Functional Analysis Of RcABI3and RcBBM Induced During The Development Of PLBs In Rosa Canina

Protocorm-like bodies (PLBs) have been the main materials in plant regeneration, cell engineering breeding and receptor of genetic transformation. PLBs are available in ninekinds of Rosa plants. Although incidence rate is high, the sprouting rate is instable and shoots germinate early.Moreover, the incidence of PLBs is extremely low in roses, which greatly restricted its application in production. We studied the function of early embryo germination-related gene, RcABI3, and gene related with generating embryonic tissue and enhancing shoot germination, RcBBM, in order to lay the theoretical foundation for regulating the dormancy, enhancing embryonic tissue generating andthe shoot germination of Rosa PLBs.ABA-insensitive 3(ABI3), initially identified in Arabidopsis thaliana, is intermediary in regulating ABA-responsive genes during embryo dormancy inception and germination developmental program. In order to study the function of ABI3 from Rosa canina, we isolated the three orthologs by a combination of degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). They are designated as RcABI3a, RcABI3b and RcABI3c. The putative amino acid sequences of them all contained an acidic activation domain A1 and the three basic domains, B1, B2, and B3. Expression analysis revealed that RcABI3 expressed in seeds and PLBs three weeks old. On a cellular level, we localized the RcABI3b::GFP and RcABI3c-GFP fusion protein to the nucleus in onion epidermal cells, while the RcABI3a::GFP spread over the cell. The RcABI3b and RcABI3cwere able to restore the Arabidopsis thalianaabi3-6 mutant embryo dormancy ability and almost completely rescued the ABA sensitivity during seed germination, while the RcABI3a cannot. Moreover, overexpression RcABI3b in WT conferred the plant delayed flowering time and male sterility. These results suggested that RcABI3b and RcABI3cmight be candidate genes for promoting PLBs dormancy. Moreover, RcABI3bmight be functional in regulating flowering time and stamen.Baby Boom (BBM)can enhance the shoot regeneration capacity in tissue culture and it is involved in the conversion from vegetative to embryogenic state. Two full-length cDNA, designated RcBBM1 and RcBBM2,were isolated from R. caninaby a combination of degenerate PCR and RACE.The predicted amino acid sequences of the two RcBBMs both contained the bbm-1 motif and all of the motifs typically conserved ineuANT lineage. The transcripts of the RcBBMs were detected in young roots, calluses andPLBs, while they wereundetectable in stems, leaves and flowers. RcBBM1-GFP and RcBBM2-GFP fusion proteins were both localized in the nucleus.35S::RcBBM1 and 35S::RcBBM2 transgenic Arabidopsis lines exhibited enhanced shoot regeneration capacity in tissue culture, shorter root, delayed flowering time and abnormal leaf, butdid not undergo spontaneous somatic embryogenesis (SE). The results suggested that RcBBMs might be candidate genes for improving the shoot regeneration efficiency of R. canina. To further investigate spatial and temporal expression of RcBBMs, we isolated a promoter fragment of RcBBM from R. canina,and analyzed the sequence using the PLACE program. We found that the sequence contained the basic components of promoter such as TATA-box and CAAT-box, and some components related to the transcriptionof embryo and endosperm specificity genes.We also fused the RcBBM promoterand the GUS reporter gene together, and then introduced it into wild-type Arabidopsis. The results showed that GUS gene expressed in the site of cell division and growth active such as cotyledon, root, hypocotyl and shoot apex.In order to further study the function ofRcABI3 and RcBBM duringthe development of PLBs in R. canina, we have initially established the regeneration system in Arabidopsisvia PLBs.This system possesses a shorter cycle and higher PLBs incidence rate. It was expected to not only provide a model system for studying the function of genes during the development of PLBs, but also greatly improve the efficiency of studying the mechanism of PLBs generating and development.