Analysis Of Spatial Auxin And Cytokinin Responses During In Vitro Establishment Of Shoot Meristerm In Rice (Oryza Sativa)

Organogenesis provides a valuable experimental system to study molecular mechanismof plant development. Almost all kinds of vegetative and reproductive organs have beenregenerated in vitro. Although the spatial and temporal distribution pattern of auxin andcytokinin during shoot regeneration was studied in detail in Arabidopsis, the distribution ofauxin and cytokinin and stem cell induction mechanism have largely unknown during shootinduction in rice.Calli were induced onto the auxin-rich callus induction medium using the mature embryoof rice as explants, after that the calli were transferred onto the cytokinin-rich shoot inductionmedium and ultimately shoots were formed. Phylogenetic analyses have revealed that the riceOsWUS is the orthologue of WUS in Arabidopsis. In this study, OsWUS transcripts weredetected in young leaf primordia with a pronounced preference for leaf margins in transgenicmaterials. However, the OsWUS signal was weak in the center of the shoot apex. In the laterstage, OsWUS was expressed in the inflorescence and branch meristems as well as spikeletmeristem. The expression pattern of OsWUS was different from that of WUS of Arabidopsisduring the shoot regeneration, suggesting that OsWUS may play different roles from WUS ofArabidopsis in the shoot regeneration and could not be served as the marker gene of shootapical meristem (SAM) in rice. OSH1was expressed in the SAM of rice plant, therefore couldbe served as a marker of SAM formation in vivo.Further, we analysis the expression of some genes related to biosyntheses and responsesof auxin and cytokinin using Real time-PCR method. The results showed that the expressionlevel of OsYUCCA1, OsYUCCA5increased and OsYUCCA3, OsYUCCA7decreased in thecytokinin-rich medium,suggesting they are the key genes of auxin synthesis during shootregeneration; the transcriptional level of OsARF6and OsARF10increased obviously, thetranscriptional level of OsARF19and OsARF23, OsARF14, OsARF15decreased notably, suggesting the auxin signaling mediate shoot induction through these factors. On the otherhand, the expression of OsRR2, OsRR4, OsRR6, OsRR7, OsRR9/10, the A-type responseregulators of cytokinin, enhanced significantly, suggesting the important roles of cytokininresponses during shoot regeneration.Furthermore, we investigated the distribution of auxin and cytokinin responses duringshoot formation. In the non-induced callus, both auxin and cytokinin gradients wereestablished in its surrounding edge region. However, these two hormones were concentratedtoward to the area where the SAM would be initiated. The relationship between thetemporal-spatial distribution patterns of auxin and cytokinin and the shoot stem cellreestablishment will be focused on in the future.