Molecular Plant Breeding 2024, Vol.15, No.5, 209-219 http://genbreedpublisher.com/index.php/mpb 213 into commercial citrus varieties has been achieved through fast-track breeding systems, significantly reducing the breeding time (Endo et al., 2020). The selection of appropriate genes and their effective combination is crucial for developing robust pest-resistant citrus varieties. 5.2 Techniques for introducing gene stacks into citrus plants Several techniques are employed to introduce gene stacks into citrus plants, each with its own set of advantages and challenges. Agrobacterium-mediated transformation is a widely used method for genetic transformation in citrus, allowing for the stable integration of multiple genes. Physical methods such as biolistics and magnetofection have also been explored for their potential to introduce gene stacks (Conti et al., 2021). Moreover, the use of inducible promoters and site-specific recombination systems has improved the efficiency of transformation and regeneration protocols, which are critical for the successful application of new breeding techniques (Poles et al., 2020). The development of transfer-DNA-free base-edited citrus plants represents a significant advancement, enabling the generation of edited, non-transgenic plants with desirable traits (Alquézar et al., 2022). 5.3 Case studies of successful multi-gene stacking in citrus breeding Several case studies highlight the successful application of multi-gene stacking in citrus breeding. One notable example is the use of CRISPR/Cas9 technology to edit the CsLOB1 gene in Duncan grapefruit, resulting in lines with significantly reduced canker symptoms (Figure 2) (Jia et al., 2017). Another example is the fast-track breeding system that incorporated CTV resistance from trifoliate orange into citrus germplasm, achieving significant resistance in a much shorter time frame compared to conventional breeding methods (Figure 3) (Endo et al., 2020). Additionally, the overexpression of flower meristem-identity genes in APETALA1 citrus plants has enabled rapid evaluation of transgenic traits, demonstrating the feasibility of stable transgene stacking in early-fruiting transgenic trees (Cervera et al., 2009). These case studies underscore the potential of multi-gene stacking strategies to enhance pest resistance in citrus breeding. Figure 2 Analysis of GFP-p1380N-Cas9/sgRNA:cslob1-transformed Duncan grapefruit (Adopted from Jia et al., 2017) Image caption: (a) six GFP-p1380N-Cas9/sgRNA:cslob1-transformed Duncan grapefruit plants (DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12) were GFP positive. The wild-type grapefruit plant did not show GFP. (b) The six transgenic lines contain Cas9/sgRNA as indicated by PCR amplification using primers 35SP-5-P1 and NosP-3-P2. Plasmid GFP-p1380N-Cas9/sgRNA:cslob1 was used as a positive control. M, 1 kb DNA ladder; WT, wild type. C. The six CsLOB1-modified lines showed differential resistance to Xcc. At 4 days postinoculation with Xcc (5 × 108 CFU/mL) using needleless syringe, canker symptoms were observed on normal grapefruit, DLOB2 and DLOB3, but absent or reduced on DLOB9, DLOB10, DLOB11 and DLOB12 (Adopted from Jia et al., 2017) 6 Field Trials and Performance Evaluation 6.1 Overview of methodologies for field trials Field trials are essential for evaluating the performance of genetically modified (GM) citrus varieties, particularly in terms of pest resistance. These trials typically involve the cultivation of GM citrus plants in natural or
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