Development of a gene transfer system for American chestnut via somatic embryogenesis.

摘要

American chestnut (Castanea dentata (Marshall) Borkhausen) was once the most important angiosperm forest tree in the eastern United States. Introduction of chestnut blight (Cryphonectria parasitica (Murrill) Barr) caused elimination of American chestnut as an important forest tree. Somatic embryogenesis provides potential to produce blight resistant American chestnut via genetic engineering. Ovules and immature zygotic embryos cultured on modified liquid Woody Plant Medium (WPM) supplemented with 2,4-D produced embryogenic cell cultures which sustained long-term potential for producing somatic embryos.; Effects of carbon source, abscisic acid (ABA), and polyethylene glycol (PEG) on accumulation of starch and storage proteins in somatic embryos were investigated. Starch content and storage protein profiles of mature zygotic embryos were compared to those of somatic embryos. Fructose was more effective than sucrose in promoting increased starch accumulation at 30 g/l, but sucrose was superior to fructose at 60 g/l. Sixty g/l levels of both sugars were more effective than 30 g/l levels in promoting increased starch accumulation. ABA at 1 mg/l increased starch accumulation compared to no ABA, but at 5 mg/l starch accumulation decreased compared to both zero and 1 mg/l. Both levels of PEG tested increased starch accumulation compared to no PEG, with 50 g/l being more effective than 25 g/l. Highest levels of starch accumulation were obtained with ABA at 1 mg/l, PEG at 50 g/l and sucrose at 60 g/l. Somatic embryos exhibited storage protein profiles different from zygotic embryos, however, no differences in storage protein content or profile could be attributed to any of the treatments from which proteins were analyzed. Plantlets were produced from somatic embryos which were maintained 8 weeks on maturation medium and given a 12 week cold treatment at 4{dollar}spcirc{dollar}C.; Suspension cultured American chestnut cells were bombarded using gold particles coated with plasmid DNA carrying the uidA gene encoding {dollar}beta{dollar}-glucuronidase (GUS), and the nptII gene encoding neomycin phosphotransferase (NPTII). Bombarded cells were placed on a medium containing kanamycin sulfate. GUS expression in kanamycin resistant cells was confirmed via fluorometric and histochemical analysis. Stable integration of the introduced uidA gene in proliferating American chestnut cells was confirmed via Southern analysis.