This study aims to establish a rapid and efficient method for qualitative and quantitative determination of plant acid hormones by ultra-high performance liquid chromatography/high resolution time-of-flight mass spectrometry (UPLC- TOF-MS). Several derivatization reagents, 2-bromoacetophenone (BP), 2-dimethylaminoethylamine (DMED), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 3-bromoactonyltrimethylammonium bromide (BTA) were selected for derivatization with abscisic acid, gibberellic acid, indoleacetic acid, jasmonic acid and salicylic acid, respectively. The effect of derivatization reagent was evaluated by comparing the response value of the derivative hormones. The plant hormone standards were diluted in gradient and reacted with the derivatization reagent, and the detection limit and quantitation limit of the derivative hormones were determined. Reagent with the best derivatization effect was reacted with the plant crude extract to test its application effect. All the four derivatized reagents could improve the MS response value of acidic hormone, among which the MS sensitivity of hormone derived by DMED is the best. Reaction with DMED was stable and reproducible. The quantitation limits of ABA-DMED, GA₃-DMED, IAA-DMED, JA-DMED and SA-DMED were 0.05, 0.2, 0.1, 0.1 and 0.5 ng∙mL^-1, respectively. Compared with non-derivatized hormones, the quantitative limits of ABA, GA₃, IAA, JA and SA were reduced by 100, 25, 50, 50 and 10 times, respectively. So, DMED derivatization increased the mass spectrometry sensitivity of several hormones by 10-100 times. The method was applied to the determination of endogenous acidic hormones in rice, wheat and broad bean, and the sensitivity was significantly higher than that before derivation. A simple, rapid and reproducible UPLC-TOF-MS method based on derivatization was established, which greatly improved the sensitivity of the determination of acidic hormones in plants.

Helenium aromaticum has a fragrant odor and its capitulum contains only disk flowers. H. aromaticum is a good model for studying development of floral patterns in Compositae. However, methods for genetic transformation of H. aromaticum is currently not available. In this study, we report the establishment of an efficient in vitro regeneration system of the H. aromaticum. The induction of adventitious buds was tested with various combinations and concentrations of 25 different phytohormones by using leaves, stem segments, and hypocotyls as explants. We found that when leaf explants were cultured on MS medium supplemented with 0.2 mg·L^-1 NAA, 1 mg·L^-1 6-BA, and 0.2 mg·L^-1 TDZ for 20 days, the induction of callus was 100% and the induction rate of adventitious buds was 62.10%. The newly-formed adventitious buds were then cultured on 1/2MS medium for 16 days and the rooting rate reached 63.33%. After culturing for an additional 14 days, floral buds initiated and eventually flowering with a rate of 93.33%. In addition, we also found that the regeneration of H. aromaticum was affected by the nature of explants, the types and concentrations of phytohormones. Whereas 2,4-D is incapable of inducing adventitious buds, the combination of appropriate concentrations of 6-BA and TDZ effectively promotes the formation of adventitious buds of H. aromaticum. This study has established an in vitro regeneration system of H. aromaticum, which is a key perquisite for the subsequent establishment of its genetic transformation system. Moreover, this method will also be an important reference for studies on the floral development patterns in Compositae.

Frost has had a prominent influence on the fruit industry in China in recent years. We need to establish a system to simulate frost treatment for fruit trees. Based on observations of frost events in field, we analyzed the characteristics of frost in terms of cooling rate, the low temperature limit, warming rate, and light conditions after frost treatment and established a system to simulate frost treatment in the laboratory. Temperature during the frost treatment in field could be divided into three stages: cooling, extreme temperature maintenance and warming. The temperature during cooling and warming stages changed in an approximately linear manner. Frost is generally followed by high intensity light. The simulated frost process was determined as followed: the temperature drops from room temperature (20°C) to 5°C in 30 min, and is maintained at 5°C for 30 min, then decreases to -2°C at a rate of 0.8°C·h ^-1, is maintained at -2°C for 2 h, then increases to 5°C at a rate of 4.7°C·h ^-1 in the dark. The recovery condition after frost was 16°C and 800 μmol·m ^-2·s ^-1.

Genetic transformation system is the foundation for gene function study and genetic improvement of Dioscorea opposita. In this study, we established an Agrobacterium-mediated genetic transformation system using microtuber in D. opposita cv. ‘Tiegun’. The transformation was performed with the DoSERK2 gene overexpression plasmid pCAMBIA1301-DoSERK2 that harboring the GUS reporter gene and the hygromycin (Hyg) resistence gene as the selectable marker, and the gene-silencing vector pART27-DoSERK2 that containing kanamycin (Kan) resistence marker. The optimal concentration of timentin (Tim) for Agrobacterium counter selection was 500 mg·L ^-1. For shoot regeneration and rooting, the optimal concentrations of Hyg were 15 mg·L ^-1 and 20 mg·L ^-1, respectively, and the optimal concentration of Kan were 120 mg·L ^-1 and 160 mg·L ^-1, respectively. Results of PCR analysis and GUS histochemical staining indicated that the exogenous genes had been integrated into the genome of transgenic plants, and are expressed in the plants. Our study provides a genetic transformation system in D. opposita cv. ‘Tiegun’, which has the advantage that explants are easy available. This method has reference value for the genetic transformation of other varieties of D. opposita.

The leaf of Sedum clavatum was used to induce callus; through callus induction, bud induction, rooting and transplatation, aseptic-frequency regeneration systems were established. The optimal medium for induction was MS+3.0 mg·L^–1 6-BA+0.1 mg·L^–1 NAA+1.0 mg·L^–1 KT; for redifferentiation 3/4MS+3.0 mg·L^–1 6-BA+0.3mg·L^–1 NAA; and for rooting 1/2MS+0.03 mg·L^–1 NAA. The callus induction, redifferentiation and rooting rate was 95.7%, 80% and 94.89%, respectively. Using perlite:vermiculite (1:2) as a matrix, the transplant survival rate was >90%. We have established a high-frequency regeneration system for S. clavatum.

The information on protein subcellular localization is important to elucidate protein function, and imaging technology is one of the important approaches to visualize protein localization. However, conventional microscopy techniques can barely resolve details of subcellular structures mainly because of the auto-fluorescence interference with their limited imaging resolution. In recent years, super-resolution optical imaging technologies have been successfully used in human and animal cell research for their 8-fold higher spacial resolution over laser confocal microscopy. Application of these technologies in plant cells has not been reported, probably due to the peculiarity of plant cells. Here, we report the successful use of DeltaVision OMX microscope technology for visualizing Zea mays sucrose synthase 1 (ZmSUS-SH1) around chloroplast grana in transgenic tobacco epidermal cells. OMX microscopy can overcome the cellular chlorophyll fluorescence interference in imaging fluorescent fusion proteins. We also developed an optimized sample preparation protocol for using DeltaVision OMX microscope technology with plant materials.