Bt Research 2024, Vol.15, No.2, 96-109 http://microbescipublisher.com/index.php/bt 98 3 Methods of Gene Stacking 3.1 Traditional breeding techniques Traditional breeding techniques have long been employed to enhance crop traits by combining desirable genes from different parent plants. One of the primary methods used in traditional breeding is marker-assisted selection (MAS), which allows for the identification and selection of plants that carry specific genes of interest. This technique has been particularly effective in gene pyramiding, where multiple genes conferring resistance to biotic and abiotic stresses are stacked together to create more resilient crop varieties (Dormatey et al., 2020). For instance, MAS has been successfully used to develop crops with improved tolerance to drought and salinity, which are critical for sustainable agricultural production in the face of climate change (Shailani et al., 2020). Despite its effectiveness, traditional breeding has limitations, particularly in the time required to achieve desired results and the complexity of combining multiple traits. The process can be slow and labor-intensive, often taking several generations to achieve the desired gene combinations. Additionally, traditional breeding is limited by the genetic diversity available within the species, making it challenging to introduce novel traits that are not present in the gene pool (José et al., 2020). These limitations have driven the development of more advanced techniques, such as genetic engineering and biotechnological methods, to overcome these challenges and achieve more precise and efficient gene stacking. 3.2 Genetic engineering approaches Genetic engineering has revolutionized the field of crop improvement by enabling the direct manipulation of plant genomes to introduce desirable traits. One of the key techniques in genetic engineering for gene stacking is the use of transgenic technology, which allows for the insertion of multiple genes into a plant's genome. This method has been used to create crops with enhanced resistance to pests, diseases, and environmental stresses (Halpin, 2005). For example, the GuanNan Stacking (GNS) system (Figure 1) utilizes Type IIS restriction enzyme-mediated Golden Gate cloning and Gateway recombination to assemble multiple gene expression cassettes into a single binary vector, facilitating the creation of transgenic plants with stacked traits (Qin et al., 2022). Figure 1 Basic Principles of the GNS System (Adapted from Qin et al., 2022) Image caption: A: Assembly of an entry vector using the backbone of the donor vector and three element modules, each containing components of the expression cassette, namely the promoter, coding sequence (CDS), or terminator; B: Stacking process using the target vector and entry vector (Adapted from Qin et al., 2022)
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