Citrus genetic transformation: an overview of the current strategies and insights on the new emerging technologies

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.

Instituto de Biotecnología

Fil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina

Fil: Conti, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Conti, Gabriela. Universidad de Buenos Aires. Faculta de Agronomía. Cátedra de Genética; Argentina

Fil: Xoconostle-Cázares, Beatriz. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional. Departamento de Biotecnología y Bioingeniería; México

Fil: Marcelino-Pérez, Gabriel. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional. Departamento de Biotecnología y Bioingeniería; México

Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina

Fil: Hopp, Horacio Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Hopp, Horacio Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Fil: Reyes Martinez, Carina Andrea. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina

Fil: Reyes Martinez, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina