The Formation Of Shoot Meristem And The Distribution Patterns Of Auxin And Cytokinin Responses During Shoot Regeneration In Maize

In maize, shoot regeneration is the base of obtaining transgenic plants. Shoots have been induced from immature embryos, suspension cells, immature stem, shoot tip, leaf and mature embryos in maize. Shoot regeneration requires special responses of auxin and cytokinin in callus. In Arabidopsis shoot regeneration process, auxin distributes in the area surrounding the shoot initiation site, and cytokinin distributes in the central area. Meanwhile, the expression of shoot apical meristem (SAM) specific genes WUSCHEL (WUS) and CLAVATA3 (CLV3) represents that shoot meristem is reformed in Arabidopsis shoot regeneration. However, the molecular mechanisms of shoot regeneration in maize are still unknown. We analyzed the function of maize SAM specific gene ZmWUSCHEL1(ZmWUS1). Then we observed the expression patterns of ZmWUS1 and another SAM specific gene KNOTTED1 (KN1) during in vitro shoot regeneration. We also studied the distribution patterns of auxin and cytokinin responses to analyze the regulation of hormones in monocotyledon shoot regeneration. The main results are as follows:(1) The morphological analysis of maize shoot regeneration using immature embryos as explants. The embryonic calli were first induced under the treatment of auxin. After the embryonic calli were transferred to the differentiation medium by removal of auxin, the SAMs were formed on the surface of the calli, which would eventually form the shoot.(2) Functional analysis of ZmWUS1. Through analyzing the phenotypes of plants, we found that the ZmWUS1-overexpressed plants have bigger plant type, shorter internodes, higher leaf density compared to wild type plants. The phyllotaxis of some ZmWUS1-overexpressed lines was changed from alternate to opposite. However, the growth and development of those ZmWUS1 down-regulated lines was slower than the wild type plants, as well as the whole plant type was smaller. So ZmWUS1 may be involved in the regulation of maize shoot development.(3) Expression patterns of ZmWUS1 and KN1 in shoot regeneration. To study the formation of SAMs during shoot regeneration, we exa mined the expression patterns of ZmWUS1 and KN1. The results showed that in the early stage of shoot regeneration, localization of the ZmWUSl transcripts was identified in a group of undifferentiated cells below L 4-6. These cells were smaller, closer, which were identified as organization center cells. Further, tissue sections showed that the KN1 signals had the similar but wider expression patterns, indicating that the SAM reformed during shoot regeneration in maize.(4) Effects of auxin and cytokinin during shoot regeneration. In the early stage of shoot regeneration in vitro, auxin distribution occurred in a specific area of callus, especially in the surface cells, where the SAM would be formed. When it began to produce coleoptile-like sheath, the auxin response signals in SAM gradually disappeared, but the signals in coleoptile-like sheath were enhanced. Auxin distribution might be involved in PINFORMED (PIN)-dependent transport. Polar localization of ZmPINFORMED1a (ZmPIN1a) was first observed in the callus where SAM would initiate. After the coleoptile-like sheath was formed, ZmPINla was further distributed in the coleoptile-like sheath.These results suggest that SAM specific genes ZmWUSl and KN1 are induced in embryonic callus regionally in maize shoot regeneration, indicating that SAM is reformed. The response signals of auxin and cytokinin exhibit monocotyledon specific distribution patterns, regulating the shoot initiation.