(Picture Courtesy of: https://threehundredandsixtysix.wordpress.com/2013/02/14/flower-african-violet/ )
BACKGROUND: Herbaceous plants are the flowering plants. They have soft stems unlike the hardwood plants that have a woody stem. There are two ways that are commonly used to propagate the African violet, Saintpaulia ionantha (Plant Finder, 2015). The two types of propagation used are micro-propagation and stem cuttings. we will look into micro-propagation in this instructional page. Stem cutting is cutting a piece of stem off of an existing piece of plant and placing it into water or soil. This process could take up to a year for first blooming. Another downfall is that you will not produce as many plants as if you were to use micro-propagation. There is a third way to “propagate” African violets. This is by use of seeds. A disadvantage of using seeds is that there is only a few cultivars to select from. With direct propagation from a plant, it allows you to propagate that specific plant and the genetics it has to offer.
WHAT YOU WILL NEED: The tools that you will need are: petri plates, plastic containers, water, a scalpel, forceps, and a tool rack. All tools MUST be sterilized before using in the propagation procedure. There are a few ways to sterilize the equipment: 1. baking supplies at 350 degrees Fahrenheit for 15 minutes, 2. place in an autoclave for 15 minutes or, 3. pressure cooker at 15 pounds for 15 minutes (Plant Propagation using African Violet Leaves, 1995). Plastic containers for holding disinfecting solutions and water. Water is needed as well. Para-film-used to seal petri dishes after explanting. Incubator for growing.
STERILIZATION: (Human) First you must remove any jewelry or irritants from the lower half of your arms, up to your elbows. Then you must wash your hands and arms up to your elbows thoroughly, as if you were “scrubbing in” for a surgery. Dry arms with disposable paper towels. DO NOT TOUCH ANYTHING with your sterilized arms and hands. If you wish or have the available tools, you can put on sterilized gloves. (Laminar Flow Hood) The laminar hood should be sterilized before yourself. Use disinfectant to clean work area prior to moving equipment in. Once equipment is in the flow hood area, sterilize the equipment. The metal tools can be sterilized by a bacti-cinerator for three seconds at a time, let cool before handling on plant tissue (Horticulture 202 Tissue Culture Lab 7 Handout).
PLANTING MEDIUM: Preparing the planting medium that you will use is extremely important. It is crucial to the plants’ growth that you include all necessary nutrients. The medium typically includes sucrose-an organic compound, mineral nutrients, and plant growth regulators (hormones). If you are to use a solid planting medium (soil) agar is needed to help the growth (Plant Propagation using African Violets, 1995). Petri plates containing 30ml sterile Murashige & Skoog (MS) basal medium supplemented with vitamins, 30mg/l sucrose, 170 mg/l NaH2PO4 x H2O, o.01 mg/l (0.05uM) Benzyladenine (BA) and 0.1 mg/l (0.5uM) Naphthalene acetic acid (NAA), plus any plant growth regulators. The medium can be solidified with 2 g/l of phytagel, the pH adjusted to 5.7, and then autoclaved for 15 minutes to sterilize. When finished, the media can be poured into petri plates (Horticulture 202 Tissue Culture Lab 7).
-Select one African violet leaf and petiole. Wash the leaf and petiole in a solution of soap and water. Drain of the soap. Place leaf and petiole into solution of 10% Clorox. Your leaf and petiole need to be submerged in the Clorox solution for 15 minutes. If you leave the plant in the solution for too long, you risk killing the explant tissue. If you leave the plant tissue in the solution for too short of time, you risk contamination. At this time, is when you should move onto sterilizing yourself. All other equipment should be set up before this stage in the procedure.
-When moving to the laminar hood, do not make sudden movements. Move with ease, for this will help keep the sterile air in the hood and the unsterile air out of the hood. Bring with you your leaf and petiole. You will have a three bin system set up to help rinse off any residue of the Clorox solution. Move from left to right when rinsing the leaf and petiole. The first one on the left will have the most Clorox solution in it from previous rinses of plant tissue. Each rinse bin will be less saturated with Clorox solution. After the third rinse is complete, place your leaf and petiole into a sterile petri dish that you have in the hood. Sterilize your forceps and scalpel prior to cutting.
-If there is damaged tissue, remove first and place in separate petri dish that you are not planning on using. Take your forceps and scalpel and cut the leaf and petiole. You should be able to obtain at least six pieces of each from each leaf-petiole tissue piece. The thinner the petiole cutting is, the less risk of disease contamination there will be in the new plant. You want to cut the petiole so that the pieces look like little disks when done. The leaf cutting pieces should be no bigger than your pinky nail. Place the cutting into a petri dish with planting medium already in it. There should be no more than six pieces of tissue in each petri dish to allow for optimal growing room. When placing the pieces in the growing medium, trying placing them in different ways: leaf-some upside down, some right-side up; petiole- some standing up like a tire, or laying down so you can see the circle when you look down on it. Gently place the lid to the petri dish to close of the petri dish. Then seal off with para-film.
-For further instruction on how to apply the para-film, watch this 2 minute video: https://www.youtube.com/watch?v=f47Hq2uiJIs .
STORAGE: (Incubator) The cells are grown in an atmosphere of 5-10% CO2 because the medium used is buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained. Culture flasks should have loosened caps to allow for sufficient gas exchange. Cells should be left out of the incubator for as little time as possible and the incubator doors should not be opened for very long. The humidity must also be maintained for those cells growing in tissue culture dishes so a pan of water is kept filled at all times (Beginning Molecular Biology Laboratory Manual, 2010). African violets should be kept in 24-25 degrees celsius for 16 hours a day (Plant Micropropagation using African Violets). If there is a contamination in the growing medium it will become apparent in 3-4 days. If there is no contamination, there should be growth within 4-6 weeks. You should monitor daily for the first week and then weekly there after. Daily monitoring the first week will make you aware of contamination and you may be able to save other propagation in that same petri dish if caught early enough.
(Picture Courtesy of: https://blog.mycology.cornell.edu/2009/10/14/taming-the-fungus/)
STAYING ALIVE: Once the 4-6 week time period has come, depending on how well your plants have grown, you may need to transfer to a larger container. Transferring to a larger container will encourage the plant to expand and grow, giving the plant more room to grow. When the plant is tall enough, you can transplant explant into normal growing medium for outdoor aesthetic appeal.
-Saintpaulia ionantha – Plant Finder. (2015, January 1). Retrieved March 15, 2015, from http://www.missouribotanicalgarden.org/PlantFinder/PlantFinderDetails.aspx?kempercode=b567
-Stephens, L. (1995, September 1). PLANT MICROPROPAGATION USING AFRICAN VIOLET LEAVES. Retrieved March 15, 2015, from http://www.biotech.iastate.edu/publications/lab_protocols/AV_Micropropagation.html
-Reddivari, L. (Director) (2015, March 5). Horticulture 202 Tissue Culture Lab 7. Lecture conducted from Pennsylvania State University, University Park.
-How to Use Parafilm with Agar Plates. (2009, December 17). Retrieved March 15, 2015, from https://www.youtube.com/watch?v=f47Hq2uiJIs
-Wolf, J. (2010, March 2). Beginning Molecular Biology Laboratory Manual. Retrieved March 15, 2015, from http://userpages.umbc.edu/~jwolf//method5.htm
-Hartmann, H., Kester, D., Davies, Jr., F., & Geneve, R. (1997). Vegetative Propagation. In Plant propagation: Principles and practices (6th ed., pp. 330-340). Upper Saddle River, N.J.: Prentice Hall.