## 1 Introduction
The _vtc4_ mutant showed less myo-inositol and AsA accumulated, indicating that VTC4 is a bifunctional enzyme that impacts both myo-inositol and AsA synthesis pathways [27]. The expression analysis of AsA related genes suggested that _GPP_ plays an important role in the regulation of AsA accumulation in tomato [28]. GalDH has a very high conversion efficiency for L-Gal, and can quickly convert exogenous L-Gal into ascorbate [10]. Therefore, the GalDH catalyzed reaction is not a rate-limiting step in the ascorbate synthesis pathway. Knock-down of the expression of _GLDH_ doesn't affect the content of ascorbate in both leaves and fruits of tomato suggesting GLDH is also not a rate-limiting enzyme for ascorbate synthesis [1]. Besides structural genes, many factors such as light, temperature, ozone, hormones, and regulatory factors can regulate AsA accumulation [29]. For instance, high light, low temperature, and combined high light and low-temperature treatment can increase AsA content by 10-50% in _Dunialella_ species [30; 31]. In tomato, seven days of shading resulted in a 50% reduction of AsA in leaves and a 10% decrease in antioxidant activity in fruit [32]. AMR1 (ascorbic acid mannose pathway regulator 1) is a regulatory factor with an F-box structure that affects the expression of genes in D-Man/L-Gal pathway and showed a negative regulation of AsA levels and ozone-nonsensitivity in _Arabidopsis_[33]. AtERF98 and SIHZ24, as transcription factors, directly bind to the promoter of _VTC1_ and _SIGMP3_ and affect the AsA accumulation in Arabidopsis and tomato, respectively [34; 35]. It is well-known that many of the plant traits are controlled by multigenes, especially for metabolites due to the complex metabolic processes of biosynthesis, degradation, and recycling [36; 37; 38]. For complex biosynthetic pathways or multiple traits; in some cases, engineering a single gene is insufficient to improve the yield of metabolic products; the assembly of multigene expression is often required. To achieve this purpose, researchers have proposed several pyramiding methods, such as cotransformation of multiple genes and transgenic pyramiding by conventional hybridization. Several examples have been developed via cotransformation with a few regulatory or structural genes of the target metabolic biosynthesis pathways, such as \(\beta\)-carotene-enriched \"Golden Rice\" [39] and anthocyanin-enriched \"Purple Tomatoes\" and \"Purple Endosperm Rice\" [36; 40]. Pyramiding by conventional hybridization is time-consuming compared with cotransformation. However, pyramiding by hybridization is technologically feasible and generate stable inherited target genes [41]. It has been also reported that transgenic pyramiding improves plant salt tolerance, disease resistance, and amino acid content [42; 43; 44]. Unlike the anthocyanin or carotenoids pathways, there is no research on the application of biosynthetic pyramiding in AsA pathway. In this study, to further investigate the AsA biosynthesis and its engineering in tomato, we pyramided structural genes (SGs) involved in d-Man/l-Gal AsA pathway by conventional hybridization. Functional characterization of different pyramiding transgenic lines showed significantly enhanced AsA levels, light sensitivity, AsA transport capacity, and tolerance to oxidative stresses.