Affordable Plant Tissue Culture for the Hobbyist

Affordable Plant Tissue Culture for the Hobbyist

or

Have Kitchen, Will Culture?

Carol M. Stiff, Kitchen Culture Kits, Inc., Lufkin, TX

www.kitchenculturekit.com [email protected]

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.

Affordable Plant Tissue Culture for the Hobbyist http://www.kitchenculturekit.com/StiffAffordablePTCforhobbyists.htm

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Where can you find these items?

There are several supply companies that willsell to schools and hobbyists. You might alsocontact the nearest university or college and

ask for small samples of things like planthormones and plant media. Many places

conducting research might assist you. [email protected] if you need help locating

supplies or user-friendly scientists. We alsohost a Yahoo listserv for "Home Plant TissueCulture" that has members from around the

world who culture just about anything and are

very willing to share information and sometimessupplies or chemicals. Membership is free – goto the website, www,kitchenculturekit.com and

click on "join a listserv".

Supplies found in your kitchen or the

local discount store:

microwave oven or pressure cookerpint and quart jarsforceps (6 " or longer)plant shoot tip, node, leaf or otherplastic or cardboard boxbaby food jars

isopropyl or ethyl alcoholmeasuring spoons (regular and"smidgen" spoons)florist’s tapekitchen knife(about 6"

long)baking sodavinegarpyrex piepan (about 8")

dish detergent (not dishwasherdetergent!)table sugarbleach and vinegarplastic (regular) pint jar caps (if youare using a microwave oven)

goggles, gloves, dusk mask, apron,and shoes (for protection)metal baby food jar caps (if using apressure cooker)

Supplies Not in Your Typical Kitchen or

Local Discount Store

Murashige and Skoog (MS) medium

Benzlaminopurine (BAP), a plant hormone that induces shootformationNaphthaleneacetic acid (NAA), a plant growth regulator thatinduces root formationPlant Preservative Mixture (PPM), a biocide that reduces

contaminationAgar, for solidifying the medium (can be substituted with othertypical items)Polypropylene baby food jar caps (if you are using a microwaveoven)pH papers

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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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 forany 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 jar20 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 ifusing a pressure cooker (the white plastic caps will surviveautoclaving though they might get a little soft)long handled spoon

pH paper

Sterile water will be needed to rinse plant pieces after they aresoaked in bleach solution. Fill pint jar 1/2 full with tap water ordistilled water. Place cap on loosely. Set aside until mediapreparation is finished.

Sterile African violet medium is needed to grow the plant parts. The following is combined in a quart jar: 1 packetMS 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 polypropylenebaby food caps on the jars (if using a microwave) and press to tighten. Polypropylene caps or the original metalcaps 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 airborneparticles carry bacteria and fungi, and can kill your plant tissues because they grow faster than the plants. A cleanarea 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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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% alcoholor 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 bewashed in soap and water for at least 20 seconds, and then wiped with 70% alcohol. Dip or soak instruments in70% 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 tothe 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 leafwith 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 otherhand, pick up the cover of the media jar and place the leaf piece in the jar. Quickly replace the cover. Wrapflorists’ tape or surgical tape around the outside of the jar. This will help to minimize the debris that gets into thejar and causes contamination of the cultures.


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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 shouldstart to swell in 2-4 weeks, and small bumps and then leaves will appear on the "mother" leaf’s surface.

The plant growth regulator, BAP induces shoots to grow from cells in the leaf. Within 4-5 weeks, small plantletswill 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 instructionsas 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


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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 andadjust 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 (aspreviously 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 smallpiece 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 incase your skin is sensitive to the culture medium. Rinse off all of the medium that is sticking to the stem and rootsunder 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 theoutside world. After 3-4 days, start opening the bag for a while, increasing the time each day until the bag can beremoved. 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 [email protected] 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 Seedto 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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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 [email protected] www.growmorebiotech.com

Ferns Micropropagation of Ferns (Platycerium Ferns)

Hibiscus http://www.hibiscus.org

Hosta 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

[email protected] 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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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. xAnnona 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 ofAnnona squamosa Linn. Plant Cell Tissue Organ Culture 3:29-40.

PineapplePineapple in vitro-plants for sale

Doris Escalante [email protected]

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

Tropical Plants Tropica Tissue Culture Laboratory (Aquatic Plants)

Tropical Plant Sources

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


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Affordable Plant Tissue Culture for the Hobbyist

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