Browsing by Author "Simon H.T. Raharjo"

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    Recovery of Avocado( Persea americana Mill. ) Plants Transformed with the Antifungal Plant Defensin Gene PDF1. 2
    (Springer-Verlag, 2008-08) Simon H.T. Raharjo; Witjaksono, N.F.N.; Miguel A. Gomez-Lim; Guillermo Padilla; Richard E. Litz
    Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l -1 phosphinothricin in liquid maintenance medium for 3-4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH4NO3 and 30.3 mg KNO3 and supplemented with 0.1 mg l -1 thiamine HCl, 100 mg l -1 myo-inositol, 30 g l -1 sucrose, 3.0 mg l -1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l -1 sucrose, 4 mg l -1 thiamine HCl, 100 mg l -1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l -1 phosphinothricin, and 6.0 g l -1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l -1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA3; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.
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    Somatic Embryogenesis and Plant Regeneration of Litchi ( Litchi Chinensis Sonn.) From Leaves of Mature Phase Trees
    (Springer-Verlag, 2007-05) Simon H.T. Raharjo; Richard E. Litz
    Embryogenic cultures were induced from leaflets from new vegetative flushes of mature ‘Brewster’ litchi trees on B5 medium containing 400 mg l -1 glutamine, 200 mg l -1 casein hydrolysate, 30 g l -1 sucrose, 4.52 µM 2,4-D, 9.30 µM kinetin and 3 g l -1 gellan gum in darkness. Embryogenic cultures consisting of proembryonic cells and masses were maintained either on semi-solid MS medium supplemented with 4.52 µM 2,4-D and 0.91 µM zeatin or as embryogenic suspension cultures in liquid medium of the same composition. Maturation of somatic embryos occurred on semi-solid MS medium with 5–20% (v/v) filter-sterilized coconut water in darkness. Recovery of plants from somatic embryos was improved with 14.4 µM GA3 on half-strength MS medium with 0.2 g l -1 activated charcoal under a 16 h photoperiod provided by cool white fluorescent lights (60–80 µmol s -1 m -2 ). Plants have been successfully acclimatized in the greenhouse.
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