CRISPR/Cas as a Genome-Editing Technique in Fruit Tree Breeding.

CRISPR (short for "Clustered Regularly Interspaced Short Palindromic Repeats") is a technology that research scientists use to selectively modify the DNA of living organisms. CRISPR was adapted for use in the laboratory from the naturally occurring genome-editing systems found in bacteria. In this work, we reviewed the methods used to introduce CRISPR/Cas-mediated genome editing into fruit species, as well as the impacts of the application of this technology to activate and knock out target genes in different fruit tree species, including on tree development, yield, fruit quality, and tolerance to biotic and abiotic stresses. The application of this gene-editing technology could allow the development of new generations of fruit crops with improved traits by targeting different genetic segments or even could facilitate the introduction of traits into elite cultivars without changing other traits. However, currently, the scarcity of efficient regeneration and transformation protocols in some species, the fact that many of those procedures are genotype-dependent, and the convenience of segregating the transgenic parts of the CRISPR system represent the main handicaps limiting the potential of genetic editing techniques for fruit trees. Finally, the latest news on the legislation and regulations about the use of plants modified using CRISPR/Cas systems has been also discussed.

Trans-grafting plum pox virus resistance from transgenic plum rootstocks to apricot scions.

Introduction: Trans-grafting could be a strategy to transfer virus resistance from a transgenic rootstock to a wild type scion. However contradictory results have been obtained in herbaceous and woody plants. This work was intended to determine if the resistance to sharka could be transferred from transgenic plum rootstocks to wild-type apricot scions grafted onto them. Methods: To this end, we conducted grafting experiments of wild- type apricots onto plum plants transformed with a construction codifying a hairpin RNA designed to silence the PPV virus and studied if the resistance was transmitted from the rootstock to the scion. Results: Our data support that the RNA-silencing-based PPV resistance can be transmitted from PPV-resistant plum rootstocks to non-transgenic apricot scions and that its efficiency is augmented after successive growth cycles. PPV resistance conferred by the rootstocks was robust, already occurring within the same growing cycle and maintained in successive evaluation cycles. The RNA silencing mechanism reduces the relative accumulation of the virus progressively eliminating the virus from the wild type scions grafted on the transgenic resistant PPV plants. There was a preferential accumulation of the 24nt siRNAs in the scions grafted onto resistant rootstocks that was not found in the scions grafted on the susceptible rootstock. This matched with a significantly lower relative accumulation of hpRNA in the resistant rootstocks compared with the susceptible or the tolerant ones. Discussion: Using transgenic rootstocks should mitigate public concerns about transgenes dispersion and eating transgenic food and allow conferring virus resistance to recalcitrant to transformation cultivars or species.

The Effect of Silver Nanoparticle Addition on Micropropagation of Apricot Cultivars (Prunus armeniaca L.) in Semisolid and Liquid Media.

Silver nanoparticles (AgNPs) are novel compounds used as antimicrobial and antiviral agents. In addition, AgNPs have been used to improve the growth of different plants, as well as the in vitro multiplication of plant material. In this work the effect of AgNPs on in vitro growth of 'Canino' and 'Mirlo Rojo' cultivars, as well as the leaf ion composition, are studied. Different concentrations of AgNPs (0, 25, 50, 75 and 100 mg L-1) were added to two culture systems: semisolid medium with agar (SSM) in jars and liquid medium in temporary immersion system (TIS). Proliferation (number of shoots), shoot length, productivity (number of shoot × average length), leaf surface, fresh and dry weight were measured. Additionally, the silver and other ion accumulation in the leaves were evaluated by inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis. The productivity of 'Canino' and 'Mirlo Rojo' decreased when increasing the concentration of AgNPs in the semisolid medium. However, the use of AgNPs in the TIS improved the proliferation and productivity of 'Canino' and Mirlo Rojo', increasing biomass production, and the concentration of nutrients in the plants, although these effects are genotype-dependent. TISs are the best system for introducing silver into shoots, the optimum concentration being 50 mg L-1 for 'Canino' and 75 mg L-1 for 'Mirlo Rojo'. Principal component analysis, considering all the analyzed ions along the treatments, separates samples in two clear groups related to the culture system used. The use of bioreactors with a liquid medium has improved the productivity of 'Canino' and 'Mirlo Rojo' in the proliferation stage, avoiding hyperhydration and other disorders. The amount of metallic silver that penetrates apricot plant tissues depends on the culture system, cultivar and concentration of AgNPs added to the culture medium. Silver ion accumulation measured in the shoots grown in the TIS was higher than in shoots micropropagated in a semisolid medium, where it is barely detectable. Furthermore, AgNPs had a beneficial effect on plants grown in TIS. However, AgNPs had a detrimental effect when added to a semisolid medium.