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Affordable Plant Tissue Culture for the Hobbyist

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

Affordable Plant Tissue Culture for the Hobbyist

or

Have Kitchen, Will Culture?


Carol M. Stiff, Kitchen Culture Kits, Inc., Lufkin, TX www.kitchenculturekit.com kck@turbonet.com

Plant tissue culture, also called micropropagation, is no longer restricted to the scientific laboratory. Now days, if you have a kitchen, or at least basic kitchen supplies, you can mass propagate your favorite plant at home! Plant tissue culture (PTC) techniques are used for growing plants in a sterile controlled environment for the purpose of mass production, germplasm preservation, plant breeding, physiological studies, and genetic engineering. By using plant hormones and other growth regulators, small plant parts can be induced to produce hundreds of small "plantlets", which can later be grown in a greenhouse, in the field, or as house plants. Use of plant tissue culture has been limited in the past by the need for expensive equipment (laminar flow hood, analytical balance, and autoclave). However, by using biocides such as PPM (Plant Preservative Mixture from Plant Cell Technology, Inc.) or NaDCC (sodium dichloro-s-triazinetrione is a spa and swimming pool disinfectant), and a simple "clean box", expensive equipment is no longer essential. Warning: this hobby can become somewhat "intoxicating" for a plant lover, and you will find you are taking over the kitchen, guest room and garage, and due to the numbers of plants produced this way, you may have to expand your home greenhouse or build a second one. To compensate for these actions, be prepared to clean up your messes, volunteer to cook, or take the family out to dinner when the kitchen is unavailable. In this article we are going to cover the basic steps in home tissue culture and include some references to tropical plants. In the subsequent articles, I give specific examples and protocols for tropicals - if you have a plant you are interested in culturing, email me and I'll see if I can include it in the next issue.

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Affordable Plant Tissue Culture for the Hobbyist

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

Supplies found in your kitchen or the local discount store:

Supplies Not in Your Typical Kitchen or Local Discount Store Murashige and Skoog (MS) medium Benzlaminopurine (BAP), a plant hormone that induces shoot formation Naphthaleneacetic acid (NAA), a plant growth regulator that induces root formation Plant Preservative Mixture (PPM), a biocide that reduces contamination Agar, for solidifying the medium (can be substituted with other typical items) Polypropylene baby food jar caps (if you are using a microwave oven) pH papers

microwave oven or pressure cooker pint and quart jars forceps (6 " or longer) plant shoot tip, node, leaf or other plastic or cardboard box baby food jars isopropyl or ethyl alcohol measuring spoons (regular and "smidgen" spoons) florists tape kitchen knife (about 6" long) baking soda vinegar pyrex pie pan (about 8") dish detergent (not dishwasher detergent!) table sugar bleach and vinegar plastic (regular) pint jar caps (if you are using a microwave oven) goggles, gloves, dusk mask, apron, and shoes (for protection) metal baby food jar caps (if using a pressure cooker)

Where can you find these items? There are several supply companies that will sell to schools and hobbyists. You might also contact the nearest university or college and ask for small samples of things like plant hormones and plant media. Many places conducting research might assist you. Contact kck@turbonet.com if you need help locating supplies or user-friendly scientists. We also host a Yahoo listserv for "Home Plant Tissue Culture" that has members from around the world who culture just about anything and are very willing to share information and sometimes supplies or chemicals. Membership is free go to the website, www,kitchenculturekit.com and click on "join a listserv".

Safety Recommendations You need to be aware of basic laboratory skills and lab safety including: the safe handling and disposal of alcohol

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Affordable Plant Tissue Culture for the Hobbyist

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

and bleach solutions, disinfecting forceps and knives with alcohol (flame sterilization is not recommended), preparation of media (depending on student age, you may need to limit this activity), and the use of protective clothing such as vinyl gloves, goggles, plastic aprons, dusk masks, and leather or tennis shoes. Material Safety Data Sheets (MSDS) provide information on the safe handling of chemicals. These are required for any chemical used in a classroom, and are obtained from the internet, manufacturers, and chemical supply stores

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


We use this as our model plant because it is readily available in discount stores, responds well in tissue culture, and is a favorite house plant. Step 1: Prepare sterile water and medium Supplies needed for one quart of medium (20 baby food jars) plus sterile water: 1 quart jar 20 baby food jars (4 or 6 oz) 2 pint jars (regular opening) 20 plastic or metal baby food jar caps 2 pint jar plastic caps or regular metal rings and lids if using a pressure cooker (the white plastic caps will survive autoclaving though they might get a little soft) long handled spoon pH paper 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. Step 2. Preparing a "clean area" 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.

These are commercial laminar flow hoods.

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Affordable Plant Tissue Culture for the Hobbyist

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

These are inexpensive clean boxes. 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. Step 3: Isolation and culture of African violet leaves 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.

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Affordable Plant Tissue Culture for the Hobbyist

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

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" leafs surface.

The plant growth regulator, BAP induces shoots to grow from cells in the leaf. Within 4-5 weeks, small plantlets will be visible on the surface of the leaves. Step 4: Transfer "plantlets" to fresh medium 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 (Peters NPK 20-20-20) 2 tablespoons sugar

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Affordable Plant Tissue Culture for the Hobbyist

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

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 its 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 Peters 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. Resources from my Species webpage Banana
Keith Benson's Banana Page Sherwood Exotics The Banana Tree

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Affordable Plant Tissue Culture for the Hobbyist

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

Banana Garden http://www.bananagarden.com Banana http://www.quisqualis.com/quisopen.html The banana photos we spoke of are at: 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.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 www.growmorebiotech.com

Ferns Hibiscus Hosta

Micropropagation of Ferns (Platycerium Ferns)

http://www.hibiscus.org

Q and Z Nursery 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.net Fax 757-424-6435 www.hostatissueculture.com Jim Anderson Rowen Gardens and Winterberry Farms TC http://RowenGardens.com www.hosta.org

Palms/Cycads

http://www.teaket.com/ Morrison's Palms and Cycads Palmdat Website

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Affordable Plant Tissue Culture for the Hobbyist

http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

SAPAD for Palm Increase

PawPaw

http://www.pawpaw.kysu.edu/webres4.html
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 Tropical Plants

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

Tropica Tissue Culture Laboratory (Aquatic Plants) Tropical Plant Sources

Happy culturing.............................carol

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