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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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
microwave oven or pressure cooker typical items)
pint and quart jars Polypropylene baby food jar caps (if you are using a microwave
forceps (6 " or longer) oven)
plant shoot tip, node, leaf or other pH papers
plastic or cardboard box
baby food jars
isopropyl or ethyl alcohol
measuring spoons (regular and
"smidgen" spoons)
florist’s tape
kitchen knife
(about 6"
long)
baking soda
vinegar Where can you find these items?
pyrex pie
pan (about 8") There are several supply companies that will
dish detergent (not dishwasher sell to schools and hobbyists. You might also
detergent!) contact the nearest university or college and
table sugar ask for small samples of things like plant
bleach and vinegar hormones and plant media. Many places
plastic (regular) pint jar caps (if you conducting research might assist you. Contact
are using a microwave oven) kck@turbonet.com if you need help locating
goggles, gloves, dusk mask, apron, supplies or user-friendly scientists. We also
and shoes (for protection) host a Yahoo listserv for "Home Plant Tissue
metal baby food jar caps (if using a Culture" that has members from around the
pressure cooker) 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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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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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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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 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

(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 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.
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 growmore@vsnl.com 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
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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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 in vitro-plants for sale

Pineapple Doris Escalante descalante@bioca.unica.cu

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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