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Production of transgenic apricot plants from hypocotyl segments of mature seeds

Author:
Petri, César, Wang, Hong, Burgos, Lorenzo, Sánchez-Navarro, Jesús, Alburquerque, Nuria
Source:
Scientia horticulturae 2015 v.197 pp. 144-149
ISSN:
0304-4238
Subject:
Agrobacterium radiobacter, Prunus armeniaca, Southern blotting, apricots, buds, coculture, genetic markers, genetic transformation, genomics, greenhouse production, hypocotyls, kanamycin, meristems, micropropagation, photosynthetically active radiation, plasmids, polymerase chain reaction, recalcitrant species, reporter genes, rootstocks, seeds, selective media, shoots, transgenes, transgenic plants
Abstract:
Apricot (Prunus armeniaca L.) is one of the most recalcitrant species for genetic transformation. In this work; we report the regeneration of transgenic apricot plants from mature seed hypocotyl segments (cv. ‘Canino’ and ‘Moniquí’) for the first time. Moreover, this is the first report of the regeneration of apricot transgenic plants by means of the selective bar gene. Agrobacterium tumefaciens strain LB4404 harbouring a binary plasmid with eyfp and nptII genes (pMOG-eyfp-IV2) or a plasmid with gus and bar genes (pFGC5941-gus), was used in this study. A gradual selection strategy with aminoglycoside antibiotics made it possible to produce transgenic plants with 1.5±0.5% efficiency using 10 μM paromomycin (PAR). When buds were isolated and placed in a meristem development medium, PAR was changed to 20μM kanamycin (KAN). As the buds developed and elongated, the concentration was increased up to 140μM KAN based on a 20μM increase every 4 weeks. When bar was the marker gene, the regeneration-inhibitory PPT concentration (75.7μM) was applied immediately after the co-culture. In this case, the PPT concentration was reduced afterwards to 10.1μM in the micropropagation medium. This strategy allowed for the regeneration of transgenic shoots with 3.8±1.4% efficiency. Transformation events were monitored by detection of the reporter gene (eyfp or gus) expression. PCR and Southern blot analysis confirmed the stable integration of the transgenes into the apricot genome. Transformed shoots were rooted on a selective medium, acclimatised, cultured and raised under greenhouse conditions. Our results confirm that the process used in this study is an efficient transformation system for the apricot species which could be used for functional genomics studies and the development of transgenic rootstocks.
Agid:
5334660