Comparison of regeneration capacity and Agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of Vitis spp. via organogenesis.

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

Qualità vivaistica di piante di fragola (Fragaria x ananassa Duch.) provenienti da propagazione in vitro e in vivo

Attualmente la tecnica della micropropagazione in fragola, secondo la regolamentazione UE, può essere utilizzata solo per la produzione di materiale esente virus e non è prevista in altre fasi della produzione vivaistica. Con lo scopo di valutare la possibilità di adottare la tecnica in vitro per la produzione vivaistica di piante madri di fragola, è stata avviata una prova sperimentale per confrontare piante madri di Alba, varietà commerciale diffusa in UE, propagate con protocolli standard in vitro e in vivo (piante frigo)

In vitro shoot regeneration from leaves of Pyrus communis L. rootstock and cultivars

The influence of TDZ on adventitious shoot regeneration from leaves of the pear cultivars Conference and Abate Fetel, and the rootstock Farold((R))87 was investigated. Our main aim was to set up efficient in vitro regeneration protocols for all these pear genotypes by using expanding leaves from elongated shoot cultures as starting plant material. Our best results in terms of percentage of regeneration were achieved by using half-strength Murashige and Skoog basal medium supplemented with 1 mu M NAA, combined with 13.5 mu M TDZ for Conference (87.3%) and Farold((R))87 (53.3%), and 9 mu M TDZ for Abate Fetel (68%). The impact on leaf organogenesis of the antibiotics timentin, cefotaxime, and carbenicillin, alone or in combination, usually used for the control of Agrobacterium overgrowth, and of kanamycin, commonly used for the selection of putatively transformed plants, were also evaluated to be exploited in future transformation trials. In general, the use of carbenicillin (475 mM), cefotaxime/carbenicillin (210/238 mM) and cefotaxime (630 mM) did not negatively affect the regeneration efficiency of Conference, Abate Fetel and Farold((R))87, respectively. The use of 4 mu M kanamycin should be suitable to select transformed shoots from Abate Fetel and Farold((R))87 leaves, while a lower concentration or a different selection strategy should be applied for Conference. We report new regeneration and selection protocols usable for the application of new biotechnologies in the genetic improvement of pear cultivars and rootstocks

Micropropagation of walnut: A real opportunity

Since 2005, Vitroplant Italia s.r.l. has been producing micropropagated walnut plants, which have been planted in demonstrative orchards from 2007 with excellent results. Following this first experience, from 2013 there has been an increasing demand for micropropagated walnut plants. The micropropagated 'Chandler' potted plants, 10 cm height (in 0.9-L pot) and 30 cm height (1.7-L pot), were compared with bare root 1-year-old 'Chandler' grafted plants, on rootstock J. regia seedlings, 1.80 m height. The plantation was made for all the plants at the end of April in a demonstrative orchard in Emilia Romagna region (45°N, Italy). Micropropagated plants in 0.9-L pots got an equivalent or higher growth than grafted plants at the third year, while the micropropagated plants in1.7-L pots overtook growing performance of grafted plants already during the first year. The cumulative production per plant since now has been equivalent or higher in micropropagated 'Chandler' orchards than in grafted ones. This result was obtained with less pruning and greater vegetative growth of micropropagated plants, which made easier the training of structured central axis. In the last few years, the most planted type of micropropagated plants was the dormant bare root type. These plants can be obtained in one growing season in the nursery, after early spring acclimation ex vitro. This type of plant was between 0.20 and 0.60 m height and easier to be managed during the first year of growth in the orchard compared to the pot plants. Therefore, in a second demonstrative field trial, 'Chandler' dormant bare root plants from micropropagation were compared with 1-year-old grafted plants on J. regia seedlings 1.80 m height. Micropropagated plants showed better growth rate and more homogeneity with minimal pruning to train the structured central axis. While, a delay in production of female flowers was found in micropropagated 'Chandler' trees during the first 3-4 years, the cumulative production per plant was higher than in grafted plants 8 years after planting

Peach (Prunus Persica L.)

Until now, the application of genetic transformation techniques in peach has been limited by the difficultiesin developing efficient regeneration and transformation protocols. Here we describe an efficient regenerationprotocol for the commercial micropropagation of GF677 rootstock (Prunus persica 7 Prunusamygdalus).The method is based on the production, via organogenesis, of meristematic bulk tissues characterizedby a high competence for shoot regeneration.This protocol has also been used to obtain GF677 plants genetically engineered with an empty hairpincassette (hereafter indicated as hp-pBin19), through Agrobacterium tumefaciens-mediated transformation.After 7\u20138 months of selection on media containing kanamycin, we obtained two genetically modifiedGF677 lines. PCR and Southern blot analyses were performed to confirm the genetic status

The bottlenecks in obtaining an efficient transformation protocol for RNAi-based sharka resistant peach

Stone fruits, especially peach (Prunus persica), are among the most important tree species grown in the Mediterranean basin subjected to viral infections, in particular caused by Plum Pox Virus (PPV), the etiologic agent of Sharka disease, which leads to significant agronomic and economic losses. At the moment, there are no means of direct struggle against such infection but only means of prevention, which are often not effective and associated to environmental sustainability issues and costs for farmers. For these reasons, many programs of genomic studies and traditional breeding are aimed at understanding the mechanisms of resistance. A possible integrative strategy to classical breeding techniques is represented by the use of genetic engineering technology to obtain transgenic plants of cultivars and rootstocks bearing resistance genes. Prunus, in particular peach, is considered one of the most recalcitrant species for what concern in vitro regeneration and transformation, especially when the starting plant material originates from mature tissues. The study here described has the main objective of transferring and adapt the protocol previously developed in grape (Mezzetti et al., 2002) to the in vitro regeneration, via organogenesis, and Agrobacterium-mediated transformation of the peach rootstock GF677 (Prunus persica x Prunus amygdalus). The ultimate purpose is the introduction of an anti PPV RNAi construct, named ihp35S-PPV194

Strategies for the induction of PPV resistance through gene silencing in cultivars and rootstocks of Prunus spp.

An innovative and efficient regeneration and genetic transformation protocol developed for grapevine is applied to different fruit species as peach and apricot. This method is used for the obtainment of virus resistance to PPV by the introduction of constructs capable of inducing a mechanism of post-transcriptional gene silencing (PTGS)