A BIOLISTIC APPROACH FOR THE TRANSFER AND EXPRESSION OF A GUSA REPORTER GENE IN EMBRYOGENIC CULTURES OF PINUS-RADIATA

The biolistic(R) particle delivery system was used for the delivery of DNA into embryogenic tissue culture cells of Pinus radiata D. Don. Se veral experiments with varying parameters were performed to increase t he delivery efficiency. Six different controlling elements were cloned upstream of the beta-glucuronidase coding sequence (gusA reporter gen e) and transient expression of the gusA reporter gene was compared thr ee days after bombardment. The results clearly indicate a decrease in transient expression as follows: pEmu-derivatives with the ocs-enhance r-element > 2x CaMV 35S (with Kozak consensus-sequence) > 2x CaMV 35S (without Kozak consensus sequence) > CaMV 35S (with Kozak consensus-se quence) > CaMV 35S (without Kozak consensus sequence). Time course exp eriments monitoring gusA expression showed a significant decrease in t he number of blue spots 10-14 days after bombardment. A few blue clump s however, were still detected 35 days after shooting. Embryo initials expressing the gusA gene in all cells were also detected. The results suggest that it will be possible to develop a reliable biolistic prot ocol for stable integration of genes into Pinus radiata embryogenic cu ltures which are capable of plant regeneration.