Biolistic transformation of chitinase and Pto genes into soybean.

摘要

Soybean [Glycine max (L.) Merrill] is one of the world's most important agronomic crops. Charcoal rot caused by the fungal pathogen Macrophomina phaseolina (Tass) and Soybean Cyst Nematode (SCN) caused by the nematode Heterodera glycines (Ichinohe) are two of the most damaging soybean diseases in the mid-west. Soybean has been the focus of extensive efforts towards pathogen resistance improvement both by conventional breeding techniques and genetic engineering. The approach was to control these diseases by engineering soybean plants for constitutive expression of pathogenesis-related (PR) protein genes and the Pto resistance gene. A rice (Oryza sativa L.) chitinase gene (chill), tobacco hornworm (Manduca sexta) chitinase gene (msc), and the Pto gene from tomato (Lycopersicon esculentum L.) were linked to the hpt gene as a selectable marker and transformed using microprojectile bombardment. Immature embryos of soybean cultivars ‘Chapman’, ‘Jack’, and ‘Fayette’ were bombarded, and several independent clones were selected on hygromycin-containing media and regenerated into plants. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of transgenes were confirmed by molecular analysis of transgenic soybean plants. Independent transformants were confirmed by the polymerase chain reaction (PCR) to contain selectable (hpt) marker gene and the gene of interest. Progeny analyses showed that the introduced genes were inherited and segregated in a 3:1 Mendelian fashion. The presence and estimated copy number of inserts were detected by Southern blot analysis. Northern blotting and Western blotting confirmed the expression of transgenes. However, the loss of the Pto gene expression was observed in early generations of transgenic plants. Progeny from soybean transgenic plants were tested for resistance to charcoal rot and the soybean cyst nematode (SCN) in the greenhouse. T ₂ progeny of transgenic soybean showed increased resistance to the charcoal rot causing fungus, Macrophomina phaseolina. Plants expressing chitinases did not have enhanced resistance to soybean cyst nematode (SCN). This is the first report of the expression of chitinase genes in soybean and enhanced resistance to M. phaseolina in transgenic soybean plants.