Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

Abstract

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 μg/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 μg/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.

Improved regeneration and transformation protocols for three strawberry cultivars

“…Our obtained protocol for strawberry transformation via Agrobacterium is in the same consensus as previous transformation protocols with different cultivars where the transformation ratio ranged between 4-11%. A transformation frequency of 0.95% has been reported for the cultivar "Rapella" 62 and 6.5% for "Red Coat" 30,31 and an efficiency of 4.22% and 10% with cultivar Chandeler 22,60 and 11% for "Firework." 63 …”

“…The transformation of Fragaria was focused on establishing the system and using genes with potential for pest and herbicide resistance, cold tolerance, and ripening. 22,[28][29][30][31][32][33][34][35][36] The aim of this study was to develop an efficient and reproducible Agrobacterium-mediated protocol for the strawberry cultivars using the juvenile leaf explants.…”

Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

“…From our results it is apparent that the transformation efficiency was enhanced with 3 days preculture of these explants on regeneration medium before cocultivation with Agrobacterium. Nehra et al (1990) reported that the preculture of inoculated leafdisks for 10days on non-selective shoot regeneration medium prior to selection on kanamycin was a key factor in obtaining transgenic shoots at high frequency. Recently, Muthukumar et al (1996) explained that the trans* formation efficiency decreased by half if two days coculture without preculture was applied in cowpea.…”

GENETIC TRANSFORMATION AND REGENERATION OF COMMON BEAN (Phaseolus vulgaris L.) USING Agrobacterium SYSTEM

“…A shoot regeneration system from leaf disks of strawberry inoculated with Agrobacterium contains nptII and gusA genes cultured on medium containing 50 mg/l kanamycin was described by Nehra et al (1990). In tobacco, leaves were wounded by microprojectile prior to inoculate with Agrobacterium strain LBA4404 containing nptII gene, and transferred to regeneration medium with 100 mg/l kanamycin for selection (Bidney et al, 1992).…”