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Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus

Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, 60131 Ancona, Italy
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Plants 2019, 8(6), 178; https://doi.org/10.3390/plants8060178
Received: 31 May 2019 / Revised: 13 June 2019 / Accepted: 15 June 2019 / Published: 17 June 2019
(This article belongs to the Special Issue 2019 Feature Papers by Plants’ Editorial Board Members)
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Abstract

Prunus spp. is one of the most recalcitrant fruit tree species in terms of in vitro regeneration and transformation, mostly when mature tissues are used as explants. The present study describes the in vitro regeneration via indirect organogenesis, and Agrobacterium tumefaciens-mediated transformation of the peach rootstock Hansen 536 (Prunus persica × Prunus amygdalus) through the use of meristematic bulks (MBs) as starting explants. Efficient adventitious shoot regeneration was obtained when Hansen 536 MBs were cultured on an optimized medium consisting of modified McCown Woody Plant medium (WPM) enriched with 4.4 μM 6-Benzyladenine (BA), 0.1 μM 1-Naphthaleneacetic acid (NAA) and 6.0 g L−1 plant agar S1000 (B&V). MB slices were used later as starting explants for Agrobacterium-mediated transformation to introduce an RNAi construct “ihp35S-PPV194” against PPV virus. Transgenic events were identified by both green fluorescent protein (GFP) screening and kanamycin selection at different concentrations (0, 17 or 42 μM). GFP-fluorescent proliferating callus lines were selected and confirmed to stably express the ihp35S-PPV194::eGFP gene construct by molecular analysis. Although shoot regeneration from these transgenic calli has not been obtained yet, this represents one of the few examples of successful attempts in peach genetic transformation from somatic tissues, and also serves as a useful in vitro system for future gene functional analysis in peach. View Full-Text
Keywords: Prunus spp.; organogenesis; genetic transformation; GFP; RNAi; PPV Prunus spp.; organogenesis; genetic transformation; GFP; RNAi; PPV
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Sabbadini, S.; Ricci, A.; Limera, C.; Baldoni, D.; Capriotti, L.; Mezzetti, B. Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus. Plants 2019, 8, 178.

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