Abstract
Fraxinus mandshurica Rupr. (hereafter “F. mandshurica”) is known as one of northeast China′s important, valuable hardwood timber species. However, tissue culture and micropropagation of the species are difficult and have low efficiency, limiting asexual propagation. In this manuscript, stem explants were utilized to establish an effective regeneration system through adventitious bud organogenesis. The factors influencing callus regeneration in vitro were determined, and callus regeneration technology was established. The mechanism of adventitious bud formation was analyzed. Thidiazuron (TDZ) played a crucial role in the formation of adventitious buds. Elevated concentrations of TDZ were beneficial to callus induction and low concentrations of 6-benzyladenine (BA) led to loose state callus formation. The order of callus induction rates for different explants was stem cotyledon (100%) > segment (98.54%) > hypocotyl (92.56%) > root (50.71%). The effects of exogenous addition of 6-BA and TDZ on the endogenous hormone content of plants during the regeneration of adventitious buds were also assessed, as well as the expression characteristics of genes related to the regeneration pathway. The comprehensive analysis results showed that the suitable medium for callus induction and adventitious bud differentiation was c12 medium (MSB5 + 30 g/L sucrose + 7 g/L Agar + 5 mg/L 6-BA + 8 mg/L TDZ + 2 mg/L glycine + 0.1 mg/L IBA + 5% coconut water). The induction rates of callus and adventitious buds were 99.15% and 33.33%. The addition of 2.4 mg/L of the DNA demethylation reagent 5-azacytidine (5-aza) and 0.15 mg/L of the histone deacetylase inhibitor trichostatin A (TSA) increased the rates of adventitious bud induction by 17.78% over the control. This further laid the foundation for large-scale cultivation of excellent varieties and genetic transformation techniques.
Highlights
F. mandshurica Rupr. is an important broad-leaved tree species in northeastern China that can be used for various types of industrial and furniture timber and has significant economic and ecological value [1,2]
The callus formed by the roots had a velvety texture with canary yellow color and severe browning of the internal callus
The results showed that trichostatin A (TSA) could inhibit HDAC enzyme activity (Figure 4) and promote the induction of adventitious buds (Figure 5d,e)
Summary
F. mandshurica Rupr. is an important broad-leaved tree species in northeastern China that can be used for various types of industrial and furniture timber and has significant economic and ecological value [1,2]. Seed germination must undergo a vernalization treatment for up to 6 months [4] These factors increase the difficulty of F. mandshurica reproduction and limit its reproduction in forest development [5,6,7]. The degree of joining between scions and the rootstock, the quality of the scions, and the method of grafting all affect the final survival rate of the plant. Because of these factors, it is difficult to gain ideal economic benefits [8]. Genetic engineering technology provides an attractive way to effectively improve current varieties [11]
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