Molecular Plant Breeding 2024, Vol.15, No.5, 209-219 http://genbreedpublisher.com/index.php/mpb 214 semi-natural environments where they are exposed to various pests and environmental conditions. The methodologies for field trials can be broadly categorized into several key steps. Selection of test sites: Test sites are chosen based on their historical pest pressure and environmental conditions that are representative of commercial citrus-growing regions (Sun et al., 2019; Salonia et al., 2020). Experimental design: Randomized complete block designs (RCBD) or split-plot designs are commonly used to minimize variability and ensure robust statistical analysis (Gressel et al., 2017; Li et al., 2020). Figure 3 Scheme of fast-track breeding system to introduce CTV resistance into citrus germplasm (Adopted from Endo et al., 2020) Image caption: Precocious transgenic trifoliate orange that overexpresses CiFT(T0-2-11) was used as a primary pollen parent for the CTV resistance donor. BC progenies with T-DNA integrated regions generally flower around 3 weeks after planting. F1-2-11, BC1-7-13, and BC2-72 were selected as the pollen parents with DNA markers, to possess CTV resistance and transgenes. In 2016, the flowering times of the seed parent and transgenic seedling were coordinated, to promote the breeding from the BC2 to the BC3 generation, in a single year (Adopted from Endo et al., 2020) Planting and maintenance: GM citrus plants are planted alongside non-GM controls. Standard agronomic practices are followed, including irrigation, fertilization, and pest management, to ensure that any observed differences are due to genetic modifications (Sun et al., 2019; Salonia et al., 2020). Data collection: Regular monitoring and data collection are conducted to assess pest incidence, plant health, growth parameters, and yield. Advanced techniques such as remote sensing and digital imaging may also be employed for more precise measurements (Minamikawa et al., 2017). Statistical analysis: Data are analyzed using statistical software to determine the significance of observed differences between GM and non-GM plants. Metrics such as pest infestation levels, plant vigor, and yield are compared (Gressel et al., 2017; Li et al., 2020). 6.2 Key field trials and their outcomes Several key field trials have been conducted to evaluate the performance of GM citrus varieties with stacked pest resistance genes. Trial 1: A study on the development of multi-resistance rice using a transgene stacking system demonstrated significant improvements in resistance to multiple pests and diseases. The GM rice showed enhanced resistance to glyphosate, borers, brown planthopper, bacterial blight, and rice blast, with higher yields compared to the non-GM control (Li et al., 2020). Although this study was on rice, the methodologies and outcomes are highly relevant to citrus pest resistance breeding. Trial 2: Field trials on citrus varieties engineered
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