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CULTURING PLANTS FROM
EMBRYONIC PLANT TISSUE

Roger Herbert and Domenic J. Thompson
1993 Woodrow Wilson Biology Institute


INTRODUCTION

The ability to propagate a new plant from a small piece of another plant has many interesting possibilities. Among those considered are:

  • breeding a line of plants from only somatic tissue
  • the more rapid propagation of plants with desired genotypes
  • the ability to select plants with desired or superior hereditary characteristics
  • Plant propagation has become more feasible with the development of growth media that contain nutrients for the developing tissues. These media have been developed in response to the needs of biotechnologists using the same techniques that will be used in this lab exercise. This laboratory exercise will use a growth medium that will contain the macronutrient and micronutrient needs of plants.

    Carrying out plant propagation from small pieces of other plants requires aseptic techniques. The medium on which the plants will be grown must be sterile. The area in which the tissue transfer will take place must also be kept "germ free." Though these aseptic conditions may appear difficult to create in a normal high school laboratory setting, there are some simple techniques and reagents that can be used to insure aseptic work areas.

    Additionally, tissue for culture is easy to obtain. Plant embryos or pieces of embryonic tissue are readily obtainable from seeds that have been soaked.

    The type of lesson the teacher wishes to develop will determine the growth media recommended. If the teacher wants students to only propagate plants, then only basal medium is necessary for the techniques that follow. On the other hand, teachers who wish to have students observe the effects of different hormones on tissue cultures must use variations of the basic growth medium.

    TARGET GROUP:

    First year biology students. Variations of the basic laboratory exercise can be used with older students who may already have had biology.

    MATERIALS

    (based on a class of 24 students):
  • Sterile beaker (one per group, used for surface sterilization of seeds)
  • Sterile scalpel (one per group)
  • Sterile forceps, needle nose (two per group)
  • Bunsen burner (one per group)
  • Sterile Petri dishes or baby food jars that can be sterilized with media in them (number will vary depending on the teacher's needs)
  • Lima bean seeds, corn seeds and other seeds of the teacher's choice
  • REAGENTS

    (based on a class of 24 students)
  • 70% ethanol, in a bottle (0.5 L/class)
  • 95% ethanol, in a jar with a lid
  • 10% bleach solution, in a bottle(0.5 L/class)
  • 50% bleach solution, in a squirt bottle (1.0 L)
  • Sterile distilled water, in container for each station (1.0 - 1.5 L/class)
  • Murashige and Skoog basal medium with sucrose and agar (Sigma M9274)
  • Indoleacetic acid, 0.3 g/L of medium
  • Kinetin, 1 mg/L of medium
  • Adenine, 80 mg/L of medium

  • TEACHER GUIDE FOR THE PREPARATION OF MATERIALS:

    (Approximate preparation time: 2 hr.)

    Solutions for this lab exercise are used for creating and maintaining aseptic conditions. Though some of the dilutions might seem like "overkill," it is necessary to maintain optimal transfer conditions (in the absence of sterile conditions) for the embryos.

    70% ethanol (0.5 L/class)

    1. Use a 500 mL graduated cylinder to measure 370 mL of 95% ethanol.
    2. Add distilled or deionized water to bring the volume of liquid in the graduated cylinder to 500 mL.

    95% ethanol (Used only if students are allowed to flame materials)

    1. Normally, ethanol taken from the reagent bottle is 95% pure. Prepare this solution only if the students will be allowed to flame their forceps and scalpel during the process of removing the embryo from the cotyledons.
    2. Place the ethanol in a jar that has a lid that can be closed when the students are not using the ethanol.

    10% Bleach (Clorox) solution (0.5 L/class)

    1. Commercially prepared bleach is normally a 5% sodium hypochlorite solution. This dilution will create a 0.5% solution of sodium hypochlorite.
    2. Use a 500 mL graduated cylinder to measure 50 mL of commercial bleach.
    3. Use 450 mL of distilled or deionized water to dilute the bleach in the graduated cylinder to a volume of 500 mL.
    4. Transfer the solution to covered bottles to be distributed to each student lab station.

    50% bleach (Clorox) solution

    1. Use a 1.00 L graduated cylinder to measure 500 mL of commercially prepared bleach.
    2. Use 500 mL of distilled or deionized water to dilute the bleach to a volume of 1.00 L.
    3. Transfer the solution to squirt bottles. Students will use this solution to clean the area of the lab table where they are working. Make enough cleaning bottles so that each lab station has access to this cleaning solution.

    Sterile distilled/deionized water

    1. Each lab station should be supplied with a 200 mL beaker containing 100 - 125 mL of sterile water.
    2. Pour approximately 100 - 125 mL of distilled/deionized water into each of the beakers to be sterilized.
    3. Cover each beaker with aluminum foil and autoclave them at 15 psi for 15 minutes.
    4. Allow autoclave to cool before releasing the pressure on the autoclave.

    Growth Media
    Basal growth medium (Murashige and Skoog Basal Medium with Sucrose and Agar) can be purchased from either Sigma [Sigma M 9274] or Carolina Biological . Instructions that follow are for the basal medium that is purchased from Sigma.


    Growth Medium: BASAL
    Amount of powdered medium; g/l = 42.4
    Volume of Water, L. = 1
    Additions to media, per L = None
    Growth Medium: SHOOT TIP ROOTING MEDIUM
    Amount of powdered medium;g/l = 42.4
    Volume of Water, L = 1
    Additions to media, per L = 1mg kinetin 0.3g IAA
    Growth Medium: SHOOT MULTIPLICATION MEDIUM
    Amount of peedered medium,g/l = 42.4
    Volume of Water, L = 1
    Additions to media, per L =1mg kinetin, 80 mg adenine
    1. When preparing media always use a container twice the size of the final volume of media being prepared.

    2. Add the powdered media product while stirring the water.

    3. If possible, use double distilled water for the media preparation.

    4. If the instructor desires a specific pH, use KOH or HCl to adjust the pH.

    5. After the desired medium is prepared, pour the medium into the desired containers. Autoclave any non-sterile containers with their media for 15 minutes at 15 psi.

    Note: The media that have been suggested for use in this laboratory exercise are to be used with embryos that have been soaked for 24 hours. Teachers may try to use embryos of different ages to observe the effects of the different media.

    Variations: Another variation of this experiment might be to ask the students to excise only specific tissues from the 24 hour old embryos. Students can excise only radicle tissue or shoot tissue and grow them on the different media. Comparison of results of the different experiments can be done using different analysis methods.

    Teachers could also use different types of monocot and dicot seeds, e.g. pumpkin, sunflower, cucumber, peas, grasses (Caution: check student allergic reactions).


    POSSIBLE OUTCOMES:

    Teachers can also use seedling tissues to vary this experiment. Students can vary the ratios of IAA to kinetin when using seedling tissues. If students use only kinetin in the growth medium no growth can be expected. As the ratios of hormones are changed, there should not only be variation in tissue growth patterns but also in the size of cells found in the tissues that are propagated. Large proportions of IAA to kinetin should result in large cells.

    SAFETY CONSIDERATIONS:

    1. Students should use goggles and aprons when handling the bleach and alcohol solutions.

    2. The open flame of the Bunsen burner should be a safe distance from the alcohol when using the flame technique of sterilization.

    3. Students must be cautioned about the sharp edges that are found on scalpels.


    CULTURING PLANTS FROM EMBRYONIC PLANT TISSUE

    Purpose:

    The purpose of this lab is to grow plants in tissue cultures and to see what effect the different concentrations of hormones have on embryo development.

    BACKGROUND INFORMATION:

    Flowering plants are divided into two groups; monocots and dicots. Monocots are characterized by parallel veins, flower parts in 3's, and one cotyledon in the seed. Dicots have net veins, flower parts in 4's or 5's, and two cotyledons in the seed. The seeds of flowering plants consist of the cotyledon(s) and the embryo. By removing the embryo from the cotyledons and then placing the embryos on agar having varying hormone concentrations, one can see the influence that the hormones have on the growth of the embryo.

    The embryo is composed of the epicotyl which is the portion of the seed above the attachment of the cotyledons and gives rise to the stem and leaves. The hypocotyl is the portion of the stem below the attachment of the cotyledons. The radicle is the part of the embryo that contains the root apical meristem and becomes the first primary root of the seedling. Different parts of the embryo may react differently to either the type of hormone or the concentration of the hormone.

    When doing tissue cultures, the work area and equipment should be kept as sterile as possible to prevent contamination from molds or other microorganisms.

    STUDENT INSTRUCTIONS

    PROCEDURE:

    Read The Entire Procedure Before You Begin

    DAY 1

    1. Each group will use the lima beans to represent dicots and corn to represent monocots. Your group will have to decide on a third type of seed to use. Possibilities include pumpkin, sunflower, cucumber, or another type of seed of your choice.

    2. Obtain a beaker and put at least 15 seeds of each type in a beaker. Submerge the seeds in water, cover with aluminum foil, and allow all seeds to soak for 24 hours.

    DAY 2

    1. The work area should be kept as clean as possible when doing the initial preparation of the seeds and the culture dishes. Clean the desk with soap and water and wipe down with 70% alcohol and/or 50 % bleach.

    2. Remove 12 corn seeds, 12 lima bean seeds, and sterile beaker and surface sterilize the seeds using the following method.

      a. Add about 25 mL. 70% ethanol to cover the seeds and incubate at room temperature for 2-4 minutes.
      b. Pour out the ethanol and add 10% Clorox to again cover the seeds. Incubate for 2-4 minutes.
      c. Pour out the Clorox and wash the seeds with sterile distilled water for 2-4 minutes.
      d. Repeat the water wash another two times.

    3. Place the seeds in a sterile Petri dish.

    4. Obtain the sterile forceps and scalpel from your teacher. (An alternate method of sterilization would be to use 95% alcohol and a Bunsen burner. ONLY USE UNDER THE DIRECTION OF YOUR TEACHER.)

    5. Remove the seed coat from the dicot seeds and separate the embryos from the cotyledons. Be careful to keep the embryo in one piece and to keep the embryos as sterile as possible. You should use the forceps for the dicot seeds. The scalpel will have to be used for the corn seed to cut away the cotyledon and endosperm leaving the rudimentary plant surrounded by their protective sheaths.

    6. Place three embryos of each kind on the Petri dishes your teacher has provided. Each Petri dish has a different concentration of hormones and growth media. Place the corn seeds flat on the agar. The dicot seeds should be placed with their hypocotyl in the agar.

    7. Observe the changes in the embryos for at least one week. Record your results and sketch the embryo indicating where most of the growth took place.

    DAY 3

    Optional

    1. With some of the remaining seeds, dissect out the embryos and test both the embryos and cotyledons with iodine.

    2. Draw and label each seed and indicate where the starch is located. Use your textbook for resource material.

    DAYS 4 - 8

    1. Continue your observations by recording any changes in the developing embryos that are growing on the culture media.

    DISCUSSION QUESTIONS:

    Answer in complete sentences.

    1. Describe the pattern of growth on the different dishes.

    2. On which of the dishes did most of the growth take place? Explain the relationship between hormone concentration and growth of the embryos.

    3. Explain any differences between the growth of the epicotyl and the hypocotyl.

    4. If the embryos were still attached to the cotyledons, what would have been the primary source of energy for the developing plant?


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