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Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods. Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties. In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization (FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations. FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes. Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations. No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines. The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations. The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.

Effect of salicylic acid (SA) on chilling injury (Cl) of sponge gourd during storage (9 days, 9°C) plus shelf life (2 days, 20°C) was evaluated in this study. SA treatment at the concentration of 1.5 mmol L−1 significantly reduced postharvest Cl of sponge gourds. Besides, the application of SA could effectively decrease the electrolyte leakage, reduce the accumulation of malondialdehyde (MDA) and total phenolics, enhance the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), and inhibit the activities of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO). The beneficial effects of SA could be attributed to preserved membrane integrity, inhibited membrane peroxidation, enhanced antioxidant system and suppressed activities of browning related enzymes. In a sense, SA as a postharvest tool may be commercially used in alleviating Cl of sponge gourd.

Locating of important agronomic genes onto chromosome is helpful for efficient development of new wheat varieties. Wheat chromosome substitution lines between two varieties have been widely used for locating genes because of their distinctive advantages in genetic analysis, compared with the aneuploid genetic materials. Apart from the substituted chromosome, the other chromosomes between the substitution lines and their recipient parent should be identical, which eases the gene locating practice. In this study, a set of chromosome substitution lines with cv. Wichita (WI) as the recipient parent and cv. Cheyenne (CNN) as the donor parent were studied for the composition of high molecular weight glutenin subunits (HMW-GS) as well as a range of agronomic important traits. Results revealed that the substitution lines of WI(CNN5D), WI(CNN6A) and WI(CNN7B) had higher plant heights than the two parents of WI and CNN, and WI(CNN3D) had later maturity than the parents. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, a substitution line WI(CNN5B) was found to contain different HMW-GS patterns from its two parents, in which 1By9 was replaced by 1By8 on chromosome 1BL. Simple sequence repeat (SSR) analysis confirmed that the variation on 1BL in WI(CNN5B) was originated from Chinese Spring (CS). It is concluded that chromosomal fragments from bridge material and donor parent were quite often retained in intracultivaral chromosome substitution lines except the substituting chromosomes.

The antifungal activity of chitosan on a common fungal phytopathogen, Sclerotinia sclerotiorum, and the control effect on sclerotinia rot of carrot were investigated. Mycelial growth and fungal biomass were strongly inhibited by chitosan. Using propidium iodide stain combined with fluorescent microscopy, the plasma membrane of chitosan-treated S. sclerotiorum mycelia was observed to be markedly damaged. Concomitantly, protein leakage and lipid peroxidation was also found to be significantly higher in chitosan-treated mycelia compared to the control. Chitosan provided an effective control of sclerotinia rot of carrot, with induction of activity of defense-related enzymes including polyphenoloxidase and peroxidase. These data suggest that the effects of chitosan on sclerotinia rot of carrot may be associated with the direct damage to the plasma membrane and lipid peroxidation of S. sclerotiorum, and the elicitation of defense response in carrot.

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins (AGPs) and hydrogen peroxide (H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 (KN199) and Xinchun 9 (XC9), together with low-frequency regeneration wheat line Chinese Spring (CS), presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L−1 AGP or 0.005 to 0.01 ‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L−1 AGP application level in callus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22 (JM22), Jingdong 18 (JD18) and Yangmai 18 (YM18), respectively; whereas at 100 mg L−1 AGP level, CS (105.44%), Chuannong 16 (CN16) (80.60%) and Ningchun 4 (NC4) (62.87%) acted the best. Moreover CS (79.05%), JM22 (7.55%), CN16 (101.87%), YM18 (365.56%), Yangmai 20 (YM20) (10.48%), and CB301 (187.40%) were more responsive to 0.005 ‰ of H2O2, and NC4 (35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overall, these two methods, inter-culture and AGP (or H2O2) application, can be further applied to wheat transgenic research.

The immature embryos (IEs) of wheat are the most widely used tissues for in vitro culture and genetic transformation due to its high regeneration competency. However, this explant can only be maintained in 4°C daily cooler for a short period time for its use in plant tissue culture or transformation experiments. This study aimed to investigate the effects of environmental temperature, cryopreservation storage temperature, and heat shock culture (HSC) temperature on the regeneration frequency of wheat IEs. Results indicated that environmental temperature significantly affected the induction of embryonic calli. The optimum total accumulated temperature (TAT) during the time of anthesis and sampling for regeneration of these tissues was around 280°C for spring wheat type cv. CB037 and approximately 300°C for winter wheat type cv. Kenong 199. Regeneration ability obviously declined when the highest environmental temperature was over 35°C for 1 d or a high temperature between 30 and 33°C lasted for 5 d during anthesis and sampling. This finding was verified by culturing the freshly isolated IEs under different temperatures from 29 to 37°C in different controlled growth incubators for 5 d; the IEs almost completely lost regeneration ability when the temperature rose to 37°C. Cryopreservation of −20°C caused the wheat samples lost ability of producing callus or embryonic callus in a few days, and cryopreservation of −10°C more than 10 d made the regeneration potential of the tissues dramatically declined. Comparatively, the temperature that best maintained high regeneration ability was −5°C, at which the materials can be maintained for around 1 mon. In addition, the preservation of the immature samples at −5 or −10°C inhibited the direct germination of the IEs, avoiding the embryo axis removing process. Our results are useful for ensuring that field collection and cryopreservation of the wheat IEs are done correctly to enable tissue culture and genetic transformation.