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Plant Biotechnology:
Controlling Tissue Differentiation

Sara Coleman and Paul Rabe
1994 Woodrow Wilson Collection


Introduction

This lab activity is designed to demonstrate hormonal control of plant tissue differentiation and relate this differentiation to gene expression. Differentiated cells in a plant usually do not divide, but can be experimentally induced to divide and to de-differentiate by extracting tissue pieces (explants) and placing them under sterile conditions on an appropriate culture medium that contains the necessary nutrients and additives (see recipe provided). The explants will develop into callus and later into random differentiation of vascular tissues (shoots or roots). The addition of cytokinin and auxin are necessary for callus growth. By manipulating the proportions of these two hormones, callus tissue can be induced to produce shoots, roots or both, resulting in a regenerated plant.

In this lab we will look at the effects of hormones (auxin and cytokinin) on the genes that control tissue differentiation in callus. Hormones are produced by organisms and control such varied activities as growth, control of cell cycle, and reproduction. Many hormones have the ability to change the type of genes that are expressed with phenotypic consequences. Thus, they play an important role in cell differentiation. It has been found that if the cytokinin-to-auxin ratio is maintained high, certain cells are produced in the callus that give rise to buds, stems, and leaves. But if the cytokinin-to-auxin ratio is lowered, root formation is favored. By choosing the proper ratio, the callus may develop into a new plant. This control is a powerful tool to select plants for genetic engineering that demonstrate resistance to drought, salt stress, pathogens, harvesting techniques, and certain herbicides.

Students will demonstrate skills of biotechnology stringent aseptic technique, statistical analysis, and form conclusions based on data collected in class.

Materials

  • complete kits are available from biological supply companies
  • explant sample; African Violet, Kalanchoe, Hosta
  • sterile glass tubes 25 mm X 150 mm or Petri dish 25 X 100 mm
  • Carolina Biological culture tubes with MS medium. Purchase correct medium for tissue used.
    MS = Murashige and Skoog medium containing a full complement of major and minor salts, vitamins, 30 g/L sucrose and ampicillin (100 mg/L), adjusted to pH 5.
  • plant hormones benzyladenine (BA) a cytokinin and 2,4-dichloro-phenoxyacetic acid (2,4-D) an auxin in a 2:1 ratio respectively
  • incubator or equivalent (25-28 oC)
  • light source - Gro-Lux or equivalent (16 hour a day photoperiod)
  • distilled water as needed
  • 70 % ethanol as needed
  • 1.5 % NaClO as needed
  • sterile scalpel or razor
  • surgical gloves
  • optional - face mask

Please note: use stringent sterile technique to minimize bacterial contamination.

References

Lewis, Ricki. 1994. Human Genetics - Concepts and Applications. Brown Pub.

Raharjo, S.H.T., Punja, Z.K. Jan. 1994. "Regeneration of Plantlets from Embryogenic Suspension Cultures of Pickling Cucumber." Vitro Cell Development Biology. 30P:16-20.

Pierch, R.L.M. 1987. In Vitro Culture of Higher Plants. Publisher unknown.

Skoog, F., C.O. Miller 1957. "Chemical regulation of growth and organ formation in plant tissues cultured in vitro." Symp. Soc. Exp. Biol. 11: 118.


Student Procedure

Students Note: Sterile Technique is needed for this procedure.

  1. Set up four test groups (or solutions) as follows:
    • Test 1: BA & 2,4-D 2: 1 concentration ratio
    • Test 2: 2,4-D alone
    • Test 3: BA alone
    • Test 4: no treatment (control)

  2. Prepare explant samples as follows; cut petiole segments 3 to 5 mm in length with sterile scalpel or razor blade. Select shoots from newly emerged leaves.

  3. Explant samples should be washed in sterile distilled water, immersed in 70% ethanol for 60 seconds, sterilized by immersion in 1.5% NaClO for 30 seconds and finally rinsed 3 times with sterile distilled water.

  4. Place explant samples onto medium and treat with selected hormone using a sterile pipette for each hormone treatment.

  5. Incubate in continuous dark for 3 weeks and transfer to the light under a 16 hour photoperiod at 25 to 28 oC.

  6. Inspect cultures weekly and record the time of cell proliferation, first organ appearance, and their numbers. At the end of the experiment determine fresh weight. This can be done by determining the volume (length x width x height) and multiplying by 1 g/cm3.

Discussion and Analysis:

  1. Collect data from the class and determine the number of cultures that exhibit shoots or roots. Suggest reasons for the observed results.

  2. Analyze the results of differentiation (shoots or roots) in class as a function of hormone treatment and graph. Suggest relationships between hormone type and gene expression.

  3. Continue to culture plantlets and transplant to soil when sufficiently developed. Observe the phenotypic relationships to parent plant and record.

  4. Comment on the social and economic effects of plant biotechnology.

  5. What risks are there to native plants with the use of biotechnological developed plant species?


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