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.
Highlights
Traditional breeding techniques applied in the genetic improvement of Prunus species, especially peach and nectarines, are characterized by a significant level of heterozygosity
A widespread problem of hyperhydricity was observed in the culture media tested, which were all supplemented with plant agar (PA) as gelling agent
Results showed a significant increase in regeneration efficiency only in medium WPMm5 when meristematic bulks (MBs) slices were cultured on media with B&V instead of PA (Table 2)
Summary
Traditional breeding techniques applied in the genetic improvement of Prunus species, especially peach and nectarines, are characterized by a significant level of heterozygosity. Virus resistance induction is among the primary goals in plant genetic improvement, and new breeding techniques (NBTs) represent a promising integrative tool to traditional methods, especially when applied on woody fruit species such as Prunus spp. RNA Interference (RNAi) has become one of the most promising new strategies applied to induce resistance in plants against different kinds of pathogens, including viruses, fungi, and insects [11,12,13,14]. This mechanism is known to regulate endogenous gene. Different successful in vitro regeneration protocols have been developed for Prunus species, in peach, using different starting tissues; among these reports, the majority used immature seeds and cotyledons as starting explants [27,28,29,30,31]
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