Levansucrase activity but not fructan accumulation in transgenic lsdA-expressing sugarcane recovered by optimized microprojectile bombardment of embryogenic calli.

Sugarcane (Saccharum spp. hybrid) emerges as an ideal crop for the cost-effective transgenic production of fructans due to its high efficiency for fixing carbon and storing the substrate sucrose. As other gramineous species, sugarcane is recalcitrant to genetic transformation. In this work, we optimized conditions for the transformation of sugarcane cv. C1051 -73 via microprojectile bombardment of embryogenic calli. The genes encoding the enhanced green-fluorescent protein (eGFP) and the neomycin phosphotransferase (nptII), both under the control of the maize ubiquitin 1 (Ubi-1) promoter, were used for the early detection of transient transformation events and for the selection of stable transfor-mants, respectively. DNA was efficiently delivered into the cell without causing drastic damages in calli bombarded at the distance of 11 cm and the argon pressure of 90 PSI. Non-mosaic transgenic plantlets were recovered by increasing the geneticin concentration from 20 mg/L during callus growth to 25 mg/L for the shooting and rooting steps. Moreover, using the optimized transformation procedure, we recovered twenty transgenic sugarcane lines carrying the Gluconacetobacter diazotrophicus levansucrase gene (lsdA) modified for vacuolar targeting of the enzyme, as a strategy for fructan production. Southern blot and PCRanalysis revealed the stable presence of the chimaeric lsdA in the primary stalk and sprouts of plants grown under field conditions. None of the transgenic lines accumulated levan in mature stems or leaves, although one of them showed evident levansucrase activity in leaf extracts. [ABSTRACT FROM AUTHOR]