Changes in structure and polyamine metabolism of litchi callus during subculture and somatic embryo development

Abstract:【Objective】Plant regeneration in vitro is the basis of transgenic breeding and rapid propagation of litchi (Litchi chinensis Sonn.). Components in the medium are the main factors that affect the ef- ficiency of litchi regeneration in vitro. At present, research on medium components has focused mainly on cytokinin and auxin, and very few studies have been performed on polyamines. In this study, the ana- tomical structure, polyamine components, and activity of associated enzymes were investigated during the subculture and somatic embryo induction of Feizixiao embryonic callus so as to optimize in vitro regeneration of litchi.【Methods】Daily proliferation was measured during the subculture of embryonic callus. Structural characteristics of embryonic callus during different stages of subculture and somatic embryo induction were studied by paraffin section, and the polyamine content was tested by GC-MS. The activity of enzymes involved in polyamine metabolism was assayed using a colorimetric method.【Results】The daily proliferation of embryonic callus remained almost unchanged during the early stage (0–6 d), after which it increased rapidly (7–15 d) to a peak at 15 d after subculture. After that, the proliferation decreased and then became relatively stable during the late stage (18–24 d). During the rapid proliferation stage, the granules of the embryonic callus were apparent. The paraffin sections of the callus showed that the cell divided vigorously, and the cell morphology was uniform with dense cytoplasm, and thus the sections were deeply stained. After inoculation on the somatic embryo induction medium, the callus was gradually dried and differentiated, and some early somatic embryos were visible at the early stage (0–10 d). Microscopic observation showed that there were apparent scleren- chyma cells in the callus, which resulted in obvious separation of the differentiated proembryos, and accumulation of many starch grains in the cytoplasm. After 15 d on the induction medium, the somatic embryo showed evident polarization. The cytoplasm of cells in the division region was dense and deeply stained, and cell division was fast. On the 20th day, the number of somatic embryos increased, and starch grains accumulated, which accelerated the division and differentiation of cells and caused cotyledon embryos to form early. Putrescine (Put) and spermine (Spm) were the main components of poly- amine (PAs) in litchi callus. The concentration of Put was much higher than that of Spm and spermidine (Spd), and the concentration of Spd was the lowest. Put content in callus in both subculture and somatic embryo induction medium maintained a high level; however, the concentration of Spd was low. Put and Spd concentrations peaked at the same time, i.e., at 6 d of subculture and 10 d of somatic embryo induction. The concentration of Spm in callus cultured on subculture medium was far higher than in the sam- ples cultured on somatic embryo induction medium. The concentration of Put determined the changing trend of Put/ (Spm+Spd), Put/Spm, and Put/Pas ratios. The values of the three ratios were all the lowest on the 15th day after subculture, and all were higher on the 6th and 21st days after subculture and on the 10th day after somatic embryo induction. The activities of polyamine oxidase (PAO) and diamine oxidase (DAO) were positively correlated with the contents of Put and PAs. PAO activity was higher in callus during somatic embryogenesis than during callus subculture; however, DAO activity showed an opposite tendency. The changes in PAO activity were consistent with the changes in the concentrations of Put, Spd, and PAs. They peaked on the 10th day after culture on somatic embryo induction medium and then decreased. DAO activity was relatively low and continued to decline during somatic embryo induction.【Conclusion】Cell division was most vigorous for callus of Feizixiao litchi after about 15 days of subculture. It is easy to produce callus tissue if the cells remain in a consistent state and if the callus at this stage is used for the proliferation. Two periods were found to be critical to somatic embryo differentiation: one around the 10th day after somatic embryo induction and the other around the 20th day. The first is essential to further differentiation and development of somatic embryos; the second is vital for development in large quantities. Put plays a crucial role in maintaining embryonic traits of the cells, and Spd plays an important role in somatic embryo differentiation. The increase in DAO enzyme activity regulated the content of PAS. High PAS content and PAO activity were beneficial to the high frequency of the litchi somatic embryo.