RcAP1, a Homolog of APETALA1, is Associated with Flower Bud Differentiation and Floral Organ Morphogenesis in Rosa chinensis.

Rosa chinensis is one of the most popular flower plants worldwide. The recurrent flowering trait greatly enhances the ornamental value of roses, and is the result of the constant formation of new flower buds. Flower bud differentiation has always been a major topic of interest among researchers. The APETALA1 (AP1) MADS-box (Mcm1, Agamous, Deficiens and SRF) transcription factor-encoding gene is important for the formation of the floral meristem and floral organs. However, research on the rose AP1 gene has been limited. Thus, we isolated AP1 from Rosa chinensis 'Old Blush'. An expression analysis revealed that RcAP1 was not expressed before the floral primordia formation stage in flower buds. The overexpression of RcAP1 in Arabidopsis thaliana resulted in an early-flowering phenotype. Additionally, the virus-induced down-regulation of RcAP1 expression delayed flowering in 'Old Blush'. Moreover, RcAP1 was specifically expressed in the sepals of floral organs, while its expression was down-regulated in abnormal sepals and leaf-like organs. These observations suggest that RcAP1 may contribute to rose bud differentiation as well as floral organ morphogenesis, especially the sepals. These results may help for further characterization of the regulatory mechanisms of the recurrent flowering trait in rose.

RcRR1, a Rosa canina type-A response regulator gene, is involved in cytokinin-modulated rhizoid organogenesis.

In vitro, a new protocol of plant regeneration in rose was achieved via protocorm-like bodies (PLBs) induced from the root-like organs named rhizoids that developed from leaf explants. The development of rhizoids is a critical stage for efficient regeneration, which is triggered by exogenous auxin. However, the role of cytokinin in the control of organogenesis in rose is as yet uncharacterized. The aim of this study was to elucidate the molecular mechanism of cytokinin-modulated rhizoid formation in Rosa canina. Here, we found that cytokinin is a key regulator in the formation of rhizoids. Treatment with cytokinin reduced callus activity and significantly inhibited rhizoid formation in Rosa canina. We further isolated the full-length cDNA of a type-A response regulator gene of cytokinin signaling, RcRR1, from which the deduced amino acid sequence contained the conserved DDK motif. Gene expression analysis revealed that RcRR1 was differentially expressed during rhizoid formation and its expression level was rapidly up-regulated by cytokinin. In addition, the functionality of RcRR1 was tested in Arabidopsis. RcRR1 was found to be localized to the nucleus in GFP-RcRR1 transgenic plants and overexpression of RcRR1 resulted in increased primary root length and lateral root density. More importantly, RcRR1 overexpression transgenic plants also showed reduced sensitivity to cytokinin during root growth; auxin distribution and the expression of auxin efflux carriers PIN genes were altered in RcRR1 overexpression plants. Taken together, these results demonstrate that RcRR1 is a functional type-A response regulator which is involved in cytokinin-regulated rhizoid formation in Rosa canina.

In vitro propagation of rose.

In vitro propagation of rose is an important tool for rapid multiplication and development of new cultivars with desirable traits. However, successful in vitro propagation requires an understanding of specific requirements and precise manipulation of various factors. Efficient protocols for different stages of micropropagation using apical buds or nodal segments are currently available. Recently, new challenges for refinements of protocols for high rate of shoot multiplication and development of cost effective methods has gained importance. Significance of the liquid static culture for shoot proliferation and root induction for rose has also gained prominence. Other distinct approaches of in vitro propagation include organogenesis and embryogenesis. These approaches are important for the successful implementation of various biotechnological techniques used for rose improvement programmes. Types of explants, media and optimization of conditions are major factors for successful regeneration of rose.

2,4,5-Trichlorophenoxyacetic acid promotes somatic embryogenesis in the rose cultivar "Livin' Easy" (Rosa sp.).

Somatic embryogenesis (SE) offers vast potential for the clonal propagation of high-value roses. However, some recalcitrant cultivars unresponsive to commonly employed SE-inducing agents and low induction rates currently hinder the commercialization of SE technology in rose. Rose SE technology requires improvement before it can be implemented as a production system on a commercial scale. In the present work, we assessed 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a synthetic auxin not previously tested in rose, for its effectiveness to induce SE in the rose cultivar "Livin' Easy" (Rosa sp.). We ran a parallel comparison to the commonly used 2,4-dichlorophenoxyacetic acid (2,4-D). We tested each auxin with two different basal media: Murashige and Skoog (MS) basal medium and woody plant medium (WPM). MS medium resulted in somatic embryo production, whereas WPM did not. 2,4,5-T induced SE over a greater concentration range than 2,4-D's and resulted in significantly greater embryo yields. 2,4,5-T at a concentration of 10 or 25 microM was better for embrygenic tissue initiation than 2,4,5-T at 5 microM. Further embryo development occurred when the tissue was transferred to plant growth regulator (PGR) free medium or media with 40% the original auxin concentration. However, the PGR-free medium resulted in a high percentage of abnormal embryos (32.31%) compared to the media containing auxins. Upon transfer to germination medium, somatic embryos successfully converted into plantlets at rates ranging from 33.3 to 95.2%, depending on treatment. Survival rates 3 months ex vitro averaged 14.0 and 55.6% for 2,4-D- and 2,4,5-T-derived plantlets, respectively. Recurrent SE was observed in 60.2% of the plantlets growing on germination medium. This study is the first report of SE in the commercially valuable rose cultivar 'Livin' Easy' (Rosa sp.) and a suitable methodology was developed for SE of this rose cultivar.

DNA-methylation alterations and exchanges during in vitro cellular differentiation in rose (Rosa hybrida L.).

DNA-methylation profiles of leaf tissues of Rosa hybrida cv. Carefree Beauty collected from in vivo-grown greenhouse plants, in vitro-grown proliferating shoots at different passages, regenerants of embryogenic callus, regenerants of organogenic callus, as well as calli from undifferentiated callus (UC), embryogenic callus, and organogenic callus were investigated using an amplified fragment-length polymorphism (AFLP)-based detection technique. Three types of AFLP bands were recovered. Type I bands were observed with both isoschizomers Msp and HpaII, while type II and type III bands were observed only with MspI and HpaII, respectively. Sequence analysis of the three types of AFLP bands revealed that a nonmethylated MspI/HpaII-recognition site 5'-CCGG-3' resulted in a type I band, while an inner 5-methylcytosine generated most type II and type III bands. About 40% of inner and 20% of outer cytosines in 5'-CCGG-3' sequences were fully methylated, and only a few hemimethylated outer cytosines were observed. Changes in types of AFLP bands among different tissues were frequently observed, including appearance and disappearance of type I, II, and III AFLP bands, as well as exchanges between either type I and type II or type I and type III AFLP bands. Methylation alterations of outer cytosines in 5'-CCGG-3' sequences triggered appearance and disappearance of type I and II AFLP bands. Methylation changes of both outer and inner cytosines resulted in either removal or generation of type III AFLP bands. Methylation alteration of an inner cytosine was responsible for exchange between type I and type II, while hemimethylation of an outer cytosine accounted for exchange between type I and type III AFLP bands. During UC induction, a significant DNA-methylation alteration was detected in both inner and outer cytosines. Variations in methylation profiles significantly differed between somatic embryogenesis and in vitro organogenesis. Demethylation of outer cytosines occurred at a high frequency during somatic embryogenesis, and most altered AFLP bands in embryogenic callus were passed on to its regenerants. However, most methylation-altered AFLP bands during organogenesis were recovered in shoot regenerants derived via organogenic callus. Seven tissue-specific bands were isolated, cloned, and sequenced. Blast search revealed that two of these might be derived from functional genes.

Dispersal and size fractionation of embryogenic callus increases the frequency of embryo maturation and conversion in hybrid tea roses.

Plant regeneration from embryogenic cells of two Rosa hybrida cultivars, Kardinal and Classy, was increased by dispersing embryogenic callus in liquid medium for 3 h followed by size-fractionation to isolate proembryogenic masses that were smaller than 530 microm. Dispersed callus of three cultivars, Kardinal, Classy, and Tineke, produced 61-135 cotyledonary-stage embryos/100 mg fresh weight (FW) as compared to intact callus that had not been dispersed, which produced only zero to three cotyledonary-stage embryos/100 mg FW. Over 500 cotyledonary-stage embryos/100 mg FW callus developed from proembryogenic masses of Kardinal, Classy, and Tineke following 2 months of culture on solidified Murashige and Skoog's basal salts medium supplemented with 0.25% activated charcoal. Cotyledonary-stage embryos of Classy that developed from both dispersed callus and fractionated cells of various sizes showed a significantly higher conversion frequency to plants (28%) than cotyledonary-stage embryos isolated from intact callus (9%). The highest conversion frequencies for Kardinal (50-58%) occurred from cotyledonary-stage embryos that developed from dispersed callus and from the fraction of cells smaller than 850 microm.