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      Plant Cellular and Molecular Biotechnology: Following Mariotti’s Steps

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          Abstract

          This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species.

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          BRASSINOSTEROIDS: Essential Regulators of Plant Growth and Development.

          Brassinosteroids (BRs) are growth-promoting natural products found at low levels in pollen, seeds, and young vegetative tissues throughout the plant kingdom. Detailed studies of BR biosynthesis and metabolism, coupled with the recent identification of BR-insensitive and BR-deficient mutants, has greatly expanded our view of steroids as signals controlling plant growth and development. This review examines the microchemical and molecular genetic analyses that have provided convincing evidence for an essential role of BRs in diverse developmental programs, including cell expansion, vascular differentiation, etiolation, and reproductive development. Recent advances relevant to the molecular mechanisms of BR-regulated gene expression and BR signal transduction are also discussed.
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            DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins

            The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.
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              CRISPR for Crop Improvement: An Update Review

              The availability of genome sequences for several crops and advances in genome editing approaches has opened up possibilities to breed for almost any given desirable trait. Advancements in genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) has made it possible for molecular biologists to more precisely target any gene of interest. However, these methodologies are expensive and time-consuming as they involve complicated steps that require protein engineering. Unlike first-generation genome editing tools, CRISPR/Cas9 genome editing involves simple designing and cloning methods, with the same Cas9 being potentially available for use with different guide RNAs targeting multiple sites in the genome. After proof-of-concept demonstrations in crop plants involving the primary CRISPR-Cas9 module, several modified Cas9 cassettes have been utilized in crop plants for improving target specificity and reducing off-target cleavage (e.g., Nmcas9, Sacas9, and Stcas9). Further, the availability of Cas9 enzymes from additional bacterial species has made available options to enhance specificity and efficiency of gene editing methodologies. This review summarizes the options available to plant biotechnologists to bring about crop improvement using CRISPR/Cas9 based genome editing tools and also presents studies where CRISPR/Cas9 has been used for enhancing biotic and abiotic stress tolerance. Application of these techniques will result in the development of non-genetically modified (Non-GMO) crops with the desired trait that can contribute to increased yield potential under biotic and abiotic stress conditions.
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                Author and article information

                Journal
                Plants (Basel)
                Plants (Basel)
                plants
                Plants
                MDPI
                2223-7747
                10 January 2019
                January 2019
                : 8
                : 1
                : 18
                Affiliations
                [1 ]Istituto di Scienze delle Produzioni Alimentari (ISPA), Consiglio Nazionale delle Ricerche (CNR), Via Monteroni, 73100 Lecce, Italy; angelo.depaolis@ 123456ispa.cnr.it
                [2 ]Istituto di Biologia e Biotecnologia Agraria (IBBA), UOS Roma, Consiglio Nazionale delle Ricerche (CNR), Via Salaria Km. 29,300, Monterotondo Scalo, 00015 Roma, Italy; giovanna.frugis@ 123456cnr.it (G.F.); donato.giannino@ 123456cnr.it (D.G.); mariaadelaide.iannelli@ 123456cnr.it (M.A.I.); giovanni.mele@ 123456cnr.it (G.M.); giulio.testone@ 123456cnr.it (G.T.); chiara.nicolodi@ 123456cnr.it (C.N.)
                [3 ]Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Via San Camillo De Lellis S.N.C., 01100 Viterbo, Italy; rugini@ 123456unitus.it (E.R.); silvestri.c@ 123456unitus.it (C.S.)
                [4 ]Istituto di Biologia e Biotecnologia Agraria (IBBA), Consiglio Nazionale delle Ricerche (CNR), Via Bassini 15, 20133 Milano, Italy; sparvoli@ 123456ibba.cnr.it
                [5 ]Dipartimento di Biologia e Biotecnologie, Sapienza Università di Roma, P.le A. Moro 5, 00185 Roma, Italy; marialuisa.mauro@ 123456uniroma1.it
                Author notes
                [* ]Correspondence: sofia.caretto@ 123456ispa.cnr.it ; Tel.: +39-0832-422605
                [†]

                These authors equally contributed to this work.

                Author information
                https://orcid.org/0000-0002-0646-4311
                https://orcid.org/0000-0002-6405-7386
                https://orcid.org/0000-0002-9634-9586
                https://orcid.org/0000-0002-3304-7548
                https://orcid.org/0000-0002-6355-3871
                https://orcid.org/0000-0002-6488-4429
                Article
                plants-08-00018
                10.3390/plants8010018
                6359066
                30634627
                ff6db0ab-b4b2-407e-9ba4-f7fbb6276b24
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 31 October 2018
                : 07 January 2019
                Categories
                Review

                plant in vitro cultures,somatic cell selection,hairy roots,rol genes,agrobacterium rhizogenes,genetic transformation,recalcitrant species,knox transcription factors,plant development,tree phase change

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