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A rosette plant of Eustoma grandiflorum requires vernalization (exposure to a period of cold temperature) and long-day conditions to promote flowering, while prolonged cold or cool temperatures in post-vernalization periods delay flowering. This study aimed to investigate the effect of growth conditions on flowering regulation in Eustoma. In Arabidopsis, vernalization suppresses a floral repressor gene, FLOWERING LOCUS C (FLC) and upregulates floral promoter genes, such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT). We identified and characterized the Eustoma homologs of these genes. In contrast to Arabidopsis FLC, Eustoma grandiflorum FLC-like (EgFLCL) expression was upregulated by cold temperature and downregulated by subsequent warm temperature exposure. The expression of Eustoma grandiflorum SOC1-like (EgSOC1L) and FT-like (EgFTL) genes was not significantly induced during vernalization, but their transcripts increased during a warm post-vernalization period in the long days. Vernalized plants grown under cool post-vernalization temperatures exhibited higher EgFLCL expression, lower EgSOC1L and EgFTL expression and flowered later than those grown under warm temperatures. Overexpression of EgFLCL cDNA repressed flowering in transgenic Arabidopsis, whereas overexpression of EgSOC1L or EgFTL cDNA promoted flowering. Our results suggest that flowering regulation by vernalization in Eustoma differs from the paradigm developed for Arabidopsis. EgFLCL is regulated by temperature and may be involved in floral repression during cold and cool seasons. Warm- and long-day conditions following vernalization are required to induce two putative floral promoters, EgSOC1L and EgFTL, effectively., (Copyright © Physiologia Plantarum 2011.)

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and alpha-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks' duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.

Eustoma grandiflorum Shinn requires vernalization for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization, the expression levels of genes encoding enzymes of GA biosynthesis, copalyl diphosphate synthetase, GA 20-oxidase and GA 3 beta-hydroxylase, were examined using two culitvars that show different responses to vernalization. The three genes were induced in a vernalization- and a cultivar-dependent manner. EgSPY, a putative negative regulator of GA signal transduction, was also induced during the vernalization period. The results suggest that the expression of the genes encoding GAs biosynthesis is regulated by vernalization. We postulate that EgSPY functions as a negative regulator of GA signal transduction during vernalization, inhibiting adventitious shoot elongation during vernalization.