Trinh Ngoc Ai

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.

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.

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.

Nitrogenous compounds enhance the growth of petunia and reprogram biochemical changes against the adverse effect of salinity

ABSTRACT This study aimed to determine ways to improve the growth and salt tolerance of petunia. Effects of polyamines (PAs; spermidine [Spd], spermine [Spm], and putrescine [Put]) and a nitric oxide (NO) donor (sodium nitroprusside [SNP]) were investigated. Initially, we screened petunia cultivars against sodium chloride (0–125 mM). The petunia cultivar Hurrah Red was identified as salt-sensitive cultivar in the basis of salt-effect on seed germination, fresh weight, and root length of seedlings. Treatment of Hurrah Red shoots with nitrogenous compounds improved the number, length, and fresh weight of roots, as well as the length and fresh weight of shoots over those of the control. Furthermore, plantlets rooted in an optimal concentration of Spd (34.5 µM), Spm (24.8 µM), Put (62.1 µM), and SNP (3.9 µM) were treated with 200 mM NaCl for 3 days to assess their tolerance level. Salt-affected plantlets showed higher level of lipid peroxidation, reduced catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO) activities and decreased contents of photosynthetic pigments and polyphenol than those of the control. PAs and SNP treatments significantly elevated photosynthetic pigments, enhanced antioxidant enzymes, and decreased lipid peroxidation in salt-stressed plantlets. Moreover, the growth and salt-tolerance response of petunia was highest when plantlets were exposed to SNP, followed by levels on treatment with Put, Spm, and Spd. Thus, the findings of this study suggested that treatment with exogenous SNP, Put, Spm, and Spd could protect petunia plants against soil salinity and improve their commercial production.

Synergistic Effect of Nano-Sliver with Sucrose on Extending Vase Life of the Carnation cv. Edun

We investigated the effects of sucrose and nano-silver (NAg) on extending the vase life of cut carnation flowers “Edun”. Sucrose (pulse treatment) suppressed ethylene production by downregulating the genes that code for its biosynthesis. Relative to the control, however, sucrose significantly promoted xylem blockage on cut stem surfaces and reduced relative fresh weight, antioxidant activity, and cysteine proteinase inhibitor gene (DcCPi) expression. Consequently, the sucrose-treated flowers had shorter vase lives than the control. In contrast, NAg suppressed ethylene production in the petal, prevented xylem blockage in the cut stem surface, and improved all the aforementioned parameters. Therefore, NAg increased flower longevity. The most effective treatment in terms of longevity extension and parameter improvement, however, was the combination of NAg and sucrose. These results suggest that sucrose can suppress ethylene production but does not necessarily extend the vase life of the flower cultivar. The role of NAg in increasing cut carnation longevity is mainly to inhibit xylem blockage rather than suppress ethylene production, and the combined effect of NAg and sucrose is most effective at prolonging cut carnation vase life, likely due to their synergetic effects on multiple modes of action.

Enhancement of Low pH Stress Tolerance in Anthocyanin-Enriched Transgenic Petunia Overexpressing RsMYB1 Gene

We investigated whether the presence of anthocyanins in plants could contribute to low pH stress tolerance using anthocyanin-enriched transgenic petunia lines (PM2, PM6, and PM8) expressing RsMYB1 and wild-type (WT) plants. We examined several physiological and biochemical factors and the transcript levels of genes involved in abiotic stress tolerance. A reduction in plant growth, including plant height and fresh weight, was observed when plants (PM2, PM6, PM8, and WT) were exposed to low pH (pH 3.0) conditions compared to growth under normal (pH 5.8) conditions. A small reduction in the growth of PM6 was observed, followed by that in PM2, PM8, and WT, reflecting the anthocyanin levels in the plants (PM6 > PM2 and PM8 > WT). An analysis of physiological and biochemical factors also supports the degree of low pH tolerance in the plants (PM6 > PM2 and PM8 > WT). In addition, an enhanced expression of the genes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), osmotin, and vacuolar H+-ATPase (V-ATPase)] was observed in the transgenic lines (PM2, PM6, and PM8). The resultant of the enhanced transcript levels of the genes could promote antioxidant activities, proline content, and pH homeostasis involved in the mechanisms underlying abiotic stress tolerance in plants. These results suggest that anthocyanin-enriched plants overexpressing RsMYB1 enhances low pH stress tolerance by elevating the transcript levels of the relevant genes.

Overexpression of Rosea1 From Snapdragon Enhances Anthocyanin Accumulation and Abiotic Stress Tolerance in Transgenic Tobacco

The co-expression of Rosea1 (Ros1) and Delila (Del) regulates anthocyanin levels in snapdragon flowers, as well as in tomato, petunia, and tobacco. However, there is little information on how Ros1 expression alone controls anthocyanin regulation and whether it is involved in the mechanism that leads to abiotic stress tolerance. In the present study, tobacco (Nicotiana tabacum ‘Xanthi’) transgenic plants overexpressing Ros1 (T2-Ros1-1, T2-Ros1-2, T2-Ros1-3, and T2-Ros1-4) promoted accumulation of anthocyanin in leaves and flowers by elevating the transcription of all key genes involved in the biosynthesis of this pigment. This promotion largely occurred through the upregulation of dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase genes in leaves and upregulation of DFR in flowers. Under normal conditions, the transgenic lines and wild type (WT) plants showed well-developed broad leaves and regular roots, whereas a reduction in plant growth was observed under cold and drought stresses. However, the transgenic T2-Ros1 lines were able to tolerate the stresses better than the WT line by inducing reactive oxygen species scavenging activities, and the expression of antioxidant-related and stress-responsive genes. In addition, phylogenetic analysis clustered Ros1 with many transcription factors (TFs) that confer tolerance to different abiotic stresses. Overall, the results obtained here suggest that Ros1 overexpression upregulates anthocyanin biosynthetic, antioxidant-related, and stress-responsive genes thereby enhancing anthocyanin accumulation and abiotic stress tolerance.

Overexpression of RsMYB1 Enhances Anthocyanin Accumulation and Heavy Metal Stress Tolerance in Transgenic Petunia

The RsMYB1 transcription factor (TF) controls the regulation of anthocyanin in radishes (Raphanus sativus), and its overexpression in tobacco and petunias strongly enhances anthocyanin production. However, there are no data on the involvement of RsMYB1 in the mechanisms underlying abiotic stress tolerance, despite strong sequence similarity with other MYBs that confer such tolerance. In this study, we used the anthocyanin-enriched transgenic petunia lines PM6 and PM2, which overexpress RsMYB1. The tolerance of these lines to heavy metal stress was investigated by examining several physiological and biochemical factors, and the transcript levels of genes related to metal detoxification and antioxidant activity were quantified. Under normal conditions (control conditions), transgenic petunia plants (T2-PM6 and T2-PM2) expressing RsMYB1, as well as wild-type (WT) plants, were able to thrive by producing well-developed broad leaves and regular roots. In contrast, a reduction in plant growth was observed when these plants were exposed to heavy metals (CuSO4, ZnSO4, MnSO4, or K2Cr2O7). However, T2-PM6 and T2-PM2 were found to be more stress tolerant than the WT plants, as indicated by superior results in all analyzed parameters. In addition, RsMYB1 overexpression enhanced the expression of genes related to metal detoxification [glutathione S-transferase (GST) and phytochelatin synthase (PCS)] and antioxidant activity [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX)]. These results suggest that enhanced expression levels of the above genes can improve metal detoxification activities and antioxidant activity, which are the main components of defense mechanism included in abiotic stress tolerance of petunia. Our findings demonstrate that RsMYB1 has potential as a dual-function gene that can have an impact on the improvement of anthocyanin production and heavy metal stress tolerance in horticultural crops.

Sodium nitroprusside stimulates growth and shoot regeneration in chrysanthemum

In this study, we demonstrate that the nitric oxide (NO) donor sodium nitroprusside (SNP) improves plant regeneration in chrysanthemums. Internode explants of three different chrysanthemum cultivars, ‘White ND’, ‘White wing × Peach ND’, and ‘Hunt × Lemon ND’, were cultured on shoot induction medium (SIM) containing various concentrations of N6–benzyladenine (2.22 or 4.44 μM BA) and SNP (0.83–6.71 μM) individually and in combination. Most combinations of BA and SNP significantly improved the morphogenetic potential of internode explants and enhanced shoot regeneration in all three chrysanthemum cultivars compared to treatment with BA alone. The cultivar ‘White wing × Peach ND’ displayed the highest regeneration response (98.3%) and shoot regeneration rate (27.3 shoots/explant) in SIM containing optimal BA (4.44 μM) and SNP (0.83 μM) concentrations within 30 days of culture. Individual shoots of ‘White wing × Peach ND’ were transferred to root induction medium (RIM) containing various concentrations of SNP (0.83–6.71 μM) alone. Shoots rooted in the presence of SNP resulted in healthy plantlets within 30 days of culture with improved root (number of roots/shoot, root length, and fresh weight) and shoot (number of leaves, shoot length, and fresh weight) growth characteristics compared to the control. In addition, the regeneration procedure described in this study only requires a short duration (60 days) to obtain rooted plantlets from internode explants of chrysanthemums. Our results suggest that supplementation of chrysanthemum regeneration medium with SNP enhances shoot regeneration and improves plant growth, overcoming problems associated with propagation and genetic transformation.

Involvement of Sodium Nitroprusside (SNP) in the Mechanism That Delays Stem Bending of Different Gerbera Cultivars

Longevity of cut flowers of many gerbera cultivars (Gerbera jamesonii) is typically short because of stem bending; hence, stem bending that occurs during the early vase life period is a major problem in gerbera. Here, we investigated the effects of sodium nitroprusside (SNP) on the delay of stem bending in the gerbera cultivars, Alliance, Rosalin, and Bintang, by examining relative fresh weight, bacterial density in the vase solution, transcriptional analysis of a lignin biosynthesis gene, antioxidant activity, and xylem blockage. All three gerbera cultivars responded to SNP by delaying stem bending, compared to the controls; however, the responses were dose- and cultivar-dependent. Among the treatments, SNP at 20 mg L-1 was the best to delay stem bending in Alliance, while dosages of 10 and 5 mg L-1 were the best for Rosalin and Bintang, respectively. However, stem bending in Alliance and Rosalin was faster than in Bintang, indicating a discrepancy influenced by genotype. According to our analysis of the role of SNP in the delay of stem bending, the results revealed that SNP treatment inhibited bacterial growth and xylem blockage, enhanced expression levels of a lignin biosynthesis gene, and maintained antioxidant activities. Therefore, it is suggested that the cause of stem bending is associated with the above-mentioned parameters and SNP is involved in the mechanism that delays stem bending in the different gerbera cultivars.