Clone Your Own Plants!
Can plants really be cloned? Perform the following procedure to find out how multiple identical clones of a carrot plant can be produced.
- Callus formation
- Sterile techniques
Botanists have long understood the concept of totipotency, or that every living cell in a plant has the genetic information in its DNA to produce an entirely new plant. In the 1950s, Frank C. Steward conducted research in his laboratory at Cornell University that eventually led to a procedure whereby a whole carrot was produced from single cells. This opened the plant world up to a whole new approach of asexual plant propagation.
Steward took small pieces of tissue, termed explants, from a carrot root. He placed these explants in a sterile liquid nutrient medium where they started to grow masses of undifferentiated cells known as calluses. Individual cells from this callus where transferred to another sterile media where they continued to divide and produce more callus cells. Manipulation of the chemical and physical environment of the callus cells (particularly with auxins and cytokins) enabled the callus to produce embryoids with root and stem meristems. These grew to form plantlets, which, when the correct nutrients were added, grew into new carrot plants (see Figure 1).
Tissue culture, or micropropagation, is a rapid and inexpensive way to produce thousands of genetically identical, individual clones from just one or several cells. During the past few decades, great strides have been made in applying the techniques of tissue culture to the commercial production of ferns, orchids, and a great variety of other plants. If a variant arises by mutation, it is no longer necessary to go through years of breeding to build up a seed stock carrying the desirable characteristic. The new plants with that trait can be propagated by the thousands the same year it arises.
Commercial micropropagation is performed in a very similar manner to the following procedure. Many greenhouses have sterile tissue culture rooms where highly skilled technicians perform plant micropropagation. The results have been so valuable that more mass production of cloned plants occurs year after year. It is an exciting process that opens up further availability of plants for the home and garden.
Sterile technique is very important in this activity and should be followed at all times during this procedure. Media rich in nutrients are used in this experiment. If bacteria or fungi come in contact with the plant tissues or the surface of the media, the culture will become contaminated. The goal is to keep both the plant tissue and the media contaminant free. All work surfaces should be wiped down and sterilized with a bleach solution. Hands should be washed before beginning the procedure and gloves should be worn. The Petri dishes should be sprayed with 70% isopropyl alcohol after culture transfers have been made. Plant tissue transfers must be done quickly to avoid additional contamination. Follow all other sterilization techniques given by the instructor.
In this activity, a basic cloning procedure will be performed to produce identical clones of an original carrot plant.
Bleach (sodium hypochlorite) solution, 5%
Ethyl alcohol, 95%, 30 mL
Beakers or other small containers, 2
Parafilm™, approximately 1.5 feet long
Petri dish (Carrot Callus Initiation)
Petri dish (Shoot Development)
Spray bottle of 70% isopropyl alcohol
Stirring rod, glass
The agar on the Petri dishes will readily support mold and bacteria. If individual Petri dishes become moldy (indicated by fuzzy areas) or support bacteria growth (slimy-looking areas) do not open the Petri dishes. The mold or bacteria may possibly be pathogenic. Ethyl alcohol and isopropyl alcohol are flammable and are dangerous fire risks. Bleach is a corrosive liquid that is moderately toxic by ingestion and inhalation. Avoid contact with acids, which can release toxic chlorine gas. The cork borer and scalpel are extremely sharp. Handle and use with care. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.
Track the development and record all observations in the Plant Cloning Data Table. Be sure to record the date of each observation and drawings of the clone development.
- Sterilize the working surface of the table by using paper towels and wiping it down with the bleach solution.
- Obtain a cork borer, a glass stirring rod, scalpel, forceps and a beaker or similar container.
- Place approximately 25 mL of 95% ethyl alcohol into the beaker or container.
- Sterilize the cork borer, glass stirring rod, scalpel and forceps by placing them into the container of ethyl alcohol. Be sure that all surfaces of the cork borer (and all of the other tools) are dipped in the ethyl alcohol to ensure total sterilization.
- Obtain a carrot from the instructor.
- Wash the carrot in warm water and soap. Remove any leaves and stems.
- Obtain a Petri dish lid or bottom and put on latex gloves at this time.
- Place the carrot in your gloved hand. Break off the large end and the small end. Discard these ends in the trash.
- Be careful not to touch any of the newly exposed surfaces of the carrot. If this new “center” section of the carrot root is longer than the cork borer, break this section in half again.
- Pour enough 95% ethyl alcohol into the Petri dish to completely cover the bottom of the dish.
- Put the center section of the carrot (large end down) into the Petri dish containing the ethyl alcohol.
- Remove the cork borer from the container of ethyl alcohol and, while holding the carrot in place with a gloved hand, push the cork borer into the cortex region outside of the core of the carrot (see Figure 2). Avoid boring the epidermis (outside section) and the central core of the carrot. Be sure the cork borer goes all the way through the carrot until it comes in contact with the Petri dish.
- Pull the cork borer out.
- Take the glass stirring rod out of the ethyl alcohol and insert the glass rod into the handle end of the cork borer. Push the core obtained from the carrot out into the Petri dish containing the ethyl alcohol.
- Remove the scalpel from the container of ethyl alcohol. Cut off about 1 cm at both ends of the core just obtained. Discard these ends.
- Using the scalpel, cut the remaining piece of core into 1- to 2-mm thick cross sections.
- Obtain the Carrot Callus Initiation Petri dish from your instructor. Be sure the lid remains closed!
- Remove the forceps from the jar of ethyl alcohol.
- Using the forceps, quickly transfer three or four of the innermost pieces of the sliced carrot core to the agar surface of the Carrot Callus Initiation Petri dish. Do so by slightly opening the lid of the dish and gently pressing the cross section of the carrot core onto the agar. Do not submerse the cross section of the carrot core. Note: Be careful not to breathe on the Petri dish when performing the transfer.
- Quickly cover the Carrot Callus Initiation Petri dish lid and wrap with a small piece of Parafilm to form a seal around the top and the bottom of the Petri dish lids (see Figure 3).
- Using a marker, label the top of the Carrot Callus Initiation Dish with your group’s initials.
- Spray the outside of the dish with 70% isopropyl alcohol.
- Place the dish in an area away from direct sunlight.
- Make sure the Petri dish remains in area that does not exceed 80 °F.
- Callus formation will take approximately one to two months.
- If the Petri dish becomes contaminated, it must be disposed of by the instructor.
- Once the callus is 1 cm or larger in diameter, it is ready to be propagated using the Shoot Emergence Procedure.
- Remove the Parafilm from the Carrot Callus Initiation Media Petri dish.
- Obtain the Shoot Development Petri dish from the instructor.
- Sterilize the forceps as done in step 4 of the Callus Formation Procedure.
- Using sterile forceps, quickly remove the callus pieces from the Carrot Callus Initiation Media and place them on the agar surface Shoot Development Petri dish. The stem sections should not be transferred in this step—only the callus. Do so by slightly opening the lid of the Shoot Development Petri dish and gently pressing the callus onto the surface of the agar. Be careful not to breathe on the Petri dish when performing the transfer.
- Seal the Petri dish, label, and sterilize as done in steps 20–22 of the Callus formation procedure.
- Place the Shoot Development Petri dish under artificial light or indirect sunlight in an area between 72 and 80 °F.
- If the Shoot Development Petri dish becomes contaminated, it must be disposed of by the instructor.
- Within one to two months, multiple green shoots should develop.
- Each shoot will develop into a mature carrot plant.
- The shoots may be placed into the soil and grown as a normal seedling.
- Consult your instructor for appropriate disposal procedures.