Towards genetic transformation in the monocot Alstroemeria L. |
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Authors: | CE van Schaik C van der Toorn MJ De Jeu CJJM Raemakers RGF Visser |
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Institution: | (1) Laboratory of Plant Breeding, Graduate School of Experimental Plant Sciences, Wageningen Agricultural University (WAU), P.O.Box 386, 6700 AJ Wageningen, the Netherlands |
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Abstract: | The successful application of plant biotechnology to Alstroemeria improvement will largely depend on the availability of an efficient regeneration/transformation system. Regeneration in Alstroemeria is accomplished from nodular 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
divided to form either a new nodule or aproembryo. Transient gene expression after particle bombardment of nodular embryogenic
callus was optimized using DNA of pAHC25. The highest β-glucuronidase expression was found when the GUS gene was under control of the maize ubiquitin promoter, the target tissue was placed 5 cm below the microcarrier launch assembly
and when the rupture disc-breakage point was between 650–900 psi. Kanamycin blocked regeneration of somatic embryos, however,
did not block growth of nodular embryogenic callus. With phosphinothricin both callus growth and regeneration were blocked.
Bombardment of nodular embryogenic callus with DNA of pAHC25 combined with selection on medium containing phosphinothricin
resulted in putative transgenic chimeric. Friable calli were selected from nodular embryogenic callus and used to initiate
suspensions. These cell suspensions were subjected to transformation by particle bombardment using DNA of pAHC25 and resulted
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 demonstrated the presence of the introduced
gene in this friable callus line.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | cell suspension monocotyledon selection somatic embryogenesis transformation |
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