Transformation of Soybean Embryogenic Cultures by Microprojectile Bombardment.

Abstract

Regenerable embryogenic suspension cultures of soybean (Glycine max [L.] Merrill, cv. Jack) were transformed by microprojectile bombardment with a β-glucuronidase (gus) gene and a hygromycin phosphotransferase (hpt) gene under control of the cauliflower mosaic virus (CaMV) 35S promoter on different plasmids mixed together. Many independent transgenic clones were obtained by selection for hygromycin resistance. GUS activity was detected in 45% of the transgenic clones showing that cotransformation occurred at high frequency. Stable integration of both the gus reporter gene and the hpt selectable marker were further confirmed by polymerase chain reaction (PCR) amplification of both genes using double primer sets together in the same reaction. Southern blot hybridization analysis also showed the presence of the foreign genes in genomic DNA. The frequency of embryo development was increased and the time span required for embryo development was reduced by the use of a liquid medium to induce embryo development. An average of 3645 GUS-positive spots and ten transgenic clones were produced per bombardment by this procedure giving a 0.27% ratio of stable to transient expression. The procedure described here can be used for modification of agronomic traits and for study of gene regulation in soybean.