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.

Adventitious Shoot Regeneration from In Vitro Leaf Explants of the Peach Rootstock Hansen 536

“…The collected shoot tips were surface sterilized by washing them in 1% (V/V) sodium hypochlorite solution for 15 min, followed by three washes with sterile distilled water. Explants were then placed on shoot multiplication medium composed of McCown Woody Plant Medium (WPM) (Duchefa Biochemie, Haarlem, The Netherlands) basal salts and vitamins [ 57 ], 30 g L −1 sucrose and 5 g L −1 plant agar S1000 (B&V, Reggio Emilia, Italy) [ 3 ] supplemented with 6.6 μM N 6 -benzylaminopurine (BAP) (Duchefa Biochemie, Haarlem, The Netherlands) and 0.1 μM α-naphtalene acetic acid (NAA) (Duchefa Biochemie, Haarlem, The Netherlands). The final pH value was adjusted to 5.7 with KOH before autoclaving at 121 °C for 20 min.…”

“…In a recent paper, we described the optimization of an in vitro regeneration and transformation protocol for the peach hybrid rootstock Hansen 536 (P. persica × P. amygdalus) using meristematic bulks as starting explants [3]. Even though the regeneration rate obtained through this system was about 80%, only transgenic callus lines were obtained that did not lead to the regeneration of any transgenic plant.…”

“…Among fruit trees, peach is recognized as one of the most recalcitrant species in terms of in vitro organogenesis [ 2 ], and only a few studies report the establishment of efficient and reproducible regeneration protocols starting from different explants of peach rootstocks and cultivars. Although the use of adult tissues is highly recommended to preserve desirable traits of peach selected clones [ 3 ], adventitious shoots regeneration starting from seed-derived material has been the main successful approach used in the past for this species [ 4 , 5 , 6 , 7 , 8 ]. However, peach adventitious shoot regeneration, avoiding the use of juvenile tissues, has also been recorded starting from meristematic tissues [ 2 , 3 , 9 ] and leaves [ 10 , 11 , 12 , 13 ].…”

“…Although the use of adult tissues is highly recommended to preserve desirable traits of peach selected clones [ 3 ], adventitious shoots regeneration starting from seed-derived material has been the main successful approach used in the past for this species [ 4 , 5 , 6 , 7 , 8 ]. However, peach adventitious shoot regeneration, avoiding the use of juvenile tissues, has also been recorded starting from meristematic tissues [ 2 , 3 , 9 ] and leaves [ 10 , 11 , 12 , 13 ]. In particular, Gentile et al (2002) described the only study reporting in vitro shoot organogenesis from leaves of Prunus persica L. cultivars (from expanded leaves, from leaves of preconditioned shoot apices and from leaves of adventitious shoots) [ 10 ].…”

Genetic Transformation in Peach (Prunus persica L.): Challenges and Ways Forward

“…Meristematic bulks (MBs), initiated from shoot tips, mechanically and chemically treated, differentiated, and regenerated adventitious shoots. After 32 weeks of selection with kanamycin, the efficiency of the procedure was relatively poor (0.3%), and when this methodology was applied to “Hansen 536”, another peach x almond hybrid rootstock, only produced transgenic callus lines [ 20 ].…”