Abstract
In this study, we analyzed differences in the enzyme activities and transcriptomes of embryogenic and non-embryogenic calli to gain insights for improving the success of tissue culture-based breeding. A total of 2,856 differentially expressed genes (DEGs; 1,632 up-regulated and 1,224 down-regulated) were identified based on RNA sequencing and verified by reverse transcription quantitative polymerase chain reaction. Gene set enrichment analysis revealed that many of the up-regulated DEGs in embryogenic callus were enriched in the photosynthesis processes. Furthermore, the enzyme activity, hormone content, and cytokinin oxidase/dehydrogenase (CKX) gene expression analyses were found to be consistent with the transcriptome results. Cytokinin biosynthesis in N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (PBU)-induced embryogenic callus increased owing to CKX repression. Measurement of endogenous hormones by high-performance liquid chromatography revealed that, compared with non-embryogenic callus, in embryogenic callus, the indole-3-acetic acid, abscisic acid and trans-zeatin riboside content had significantly higher values of 129.7, 127.8 and 78.9 ng/g, respectively. Collectively, the findings of this study will provide a foundation for elucidating the molecular mechanisms underlying embryogenic callus differentiation and can potentially contribute to developing procedures aimed at enhancing the success of callus-based plant regeneration.
Highlights
Species of Eucalyptus, belonging to the Myrtaceae family of dicotyledonous plants, are among the most commonly cultivated plantation trees worldwide
We found the growth of embryogenic callus to be more vigorous than that of non-embryogenic callus
Among the four hormone examined, we found that the contents of indole-3-acetic acid (IAA), abscisic acid (ABA), and trans-zeatin riboside (TZR) were significantly higher in the embryogenic callus than in non-embryogenic callus, whereas in contrast, the levels of gibberellic acid (GA3) were considerably higher in non-embryogenic callus, indicating that GA3 may reduce callus differentiation capacity and that IAA, ABA and TZR may contribute to enhancing embryogenic callus formation, including green callus induction and somatic embryogenesis
Summary
Species of Eucalyptus, belonging to the Myrtaceae family of dicotyledonous plants, are among the most commonly cultivated plantation trees worldwide. The growth of Eucalyptus trees tends to be superior to that of other trees used for plantation, in that these species adapt well to tropical and subtropical regions, and its wood can be used for multiple purposes, including veneer, firewood, and the production of essential oil (Pinto et al, 2010). Genetic engineering of Eucalyptus can be used to effectively improve germplasm resources (Girijashankar, 2011; Ouyang & Li, 2016). For most plants, Agrobacterium tumefaciens-mediated transformation depends on the effectiveness of the tissue culture methods used, of which callus induction is the initial step (Li & Luo, 2001). In this regard, few studies have reported the successful regeneration of E. grandis × E. urophylla via callus propagation (Ouyang et al, 2012; Ouyang & Li, 2016)
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