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.