GhSEM-1 Marker Potentially Associated with Regeneration Ability in Cotton

Abstract

A marker protein for embryogénie potential could be useful in determining if target tissue for Agrobacterium tumefaciens or microprojectile bombardment has the ability to regenerate plants. Certain varieties of cotton, especially Coker 312, are known to form somatic embryos readily, while others are more recalcitrant. Callus induced from Coker 312–17wa s found to be more embryogénie than that from TM-1. To investigate the molecular basis for this difference in somatic embryo potential, total RNA was isolated from globular stage callus embryos, calli with heart-stage embryos, and non-embryogenic calli. Northern analyses were performed using a developmentally regulated cotton fiber gene as a probe. This probe hybridized to a single band, GhSEM-1 (Gossypium hirsutum somatic embryogénie marker 1), only in globularstage embryos. Genomic DNA was also isolated from young leaf tissue of both embryogénie (Coker 312–17) and non-embryogenic (TM-1) lines and a Southern analysis using the same probe was performed. No differences were found between the genomic DNA of both embryogenie and non-embryogenic lines, indicating that this gene was structurally present in both lines but only expressed in Coker 312–17 at the globular stage. Antibodies against GhSEM-1 were developed and used in immunoblots after SDS-PAGE separation of proteins from embryogenie and non-embryogenic calli. The antibody recognized a -15 kDa protein in the globular and heart-stage somatic embryos from Coker 312–17 and was designated as GEP15 (15 kDa Gossypium embryogénie protein). This protein could be used as a marker for determining if tissue has embryogénie potential. Our results represent the first reported potential association of a marker gene (GhSEM-1) and protein (GEP15) with somatic embryogenesis in cotton.