Transgenic alfalfa : development and characterization

Abstract

Alfalfa (Medicago sativa L.) is the world's premier forage crop because of its nutritional quality, performance and broad adaptation. Several problems exist in the production of alfalfa, including disease and insect damage, herbicide sensitivity and limited nutritional quality. Plant transformation (the introduction of foreign genes into a species) has been proposed as a potential solution to each of these problems. One consideration in alfalfa transformation research which has not been adequately addressed is the inheritance and expression stability of foreign genes in transgenic alfalfa. This study is the initial phase of a project to examine this question. Transgenic alfalfa plants were generated and characterized prior to the initiation of crossing studies. In particular, the usefulness of polymerase chain reaction sequence amplification (PCR) in characterizing transgenic alfalfa was examined. Three Montana-adapted alfalfa cultivars were transformed using Agrobacterium-mediated transformation. The transformation vector used contained two linked genes: the NPT II gene conferring kanamycin resistance and the GUS gene, a commonly used expression reporter gene. Contrary to expectations, 22% of the transgenic alfalfa plants failed to integrate the GUS gene into their genomes although they did integrate the NPT II gene. Further, 29% of the transgenic alfalfa plants which contained GUS gene failed to express it. This data was based on Southern blot analysis but similar results were obtained using the more rapid PCR method. The GUS reporter gene system functions in transgenic alfalfa and provides a rapid way to score progeny. PCR also promises to be useful in the continuing inheritance studies of these transgenic alfalfa.