Physiological And Biochemical Aspects Of Picloram Regulates The Embryonic Potential Of Callus In Agapanthus

Picloram(PIC) as a kind of exogenous auxin can effectively induce Agapanthus explants to form No Embryogenic culture(NEC) and further develop into embryonic callus(EC). However, EC induced by different concentrations of PIC has different embryonic potentiality in Agapanthus. In order to investigate physiological and biochemical aspects of different concentrations of picloram regulates the embryonic potential of callus in Agapanthus,This study observed the morphological and PCD features, measured the contents of IAA,energy source,ROS and antioxidant system,detectd the DNA methylation levels,and analyzed the relative quantitative expression of 46 related genes by q RT-PCR. The results were as follows:(1) The morphology observation exhibited that the NEC cell was larger, smaller nuclear and abundant intracellular starch, however, the EC cells showed denser than NEC. Endogenous IAA concentration of NEC maintained at very low levels compared to EC, and tit decreased with the exogenous PIC concentration increasing. These results suggested that auxin signaling were involved in regulation the embryonic obtaination of EC, and a competition between exogenous PIC concentration and endogenous IAA signal during somatic embryogenesis in Agapanthus. Ten kinds of fatty acid components were detected in NEC and EC by Browse and Larkindale’s method, including 5 saturated fatty acids: palmitic acid, stearic acid, eicosanoic acid, behenic acid and lignoceric acid; and 5 unsaturated fatty acids: palmitoleic acid, linoleic acid, linolenic acid, oleic acid, eicosenoic acid. The contents of oleic acid, stearic acid, behenic acid and lignoceric acid in EC were significantly higher than NEC, and the double-bond index of fatty acids of EC was obviously lower than NEC.(2) The balance between ROS(O2·-,OH·and H2O2) and antioxidant system(SOD,POD and CAT) promoted the differentiation of somatic cell and development of embryonic cells. The aggregation and diffusion of ROS components can induct PCD to remove suspensor organisms and establish embryonic polarity at the begin stage to promote somatic embryogenesis.(3) Using MSAP(Methylation Sensitive Amplified Polymorphism) technique to detect DNA methylation level, and found that total methylation rate reduced in the process of conversion of NEC to EC. With the change of concentrations of PIC, DNA methylation level increased. Relatively low level of DNA methylation level was essential to the induction and maintenance of embryonic cells.(4) q RT-PCR technique was performed to analysed the expression of 46 genes related to the acqure of embryonic potential in NEC and EC of Agapanthus. Results indicated that auxin signal and ethylene signal enhanced, chromatin structure changes a lot and regulation of epigenetic modifications obviously enhance. The cell wall and secondary metabolic processes weaken obviously during embryonic induction stage. Furthermore, sugar components, fatty acid composition, hormone signaling, cell division, cell cycle, transcription factors and epigenetic process exhibite significant changes during EC embryonic-maintianed period.