Study On Optimization Of Immature Embryo Culture System And The Correlation Between Nitrate Metabolism And Immature Embryo Culture In Maize

To resolve the problem of Maize inbred lines induced high quality callus hardly, we used three inbred lines, such as Zheng22, Ye478 and Zheng58, then induced them on different mediums adding with2,4-D, dicamba and two mixed use for different concentrations to research the influences of hormones on the induction and growth of callus. The results showed that the induction rate and the quality of callus were different among different genotypes; the materials that could produce high quality callus easily, such as Zheng22, could be induced and cultured on medium with 2mg?L-12,4-D; other materials such as Ye478 and Zheng58 that were difficult to induce callus could be induced by 2～3mg?L-1 dicamba and cultured on the medium adding with 2mg?L-12,4-D.It was confirmed similarly that the growth capacity and plant regeneration rate of long-term subcultured Maize callus decreased obviously and the growth capacity and plant differentiation ratio of inbred line were prevalently lower than one of cross combination; but the growth capacity and plant regeneration rate of typeⅡcallus, such as Qi319, had no dramatic decline. To keep the embryogenisis and improve the plant regeneration rate of long-term subcultured Maize callus, 48h dehydration to the callus was carried on before plant differentiation. The results indicated the plant differentiation ratio of the callus after 48h dehydration was averagely increased by 57.39% compared with the control, the highest was 11times that of the control. In addition, the callus was pretreated by sorbitol, ABA and 48h dehydration before subculture and plant differentiation. The results suggested that suitable concentration of sorbitol could accelerate the growth of the callus and its suitable concentration was 4% or 5%; ABA had no obvious effect on the growth of the callus; the growth capacity of each genotype was increased obviously after 48h dehydration. (2) The plant differentiation ratio of each treatment was higher than the control and the effect of 48h dehydration was the best; the effect of ABA on raising plant differentiation ratio was better and its concentration of the highest plant regeneration rate was 3 mg?L-1. Three treatments could speed up the regeneration process more or less. After comprehensively analyzing the aspect of improving the growth capacity, plant differentiation ratio and regeneration process the 48h dehydration was optimal way, the operation of which was simple and there were no differences among various genotypes. So 48h dehydration treatment had a broad adaptability. Because ABA is superior to sorbitol on improving plant regeneration, we compared the effects of pretreating the callus for a week with ABA with growing on the subcultured medium and plant differentiation medium containing ABA. When subcultured on the medium containing ABA, the growth capacity was low; After pretreating with ABA for a week, the growth capacity of most genotypes were higher than the control obviously. Especially for the pretreatment of 3mg?L-1ABA, the growth capacity were higher than other concentrations, the plant regeneration ratio could improved by 25.71%, and the average plant regeneration ratio were 22.6% on the second week. So we could get the result that pretreating with 3mg?L-1ABA for a week can improve the growth capacity, plant differentiation ratio and regeneration process.Nitrogen metabolism is one of the basic metabolisms for plants, the accumulation of nitrous acid is harmful to cell. It is not reported that the effects of Nitrogen metabolism and other physiological factors on the growth and plant regeneration of callus. In this paper, the Nitrate metabolism of Maize inbred line was studied during the process of callus induction, subculture and regeneration respectively and the Nitrate metabolism of different types of calli of one genotype and the 48h dehydration treatment were compared. The results were as follows: (1) The Nitrate metabolism had some differences among different genotypes during the process of callus induction, there were no significant differences on NO3- content among different genotypes; the nitritase activity(NiRA) of Bendibai was the lowest, and the NiRA of other genotypes was 2.0 to 2.7 times that of Bendibai. In addition, the NO2- content of Bendibai was obviously higher than other genotypes. (2) Both the NiRA and the NO2- content of Ye478 were higher during the process of subculture and regeneration. Although the callus of Ye478 could be subcultured for long time, the quality and the rate of embryogenic callus were low. (3) There were no significant differences on nitratase activity between typeⅠand typeⅡcallus of Qi319 during the process of subculture and regeneration, but the NiRA of typeⅡcallus were higher than the typeⅠcallus all the time; NO3-and NO2- were substrates assimilated by NR and NiR separately, their content in the callus affected the activity of enzymes. The NO2-accumulation of typeⅡcallus was little higher than typeⅠcallus before subculture and regeneration, but the net NO2- accumulation of typeⅠwas higher than that of typeⅡbefore next subculture and after the 20th day of regeneration. (4) Compared with the control, the NiRA was higher and the NO2- content was lower in the callus after 48h dehydration.