J. doi: 10.1371/journal.pbio.1001877, Voytas, D. F. (2013). Similarly, Gaudelli et al., 2018 reported adenine base editors (ABEs) converting A.T to G.C. Plant Sci. New plant breeding techniques State-of-the-art and prospects for commercial development - Study EU publications. doi: 10.1104/pp.103.026039, Espley, R. V., Butts, C. A., Laing, W. A., Martell, S., Smith, H., McGhie, T. K., et al. 17, 1928–1937. Hortic. Acad. doi: 10.1093/aob/mcz029, Albani, M. C., Coupland, G. (2010). J. A critical point of discussion involves two aspects, considering either the used method or the characteristics of the end product. doi: 10.1007/s11295-013-0675-z, Friedrichs, S., Takasu, Y., Kearns, P., Dagallier, B., Oshima, R., Schofield, J., et al. Similarly, in apple, MYB10 overexpression induced the large accumulation of anthocyanin in the flesh, and resulted in reduced inflammatory markers in mice (Espley et al., 2014). Front. (2004). doi: 10.1007/s00299-001-0399-7, Guo, W., Duan, Y., Fuster, O. O., Wu, Z., Arias, C. R., Burns, J. K., et al. Rasthali genome. Int. Talon, M.,., Caruso, M., Gmitter, F. G. (Cambridge, UK: Elsevier), 495–511. Natl. Mutation mapping approaches have shown how similar mutations in orthologue genes among species that are not genetically related, exert similar effects on the phenotype. Moreover, the effect of grafting on the precious floral induction has been reported, up to now, in apple, where the M9 rootstock (a spontaneous mutant overexpressing FT genes) induces early flowering in juvenile apple scion (Foster et al., 2014). Citrus could take advantage of NPBTs because of its complex species biology (seedlessness, apomixis, high heterozygosity, and long juvenility phase) and aptitude for in vitro manipulation. Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars. Plant Sci. Traditional breeding methods used for genetic improvement of citrus cultivars and create new varieties, or to improve desired traits require a long time and is hampered by many difficulties and restrictions related with plant size and long juvenile phase of citrus, as well as female and/or male sterility, polyembryony, heterozygosity, and parthenocarpy. 14, 54–61. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). FS prepared the main parts of the manuscript being the application of new plant breeding techniques in citrus the scope of his PhD project. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. This knowledge allowed to replicate a point mutation in the ALS gene, by base editing, to produce herbicide-resistance crops (Shimatani et al., 2017); iii) finally, attention has been paid to the Terminal Flower gene family (FT/FTL), whose function is to guide the transition from vegetative to floral meristem and is preserved in many species (Pillitteri et al., 2004). the ag- 8, 2135. doi: 10.3389/fpls.2017.02135, Jia, H., Zhang, Y., Orbovic, V., Xu, J., White, F. F., Jones, J. During the last decade new plant-breeding techniques have been developed. Innov. The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Traditional breeding is one of the main strategies used to improve agronomic traits. New dimensions for VIGS in plant functional genomics. The working group . J. Mol. Plant 11, 623–626. 6, 286. doi: 10.3389/fpls.2015.00286, La Malfa, S., Distefano, G., Domina, F., Nicolosi, E., Toscano, V., Gentile, A. The Citrus canker epidemic in Florida: the scientific basis of regulatory eradication policy for an invasive species. doi: 10.1371/journal.pone.0177966, Nishitani, C., Hirai, N., Komori, S., Wada, M., Okada, K., Osakabe, K., et al. (2014) highlighted the usefulness of the overexpression of early flowering genes as a tool to accelerate the evaluation of traits related to fruit quality. CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry. Recombination/excision systems have been shown to effectively produce SMG-free apple (Kost et al., 2015; Krens et al., 2015), apricot (Petri et al., 2012), and citrus (Zou et al., 2013). 8, 339–374. Unlike traditional breeding, and similarly to transgenesis approaches, NPBTs do not alter the genetic background and this is particularly important for elite cultivars. doi: 10.1038/NPBT.2647, Hu, Y., Zhang, J., Jia, H., Sosso, D., Li, T., Frommer, W. B., et al. NPBTs produce targeted and minimal modifications to selected genotypes, such as elite cultivars, which are highly valued by consumers for their quality and productivity but that can be even further improved. doi: 10.2791/54761, Lyznik, L. A., Mitchell, J. C., Hirayama, L., Hodges, T. K. (1993). Two gRNAs were synthesized to edit the PDS gene and five transgenic lines exhibited targeted mutagenesis sites, resulting in a global and mosaic albino phenotype (Zhu et al., 2019). 30, 437–446. The resolution of critical issues on both aspects enables to process of NPBT or product assimilated to be considered, and, as consequence, ruled as a GMO or not. Plant Biotechnol. What are New Plant Breeding Techniques (NPBTs) April 19, 2018 / in news /. Appendix 1 provides details of the membership of the panel. Biotechnol. Changes in anthocyanin production during domestication of citrus. doi: 10.1111/pbi.12110, Varkonyi-Gasic, E., Wang, T., Voogd, C., Jeon, S., Drummond, R. S. M., Gleave, A. P., et al. Nat. A., Pino, L. E., Boscariol, R. L., Rodriguez, A. P. M., Mendes, B. M. J. Nature 551, 464–471. New plant breeding techniques State-of-the-art and prospects for commercial development Publication metadata Download and languages Close. doi: 10.1016/j.biori.2019.07.001, Gaj, T., Gersbach, C. A., Barbas, C. F. (2013). Plant Sci. (2015). Front. Gene targeting and editing in crop plants: a new era of precision opportunities. Introduction of the Xa21, Xanthomonas resistance gene from rice, into ‘Hamlin’ sweet orange [Citrus Sinensis (L.) Osbeck] using protoplast/GFP co-transformation and single plasmid transformation. Harmonised EU legislation regulating organisms produced by modern bio-techniques (GMOs) goes back to the year 1990 and the definition of GMOs was not up-dated since. The regulation of NPBTs has been a topic of worldwide discussion. What Does Young Generation Think About Gene Editing? Funct. (2011). The objective of the present review was to describe the current state of the art and recent advances on the use of NPBTs, in particular regarding genome editing and cisgenesis, on citrus, and other fruit tree species. Production of transgenic adult plants from clementine mandarin by enhancing cell competence for transformation and regeneration. This phenomenon is likely due to the non-specific design of the gRNA and represents a critical limiting factor considering that Cas9 tolerates mismatches between gRNA and target DNA at different positions in a sequence-dependent manner (Hsu et al., 2013). 3 For convenience, the Scientific Committee has defined some of its terms in the glossary in Appendix VI. (2000). A., et al. Cell. (2008). GENIUS addresses the use of genome editing for traits involved in the reduced use of pesticides and in the mitigation of climate change for more sustainable agriculture. 16, 1424–1433. Plant Genomics, 1–17. Hort. (2019). 57 (1), 17–42. doi: 10.3945/jn.113.182659, Fauser, F., Schiml, S., Puchta, H. (2014). Plant Sci. Transgene-Free genome editing in tomato and potato plants using agrobacterium-mediated delivery of a crispr/cas9 cytidine base editor. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Trends Plant Sci. (2017). Yuan, J. M., Stram, D. O., Arakawa, K., Lee, H. P., Yu, M. C. (2003). Sci. Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. Genet. 15, 1509–1519. Base editing has been adopted in plant species (Figure 2), such as watermelon (Li et al., 2017; Lu and Zhu, 2017; Ren et al., 2018; Tian et al., 2018), to produce transgene-free homozygous als (acetolactate synthase) mutant plants, resistant to the herbicide tribenuron (Tian et al., 2018). In particular, the presence of cytokinin in the callus-induction medium promotes callus formation (Guo et al., 2002; Khan et al., 2009). Genetic engineering is the best approach to induce resistance to CBC (Zhang et al., 2010). B., et al. Efficient targeted mutagenesis in apple and first-time edition of pear using the CRISPR-Cas9 system. Mutations in both EBEs alleles (type I and type II) of CsLOB1 in “Duncan” grapefruit and “Wanjincheng” orange, result in reduced symptoms in transformed plants caused by wild-type Xcc infection (Jia et al., 2016; Jia et al., 2017b; Peng et al., 2017). Dev. In citrus, the use of embryogenic cell culture and of protoplast technology has been exploited since 1990; many somatic hybrids have been generated through protoplast fusion (Grosser and Gmitter, 1990; Grosser and Gmitter, 2011; Grosser et al., 1992; Grosser et al., 2000) and transgenic plants have been recovered from transformed protoplasts (Guo et al., 2005; Omar et al., 2007; Dutt et al., 2018b; Omar et al., 2018) and calli (Dutt et al., 2018b). (2017). Evaluation of parameters affecting Agrobacterium-mediated transient expression in citrus. doi: 10.1038/sj.embor.7400769, Senthil-Kumar, M., Mysore, K. S. (2011). J. (Hoboken, NJ, USA: John Wiley & Sons, Inc), 109–155. Many factors affect the stable transformation process mediated by A. tumefaciens that usually involves explants pre-incubation, co-cultivation with the engineered bacteria, callus induction, and selection of the regenerated shoots. Knowledge on the gene(s) controlling a specific trait is essential for the use of NPBTs, such as genome editing and cisgenesis. In particular, F. hindsii needs around 5 months from seeds to T0 plantlets and about another 10 months to obtain the following T1 generation after the editing in PDS gene, supporting the evidence to be really promising using F. hindsii for functional studies (Zhu et al., 2019). Eds. 3. fell under Directive 2001/18/EC, but remained divided on the regulatory status of m ost (2002). doi: 10.1094/PHYTO.2001.91.1.30, Grosser, J. W., Gmitter, F. G., Jr. (2011). doi: 10.1371/journal.pone.0040715, Steinert, J., Schiml, S., Fauser, F., Puchta, H. (2015). 3 . 110, 337–346. B., Wang, N. (2016). Efficiency optimization of CRISPR/CAS9-mediated targeted mutagenesis in grape. doi: 10.1105/tpc.111.095232, Butelli, E., Garcia-Lor, A., Licciardello, C., Las Casas, G., Hill, L., Reforgiato Recupero, G., et al. An EU perspective on biosafety considerations for plants developed by genome editing and other new genetic modification techniques (nGMs). J. Amer. Privacy Policy In “cisgenesis” the new trait is derived from a sexually compatible species and it is transferred as it is, with its promoter and terminator, to the recipient. It is not yet clarified how in purely legal terms we shall regulate these new techniques. Recently, new sources of resistance to HLB have been identified in Eremocitrus and Microcitrus spp. (2019). doi: 10.1111/pbi.12884, Wang, L., Chen, S., Peng, A., Xie, Z., He, Y., Zou, X. These include (I) zinc finger nuclease technology, (II) oligonucleotide directed mutagenesis, (III) cisgenesis, (IV) intragenesis, (V) RNA-dependent DNA methylation, (VI) grafting on genetically modified rootstock, (VII) reverse breeding, (VIII) agro-infiltration, and (IX) synthetic genomics (Lusser et al., 2011). New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for fruit tree species, in order to obtain new varieties with improved organoleptic traits and resistance to biotic and abiotic stress, and to maintain fruit quality achieved over centuries by (clonal) selection. 207, 478–485. New plant breeding techniques make use of steps that qualify as genetic modification as at some point in time (recombinant) DNA is introduced into the plant. doi: 10.1016/j.scienta.2018.07.026, Dutt, M., Zambon, F. T., Erpen, L., Soriano, L., Grosser, J. Citrus genomics. (2016). doi: 10.1111/pbi.12555, Vives, M. C., Martín, S., Ambrós, S., Renovell, Á., Navarro, L., Pina, J. 128, 91–100. Plant Biotechnol. More recently, a number of new techniques has been developed and classified as new plant breeding techniques (NPBTs). All the shoots or embryos, obtained from regeneration of transformed explants, are grafted onto vigorous rootstocks in order to induce early flowering, with the final aim to shorten the evaluation of the modified traits. Moreover, in many cases, after breeding, backcrosses are required to recover elite features of the improved cultivar, lengthening even more breeding programs. Mol. “Citrus and health,” in The Genus Citrus, 1st edition. Int. (2019). doi: 10.1007/978-1-4939-3061-6_13, Omar, A. Grafting as a strategy to increase flowering of cassava. In citrus, 65% efficiency was demonstrated for ‘Pineapple’ sweet orange plant; however, the results were not appropriate for the ‘Carrizo’ citrange, due to the genotype specificity of this approach (Ballester et al., 2008). (2019). J. doi: 10.1038/nature17945, Foster, T. M., Watson, A. E., van Hooijdonk, B. M., Schaffer, R. J. (2017). J. Genomics 2016, 1–10. お手本を使わない. Chemopreventive agents and inhibitors of cancer hallmarks: may citrus offer new perspectives? J. Plant Biotechnol. In the framework of biotechnological uses, it may be possible to reduce the duration of the breeding cycle acting on the endogenous genetic flowering pathways by using inducible promoters that activate transgene expression, in addition to several approaches used to transmit the transgenic stimulus through grafting (van Nocker and Gardiner, 2014). The New Breeding Techniques (NBT) Platform is a coalition of SMEs, large industry and prominent academic and research institutes, which strives to achieve regulatory clarity on whether or not New Breeding Techniques (‘NBTs’) are to be defined as leading to a genetically modified organism (‘GMO’). Seasonal and developmental timing of flowering. Activity of yeast FLP recombinase in maize and rice protoplasts. Nat. Plant Biotechnol. NPBTs are powerful tools used to improve quality traits without modifying the characteristics of elites in citrus, and to protect the world citriculture from the most aggressive and devastating disease, like HLB. [2] JRC/IPTS/IHCS. Sci. doi: 10.1038/nbt.2954, Velázquez, K., Agüero, J., Vives, M. C., Aleza, P., Pina, J. doi: 10.1016/j.cub.2018.11.040, Cao, H. X., Wenqin, W., Hien, T. T. L., Giang, T. H. V. (2016). Long-distance, graft-transmissible action of Arabidopsis FLOWERING LOCUS T protein to promote flowering. There is debate over whether some of these techniques constitute genetic modification (GM) as defined in EU Directive 2001/18 and are thus subject to the various EU GM regulations. Nat. New developments in genetic engineering raise questions about which crops will be treated as genetically modified (GM) for the purposes of regulation. Front. (2020). Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Other major issues addressed in this review are related to the requirement for marker-free systems and shortening the long juvenility phase. (2019). J. (2007). Hortic. 20, 3719. doi: 10.3390/ijms20153719. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. (2010). Plant Sci. 5, 13. doi: 10.1038/s41438-018-0023-4, Cirmi, S., Ferlazzo, N., Lombardo, G., Maugeri, A., Calapai, G., Gangemi, S., et al. (2000). Considering the bacterium, X. citri strains are characterized by specific pathotypes (PthA4, PthA*, PthAw, PthB, and PthC), which are distinguished based on the conservation of repeated variable diresidues (RVDs), encoding transcription activator-like (TAL) effectors. (2018). 154, 304–311. Plant Sci. A., Ran, F. A., Konermann, S., Agarwala, V., et al. 12, e1001877. 82 (6), 914–923. A review of genetic improvement of main fruit trees through modern biotechnological tools and considerations of the cultivation and research of the engineered plant restrictions. J. Biotechnol. In particular, genome editing approaches could display their effectiveness especially for those characters for which the down regulation or the reduction of the expression levels of a single gene could determine a modification of a given trait. Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini-Citrus (Fortunella hindsii). New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for fruit tree species, in order to obtain new varieties with improved organoleptic traits and resistance to biotic and abiotic stress, and to maintain fruit quality achieved over centuries by (clonal) selection. Traditionally bred plants: strategies and applications beyond genome editing in kiwifruit * Correspondence: Stefano La Malfa Licciardello! 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