HIGH-EFFICIENCY, MICROPROJECTILE-MEDIATED COTRANSFORMATION OF SUGARCANE, USING VISIBLE OR SELECTABLE MARKERS

Transient expression of the maize anthocyanin regulatory elements, R a nd C1, was used to optimise parameters for microprojectile-mediated de livery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pul se increased the frequency of transient expression to 5-8 x 10(3) cell s per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthoc yanin-pigmented proembryoids which subsequently regenerated into plant lets. However, constitutive expression of R and C1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing lucifer ase activity with antibiotic selection on medium containing G418 or ph osphinothricin, upon bombardment of callus with constructs driving str ong expression of luc, aphA or bar genes. Selective subculture based o n luciferase activity enabled recovery of 1.4 +/- 0.5 independent tran sgenic plants per bombardment, compared to 19.8 +/- 3.7 independent tr ansgenic plants per bombardment from an optimised G418 selection regim en, and no transformed plants from phosphinothricin selection. When lu c and aphA on separate plasmids were coprecipitated onto microprojecti les before bombardment, 67-79% of callus lines selected for G418 resis tance also showed luciferase activity detectable under a low-light cam era. Southern analysis confirmed a very high cotransformation frequenc y, with variable copy numbers of introduced genes. The high efficienci es of gene transfer, selection and cotransformation in the optimised s ystem, coupled with the simple initiation and regeneration of embryoge nic callus, provide an effective tool for practical genetic transforma tion of sugarcane.