Characterization of increased cuticular wax mutant and analysis of genes involved in wax biosynthesis in Dianthus spiculifolius

Cuticular wax formation on the surface of plant leaves is associated with drought-stress tolerance.The identification of wax biosynthesis-related genes will contribute to the genetic improvement of drought resistance in plants.In this study,we characterize a novel Dianthus spiculifolius mutant with increased cuticular wax.The mutant exhibited stronger drought resistance as indicated by less leaf wilting and death,higher leaf relative water content and water retention capacity,and slower water loss and chlorophyll extraction than did the wild type during drought treatment.In the mutant leaves,2730 upregulated and 2151 downregulated differentially expressed genes(DEGs)were identified by transcriptome sequencing.A wax biosynthesis pathway of the identified DEGs was significantly enriched.Finally,three key genes(DsCER1,DsMAH1,and DsWSD1)involved in wax biosynthesis were identified and verified by qPCR.These results suggest that differential expression of DEGs involved in wax biosynthesis may be associated with the increase in cuticular wax in the mutant.Taken together,our results help elucidate wax formation patterns in D.spiculifolius.Furthermore,the DEGs involved in wax biosynthesis identified here may be valuable genetic resources for improving plant stress tolerance through increased accumulation of cuticular wax.

Reliable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of Dianthus spiculifolius

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.