Towards genetic transformation in the monocot Alstroemeria L.

The successful application of plant biotechnology to Alstroemeria improveme nt will largely depend on the availability of an efficient regeneration/tra nsformation system. Regeneration in Alstroemeria is accomplished from nodul ar embryogenic callus initiated from zygotic embryos. Histological studies of embryogenic callus initiation from 4-weeks old cultured ovules revealed that the outermost layers of the protoderm of the embryogenic nodules divid ed to form either a new nodule or a proembryo. Transient gene expression af ter particle bombardment of nodular embryogenic callus was optimised using DNA of pAHC25. The highest beta-glucuronidase expression was found when the GUS gene was under control of the maize ubiquitin promoter, the target tis sue was placed 5 cm below the microcarrier launch assembly and when the rup ture disc-breakage point was between 650-900 psi. Kanamycin blocked regener ation of somatic embryos, however, did not block growth of nodular embryoge nic callus. With phosphinothricin both callus growth and regeneration were blocked. Bombardment of nodular embryogenic callus with DNA of pAHC25 combi ned with selection on medium containing phosphinothricin resulted in putati ve transgenic chimeric. Friable calli were selected from nodular embryogeni c callus and used to initiate suspensions. These cell suspensions were subj ected to transformation by particle bombardment using DNA of pAHC25 and res ulted in a stable transformed friable callus line after selection based on luciferase activity. Even after 2 years of maintenance this callus line was luciferase positive and the Polymerase Chain Reaction analysis demonstrate d the presence of the introduced gene in this friable callus line.