Cloning Plants:

Tissue Culture
Horticulture and the Science of Plants
  Youth Adventure Program

David Wm. Reed, Instructor

Department of Horticultural Sciences
Texas A&M University

BACKGROUND

Tissue culture (often called micropropagation) is a special type of asexual propagation where a
very small piece of tissue (shoot apex, leaf section, or even an individual cell) is excised (cut-
out) and placed in sterile (aseptic) culture in a test tube, petri dish or tissue culture container
containing a special culture medium. 
 Overview of the Tissue Culture Process

The culture medium contains a gel (agar) with the proper mixture of nutrients, sugars, vitamins
and hormones, which causes the plant part to grow at very rapid rates to produce new plantlets. 
It has been estimated that one chrysanthemum apex placed in tissue culture could produce up to
1,000,000 new plantlets in one year.  Thus, tissue culture is used for rapid multiplication of
plants.  A very specialized laboratory is required for tissue culture.  All the procedures are done
in a laboratory and special ventilated cabinet that is as sterile as an operating room.

Steps in Tissue Culture

(images courtesy of  Dr. Dan Lineberger, aggie-
horticulture.tamu.edu/tisscult/microprop/microprop.html)

Explant:  Cut-out Plant Tissue and Place in  Tissue Culture Container
The first step is to obtain what is called and explant.  This means to simply cut-out a very small
piece of leaf or stem tissue, or even isolate individual cells, and place them in a tissue culture
container.  The tissue has to be sterilized so it will not have any contaminating bacteria or
fungus.  It is then placed inside the tissue culture contain on a gel called agar.  In the agar is
dissolved all the sugar, nutrients and hormones the plant needs.

 
Explants can be pieces of  any part of the plant (leaves, stems, flowers, etc.),
or even individual isolated cells.
Multiplication:  Tissue Grows and Produces Small Plants
The tissue will begin to grow.  It may make a big blob of tissue called callus, or it may make new
shoots directly from the explant tissue that was inserted in the container.

A mass of callus tissue is formed that is just starting to make new plantlets.

New plantlets (shoots with leaves) are forming.

If the conditions are right a small "forest" of plants

will develop in the tissue culture container.
Rapid Multiplication by Transfer of Cultures
Once the plantlets start developing, some can be removed and placed in new tissue culture
containers.  Thus, another "forest"' of plants is produced.  This results in a rapid multiplication of
the cultures and many thousand of plants can be produced in a few months.

Some of the small plantlets can be removed and transferred to new tissue culture
containers.  These will produce more shoots and fill the container.

Transplanting
When the plantlets are large enough, they can be removed from the tissue culture container and
transferred into pots with potting soil.  The young plants are growth in a greenhouse just like you
would any young seedling or cutting.

When the small plant clones are removed from the culture containers, they must be transplanted
into some type of acclimation container or  kept under a mist system until the acclimate to the
ambient environment.
 After acclimation, the young plants can be transplanted
and grown in pots in a greenhouse to produce new plants.

TAKE HOME LESSONS

1) To become familiar with how plants can be cloned from very small pieces of  leaf, stem or
other plant tissue.
2) To transfer plants out of a tissue culture container into a new tissue culture container.
3) Bring the new tissue culture clone home and transplant it into soil in a pot when it gets large
enough.

MATERIALS NEEDED
Plantlets in a tissue culture container ready to be transferred.
Tissue culture lab and transfer hood.
Tissue culture container with fresh tissue culture medium.

PROCEDURES
View a Video on Cloning with Tissue Culture and Observe a Variety of Tissue Cultures
The class go to a tissue culture lab and view a video on tissue culture.
The class will view a variety of tissue culture containers with cloned plantlets.
Transfer Cloned  Plantlets to Fresh Tissue Culture Container
The class will be instructed on the proper procedure to:

 Work in a tissue culture transfer hood and proper safety procedures..

 Learn how to sterilize all the tools that will be used.
 Learn how to keep the entire working area sterile.
 Open a tissue culture container and divide the clumps of  plantlets.
 Transfer a plantlet to a fresh tissue culture container.
 Seal the tissue culture container to keep it sterile.

Transfer Protocol:  Each student will then carry-out the procedure and transfer a tissue culture
plantlet to a fresh tissue culture container.  Each student will bring their tissue culture home.

TRANSFER PROTOCOL
Transfer African violets from multiplication culture tube to culture containers that will grow the
clones to a size that can be transplanted.

Sterilize the surfaces of the transfer hood.

Sterilize all tools that touch the plants by first
dipping in alcohol them flaming.

African violet clones in a shoot multiplication

tube.

Remove the cluster of plants in the culture.

 Insert the cluster of plants into the new culture
container.

Break up the cluster of plants and spread them out.

Seal the container with paraffin film.

The culture before transfer (left) and after transfer

(right).