least 25.4 mm (about 1 inch) high. If you are planning sow the seeds and re-plant into another flask use 12.7 mm of medium (about ½ inch) Do not be tempted to make a thin layer of medium.
2. Put the lid on the flask and put the flask into the pressure cooker. Pour about 2 inches of water into the cooker and cook the flask for 15 minutes @ 15 psi. A microwave can also be used to instead of a pressure cooker but a dish of water must be added under the flask so it does not boil over.
3. Place all tools and the flask inside glove box (knife, bleach, container of water). Spray the glove box with a mist of 10% bleach solution all over the inside of the case and wait five minutes for the solution to kill of contaminants.

Tissue culture

1. Using the forceps remove the shoot and dip it in the 70% ethanol. Then immeaditely transfer it to the 0.5 sodium hypoclorate solution and occasionally stir it for 10 to 15 minutes.
2. Then transfer the shoot to a solution of 1% sodium hypoclorate and stir it for 15 to 20 minutes and then dip it in the 70% ethanol and rinse it in DH₂O four times.
3. Embryonic leaves should then be stripped from shoots
4. The shoot should then be cut into 0.5 to 1 cm segments and are placed in a culture medium with stainless steel rod.
5. Seal the flask

References

• H. S., Chawla. Introduction to Plant Biotechnology. Science Publishers, 2002.
• Banks, David P.. Orchid Grower's Companion: Cultivation, Propagation, and Varieties. Timber Press, 2005.
• Elvin, McDonald, and Ortho Books. Ortho's All About Orchids. Meredith Books, 1999.
• D. G. A. WALKEY & J. M. G. WOOLFITT. "Clonal Multiplication of Nicotiana rustica L. from Shoot Meristems in Culture". Nature 220(1968)
• Dwight M. Sato, Delia Barnes and Toshio Murashige. " Bibliography on clonal multiplication of orchids through tissue culture". Methods in Cell Science 4(1978)