Tissue Culture Col 1 page 2

Cover

Contents

Plant Tissue Culture of African Violet (Our "model plant")

Sterile water will be needed to rinse plant pieces after they are soaked in bleach solution. Fill pint jar 1/2 full with tap water or distilled water. Place cap on loosely. Set aside until media preparation is finished.

Sterile African violet medium is needed to grow the plant parts. The following is combined in a quart jar: 1 packet MS medium, 2 tablespoons table sugar, 1 mg BAP, 1 ml PPM, distilled water. Using pH paper, vinegar, and baking soda, adjust the pH to about 5.5 to 6.0. Note that hormones solutions can be purchased in concentrations of 1 mg/ml and measured with a simple baby dropper hence you will not need an expensive balance to weigh them.

Add 3 tablespoons of liquid medium to each baby food jar using a plastic measuring tablespoon. Add one half level "pink Baskin-Robbins" spoon OR one level "pinch" spoon of agar to each baby food jar. Place polypropylene baby food caps on the jars (if using a microwave) and press to tighten. Polypropylene caps or the original metal caps can be used in a pressure cooker.

Sterilization of water and media can be done with either a microwave oven or a pressure cooker.

The purpose of the clean area is to limit the number of particles that fall into your tissue culture jar. These airborne particles carry bacteria and fungi, and can kill your plant tissues because they grow faster than the plants. A clean area can be made from a plastic-lined cardboard box or a plastic storage box with a "window" cut out.

The inside of the clean box and the surface of the clean area should be wiped down, or sprayed, with 70% alcohol or a 10% bleach solution. All items that are put into the clean area (media jars, bleach container, sterile water jar, "dipping" alcohol) need to be wiped down, or sprayed, to get rid of possible contaminants. Hands should be washed in soap and water for at least 20 seconds, and then wiped with 70% alcohol. Dip or soak instruments in 70% alcohol.

Pick up a leaf with forceps and dip into the 70% alcohol for a few seconds. This will remove some debris and wax. Place leaf in 10% bleach solution and allow to soak for 10 minutes. Stir occasionally. Move the bottle with leaves to the clean box. Transfer leaves to sterile water using the forceps, and allow to soak for 1-5 minutes.

Inside the clean box, wipe a small salad plate with 70% alcohol. Note that you could also use a paper towel laid directly on the table surface; spray it down with alcohol and you have a sterile surface. Dip the forceps in 70% alcohol and transfer one leaf to the plate. Dip the kitchen knife in 70% alcohol. Holding the petiole end of the leaf with the forceps, cut the edges of the leaf away. Then cut the leaf into two pieces.

Loosen the caps on 2 baby food jars. Dip the forceps in 70% alcohol. Pick up one leaf piece. With your other hand, pick up the cover of the media jar and place the leaf piece in the jar. Quickly replace the cover. Wrap florists’ tape or surgical tape around the outside of the jar. This will help to minimize the debris that gets into the jar and causes contamination of the cultures.


Various containers can be used for culture: Gladware (left-top), baby food jars and mason jars (right-top), Combiness vessels (left-bottom) and "Quesa" jars (right-bottom).

Put the cultures in a bright room out of direct sunlight or set the cultures on shelves with cool-white fluorescent lights positioned about 9-12 inches from the shelf below. Lights should be on 16 hours per day. The leaves should start to swell in 2-4 weeks, and small bumps and then leaves will appear on the "mother" leaf's surface.

The newly developing plantlets will grow better if they are transferred to fresh medium without growth regulators. The growth regulators can inhibit elongation of the shoots and the formation of roots.

After 4-6 weeks, make fresh medium using the "Home Style Medium" recipe below. Follow the same instructions as you did for the original medium using these ingredients:

"Home Style Medium"
In a quart jar filled with water, mix:
1 teaspoon hydroponic fertilizer
(Peter’s NPK 20-20-20)
2 tablespoons sugar
a multivitamin pill
1 ml PPM

Mix well. The vitamin pill will not completely dissolve. It can be removed after a couple of minutes. Test pH and adjust as you did in the first batch of medium.

Measure 3 tablespoons medium into each baby food jar. Add two cotton balls, or 1/2 teaspoon gelatin, or agar (as previously described). Cap with polypropylene caps, or metal baby food jar caps if using a pressure cooker. Sterilize as described earlier.

In the clean box, dip the forceps in 70% alcohol and carefully remove the plant culture from it’s jar and place on the alcohol wiped plate. Cut into sections or pull apart plantlets using sterile forceps and knife. Place each small piece or plantlet into fresh medium. Recap and seal.

Step 5: Transfer rooted plantlets to soil

When plants have developed shoots and roots, they are ready for transfer to sterile soil or soil less medium (found at your local discount store). Gently remove the plants from the jar. Gloves should be worn when doing this in case your skin is sensitive to the culture medium. Rinse off all of the medium that is sticking to the stem and roots under lukewarm running water. Plant the tissue cultured plantlet in the moist soil. Water with a liquid fertilizer such as Peter’s or Miracle Gro.

Cover the pot with a plastic bag. A high humidity is necessary for the plant until it hardens off and adjusts to the outside world. After 3-4 days, start opening the bag for a while, increasing the time each day until the bag can be removed. Now you can treat your new plant like any other normal plant purchased from a store or grown in your greenhouse.

References to items in this article can be found at my website: www.kitchenculturekit.com The poster that this article is based on is at: http://www.kitchenculturekit.com/sivbposter.htm. Email me at kck@turbonet.com with your questions or suggestions for tropical plant tissue culture protocols and I'll try to address them in the next issue .......................... Carol

Helpful Resources
Dirr, Michael A., and Charles W. Heuser, Jr. 1987. The Reference Manual of Woody Plant Propagation: From Seed to Tissue Culture. Varsity Press, Inc. 239 p.
Kyte, Lydiane, and John Kleyn. 1996. Plants from Test Tubes: An Introduction to Micropropagation (Third Edition). Timber Press, Inc. Portland, Oregon. 250 p.
Basic plant tissue culture information, resources, and listservs are located at: http://www.kitchenculturekit.com.

<--Back to First Page of Article

Resources from my Species webpage
Banana	Keith Benson's Banana Page http://www.geocities.com/RainForest/2011/MicropropBAN.html
	Sherwood Exotics http://www.sherwood.exotics.btinternet.co.uk/
	The Banana Tree http://www.banana-tree.com/
	Banana Garden http://www.bananagarden.com
	Banana seeds and information http://www.quisqualis.com/quisopen.html Rare banana photos: http://www.quisqualis.com/rareban00.html
	Morocco - banana trees, potato seed, paulownia trees and date palm. http://www.cropdevelopment.org
	INIBAP Technical Guidelines No.6, Appendix 1, p29. http://www.inibap.org/publications/publications_eng.htm

Bamboo	American Bamboo Society http://www.americanbamboo.org
	http://www.inbar.int/publication/txt/INBAR_Technical_Report_No27.htm
	John Woods http://esi.athenstn.com/wwt/wwt.html
	International Network for Bamboo and Rattan http://www.inbar.int/
	Equipment, Methods and Protocols for Tissue culture http://users.cwnet.com/three4al/bamboo/Technical.htm
	Dr N. Barathi, Director, Growmore Biotech Limited 41 B, Sipcot Phase II, Hosur, Tamil Nadu, India 635 109 Phone +91 4344 560564 fax +91 4344 560560 E-Mail growmore@vsnl.com http://www.growmorebiotech.com

Ferns	Micropropagation of Ferns (Platycerium Ferns) http://www.geocities.com/RainForest/2011/MicropropSHF.html

Hibiscus	http://www.hibiscus.org

Hosta	Q and Z Nursery http://www.qandznursery.com/
	Hosta Haven
	http://www.gardensights.com/MissVitro/
	http://www.HostaLibrary.org
	http://www.shadyoaks.com/home.html
	http://www.winterberryfarms.com/
	BA induces shoot formation in hosta: http://home.okstate.edu/Okstate/dasnr/hort/hortlahome.nsf/toc/cole4
	Wessel Nursery Virginia Beach, VA. 23464 jwessel@infi.ne t Fax 757-424-6435 http://www.hostatissueculture.com
	Jim Anderson Rowen Gardens and Winterberry Farms TC http://RowenGardens.com
	http://www.hosta.org

Palms/Cycads	http://www.teaket.com/
	Morrison's Palms and Cycads http://www.cycadsandpalms.com/
	Palmdat Website http://www.palmdat.com/
	SAPAD for Palm Increase http://sapad.com.sa/

PawPaw (Asimina sp.)	http://www.pawpaw.kysu.edu/webres4.html
Sugarapple Atemoya Annona sp.	Nair, S., P.K. Gupta, and A.F. Mascarenhas. 1984a. In vitro propagation of Annona hybrid (Annona squamosa L. x Annona cherimola L.). Indian J. Hort. 41:160-165. Nair, S., P.K. Gupta, M.V. Shirgurkar, and A.F. Mascarenhas. 1984b. In vitro organogenesis from leaf explants of Annona squamosa Linn. Plant Cell Tissue Organ Culture 3:29-40.

Pineapple	Pineapple in vitro-plants for sale Doris Escalante descalante@bioca.unica.cu

Plumeria	http://www.ghgcorp.com/beyer/plumeria.htm

Tropical Plants	Tropica Tissue Culture Laboratory (Aquatic Plants) http://www.tropica.dk/tiscul.htm
	Tropical Plant Sources http://www.zoneten.com/index.htm

Information on the web comes and goes very quickly, if you discover a bad link please advise us so we can update.

<--Back to First Page of Article


These are commercial laminar flow hoods.

Plant Tissue Culture of African Violet (Our "model plant")

Happy culturing.............................

Carol