Development of efficient regeneration and transformation systems in Alstroemeria

This thesis describes an alternative regeneration system via somatic embryogenesis of leaves with axil tissue and the application of this system for genetic modification in Alstroemeria . Both compact embryogenic callus (CEC) and friable embryogenic callus (FEC) were induced from leaves with axil tissues. Alstroemeria plants were regenerated from somatic embryos induced from CEC and FEC within six to seven months. Protoplasts were successfully and efficiently isolated from FEC culture. After culture of protoplasts in liquid culture and micro-colony formation in solid medium, embryogenic callus cultures were obtained with high efficiency from isolated protoplasts. Plants with healthy roots were then produced and established in the greenhouse after 8 months of protoplast isolation. FEC cultures were transformed using the Alstroemeria mosaic virus (AlMV) coat protein gene and a 3'-untranslated region sequence generated by particle bombardment. The presence of transgenes was confirmed by the expression of the GUS gene, luciferase, as well as by PCR. Over 20 independent transgenic virus-resistant lines were obtained by particle bombardment protocol developed. Agrobacterium -mediated transformation was made to transform FEC of Alstroemeria by using a high efficient protocol. Finally, the FEC culture system was subjected to particle bombardment and Agrobacterium -mediated transformation in Alstroemeria