BACKGROUND
      Plant tissue culture is the propagation of plants through "cloning," an asexual method of reproduction.
A portion (explant) of a desired plant is cultured in vitro on a defined medium which promotes rapid multiplication. As the new plants develop, they are removed from culture and transferred to a standard
potting medium.
      Tissue culture is based on the theory of totipotency; that is, the genetically based ability of a
nonembryonic organ or cell to develop along a pathway, similar to that of a zygote, leading to the
formation of a new entire organism identical to the original. Currently, tissue culture is being used
in both research and commercial applications. Tissue culture not only provides a method of mass
propagation, but also makes possible the production of disease-free plants, mutants, and secondary
plant products. A new and important use is in the genetic engineering of plants. A single plant cell
can be genetically modified and grown into a mature plant or plants having new characteristics.

Part I: Carrot Tissue Culture
      In this exercise students use tissue (an explant) from a carrot taproot to observe several stages
of plant tissue culture. The carrot tissue is taken from its normal relationship to the other plant parts
(in vivo) and is placed under test tube (in vitro) culture conditions. As an explant becomes established,
its cells are stimulated to divide and form an undifferentiated, irregular mass of cells called a callus.
The callus is then subcultured to induce shoot and root formation. When the plantlets are 2.5 to 5 cm
tall, they can be removed from culture and transferred to potting medium.
      The cultures should be maintained at 24° to 27° C. Ideally, the photoperiod should be 16 hours
of light and 8 hours of darkness. Use bright indirect natural light or fluorescent plant growth lights. In
no case should direct sunlight be used, as this will probably kill the cultures.

PROCEDURE
A: Production of Carrot Callus
1. Each lab group will need to obtain 2 sterile petri dishes. One of the petri dishes should be labeled
      “carrot callus”, and should also have the names of each member of the lab group. Pour some
      “carrot callus” agar into the petri dish and allow the agar to solidify. The second petri dish should
      simply be left next to the first until needed.

2. Each lab group will need a lab station with the following equipment.
      - a box or other container (serve as a sterile area for transferring tissue cultures)
      - spray bottle with alcohol or bleach solution (spray inside of box before using to sterilize area)
      - container filled with alcohol (holds and sterilizes forceps, glass rods, cork borers, etc.)

3. When a lab station is available each lab group will need to:
      Person #1: (This person assists partner, but does not enter the sterilized box)
            Step 1 - Wash a raw carrot using the bleach water solution provided (in a large tub)
            Step 2 – While holding one end of the carrot with a pair of tongs, dip the entire carrot into
                        alcohol, touch it to a flame, and let the alcohol burn off. (This step sterilizes the
                        carrot.) Still using the tongs, immediately carry the sterilized carrot to the sterilized
                        box and place it inside.
                BE AVAILABLE FOR ANY ASSISTANCE NEEDED BY YOUR LAB PARTNER.
           Step 3 – When partner is finished putting carrot pieces into agar plate labeled “carrot callus”,
                       obtain a piece of Parafilm and stretch/wrap it around the plate to seal out air (and bacteria)
           Step 4 – Place the finished “carrot callus” plate under the light provided in the back of the room.
      Person #2: (This person will enter the sterilized box)
           Step 1 – Spray inside of box with the spray bottle to sterilize it. Spray liberally (wet all surfaces)
           Step 2 – Obtain the two sterile petri dishes prepared earlier and set both just inside the edge
                        of the box.
           Step 3 - Clean hands using bleach and water solution provided (in a large tub).
           Step 4 – Go to lab station and keep hands inside box. When sterilized carrot is delivered by
                        partner complete the following steps:
                 (NOTE – These steps can be seen on the diagram provided)
                            a) Place the carrot into the empty sterile petri dish. Cut the carrot into several pieces
                                using forceps. Remember which ends were nearer top of carrot.
                            b) Use cork borer to bore several holes in carrot. Remember which ends were nearer
                               top of carrot.
                            c) Cut each bore into smaller pieces (about half the thickness of an aspirin/tylenol pill).                                       Remember which ends were nearer top of carrot.
                            d) Place 3-5 of these small pieces of carrot onto the “carrot callus” agar plate and use
                                forceps to push them slightly into the agar. Each piece should be positioned
                                with the end that originally was nearer the top of the carrot still on top.
           Step 5 – Hand the finished “carrot callus” plate to your lab partner. Remove the other petri dish
                        and unused carrot pieces and throw them in the trash.
           Step 6 – Place all equipment back where it was when you began. (Make sure scalpel, cork
                        borer, forceps, and glass rod are back in alcohol)
      In one to two weeks, callus will begin to form on the explants. In four to six weeks, the callus should
be 1 cm in diameter. At this point the callus can be transferred to shoot development medium to induce
shoot and root formation. In Part B you will accomplish this transfer.

B: Carrot Root & Shoot Production
1. Each lab group will need to obtain a sterile tissue culture tube (have blue lids). Label this tube
      “carrot development” and add the names of each member of the lab group. Pour some “carrot
      shoot & root development” agar into the tube and allow the agar to solidify.

2. Each lab group will need a lab station with the following equipment.
      - a box or other container (serve as a sterile area for transferring tissue cultures)
      - spray bottle with alcohol or bleach solution (spray inside of box before using to sterilize area)
      - container filled with alcohol (holds and sterilizes large forceps)

3. When a lab station is available each lab group will need to:
      Person #1: (This person assists partner, but does not enter the sterilized box)
           Step 1 – Locate a petri dish that contains pieces of carrot callus. (This dish was prepared
                        by teacher) Bring this dish to a lab station and place it just inside the edge of the box.
           Step 2 – Bring tissue culture tube (prepared earlier) and place it just inside the edge of the box.
               BE AVAILABLE FOR ANY ASSISTANCE NEEDED BY YOUR LAB PARTNER.
           Step 3 – When partner is finished putting a piece of carrot callus into the tissue culture tube,
                        place the  tube under the light provided in the back of the room.
      Person #2: (This person will enter the sterilized box)
            Step 1 – Spray inside of box with the spray bottle to sterilize it. Spray liberally (wet all surfaces)
           Step 2 - Clean hands using bleach and water solution provided (in a large tub).
           Step 3 – Go to lab station and keep hands inside box. When tissue culture tube and petri dish
                        of carrot callus are delivered by partner complete the following step:
                 (NOTE – This step can be seen on the diagram provided)
                        Using the LARGE forceps lift one piece of carrot callus from the petri dish that contains it.
                        Open the tissue culture tube and place the carrot callus onto the agar inside.
                        Still using the forceps, push the callus slightly into the agar. Quickly put the lid
                              back on the tube, and hand it to your lab partner.
           Step 4 – Place all equipment back where it was when you began. (Make sure forceps are back
                        in the alcohol)

Part II: African Violet Tissue Culture
      In this exercise students use a portion of African violet or gloxinia leaf to observe the four stages
of plant tissue culture. Stage I takes the plant part from in vivo ("life") to in vitro ("glass"). This means
simply that the explant is taken from its normal relationships to the other plant parts and is placed
under "test-tube" culture conditions. Stage II is the multiplication stage. The explant undergoes
rapid tissue or shoot multiplication. This process can be repeated, depending upon how many plants
are ultimately desired. Stage III is the rooting stage. A different growth medium is used to induce root
formation from Stage II plants. Stage IV is the transfer of the plants to a potting medium.
      The media used in this lab are Murashige African Violet/Gloxinia Multiplication Medium and
Murashige African Violet/Gioxinia Pretransplant Medium. These media and most other plant tissue
culture media are composed of inorganic salts, vitamins, hormones, a carbon source, and agar.
     The inorganic salt base will provide all the essential macro- and micronutrients. To this base
other items can be added to enhance a particular plant or stage. The vitamins in these media are
thiamine, i-Inositol, and adenine. The adenine enhances shoot production; the pretransplant
medium lacks adenine because roots rather than shoots are desired. Two types of hormones
are contained in these media—auxins (IAA) and cytokinins (kinetin). IAA stimulates root
production, kinetin promotes shoot production, and both stimulate cell division. Sucrose is used
in the media as a carbon or energy source. (There is too little light for the plant to carry on normal photosynthesis or there may not be any chlorophyll present.) Agar may or may not be used
depending on the tissue. When agar is used, it must be soft enough to not damage the tissue
and yet hard enough to hold the tissue in place.