Submitted to: Journal of Crop Improvement
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2006
Publication Date: 6/1/2006
Citation: Atlaf-Khan, M., Kim, H.J., Myers, G.O., Triplett, B.A. 2006. GhSEM-1 marker potentially associated with regeneration ability in cotton.. Journal of Crop Improvement. 16. p. 21-35. Interpretive Summary: To produce transgenic crops, large quantities of viable embryos from non-reproductive tissue need to be produced. Formation of these "somantic embryos" in tissue culture is largely an empirically-derived set of conditions. In many plant species, including cotton, different varieties produce somatic embryos more readily than other genetic varieties. Many of the genetic lines that readily form somatic embryos in culture are not commercially important and commercially important lines are recalcitrant to somatic embryo formation. We have cloned genes from cotton with sequence similarity to genes identified in other species as potential markers for somatic embryo formation. Expression of the marker gene transcript and production of the marker protein were monitored in two cotton varieties that differ in the ease of forming somatic embryos. The gene transcript and marker protein accumulate in the genetic line that easily forms somatic embryos and do not accumulate in the genetic line that does not form somatic embryos. Analysis of the deoxyribonucleic acid (DNA) indicates that the marker gene is present in both genetic lines. The function of this cotton somatic embryo marker 1 protein (GhSEM1) in cotton embryo formation will be the focus of future studies to improve the process of introducing novel genes into cotton plants.
Technical Abstract: A marker protein for embryogenic 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 312-17 was found to be more embryogenic 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 embryogenic marker 1), only in globular stage embryos. Genomic DNA was also isolated from young leaf tissue of both embryogenic (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 embryogenic 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 embryogenic 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 embryogenic protein). This protein could be used as a marker for determining if tissue has embryogenic potential. Our results represent the first reported potential association of a marker gene (GhSEM-1) and protein (GEP15) with somatic embryogenesis in cotton.