tools in plant biotechnology, which includes genomics and proteomics, are gel electrophoresis, polymerase chain reaction (PCR), and DNA fingerprinting. To incorporate these techniques into middle and high school and college curricula, content instruction
R.N. Trigiano, B.H. Ownley, A.N. Trigiano, J. Coley, K.D. Gwinn, and J.K. Moulton
The apical meristems of Calanthe orchid embryos were exposed to 1 mg/ml pBI-121 DNA in an electric field. pBI-121 contains the GUS marker gene glucoronidase under the control of the 35 S cauliflower mosaic virus promoter. A pipette containing 0.3% agarose and acetate buffer containing the DNA was placed on one end of the embryo; while the opposite end was in contact with a pipette containing only buffer and agarose. Uptake of the DNA into the meristem was monitored by 4′6-diamidino-2-phenylindole (DAPI) fluorescence. Optimal uptake occurred after 10 min of electrophoresis at 10 volts and 0.5 milliamps. Under these conditions, 55% of the embryos survived the treatment and 57% of those which survived were transformed as measured by GUS-positive staining. Leaves from 6 month old plants which developed from the transformed embryos expressed specific patterns of GUS staining.
Isabel Trujillo, Luis Rallo, and Pere Arús
Pollen samples of 155 olive (Olea europaea L.) cultivars from different origins were analyzed to study isoenzymatic variability in five enzyme systems: alcohol dehydrogenase (ADH), esterase (EST), glucose phosphate isomerase (GPI), leucine aminopeptidase (LAP), and malic enzyme (ME) using starch gel electrophoresis. Polymorphism was observed in all of the isozyme systems. ME, GPI, EST, and LAP were the most useful systems for identification of cultivars. Different combinations of banding patterns of these systems allowed us to identify 85% of the cultivars. The remainder were separated into groups of two or three cultivars that could be identified using morphological characteristics. No intracultivar polymorphisms were observed.
A.R. Dixon, R.B. Boone, A. Gardea, L.S. Daley, and T.L. Righetti
A microcomputer-based image processing system was used to simplify the large number of visual comparisons required to identify various Corylus spp., cultivars, and clonal accessions using polyacrylamide gel electrophoresis isozyme patterns. Photographs of gels stained for peroxidase, acid phosphatase, and phenol oxidase were digitally captured and selected lanes were enhanced and scanned. The scan data were analyzed to locate bands and normalize their position to that of standards. Such data were plotted and a computer-generated isozyme pattern was displayed. Compressed image data were then stored in a database for subsequent automated isozyme pattern comparisons. Photographic records that were previously used in published reports were reevaluated with the computerized system. Species, cultivars, or clones that were characterized in visual evaluations were similarly characterized using the computer method. Computer evaluations usually identified more bands. Band positions were only rarely different and probably resulted from better normalization relative to standard bands when using the computerized procedure.
R.J. Griesbach and J. Milo
Most methods developed to transform plants either require tissue culture to regenerate a whole plant from transformed tissue or the transformation of reproductive tissue. These approaches are not applicable to vegetatively propagated crops or crops that can not be tissue-culture propagated. We have modified a procedure that was developed to transform zygotic meristems for use with whole plant meristems. We developed a method to introduce DNA via electrophoresis into the cells of axillary meristems of whole plants growing in soil. About half of the treated meristems developed into shoots. Of those shoots, up to half had some level of GUS marker gene expression. We were able to transform Cercis, Chrysanthemum, Capsicum, and Prunus. In Capsicum, the GUS gene was inherited.
Larry S. Kennedy and Paul G. Thompson
The enzymes alcohol dehydrogenase, diaphorase, esterase, glutamate dehydrogenase, glucosephosphate isomerase, isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, 6-phosphogluconate dehydrogenase, phosphoglucomutase, shikimate dehydrogenase, and xanthine dehydrogenase were analyzed by starch gel electrophoresis of leaf tissue from nine sweetpotato [Ipomoea batatas (L.) Lam.] cultivars. Bands of most enzymes were well-defined. Polymorphisms were found in nine enzymes, and cultivars were identified by comparing polymorphisms.
J. Tous, C. Olarte, M.J. Truco, and P. Arús
The variability of isozymes in nine enzyme systems was studied in 25 carob (Ceratonia siliqua L.) cultivars using starch gel electrophoresis of leaf extracts. Five enzymes (phosphoglucomutase, phosphoglucoisomerase, aspartate aminotransferase, shikimic dehydrogenase, and aconitase) were polymorphic, making it possible for the 25 cultivars to be classified into eight phenotype categories.
C.E. Greer, R.E. Schutzki, A. Fernandez, and J.F. Hancock
Starch gel electrophoresis was used to fingerprint 55 Taxus plants, listed as 21 species and/or cultivars. Plants were analyzed for six enzymes, representing eight putative loci. Within many of the cultivars, different fingerprints were observed, indicating nomenclatural errors in Taxus.
Robert D. Marquard
Six phosphoglucomutase phenotypes were observed in pecan [Carya illinoensis (Wangenh.) K. Koch] progeny after controlled pollinations. At least one locus (Pgm-1) is present that controls polymorphism of phosphoglucomutase (PGM) isozymes in pecan. The inheritance appears simple with three observed alleles. However, progeny produced from two crosses resulted in significant deviation from the expected segregation ratios. Out of 65 named cultivars, 61 were of a single phenotype, and two of six possible phenotypes were not observed. Only one region of PGM activity was consistently expressed by gel electrophoresis from pecan tissue.
R. Messina, R. Testolin, and M. Morgante
The usefulness of isozyme banding patterns as genetic markers in kiwifruit [Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson] was investigated using starch gel electrophoresis. Fifty-four entries putatively belonging to seven female and two male kiwifruit cultivars were examined for 13 enzyme systems (AAT, ACO, GDH, G6PDH, IDH, MDH, ME, MNR, NDH, 6PGD, PGI, PGM, and SKDH). Four enzyme systems, ACO, MDH, NDH, and SKDH, showed identical banding patterns in all clones surveyed. Of the remaining enzymes, AAT, PGI, and PGM had the best discriminating power. Six enzyme systems (GDH, G6PDH, IDH, ME, MNR, and 6PGD), though showing polymorphic banding patterns, were poorly resolved. All the New Zealand cultivars were uniquely identified by the simultaneous comparison of the AAT, PGI, and PGM zymograms. Some enzyme systems were also polymorphic among plants within the same cultivar, thus proving the heterogeneity of kiwifruit material introduced into Europe in the early 1970s.