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

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 M 6-Benzyladenine (BA), 0.1 M 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 M). 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.

Keywords: GFP; PPV; Prunus spp.; RNAi; genetic transformation; organogenesis.

Figure 1

Hansen 536 regeneration via organogenesis: (a) adventitious shoot regeneration after 30 days of culture on WPMm5 medium. (b) MB slices (1 cm², 2 mm thick) used as starting explant for the transformation experiment (bar = 1 cm).

Figure 2

Hansen 536 GFP fluorescing spots and/or Lzs detected on MB slices at three weeks after co-culture on medium with 0, 17 or 42 μM Kan. White and yellow arrows indicate examples of GFP spots and large zones of fluorescent actively growing calli, respectively. Uniform bright green fluorescence was observed under UV light. Upper and lower panels show images taken under UV and white light, respectively (bar = 2 mm).

Figure 3

Hansen 536 GFP fluorescing spots and/or Lzs detected on MB slices at three weeks and 12 weeks after co-culture on medium with 17 μM Kan: (a,b) MB slices obtained from two independent MBs presenting Lzs of GFP expression actively growing within 12 weeks of culture. Uniform bright green fluorescence was observed under UV light. Upper and lower panels show images taken under UV and white light, respectively (bar = 2 mm).

Figure 4

Hansen 536 transgenic callus lines and molecular analysis of transformants: (a) Callus line proliferated for five months after Agrobacterium infection. (b) Transgenic callus expressing uniform GFP fluorescence. (c) Amplification of ihp35S-PPV194 (456 bp) and eGFP (701 bp) genes fragments from the genomic DNAs of four transgenic (T1-T4) and one wild-type (WT) callus lines obtained from different MBs, and pK7WG2-ihp35S-PPV194::eGFP “(+)”. The lane labelled “(−)” shows the PCR result using water as negative control. M, DNA marker (1Kb Plus DNA Ladder, Invitrogen, Carlsbad, CA, USA). (d) RT-PCR analysis of eGFP (701 bp), ihp35S-PPV194 (456 bp) sequences of four transgenic (T1-T4) and one wild-type (WT) callus lines obtained from different MBs. The 129 bp fragment of cDNA from housekeeping gene Tef2 was amplified as control to validate RT-PCR results. The lane labelled “T1c” shows the PCR result using total RNA of the line T1 as template control (RNA sample treated with DNase but without reverse transcription was used as PCR template to evaluate DNA contamination).