Open tissue culture technique could be simplified by using different bacteriostatic agents. There is a great difference in the bacteriostatic effects of different antimicrobial agents on various explants. However, there is no report about the effective bacteriostatic agent for open tissue culture of Acacia auriculiformis. We carried out the bud induction trials under open conditions to screen out an effective antibacterial agent for open tissue culture of A. auriculiformis. The results showed that the suitable type and concentration of bacteriostatic agent was 0.2 g L−1 Chlorothalonil, and the suitable explant type was middle shoot section with leaves (the shoot section with third to fifth axillary bud). The treatment of 0.8 g L−1 Carbendazim for 3 min was the most suitable strategy for explants disinfection, and October was the best time for explants collection. The suitable bud induction medium was 1/8 MS + agar 7 g L−1 + Chlorothalonil 0.2 g L−1 + 6-BA 1.5 mg L−1, and the bud induction rate was 99.54%. Our results revealed that Chlorothalonil is an effective bacteriostatic agent for bud induction of A. auriculiformis under open condition. These results would be very helpful for further establishment of open tissue culture technology for A. auriculiformis.

Acacia mangium is well known as a valuable commercial tree species in the Acacia genus. A. mangium was recently found to be self-incompatible (SI), but its SI mechanism is not clear, which has hindered the progress of genetic improvement of A. mangium with strong resistance. To confirm the SI type of A. mangium, pollen germination was observed via fluorescence microscopy at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h after self-pollination. We found over ninety percent of the pollen grains produced no pollen tube growth on the stigma/style. To further explore the SI molecular mechanism of A. mangium, tests of the transcriptome and metabolome were carried out after self-pollination. Observations of pollen germination after self-pollination using fluorescence microscopy suggested that the SI type of A. mangium is gametophytic self-incompatibility (GSI). A combined transcriptomic and metabolomic analysis showed that DEGs (differentially expressed genes) related to SI (6 S-glycoproteins, 93 F-box proteins, 69 26S proteasomes, 38 calcium-dependent protein kinases/calmodulin and 41 thioredoxin genes) were significantly enriched in six KEGG (sulfur metabolism, tyrosine metabolism, phenylalanine metabolism, butanoate metabolism, and valine, leucine, and isoleucine degradation). Further analysis of these six pathways revealed the enrichment of SI-related DEGs corresponding to succinate, methylmalonate, and 3-hydroxypropane. These three metabolites were significantly downregulated. The analysis of transcripts and metabolites suggested that transcripts of SI-related gene families (thioredoxin and F-box protein) were significantly upregulated under the regulation of transcription factors (TFs) after self-pollination, leading to a decrease in metabolites (such as succinate, methylmalonate, and 3-hydroxypropionate). We also further speculated that TFs (MYB, HB-HD-ZIP, AP2/ERF-ERF, and bZIP) and gene families (thioredoxin and F-box protein) were important factors related to the SI of A. mangium.

Background: Autoclaving is used to eliminate contamination during tissue culturing, however, it is a complicated process, time-consuming and costly. Chemical sterilization of tissue culture can effectively eliminate contamination, is a simple procedure, and cost effective. However, studies on the chemical sterilization mostly focus on bud induction, while the effects of chemical sterilization overall process of tissue culture, including bud induction, proliferation, and rooting, remain to be determined. Here, we investigate the effect of chemical sterilization on bud induction, proliferation, and rooting of Acacia mangium × A. auriculiformis.Results: The results showed that chlorothalonil (0.2 g/L) was a suitable chemical sterilant, and bud induction medium was 1/8 Murashige and Skoog medium + agar 7 g/L + chlorothalonil 0.2 g/L + 6-benzylaminopurine 0.5 mg/L The highest induction rate (99.54%) was observed in the third to fifth buds’ stem segments collected in October treated with 0.8 g/L carbendazim for 3 min, with a contamination rate of 0. The rooting medium was agar 7 g/L + chlorothalonil 0.2 g/L+ indolebutyric acid 1.5 mg/L + naphthylacetic acid 0.5 mg/L, and the rooting rate was 97.62%. The proliferation rate and subculture duration showed a positive correlation, while the proliferation rate was 3.58 times higher at the fourth subculture rooting. Conclusions: Our results suggest that chlorothalonil can effectively replace autoclaving during bud induction, proliferation, and rooting of A. mangium × A. auriculiformis. The findings of this study provide technical support for rapid seedlings propagation, accelerates the breeding process of Acacia, and can be applied in other tree species.

Stem segments of Acacia mangium and A. auriculiformis containing full axillary buds were used to study the effects of reduced amounts of the main nitrogen source in the growth media. This condition, referred to as nitrogen deficiency in this article and denoted as -N, involved the omission of ammonium nitrate and potassium nitrate from MS media, and its impact on bud induction was assessed. The results show that in media lacking nitrogen, the bud induction rate, contamination rate, browning rate, stem length, and leaf number of induced buds of A. mangium and A. auriculiformis varied depending on the different culture media used. The optimal bud induction medium for A. mangium and A. auriculiformis was as follows: 1/4MS (-N) + 1.0 mg·L⁻¹ 6-BA + 0.2 g·L⁻¹ chlorothalonil + 5 g·L⁻¹ AGAR. The bud induction rates were 72.6% and 100.0%, respectively. There were no significant differences in the rooting rates of the induced buds between the -N treatment and the complete nutrient treatment. We found that the buds induced in the -N media did not show obvious symptoms of nitrogen deficiency, and their growth status was not significantly different from those induced in the complete nutrient media, which indicates that nitrogen is not essential for the bud induction of A. mangium and A. auriculiformis. The results of this study provide an important reference for conducting related research on other plants and have are greatly significant for the sustainable development of tissue culture technology in the future.