References

Remington R.D. and Schork M.A. 1985. Statistics with applications to the biological and health sciences. Prentice-Hall, Englewood Cliffs, New Jersey.

s we discussed in Chapter 2, two methods of obtaining a mutant are by directed screening for plants with a certain phenotype and by knocking out a gene of interest. The phenotypes of knock-out mutants are difficult to predict, and it is also quite common for mutants, first isolated because of a specific phenotype, to have other, pleiotropic defects, i.e., multiple defects in addition to that of primary interest. Thus, it is often necessary to characterize a variety of phenotypic parameters. This is particularly important as the field of molecular genetics expands. If a number of groups, each looking for a different phenotype, independently identify different mutations in the same gene, an awareness of additional defects will often show that the new mutation is allelic to existing

Before embarking on an extensive phenotypic characterization, it is important to confirm that the various phenotypes of an individual are genetically linked. Strictly speaking, all of the phenotypes should be mapped, but it is normally sufficient to backcross a newly isolated mutant about five times to remove defects due to unlinked mutations. Phenotype mapping becomes more important, however when dealing with a mutant that has been isolated by site-selected mutagenesis (T-DNA or transposon knock-out). In this case, it must be shown that any phenotype cosegregates with the induced mutation. Definitive proof that a phenotype is indeed due to the disrupted gene can only come from complementing the mutant with a wild-type copy of the disrupted gene or by constructing trans-heterozygotes carrying two independently isolated mutations.

mutants.

The number of phenotypes that can be investigated is limited only by the imagination of the investigator. The following is a collection of protocols designed to investigate the more obvious phenotypes.

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