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.

Application of Ascophyllum nodosum-Based Soluble Extract on Micropropagation and Regeneration of Nicotiana benthamiana and Prunus domestica.

In the present study, the effect of a commercial extract of the seaweed Ascophyllum nodosum on in vitro micropropagation, shoot regeneration, and rhizoghenesis were studied in Nicotiana benthamiana and Prunus domestica. Results showed that the MS medium supplemented with various concentrations of the Ascophyllum extract (5, 10, 50, and 100 mg L-1) significantly enhanced the number of regenerated buds from N. benthamiana leaf discs to the conventional MS regenerating medium. Increases ranged from 3.5 to 6.5 times higher than the control. The effect of the Ascophyllum extract on N. benthamiana micropropagation was assessed through the measurement of some plant growth parameters. Results showed that the extract alone could not replace the micropropagation medium since shoot length, shoot diameter, root length, and leaf area were significantly reduced. However, its combination with a half-strength MS medium enhanced these parameters. Its effect was also evaluated on regeneration from plum hypocotyl slices. When added to the shoot regeneration medium without any plant growth regulators, the Ascophyllum extract alone could induce shoot regeneration. However, the percentage of bud regeneration and number of regenerated buds were lower than with the conventional shoot regeneration medium containing complete growth regulators. In contrast, the Ascophyllum extract drastically promoted rhizogenesis from plum hypocotyl slices. These results pave the way for the possible use of A. nodosum extracts in in vitro mass propagation of higher plants.

Interplay among Antioxidant System, Hormone Profile and Carbohydrate Metabolism during Bud Dormancy Breaking in a High-Chill Peach Variety.

(1) Background: Prunus species have the ability to suspend (induce dormancy) and restart growth, in an intricate process in which environmental and physiological factors interact. (2) Methods: In this work, we studied the evolution of sugars, antioxidant metabolism, and abscisic acid (ABA) and gibberellins (GAs) levels during bud dormancy evolution in a high-chill peach variety, grown for two seasons in two different geographical areas with different annual media temperature, a cold (CA) and a temperate area (TA). (3) Results: In both areas, starch content reached a peak at ecodormancy, and then decreased at dormancy release (DR). Sorbitol and sucrose declined at DR, mainly in the CA. In contrast, glucose and fructose levels progressively rose until DR. A decline in ascorbate peroxidase, dehydroascorbate reductase, superoxide dismutase and catalase activities occurred in both seasons at DR. Moreover, the H2O2-sensitive SOD isoenzymes, Fe-SOD and Cu,Zn-SOD, and two novel peroxidase isoenzymes, were detected. Overall, these results suggest the occurrence of a controlled oxidative stress during DR. GA7 was the major bioactive GA in both areas, the evolution of its levels being different between seasons and areas. In contrast, ABA content decreased during the dormancy period in both areas, resulting in a reduction in the ABA/total GAs ratio, being more evident in the CA. (4) Conclusion: A possible interaction sugars-hormones-ROS could take place in high-chill peach buds, favoring the DR process, suggesting that, in addition to sugar metabolism, redox interactions can govern bud DR, regardless of chilling requirements.

Hydrogen peroxide generated by over-expression of cytosolic superoxide dismutase in transgenic plums enhances bacterial canker resistance and modulates plant defence responses.

H2O2 generated during the oxidative burst, plays important roles in plant defenses responses against pathogens. In this study we examined the role of H2O2 on bacterial canker resistance in transgenic plums over-expressing cytosolic superoxide dismutase. Three transgenic lines (C64, C66 and F12) with elevated levels of H2O2 accumulation showed enhanced resistance against bacterial canker disease caused by Pseudomonas syringae pv. syringae, when compared to the non-transformed control. Analysis of the expression of several genes involved in the plant-pathogen interaction showed that the expression of those involved in SA pathway (pr1 and npr1) and JA (lox3) were activated earlier and transiently in transgenic lines C66 and F12 when compared to the wild type. However, the expression of genes involved in anthocyanin synthesis (chi, chs, f3h, dfr, atcs, myb10) and ethylene (acs) was induced at very low levels whereas it was activated by the pathogen at exaggerated levels in the non-transformed line. These results suggest that resistance observed in transgenic lines over-producing H2O2 is correlated with an early and transient induction of defense genes associated with the SA and JA pathways and inhibition of gene expression associated with ethylene and anthocyanin biosynthesis.

Current achievements and future directions in genetic engineering of European plum (Prunus domestica L.).

In most woody fruit species, transformation and regeneration are difficult. However, European plum (Prunus domestica) has been shown to be amenable to genetic improvement technologies from classical hybridization, to genetic engineering, to rapid cycle crop breeding ('FasTrack' breeding). Since the first report on European plum transformation with marker genes in the early 90 s, numerous manuscripts have been published reporting the generation of new clones with agronomically interesting traits, such as pests, diseases and/or abiotic stress resistance, shorter juvenile period, dwarfing, continuous flowering, etc. This review focuses on the main advances in genetic transformation of European plum achieved to date, and the lines of work that are converting genetic engineering into a contemporary breeding tool for this species.

Silencing of Agrobacterium tumefaciens oncogenes ipt and iaaM induces resistance to crown gall disease in plum but not in apricot.

BACKGROUND: In this study, two vectors with short-length chimeric transgenes were used to produce Prunus rootstocks resistant to crown gall disease through RNA-interference-mediated gene silencing of the Agrobacterium tumefaciens oncogenes ipt and iaaM. RESULTS: Transgenic plum and apricot lines were produced with efficiencies of up to 7.7 and 1.1% respectively. An in vitro evaluation method allowed identification of susceptible lines and reduction in the number of lines to be evaluated in the greenhouse. Five transgenic plum lines, expressing transgene-derived small interfering RNA (siRNA) and low levels of transgene hairpin RNA (hpRNA), showed a significant reduction in the development of the disease after infection with Agrobacterium strains C58 and A281 under greenhouse conditions. However, unexpectedly, all transgenic apricot lines were gall susceptible. The infection of apricot plants with a binary vector containing only the 6b oncogene demonstrated that the expression of this gene is involved in the induction of tumours in the apricot species. CONCLUSION: RNAi-mediated gene silencing can be used for inducing crown gall resistance in plum rootstocks. These could be used to graft non-genetically modified commercial fruit cultivars reducing, or eliminating, the disease symptoms. © 2017 Society of Chemical Industry.

A new strategy to enhance the biosynthesis of trans-resveratrol by overexpressing stilbene synthase gene in elicited Vitis vinifera cell cultures.

In this work, transgenic lines of suspension cultured cells of Vitis vinifera cv. Monastrell containing the plasmid pMOG800-sts have been obtained. The cell growth of these transgenic cell lines decreased slightly as compared to non-transgenic suspension cultured cells, while cell viability was not affected. In addition, the elicitation with cyclodextrins and methyl jasmonate enhanced the production of trans-resveratrol, observing the highest levels of this compound in sts-expressing transgenic Vitis suspension cultured cells with the sts expression cassette in the forwards orientation. Moreover, the forwards 2 (F2) transgenic cell line produced the greater levels of trans-resveratrol in comparison with the non-transgenic cell line. In fact, when suspension cultured cells were treated with both elicitors, the accumulation of trans-resveratrol outside the cells in the F2 transgenic suspension cultured cells increased twice (1458 mg.L-1) as compared to non-transgenic cell lines (724 mg.L-1). In both cases, the levels of trans-resveratrol detected in the treatment with cyclodextrins and methyl jasmonate were greater than the sum of the individual treatments, and therefore we observed a synergistic effect in the presence of both elicitors. Moreover, the expression profile of sts gene in transgenic V. vinifera cell lines was similar to the expression profile detected for the endogenous sts gene in non-transgenic V. vinifera cell lines, being the expression levels greater in the treatment with methyl jasmonate and cyclodextrins, which was related to the high levels of trans-resveratrol found in the presence of both elicitors.

Antimicrobial Activity of two Semisynthetic Triterpene Derivatives from Euphorbia officinarum Latex against Fungal and Bacterial Phytopathogens.

This study evaluated the in vitro antimicrobial effect of 3ß-acetoxy-norlup-20-one (1) and 3-chloro-4a,14a-dimethyl-5a-cholest-8-ene (2), triterpene derivatives from Euphorbia officinarum latex against fungal and bacterial phytopathogens. Results showed that although mycelial growth of several strains of Vericillium dahlia, and Fusarium oxysporum fsp. melonis and Penicillium expansum was affected only moderately, the two compounds were able to reduce highly conidia formation and germination, suggesting that they act as fungistatic compounds. Their antibacterial activity was tested against Pseudomonas syringae pv. syringae (Pss), P. syringae pv. tabacci (Pst), Erwinia amylovora (Ea) and Agrobacterium tumefaciens (At) using disc diffusion method. Results showed that compound 2 was more effective in inhibiting the growth of Pss, Pst and Ea than compound 1.

Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters.

Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016.

Apricot (Prunus armeniaca L.).

A protocol for Agrobacterium-mediated stable transformation of whole leaf explants of the apricot (Prunus armeniaca) cultivars 'Helena' and 'Canino' is described. Regenerated buds were selected using a two-step selection strategy with paromomycin sulfate and transferred to bud multiplication medium 1 week after they were detected for optimal survival. After buds were transferred to bud multiplication medium, antibiotic was changed to kanamycin and concentration increased gradually at each transfer to fresh medium in order to eliminate possible escapes and chimeras. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines, was 5.6%. Green and healthy buds, surviving high kanamycin concentration, were transferred to shoot multiplication medium where they elongated in shoots and proliferated. Elongated transgenic shoots were rooted in a medium containing 70 µM kanamycin. Rooted plants were acclimatized following standard procedures. This constitutes the only transformation protocol described for apricot clonal tissues and one of the few of Prunus.

Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants.

A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.