![]() In this review, we discuss (i) the new findings in gene editing technologies, (ii) a comparison between them, and (iii) their applications for genetic analysis and manipulation of fruit crops. (CRISPR) associated with Cas proteins are offering new possibilities for crop improvement and new insights for functional genomics. Plant genome editing tools as Zinc-Finger Nucleases (ZFNs), Trans Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats In particular, the current state of the rapid crop cycle breeding system and its applications will be presented. In addition to these methods, which are primarily aimed at increasing transformation efficiency, improving the precision of genetic modification and reducing the time required, methods are also discussed in which genetically modified plants are used for breeding purposes. For example, it was recently possible for the first time to generate a homohistont GE line into which a biallelic mutation was specifically inserted in a target gene. Success has also been achieved in establishing methods for targeted genome editing (GE). Besides the classical strategies of RNAi-based silencing by stable transformation with hairpin gene constructs, optimized protocols for virus-induced gene silencing (VIGS) and artificial micro RNAs (amiRNAs) have emerged as powerful technologies for silencing genes of interest. Further, we highlight targeted gene silencing applications. In addition to the use of different tissues and media for transformation, agroinfiltration, as well as pre-transformation with a Baby boom transcription factor are notable successes that have improved transformation efficiency in apple. This review article attempts to summarize recent developments in the Agrobacterium-mediated transformation strategies of apple. Nevertheless, there have been some developments, especially in recent years, which allowed for the expansion of the toolbox of breeders and breeding researchers. Although the genetic transformation of apple using Agrobacterium tumefaciens has been possible since 1989, only a few research groups worldwide have successfully applied this technology, and efficiency remains poor. ![]() One of such plants is the apple (Malus spp.), the most important fruit of the temperate climate zone. This applies not only to model plants such as Arabidopsis thaliana but also increasingly to cultivated plants, where the establishment of transformation methods could still pose many problems. Genetic transformation has become an important tool in plant genome research over the last three decades. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. ![]() In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants.
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