The success of in vitro plant regeneration and the competence of genetic transformation greatly depends on the genotype of the species of interest. In previous work, we developed a method for the efficient Agrobacterium-mediated genetic transformation via organogenesis of V. vinifera cultivar Thompson Seedless, by using meristematic bulk (MB) as starting tissue. In this study, we applied this method for the regeneration and transformation of MBs obtained from the Italian cultivar Ciliegiolo and two of the commonly used Vitis rootstocks, 110 Richter and Kober 5BB, in comparison with Thompson Seedless. The A. tumefaciens strain EHA105, harbouring pK7WG2 binary vector, was used for the transformation trials, which allowed selection through the enhanced-green fluorescent protein (eGFP) and the neomycin phosphotransferase (nptII) gene. Putative transformed tissues and/or shoots were identified by either a screening based on the eGFP expression alone or its use in combination with kanamycin in the medium. MBs obtained from Thompson Seedless showed the highest regeneration and transformation cell competence, which subsequently allowed the recovery of stably transformed plants. Ciliegiolo, 110 Richter, and Kober 5BB, produced actively growing transgenic calli showing eGFP fluorescence, more consistently on selective media, but had no regenerative competence.
Almost 30 years have passed since the first publication reporting regeneration of transformed peach plants. Nevertheless, the general applicability of genetic transformation of this species has not yet been established. Many strategies have been tested in order to obtain an efficient peach transformation system. Despite the amount of time and the efforts invested, the lack of success has significantly limited the utility of peach as a model genetic system for trees, despite its relatively short generation time; small, high-quality genome; and well-studied genetic resources. Additionally, the absence of efficient genetic transformation protocols precludes the application of many biotechnological tools in peach breeding programs. In this review, we provide an overview of research on regeneration and genetic transformation in this species and summarize novel strategies and procedures aimed at producing transgenic peaches. Promising future approaches to develop a robust peach transformation system are discussed, focusing on the main bottlenecks to success including the low efficiency of A. tumefaciens-mediated transformation, the low level of correspondence between cells competent for transformation and those that have regenerative competence, and the high rate of chimerism in the few shoots that are produced following transformation.
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.
Somatic embryogenesis is the most common regeneration method for the application of new genomic techniques like cisgenesis/intragenesis, genome editing, and RNAi. However, some local important genotypes show recalcitrance to this morphogenetic strategy, which represents an obstacle for the application of genetic engineering techniques. Whole flowers, stamens, and pistils of three different Italian Vitis vinifera L. cultivars (Ancellotta, Glera, and Lambrusco Salamino), and four hybrid rootstocks (110 Richter, 17.37, SO4, Star 50) have been tested in several culture media with changing basal salts (NN and MS), different combinations of growth regulators (BAP, 2,4-D, NOA, PIC, and NAA), and gelling agents, to initiate somatic embryogenesis. The formation of embryogenic calli was observed mainly from whole flowers cultured on PIV medium (NN salts, B5 vitamins, 3 g L−1 gelrite, 60 g L−1 sucrose, 8.9 µM BAP, and 4.5 µM 2,4-D), and stamens on MS1 medium (MS salts and vitamins, 7 g L−1 plant agar, 20 g L−1 sucrose, 4.5 µM BAP, and 5 µM 2,4-D), in the cv. Ancellotta, Lambrusco Salamino, and all the rootstocks, except for Star 50, which showed the best embryogenetic response from pistils placed on MS1. In a recalcitrant cv. as Glera, pistils placed on MS medium supplemented with 1 µM BAP, 5 µM 2,4-D, and gelrite as gelling agent, showed the highest percentage of embryogenesis. In addition, a two-step protocol was efficiently optimized for further induction of secondary embryo production for the above-listed grapevine genotypes, which guaranteed the long-term maintenance of embryogenic cultures from clusters or single somatic embryos.
In the present study, an efficient system for the in vitro regeneration of adventitious shoots from the peach rootstock Hansen 536 leaves has been established. Twenty regeneration media containing McCown Woody Plant Medium (WPM) as a basal salt supplemented with different concentrations and combinations of plant growth regulators (PGRs) were tested. Expanded leaves along with their petiole from 3-week-old elongated in vitro shoot cultures were used as starting explants. The highest regeneration rate (up to 53%) was obtained on WPM basal medium enriched with 15.5 μM N6-benzylaminopurine (BAP). The influences on leaf regeneration of the ethylene inhibitor silver thiosulphate (STS) and of different combinations of antibiotics added to the optimized regeneration medium were also investigated. The use of 10 μM STS or carbenicillin (238 μM) combined with cefotaxime (210 μM) significantly increased the average number of regenerating shoots per leaf compared to the control. In vitro shoots were finally elongated, rooted and successfully acclimatized in the greenhouse. The results achieved in this study advances the knowledge on factors affecting leaf organogenesis in Prunus spp., and the regeneration protocol described looks promising for the optimization of new genetic transformation procedures in Hansen 536 and other peach rootstocks and cultivars.
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