Chang Kil Kim

Expression of an Arabidopsis Ca2+/H+ antiporter CAX1 variant in petunia enhances cadmium tolerance and accumulation.

Phytoremediation is a cost-effective and minimally invasive technology to cleanse soils contaminated with heavy metals. However, few plant species are suitable for phytoremediation of metals such as cadmium (Cd). Genetic engineering offers a powerful tool to generate plants that can hyperaccumulate Cd. An Arabidopsis CAX1 mutant (CAXcd), which confers enhanced Cd transport in yeast, was ectopically expressed in petunia to evaluate whether the CAXcd expression would enhance Cd tolerance and accumulation in planta. The CAXcd-expressing petunia plants showed significantly greater Cd tolerance and accumulation than the controls. After being treated with either 50 or 100μM CdCl(2) for 6 weeks, the CAXcd-expressing plants showed more vigorous growth compared with controls, and the transgenic plants accumulated significantly more Cd (up to 2.5-fold) than controls. Moreover, the accumulation of Cd did not affect the development and morphology of the CAXcd-expressing petunia plants until the flowering and ultimately the maturing of seeds. Therefore, petunia has the potential to serve as a model species for developing herbaceous, ornamental plants for phytoremediation.

Modest calcium increase in tomatoes expressing a variant of Arabidopsis cation/H+ antiporter

The over-expression of Arabidopsis CAX1 and CAX2 causes transgenic tomato plants to reveal severe Ca2+ deficiency-like symptoms such as tip-burn and/or blossom end rot, despite there being sufficient Ca2+ in each plant part. To correct the symptoms and to moderately enhance the calcium level, a worldwide vegetable tomato was genetically engineered using a modified Arabidopsis cation/H+ antiporter sCAX2A, a mutant form of Arabidopsis CAX2. Compared with the wild-type, the sCAX2A-expressing tomato plants demonstrated elevated Ca2+ levels in the fruits with almost no changes in the levels of Mn2+, Cu2+, and Fe2+. Moreover, expression of sCAX2A in tomato plants did not show any significant alterations in their morphological phenotypes. Unlike 35S::sCAX1 construct, sCAX2A antiporter gene driven by 35S promoter can be a valuable tool for enriching Ca2+ contents in the tomato fruit without additional accumulation of the undesirable cations.

Elimination of Chrysanthemum stunt viroid (CSVd) from meristem tip culture combined with prolonged cold treatment

Chrysanthemum production in Korea has recently been greatly affected by the spread of Chrysanthemum stunt viroid (CSVd) infection, necessitating the use of CSVd-free stocks to ensure successful chrysanthemum cultivation. We investigated the effects of low temperature (4°C), antiviral chemicals (ribavirin and amantadine) and a combination of these treatments on CSVd elimination by meristem tip cultures using plantlets that originated from CSVd-infected chrysanthemum ‘Ency’. Neither antiviral agents led to CSVd elimination, despite the suppression of meristem tip growth in a concentration dependent manner. However, the CSVd elimination rate increased up to 42.8% when meristem tips were excised after storage at 4°C for two months. The most effective results were obtained from a combination of low temperature for three months at 4°C followed by meristem tip culture on media containing 50 and 100 mg·L−1 ribavirin. These results suggest that antiviral agents can also be useful for CSVd elimination if their treatment is combined with prolonged periods of low temperature. This is the first report of eradication of viroids from spray type chrysanthemum bred in Korea.

Overexpression of snapdragon Delila ( Del ) gene in tobacco enhances anthocyanin accumulation and abiotic stress tolerance

BackgroundRosea1 (Ros1) and Delila (Del) co-expression controls anthocyanin accumulation in snapdragon flowers, while their overexpression in tomato strongly induces anthocyanin accumulation. However, little data exist on how Del expression alone influences anthocyanin accumulation.ResultsIn tobacco (Nicotiana tabacum ‘Xanthi’), Del expression enhanced leaf and flower anthocyanin production through regulating NtCHS, NtCHI, NtF3H, NtDFR, and NtANS transcript levels. Transgenic lines displayed different anthocyanin colors (e.g., pale red: T0-P, red: T0-R, and strong red: T0-S), resulting from varying levels of biosynthetic gene transcripts. Under salt stress, the T2 generation had higher total polyphenol content, radical (DPPH, ABTS) scavenging activities, antioxidant-related gene expression, as well as overall greater salt and drought tolerance than wild type (WT).ConclusionWe propose that Del overexpression elevates transcript levels of anthocyanin biosynthetic and antioxidant-related genes, leading to enhanced anthocyanin production and antioxidant activity. The resultant increase of anthocyanin and antioxidant activity improves abiotic stress tolerance.

Factors influencing in vitro shoot regeneration from leaf segments of Chrysanthemum

The objective of this research was to develop an efficient protocol for shoot regeneration from leaf segments of the Chrysanthemum cv. Vivid Scarlet by examining the effects of plant growth regulators, dark incubation period, gelling agents, and silver nitrate. The highest number of shoots per explant (12.3) was regenerated from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with a combination of 1 mgL(-1) of 6-benzyladenine (BA) and 2 mgL(-1) of α-naphthaleneacetic acid (NAA) under light conditions without any initial dark period. Gelrite was the most effective gelling agent for shoot regeneration among those tested, whereas the presence of silver nitrate distinctly inhibited shoot regeneration. Superior plant growth and rooting was observed on a hormone-free MS medium solidified with Gelrite. Flow cytometry analysis revealed no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The established protocol was applicable to shoot regeneration for four out of six cultivars tested. This research will facilitate the genetic transformation and micropropagation of Chrysanthemum cultivars.

The usage of snapdragon Delila ( Del ) gene as a visible selection marker for the antibiotic-free transformation system

This study was conducted to assess the suitability of the Delila gene (Del) from snapdragon for use as a visible selectable marker, under the control of the cauliflower mosaic virus (CaMV) 35S promoter, to develop a plant genetic transformation system that helps to avoid using antibiotic- or herbicide-resistance genes, such as the gene for resistance against kanamycin or PPT. Following transformation, tobacco shoots showing red coloration always contained the Del gene, which was confirmed by PCR analysis. No chimerism or ploidy variation was observed during genetic transformation. In addition, the integration ratio of the transgene to the T1 progeny was 3:1, following typical Mendelian fashion. By anthocyanin analysis, the plants containing the Del gene were shown to have 80 times higher anthocyanin content than the control plants. Thus, we conclude that strong anthocyanin accumulation, as a result of the snapdragon Del gene, can be used as a visible selectable marker for genetic transformation in the tobacco plant, replacing the use of antibiotic- or herbicide-resistance genes.

Erratum to: Primary and secondary somatic embryogenesis in Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’ and assessment of ploidy stability of somatic embryogenesis process by flow cytometry

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from petal explant of Chrysanthemum (Chrysanthemummorifolium) cv. ‘Baeksun’. Somatic embryogenesis was induced from petal explants on the Murashige and Skoog (MS) medium supplemented with 1.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 3.0 mg l−1 6-benzyladenine (BA), yielding the highest mean number of embryos (56.3) per explant after 5 weeks of culture. We evaluated the effects of basal medium and various concentrations of sucrose on the proliferation of secondary somatic embryos. MS medium was observed to be more effective in promoting the proliferation of somatic embryos than half-strength Murashige and Skoog (1/2MS). In addition, 1 % sucrose was also found to be the best in induction of secondary embryogenesis. The highest germination rate (70 %) of the somatic embryos was observed on the MS medium containing 0.2 mg l−1 α-naphthalene acetic acid and 1 g l−1 activated charcoal (AC). Shoots elongated rapidly and roots developed well on hormone-free MS medium with 1 g l−1 AC and successfully acclimated in the greenhouse. Flow cytometric analysis of the primary somatic embryos, secondary somatic embryos, and the somatic embryo-obtained plants along with the parent grown in the greenhouse showed that they all had same identical peaks, indicating that there was no variation of ploidy level during the regeneration process. We expect that our report would be useful for micropropagation and Agrobacterium-mediated genetic transformation studies of this cultivar.

Genome-Wide Identification, Characterization and Expression Profiling of ADF Family Genes in Solanum lycopersicum L.

The actin depolymerizing factor (ADF) proteins have growth, development, defense-related and growth regulatory functions in plants. The present study used genome-wide analysis to investigate ADF family genes in tomato. Eleven tomato ADF genes were identified and differential expression patterns were found in different organs. SlADF6 was preferentially expressed in roots, suggesting its function in root development. SlADF1, SlADF3 and SlADF10 were predominately expressed in the flowers compared to the other organs and specifically in the stamen compared to other flower parts, indicating their potential roles in pollen development. The comparatively higher expression of SlADF3 and SlADF11 at early fruit developmental stages might implicate them in determining final fruit size. SlADF5 and SlADF8 had relatively higher levels of expression five days after the breaker stage of fruit development, suggesting their possible role in fruit ripening. Notably, six genes were induced by cold and heat, seven by drought, five by NaCl, and four each by abscisic acid (ABA), jasmonic acid (JA) and wounding treatments. The differential expression patterns of the SlADF genes under different types of stresses suggested their function in stress tolerance in tomato plants. Our results will be helpful for the functional characterization of ADF genes during organ and fruit development of tomato under different stresses.

An efficient protocol for Agrobacterium-mediated genetic transformation of recalcitrant chrysanthemum cultivar Shinma

Chrysanthemum cultivar Shinma is a standard cultivar and has a large flower size, long vase life, and strong resistance to the white rust disease; thus, it is an important commercial cut flower in the flower markets of Korea. However, its flower color (white) is simple, so variation in flower color by using genetic transformation is necessary to increase its value in flower markets worldwide. Success of genetic transformation in chrysanthemum is dependent on many factors. In this study, factors that affect the efficient genetic transformation of this chrysanthemum were assessed, transgenic plants with the RsMYB1 anthocyanin regulatory gene were produced, and the presence of the transgene and its stable expression were confirmed using PCR and reverse transcription-PCR. Co-cultivation temperature and Agrobacterium strains were observed to be the main factors that affected higher transformation efficiency. However, the protocol developed by a combination of all optimized factors yielded eight- or fourfold higher transformation efficiency than the simple (un-optimized) protocol or individually optimized factors. The herbicide resistance assay revealed that PCR-positive transgenic shoots have stronger resistance to Basta™ than the wild type (WT). We expect that the efficient protocol developed in this study will facilitate the genetic transformation of genes of interest in this cultivar and that the anthocyanin regulatory gene will help in modifying the flower color.

The effect of antifreeze protein on the cryopreservation of chrysanthemums

Abstract To our knowledge, this is the first study to show the application of antifreeze protein (AFP) in the cryopreservation of ornamental plants. We studied the effect of AFP on the cryopreservation of Chrysanthemum grandiflorum (Ramat.) Kitam ‘Borami’, ‘Secret Pink’, and ‘Yellow Cap’. For all cultivars, survival and regrowth rates were higher in shoot tips vitrified with plant vitrification solution 3 (PVS3) containing various concentrations of AFP than in those vitrified with PVS3 alone. The optimal AFP concentration was genotype-dependent. Scanning electron microscopy revealed that shoot tips vitrified with PVS3 containing AFP had less freezing injuries than those treated with PVS3 alone. Analysis of the enthalpy in each sample by differential scanning calorimetry supported the finding that AFP is a potent cryoprotectant that can reduce the freezing point of water in plant tissues. We expect that our results will facilitate the successful application of AFPs in the cryopreservation of rare and commercially important plant germplasm.