Shipping of in vitro micro-cuttings in tubes or jars is a frequently used method as the plants are more likely to quickly reproduce and comply with quarantine regulations in plant germplasm distribution. However, these containers are fragile during transportation. To diminish the risk associated with the long-distance shipping of in vitro plants, a safe and widely applicable packing and conservation technique based on microplate and slow growth was developed in this study. Potato cultivar ZHB and ginger cultivar G-2 were used to optimize the system with microplates (96 wells), vacuum-sealed packaging, and slow-growth techniques. Under regular culture conditions, packing in vacuum-sealed microplates reduced the survival of ZHB and G-2 micro-cuttings to 85.8% and 20.0%, respectively, and regeneration to 61.8% and 0%, respectively. Reducing the temperature to 10 °C maintained the survival of ZHB and G-2 micro-cuttings in the range of 83.3–100% after 60 days. Exposure to darkness decreased the survival of G-2 and inhibited regrowth. Thus, conservation in darkness at 10 °C is suggested. The effects of iron concentration and plant growth retardants were further assessed. The addition of 1/4 MS medium combined with 100 mg/L chlormequat chloride (CCC) resulted in full survival and growth inhibition of plantlets, without malformation identified. Finally, incubation with 1/4 MS medium supplemented with 100 mg/L CCC in vacuum-sealed microplates at 10 °C in the dark resulted in high survival and suppressed germination. Sweet potato HXS was incubated as well to test the broad-spectrum applications of the technique; 100% survival and 6.7% germination was gained. Morphological indices of released cuttings recovered to control levels after two cycles of subculture in MS medium. A 0.1–0.2% genetic variation was detected by SSR and ISSR, suggesting genetic stability of the conserved samples. Finally, micro-cuttings were safely transported to cities located thousands of kilometers away without package and sample damage. Our results enable easy distribution of in vitro plant germplasms.

Radish (Raphanus sativus L.) is an economically important vegetable worldwide, but its sustainable production and breeding are highly threatened by blight disease caused by Alternaria brassicae. Melatonin is an important growth regulator that can influence physiological activities in both plants and microbes and stimulate biotic stress resistance in plants. In this study, 0-1500 μM melatonin was exogenously applied to healthy radish seedlings, in vitro incubated A. brassicae, and diseased radish seedlings to determine the effects of melatonin on host, pathogen, and host-pathogen interaction. At sufficient concentrations (0-500 μM), melatonin enhanced growth and immunity of healthy radish seedlings by improving the function of organelles and promoting the biosynthesis of antioxidant enzymes, chitin, organic acid, and defense proteins. Interestingly, melatonin also improved colony growth, development, and virulence of A. brassicae. A strong dosage-dependent effect of melatonin was observed: 50-500 μM promoted host and pathogen vitality and resistance (500 μM was optimal) and 1500 μM inhibited these processes. Significantly less blight was observed on diseased seedlings treated with 500 μM melatonin, indicating that melatonin more strongly enhanced the growth and immunity of radish than it promoted the development and virulence of A. brassicae at this treatment concentration. These effects of MT were mediated by transcriptional changes of key genes as identified by RNA-seq, Dual RNA-seq, and qRT-PCR. The results from this work provide a theoretical basis for the application of melatonin to protect vegetable crops against pathogens.

Plasmodiophora brassicae causes a serious threat to cruciferous plants including radish (Raphanus sativus L.). Knowledge on the pathogenic regularity and molecular mechanism of P. brassicae and radish is limited, especially on the metabolism level. In the present study, clubroot-susceptible and clubroot-resistant cultivars were inoculated with P. brassicae Race 4, root hairs initial infection of resting spores (107 CFU/mL) at 24 h post-inoculation and root galls symptom arising at cortex splitting stage were identified on both cultivars. Root samples of cortex splitting stage of two cultivars were collected and used for untargeted metabonomic analysis. We demonstrated changes in metabolite regulation and pathways during the cortex splitting stage of diseased roots between clubroot-susceptible and clubroot-resistant cultivars using untargeted metabonomic analysis. We identified a larger number of differentially regulated metabolites and heavier metabolite profile changes in the susceptible cultivar than in the resistant counterpart. The metabolites that were differentially regulated in both cultivars were mostly lipids and lipid-like molecules. Significantly regulated metabolites and pathways according to the P value and variable important in projection score were identified. Moreover, four compounds, including ethyl α-D-thioglucopyranoside, imipenem, ginsenoside Rg1, and 6-gingerol, were selected, and their anti-P. brassicae ability and effects on seedling growth were verified on the susceptible cultivar. Except for ethyl α-D-thioglucopyranoside, the remaining could inhibit clubroot development of varing degree. The use of 5 mg/L ginsenoside Rg1 + 5 mg/L 6-gingerol resulted in the lowest disease incidence and disease index among all treatments and enhanced seedling growth. The regulation of pathways or metabolites of carbapenem and ginsenoside was further explored. The results provide a preliminary understanding of the interaction between radish and P. brassicae at the metabolism level, as well as the development of measures for preventing clubroot.

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