Teacher Notes

Clone Your Own Plants!

Student Laboratory Kit

Materials Included In Kit

Bleach (sodium hypochlorite) solution, 5%, 475 mL
Ethyl alcohol, 95%, 500 mL
Isopropyl alcohol, 70%, 500 mL
Shoot development media, melt-and-pour bottle, 200 mL
Parafilm™, 2" x 24"
Petri dishes, 40

Additional Materials Required

Water, sterile
Autoclave or pressure cooker (one per classroom)
Beaker or container for sterilization
Boiling water bath setup (one per classroom)
Carrot
Cork borer
Disposal container
Forceps
Gloves, heat-resistant
Gloves, latex
Paper towels
Ruler
Scalpel
Spray bottle
Stirring rod, glass

Prelab Preparation

  1. All culture plates should be prepared at least one day prior to initiating the cultures.
  2. Pour the media one day prior to each step (callus initiation and shoot development), respectively.
  3. Loosen the cap of the desired media bottle to allow for air expansion in the bottle.
  4. Melt the agar by placing the bottle in a boiling water bath.
  5. When the agar in the bottle melts completely, remove the bottle from the boiling water bath.
  6. Allow the bottle to cool for a minute or two.
  7. Pour just enough of the melted agar to cover the bottom of a Petri dish. Quickly place the cover on the Petri dish. Prepare one Petri dish of each medium for each student group.
  8. Label each Petri dish with the appropriate medium designation (callus initiation or shoot development).
  9. Allow the media to “set up” for one day before use.

Safety Precautions

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. Consult the disposal procedure. 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 with care. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please consult current Material Safety Data Sheets for additional safety, handling, and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The best way to dispose of contaminated Petri dishes is to place the dishes in an autoclavable biohazard bag and sterilize in a pressure cooker or autoclave. See Flinn Suggested Biological Disposal Method Type I.

Teacher Tips

  • Enough materials are provided in this kit for 30 students working in pairs or 15 groups of students.

  • Forty Petri dishes are given in this kit. Thirty of the dishes should be used for the Carrot Callus Initiation and Shoot Development Medias. The other 10 dishes may be split up and used in step 7 of the Callus Formation Procedure.
  • Keep the sleeves of Petri dishes closed after they are initially opened to prevent contamination.
  • The Carrot Callus Initiation and Shoot Growth Development Media should be kept in a refrigerator for maximum shelf life.
  • The entire procedure will take 2 to 3 months to complete. Plan ahead accordingly. If time does not allow, the young plants developed from the callus material may be rinsed with water to remove agar remnants and may be taken home by students and placed in potting soil for full development.
  • Some of the steps, such as seed sterilization and seed germination, can be done by the instructor.
  • Additional copies of the Plant Cloning Data Table may be made if space for additional observations is needed.
  • Sterile technique is of the utmost importance in this activity. The following tips will help ensure a sterile work environment:

    1. The laboratory should be kept swept and mopped.
    2. All work surfaces should be cleaned with a bleach solution before the procedures are followed.
    3. All fans and exhaust systems in the classroom should be turned off.
    4. It would be best for students to perform plant tissue transfers in a sterile, confined area such as a fume hood or a transfer cabinet. A cardboard box lined with plastic or a sterile aquarium are great places to minimize contamination during transfer.
    5. Spray bottles containing 70% isopropyl alcohol should be provided to students.
    6. Sterile forceps and scalpels will be needed during the procedure. A container of 95% ethyl alcohol should be provided to each student group. Forceps and scalpels should be sterilized in the ethyl alcohol before use. Beakers work well as sterilization and rinsing containers.
    7. The students’ hands should be washed before performing the experiment.
    8. Plant tissue transfers should be performed quickly to minimize contamination.

  • Even when all sterile techniques are followed, success is not guaranteed. Success may have to be measured by knowledge gained rather than by the number of sterile cultures.
  • If a group’s Petri dish becomes contaminated, the Petri dishes of other student groups should be observed.
  • Lysol® or other bleach cleaners may be used to sterilize the countertops. Rubbing alcohol may also be used in place of 70% ethyl alcohol.
  • Please consult the disposal method if mold or bacterial cultures develop.
  • Cultures should be stored in a well-lit area out of direct sunlight. 16 hours of light and 8 hours of darkness are the ideal growing conditions.

References

Schumann, D. N. Living with Plants; Mad River Press: Eureka, CA; 1992

Student Pages

Clone Your Own Plants!

Introduction

Can plants really be cloned? Perform the following procedure to find out how multiple identical clones of a carrot plant can be produced.

Concepts

  • Cloning

  • Callus formation
  • Sterile techniques

Background

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).

{10668_Background_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.

Preparation

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.

Experiment Overview

In this activity, a basic cloning procedure will be performed to produce identical clones of an original carrot plant.

Materials

Bleach (sodium hypochlorite) solution, 5%
Ethyl alcohol, 95%, 30 mL
Beakers or other small containers, 2
Carrot
Cork borer
Forceps, sterile
Gloves, latex
Marker
Paper towels
Parafilm™, approximately 1.5 feet long
Petri dish (Carrot Callus Initiation)
Petri dish (Shoot Development)
Ruler
Scalpel, sterile
Spray bottle of 70% isopropyl alcohol
Stirring rod, glass

Safety Precautions

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.

Procedure

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.

Callus Formation

  1. Sterilize the working surface of the table by using paper towels and wiping it down with the bleach solution.
  2. Obtain a cork borer, a glass stirring rod, scalpel, forceps and a beaker or similar container.
  3. Place approximately 25 mL of 95% ethyl alcohol into the beaker or container.
  4. 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.
  5. Obtain a carrot from the instructor.
  6. Wash the carrot in warm water and soap. Remove any leaves and stems.
  7. Obtain a Petri dish lid or bottom and put on latex gloves at this time.
  8. Place the carrot in your gloved hand. Break off the large end and the small end. Discard these ends in the trash.
  9. 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.
  10. Pour enough 95% ethyl alcohol into the Petri dish to completely cover the bottom of the dish.
  11. Put the center section of the carrot (large end down) into the Petri dish containing the ethyl alcohol.
  12. 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.
{10668_Procedure_Figure_2}
  1. Pull the cork borer out.
  2. 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.
  3. 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.
  4. Using the scalpel, cut the remaining piece of core into 1- to 2-mm thick cross sections.
  5. Obtain the Carrot Callus Initiation Petri dish from your instructor. Be sure the lid remains closed!
  6. Remove the forceps from the jar of ethyl alcohol.
  7. 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.
  8. 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).
{10668_Procedure_Figure_3}
  1. Using a marker, label the top of the Carrot Callus Initiation Dish with your group’s initials.
  2. Spray the outside of the dish with 70% isopropyl alcohol.
  3. Place the dish in an area away from direct sunlight.
  4. Make sure the Petri dish remains in area that does not exceed 80 °F.
  5. Callus formation will take approximately one to two months.
  6. If the Petri dish becomes contaminated, it must be disposed of by the instructor.
  7. Once the callus is 1 cm or larger in diameter, it is ready to be propagated using the Shoot Emergence Procedure.

Shoot Emergence

  1. Remove the Parafilm from the Carrot Callus Initiation Media Petri dish.
  2. Obtain the Shoot Development Petri dish from the instructor.
  3. Sterilize the forceps as done in step 4 of the Callus Formation Procedure.
  4. 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.
  5. Seal the Petri dish, label, and sterilize as done in steps 20–22 of the Callus formation procedure.
  6. Place the Shoot Development Petri dish under artificial light or indirect sunlight in an area between 72 and 80 °F.
  7. If the Shoot Development Petri dish becomes contaminated, it must be disposed of by the instructor.
  8. Within one to two months, multiple green shoots should develop.
  9. Each shoot will develop into a mature carrot plant.
  10. The shoots may be placed into the soil and grown as a normal seedling.
  11. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10668_Student1.pdf

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