Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

Abstract

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.

Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri

“…However, it should be noted that agrobacterium infiltration is not used widely in citrus due to its low efficiency, therefore, there is a need for a more robust transient expression system in citrus leaves 44 , 45 . Additionally, the stable expression of scFvH6 in plants can help prove the ability of this antibody to control Xcc , which requires a time- and labor-intensive procedure and may not be provided by agrobacterium-mediated transformation 46 . Hence, our findings can provide preliminary insight into the development of more tolerant plants to Xcc in the future.…”

A promoter trap in transgenic citrus mediates recognition of a broad spectrum of Xanthomonas citri pv. citriTALEs, including in planta‐evolved derivatives

“…However, we failed to observe transgenic citrus lines for ProBs3 14EBE :avrGf1 (Shantharaj et al ., 2017) while we eventually identified one line containing the AvrGf2‐based Xcc‐TALE‐trap (Figure S2). The low overall transformation success rate might reflect the fact that the transformation of citrus is still challenging (Conti et al ., 2021). However, recent studies have shown that transgenic application of the rice executor gene Xa23 leads reduced bacterial growth, probably due to upstream promoters at the specific transgene integration site (Ji et al ., 2022).…”

CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane

“…Citrus species can be transformed through different techniques ( Conti et al., 2021 ) ( Figure 3 ), although Agrobacterium -mediated gene transfer is the most commonly used transformation method ( Boscariol et al., 2006 ; Dutt and Grosser, 2009 ; Caserta et al., 2014 ; Souza-Neto et al., 2022 ). Different explants can be used to transform citrus species, mostly obtained from juvenile tissues, such as epicotyls and embryogenic cells, besides protoplasts ( Omar et al., 2007 ; Dutt et al., 2020 ; Li et al., 2022 ; Mahmoud et al., 2022 ; Su et al., 2023 ).…”