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- Author or Editor: A.N. Trigiano x
- HortTechnology x
A tissue culture laboratory exercise illustrating regeneration of whole plants from leaf segments of chrysanthemum by organogenesis is described. Using simple, common media, shoots can be generated in 5 weeks and rooted after an additional 3 weeks. Acclimatization of plants can be accomplished in a simple mistbed in the greenhouse. The exercise is adaptable to depict genotype differences among cultivars, optimization of shoot induction, effects of growth regulators, and experimental design. Callus is typically not formed during shoot formation; however, co-cultivation of leaf segments with a virulent strain of Agrobacterium tumefaciens produces callus with a strain of disarmed A. tumefaciens harboring NPTII construct affects regeneration of plants resistant to kanamycin.
One of the most frequently used tools in plant biotechnology, which includes genomics and proteomics, is gel electrophoresis. Our experience with middle and high school students as well as teachers and undergraduate students is that they have very little, if any, hands-on experience with this technique. These exercises were developed to demonstrate the principles of electrophoresis and DNA fingerprinting in middle and high school and university laboratories with minimal expense and equipment. The experiments have been tested by middle and high school students, as well as by teachers, and undergraduate and graduate students. The first exercise, electrophoresis of common food dyes, is primarily designed for secondary and undergraduate students, but can be used as an inexpensive means for introducing the main concepts of electrophoresis to anyone who has little or no experience, including graduate students. Popular brands of food dyes (red, blue, yellow, and green) purchased at local markets are mixed into a 60% glycerol/water solution and are separated on 1% agarose gels using 100 V for 35 min. Mixed colors are separated into primary colors (e.g., green into blue and yellow) and some apparently single dyes often have extra “surprise” components. A simple exercise illustrating forensic use of gel electrophoresis with dyes is also included. Over 100 students and teachers have completed this experiment successfully. The second laboratory exercise requires more extensive equipment and a more advanced set of skills; however, the exercise has been completed successfully by middle school-level through graduate-level students and by teachers. In this exercise, the internally transcribed spacer region of the ribosomal subunit for a fungus, plant, and insect are amplified and separated electrophoretically on agarose gels. A simple crime is solved using polymerase chain reaction (PCR) and DNA fingerprinting. The experiment protocol provides students with hands-on activities that include assembling master mixes for PCR, practice using pipettes, and performing the various steps involved in PCR amplification. Instructions for both exercises are formatted in easy-to-follow procedure boxes, and a downloadable presentation is available on the web. The cost of the expendables is about $1 per student, making these exercises relatively inexpensive to conduct, assuming that hardware and DNA are available.