Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae)

Abstract

KEY MESSAGE : Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species. Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants.

De novo Sequencing, Characterization, and Comparison of Inflorescence Transcriptomes of Cornus canadensis and C. florida (Cornaceae)

“…The putative DEGs (including the species-specific ones and those shared by ccBud and cfBud, but with significantly different RPKM values in the two species) will serve as our best initial choices of candidate genes for analyses using qRT-PCR and in situ hybridization to characterize, in detail, their expression patterns in different dogwood species to evaluate their potential roles. Those displaying differences in expression pattern among species would then serve as the candidates for functional analyses through in vivo gene transformation using the systems established in C. canadensis [40], [41] and Arabidopsis [42].…”

Evolution and developmental genetics of floral display—A review of progress

“…Developing transformation systems in non-model species is in great need to facilitate evo-devo studies beyond the realm of morphology of the model plants. Stable transformation systems in non-crop woody genera are especially rare and developed in Populus (Tsai et al, 1994), Citrus (Ju arez, 2001), Malus zumi (Xu et al, 2009), and Cornus (Liu et al, 2013c). The recently developed CRISPR/Cas9 technology brought great promise to the analysis of gene functional due its ability to allow editing of the genome (Feng et al, 2013;Jiang et al, 2013;Li et al, 2013).…”

“…Evidence from comparative analyses of developmental processes, gene expression patterns, and functional characterization has prompted efforts to integrate expression patterns of key regulatory genes and the regulation network into models that explain variation in inflorescence structures, such as the unifying transient inflorescence model in flowering plants, the zigzag model in tomato, the FOS‐GRN (floral organ cell fate specification‐gene regulatory network) model in Arabidopsis , and the TFL1 ‐ and AP1 ‐based model in Cornus (Prusinkiewicz et al, ; Périlleux et al, ; Davilavelderrain et al, ; Ma et al, ). To understand the molecular mechanisms responsible for the evolutionary modification of inflorescence architecture, studies on closely related plant lineages exhibiting divergent inflorescence structures are crucial for identifying the evolutionary changes in developmental morphology and gene regulation that were responsible for modifying the inflorescence, as shown by work in the Cornus (Feng et al, , ; Liu et al, , , 2016; Ma et al, ). Functional characterization of key candidate regulators using transgenic experiments in planta will ultimately be essential in validating the phenotypic effects of genes and in developing models that account for the observed variation in inflorescence structure.…”

Down regulation of APETALA3 homolog resulted in defect of floral structure critical to explosive pollen release in Cornus canadensis

“…3.3 Confirmation of insertion of CorcanAP3 hairpin structure and gene expression analysis in transgenic plants Following previous protocol (Liu et al, 2013), CorcanAP3 hairpin structure (forward fragment -gus linker-inverted repeat fragment) was successfully introduced into explants of C. canadensis. Forty eight putative transgenic plants regenerated from seven selected calli (C206, C208, C209, C210, C501, C509a, and C509b) were transplanted into pots with gravel/peat moss mix (2:1).…”

“…We cloned and characterized AP3 homologs from six species of different lineages of Cornus . A hairpin construct ( hp ) of euAP3 homolog from C. canadensis was introduced into wild type plant of C. canadensis (WT) using a stable Agrobacterium ‐mediated transformation system (Liu et al, ) to down‐regulate the expression of endogenous AP3 homolog. We observed reduced expression of the gene and abnormal development of flowers in transgenic plants.…”