Aung Htay Naing

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.

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.

Elimination of chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid from infected chrysanthemum by cryopreservation

Chrysanthemum morifolium ‘Borami’ and ‘Secret Pink’ showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and ‘Yellow Cap’ showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected ‘Borami’ collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment. The solution (PVS2) and low-temperature treatment were found to be critical factors determining the efficacy of viroid elimination. We optimized the protocol by combining of all resulted optimal factors and tested the applicability of the protocol in ‘Borami’ collected from Chilgok and Gyeongsan and in ‘Secret Pink’ from Chilgok, Gumi, and Gyeongsan, which displayed different viroid concentrations. We found that the elimination rates varied depending on the cultivar and region of origin. Similar results were observed when the protocol was applied to eliminate CChMVd from the ‘Yellow Cap’ collected from the same regions. Finally, we found that nested PCR is more reliable for viroid detection than RT-PCR. Overall, cryopreservation can be used to eliminate viroids from infected chrysanthemums; however, the efficacy depends on genotype and initial viroid concentration.

Influence of Genotype, Explant Source, and Gelling Agent on in Vitro Shoot Regeneration of Chrysanthemum

The capacity for shoot regeneration of leaf, petiole, and stem explants of eleven chrysanthemum cultivars was examined on the MS medium containing 1 μM naphthaleneacetic acid and 10 μM 6-benzyladenine solidified with 0.8% Agar, 0.4% Agarose, or 0.25% Gelrite. Significant differences in frequency of callus formation and regeneration from the different explants were observed among the different cultivars when grown on the media solidified with the different gelling agents. Gelrite was found to be the most effective gelling agent in promoting of the shoot. Of the different explants used, in general, stem exhibited the highest frequencies of shoot organogenesis and mean number of shoots per explant regardless of cultivar and gelling agent. However, the highest frequency of regeneration (11.67 shoots per explant) was noted from leaf explants of cv. Borami followed by (4.33 shoots per explant) from stem explants of cv. Yes Nuri. Shoots were directly developed from the surface of explants, not through callus formation. Low frequencies of shoot organogenesis were observed for the remaining cultivars except for cvs. Yes Time and Yes Star, which exhibits no shoot formation at all. In this study, we have developed an efficient in vitro protocol for cvs. Borami and Yes Nuri from suitable explant.

Combined effects of supplementary light and CO2 on rose growth and the production of good quality cut flowers

Abstract: The effects of supplementary lighting with high-pressure sodium (HSP) lamps alone or in combination with carbon dioxide (CO2) on the growth, yield, and flower stem quality of two rose cultivars (“Loving Heart” and “Top Grace”) were studied. Compared to natural lighting (control), supplementary lighting alone was beneficial for plant growth, and it increased plant height, stem diameter, and the number of axillary shoots. Furthermore, increases in flower stem yield (>70 cm), flower stem diameter, fresh weight, and the number of petals per flower were also observed. The combination of supplementary lighting and CO2 significantly enhanced all of the studied parameters compared to supplementary lighting alone. Moreover, stomatal density and chlorophyll fluorescence were seemingly affected by either supplementary lighting alone or in combination with CO2. This is the first study to examine the beneficial effects of combined supplementary lighting and CO2 conditions, and the resulting information is essential to rose growers and commercial production.

Overexpression of RsMYB1 enhances heavy metal stress tolerance in transgenic petunia by elevating the transcript levels of stress tolerant and antioxidant genes

The RsMYB1 transcription factor (TF) controls the regulation of anthocyanin in radish (Raphanus sativus), and its overexpression in tobacco and petunia strongly enhances anthocyanin production. However, no data exists on whether RsMYB1 is involved in the mechanism that leads to abiotic stress tolerance. Under normal conditions, transgenic petunia plants expressing RsMYB1 and WT were able to thrive by producing well-developed broad leaves and regular roots. In contrast, a reduction in plant growth was observed when they were exposed to heavy metals (CuSO4, ZnSO4, MnSO4, and K2Cr2O7). However, RsMYB1-overexpressing plants were found to be more tolerant to the stresses than the WT plants because the expressions of stress tolerant genes (GSH and PCs) and antioxidant genes (SOD, CAT, and POX) were enhanced. In addition, according to the phylogenetic analysis, RsMYB1 has a strong sequence similarity with other MYB TFs that confer different abiotic stresses. These results suggest that overexpression of RsMYB1 enhances the expression levels of metal-induced stress tolerance genes and antioxidant genes, and the resultant increase in gene expression improves heavy metal stress tolerance in petunia.