Young-Min Goo

Expressional characterization of dehydroascorbate reductase cDNA in transgenic potato plants

In plants ascorbic acid (AsA) is a strong antioxidant or reductant that can be converted to dehydroascorbate (DHA) by oxidation. DHA, a very short-lived chemical, can either be hydrolyzed irreversibly to 2,3-diketogulonic acid or recycled to AsA by dehydroascorbate reductase (DHAR).DHAR cDNA, isolated from sesame hairy roots, was inserted into two plant expression vector syrtems with theCaMV35S promoter (CaMV35S : :DHAR) and a potato tuber-specific promoter,Patatin (Patatin : :DHAR). Southern and northern blot hybridization analyses indicated thatDHAR cDNA was successfully integrated into the potato genome and actively transcribed. High levels of sesameDHAR transcript and DHAR enzyme activity were determined, by thePatatin promoter, in regenerated potato tubers, but their levels in leaves were very low. In contrast, much higher amounts of transcript were accumulated in the leaves of CaMV35S : :DHAR regenerants than in the tubers while the activity of DHAR enzyme was higher in the latter. AsA content in the tubers of Patatin : :DHAR transgenic lines was also increased (1.1- to 1.3-fold) compared with that of non-transgenic plants. However, this was not true for the transgenic leaves. In contrast, theCaMV35S promoter was associated with AsA accumulations in both the tubers (up to 1.6-fold) and the leaves (up to 1.5-fold). However, more detailed analyses indicated that this increased enzyme activity was not always accompanied by an elevation in AsA content from transgenic plants. This suggests that other factors may limit the accumulation of vitamin C via ascorbate-recycling in transgenic potato plants.

Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato.

In a previous study, we have evidenced that the overexpression of the IbOr gene isolated from sweet potato conferred a tolerance activity against salinity and methyl viologen (MV) treatment in transgenic sweet potato calli along with an enhanced carotenoid content. In this study, to further examine the function of the IbOr gene in heterologous organism, we transformed the IbOr gene into potato under the direction of SWPA2 promoter, a strong inducible promoter upon treatment with various environmental stresses. Consistently with our previous study of sweet potato calli, the level of total carotenoid was elevated up to 2.7-fold (38.1 μg g(-1)DW) compared to the non-transgenic control, Atlantic cultivar. However, the composition of carotenoid was not influenced by the overexpression of the IbOr gene since only pre-existing carotenoids in the non-transgenic control including violaxanthin, lutien and β-carotene were elevated at a similar level of total carotenoids. In general, the transcript levels for most of carotenogenesis-related genes were elevated in transgenic tuber, whereas they remained at similar levels in transgenic leaf tissues compared to those of non-transgenic controls. The increased levels of carotenoid content in the leaf or tuber tissue of transgenic lines were correlated with the enhanced tolerance activity against salt- or MV-mediated oxidative stresses and DPPH radical-scavenging activity. Our preliminary results suggest that further investigation is required for the development of a crop tolerant to salinity and other environmental stresses through the overexpression of the IbOr gene.

Development of molecular markers, based on chloroplast and ribosomal DNA regions, to discriminate three popular medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum

Identification of plant species is important for standardizing herbal medicine. Cynanchum wilfordii (Baekshuoh in Korean) and Polygonum multiflorum (Hashuoh in Korean) are important oriental medicinal herbs in Korea, Japan, and China. Cynanchum auriculatum is a faster growing and more productive plant than C. wilfordii; and, it is not recognized as a medicinal plant in the Korean Pharmacopoeia. C. wilfordii, P. multiflorum, and C. auriculatum are often misidentified in the Korean herbal medicine marketplace due to their morphological similarities and similar names. In this study, we investigated molecular authentication of these three medicinal plants using DNA sequences in the TrnL-F chloroplast intergenic region. Specific species identification was achieved by detecting allelic variations of single nucleotide polymorphisms (SNPs) using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and high resolution melting curve analysis. Our results demonstrate that the intraspecific genetic distance between C. wilfordii and C. auriculatum is relatively low. We also developed a quantitative PCR assay using species-specific TrnL-F primers, which allowed us to estimate the ratio of C. wilfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. Additionally, to identify species in hybrid plants produced by cross-fertilization, we analyzed nuclear ribosomal DNA internal transcribed spacer regions in C. wilfordii and C. auriculatum by ARMS-PCR. Our results indicate that SNP-based molecular markers, usable to barcode tools could provide efficient and rapid authentication of these closely related medicinal plant species, and will be useful for preventing the distribution of products contaminated with adulterants.

The sweetpotato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter confers high-level expression of the GUS reporter gene in the potato tuber

Molecular farming refers to the process of creating bioengineered plants with the capability of producing potentially valuable products, such as drugs, vaccines, and chemicals. We have investigated the potential of the sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter and its transit peptide (TP) as an expression system for the mass production of foreign proteins in potato. The ibAGP1 promoter and its TP sequence were transformed into potato along with beta-glucuronidase (GUS) as a reporter gene, and GUS activity was subsequently analyzed in the transgenic potato plants. In tuber tissues, GUS activity in transgenic plants carrying only the ibAGP1 promoter (ibAGP1::GUS) increased up to 15.6-fold compared with that of transgenic plants carrying only the CaMV35S promoter (CaMV35S::GUS). GUS activity in transgenic plants was further enhanced by the addition of the sweetpotato TP to the recombinant vector (ibAGP1::TP::GUS), with tuber tissues showing a 26-fold increase in activity compared with that in the CaMV35S::GUS-transgenic lines. In leaf tissues, the levels of GUS activity found in ibAGP1::GUS-transgenic lines were similar to those in CaMV35S::GUS-lines, but they were significantly enhanced in ibAGP1::TP::GUS-lines. GUS activity gradually increased with increasing tuber diameter in ibAGP1::GUS-transgenic plants, reaching a maximum level when the tuber was 35 mm in diameter. In contrast, extremely elevated levels of GUS activity - up to about 10-fold higher than that found in CaMV35S::GUS-lines - were found in ibAGP1::TP::GUS-transgenic lines at a much earlier stage of tuber development (diameter 4 mm), and these higher levels were maintained throughout the entire tuber developmental stage. These results suggest that the sweetpotato ibAGP1 promoter and its TP are a potentially strong foreign gene expression system that can be used for molecular farming in potato plants.

Antioxidant and Antimicrobial Activities of Ethanol Extract from the Stem and Leaf of Impatiens balsamina L. (Balsaminaceae) at Different Harvest Times

The aim of this study was to investigate the total phenolic content, total flavonoid contents, antioxidant activity and antimicrobial activity of ethanolic extract from stems (S) and leaves (L) of Impatiens balsamina L. (Balsaminaceae), which were harvested in Korea on March 10, 2011 (S1 and L1), May 14, 2011 (S2 and L2), and July 5, 2011 (S3 and L3), respectively. Our results revealed that the total phenolic (79.55–103.94 mg CE/g extract) and flavonoid (57.43–104.28 mg QE/g extract) contents of leaf extract were higher (p < 0.01) than those of stem extract. Leaf extracts (L1, L2, and L3) exhibited stronger (p < 0.01) free radical scavenging activity (66.06, 63.71, and 72.19%, respectively) than that of the positive control. In terms of antimicrobial activity, leaf extracts showed higher inhibitory effects against microorganisms than those of stem extracts (S1, S2, and S3). Among the leaf extracts at different harvest times, L3 showed the greatest antimicrobial activity against both Gram negative and Gram positive strains. From these results, the leaf extract from I. balsamina L. might be a valuable bioactive resource, and would seem to be applicable as a natural antioxidant in food preservation.

Comparison of Isoflavone Contents and Antioxidant Effect in Cheonggukjang with Black Soybean Cultivars by Bacillus subtilis CSY191

BACKGROUND: Soybeans are the rich sources of isoflavones. To date, the changes of isoflavone contents in various black soybeans cheonggukjang during fermentation by Bacillus subtilis CSY191 has not been investigated.METHODS AND RESULTS: This study investigated the changes of total phenolic and isoflavone contents and antioxidant effects during cheonggukjang fermentation made with four black soybean (BS) cultivars including Cheongja, Cheongja#3, Geomjeong#5, and Ilpumgeomjeong with a potential probiotic Bacillus subtilis CSY191. The total phenolic contents, isoflavone-malonylglycoside and -aglycone contents, and antioxidant activity were increased in cheonggukjang at 48 h fermentation, while the content of isoflavone-glycosides was decreased during cheonggukjang fermentation. In particular, the Cheongja#3 soybean fermented at 37℃ for 48 h displayed the highest antioxidant activities, compared to those of the other BS cultivars tested. Also, the highest levels of total phenolic, daidzein, glycitein, and genistein were present at concentrations of 17.28 mg/g, 283.7 g/g, 39.9 g/g, and 13.2 g/g at the end of Cheongja#3 soybean fermentation.CONCLUSION: The results from this study suggested that the enhanced antioxidant activity of cheonggukjang of BS might be related to increased levels of total phenolic, isoflavon-aglycone, and malonyl-glycoside contents achieved during fermentation. Furthermore, fermented Cheongja#3 soybean showed the highest levels of enhanced antioxidant activities than the other BS cultivars.

Development of transgenic potato with high content of sulphur-containing essential amino acids

Potato is the 4th important crop along with rice, wheat and maize. It contains high quality of starch with relatively high content of vitamin C and protein. However, there is a nutritionally limiting factor due to a low level of sulphur-containing essential amino acid including methionine and cysteine. Recently, recombinant DNA technology and metabolic engineering with genes involved in the bio-synthetic pathway have been applied to enhance the level of these essential amino acids. In this report, it has been discussed about the current status and bottleneck on the development of transgenic potato containing high level of sulphur-containing essential amino acids.

Effect of Volatile Organic Chemicals in Chrysanthemum indicum Linné on Blood Pressure and Electroencephalogram

This study identified the volatile organic compounds in the essential oils that are extracted from Chrysanthemum indicum Linné (C. indicum Linné) and investigated the effects of the inhalation of these compounds. We detected a total of 41 volatile organic compounds, including 32 hydrocarbons, four acids, three alcohols, two ketones, and one aldehyde. In a sniffing test, seven types of volatile organic compounds were identified. Furthermore, the volatile organic compounds in C. indicum Linné that were identified were found to be derived from 1,8-cineole and camphor. After inhalation of the essential oils, the subjects’ systolic blood pressure and heart rate decreased. This indicates that inhalation of the essential oils extracted from C. indicum Linné provides mental and physical relaxation. We examined the changes in electroencephalogram findings that are observed after C. indicum Linné essential oil inhalation. An increase in theta and alpha waves, which usually appear during relaxation, as well as a decrease in beta and gamma waves, which appear during brain activity such as excessive attention, were noted. These results indicate that C. indicum Linné essential oil inhalation helps to reduce blood pressure and may provide mental and physical relaxation.

Proteomic analysis of dehydroascorbate reductase transgenic potato plants

Ascorbic acid (AsA) is a strong antioxidant/reducing agent that can be converted to dehydroascorbate (DHA) by oxidation in plants. DHA, a very short-lived chemical, is recycled to AsA by dehydroascorbate reductase (DHAR). Previously, DHAR cDNA was isolated from the hairy roots of the sesame plant, and DHAR-overexpressing transgenic potato plants were generated under the control of the CaMV35S promoter (CaMV35S::DHAR). An increase in transgene expression and ascorbate levels were observed in the transgenic plants. In the present study, proteomic analysis revealed that transgenic plants not only accumulated DHAR in their cells, but also induced several other antioxidant enzyme-related proteins during plant growth. These results suggest that DHAR is important for stress tolerance via induction of antioxidant proteins, and could improve stress tolerance in transgenic potato plants.

An efficient transformation method for a potato (Solanum tuberosum L. var. Atlantic)

Abstract We found that a long period of in vitro culture is a critical factor on the low transformation rate for a specific potato genotype, Solanum tuberosum L. var. Atlantic when phosphinothricin (PPT) was added to select putative trans-formants in a solid media. The fresh explants of the newly produced plants from a micro-tuber was able to increase the transformation rate significantly while the old explants prepared from a plant maintained for longer than 6 months in vitro by sub-culturing every 3 ~ 4 weeks resulted in a very low transformation frequency. However, Jowon cultivar was not so much influenced by the period of in vitro culture with high transformation rate (higher than 10.0%). Further research need to be explored for the reason why a particular potato genotype, Atlantic is more vulnerable than the Jowon cultivar during the regeneration stage resulting in the low transformation frequency. Introduction During the last fifteen years, the total cultivation area of genetically modified (GM) crops has been gradually increased and in year 2012, they were cultivated in 170,000,000 ha all over the world. Maize, soybean and cotton bearing insect- resistant gene or/and herbicide-resistant gene were the major GM crops cultivated so far (James 2013). Recently, GM potato such as amylose-free line created through RNAi technology has also been approved for the commercialization (Abdallah 2010). Moreover, potato has been considered as a good candidate crop for mass production of industrially valuable protein or pharmaceutically useful substances by using recombinant DNA technology since potato tuber tissue contains well known mechanism to accumulate proteins in protein body (Pots et al. 1999; Shewry 2003; Gerszberg et al. 2012; Ahmad et al. 2012). However, there have been several reports that a particular genotype of potato exhibited very low transformation frequency and became a stumbling block for the development of a new GM potato. Although De Block (1988) reported a genotype- independent method was developed for potato, there have been several claims that to some extent, the efficiency of transformation is dependent on genotypes. For example, Dale and Hampson (1995) reported that out of 34 varieties, only half of them were successful for transformation by using tuber discs and some varieties were not able to transform by using either tuber discs or leaf explants. In addition, most of cultivars in Latin America, having andigena genetic background have been very inefficient until genotype-specific method was developed (Rodriguez et al. 2000; Trujillo et al. 2001). There are several other critical factors influencing the transformation rate including a kind of growth regulator and carbon sources such as sucrose or glucose in the regeneration media, tissues of explants and selective marker such as nptII and bar gene. We have also experienced same problems with some potato cultivars bred in South Korea including Namseo, Chuback, Jopoong, Jasim and Jowon (Lee et al. 2003). In detail, all of them except Jowon cultivar exhibited very low transformation ratio by using either leaf or internode explants. Interestingly enough, we were able to increase the transformation efficiency up to 80% with Jowon cultivar by using available method but most of other cultivars were lower than 5.0%. Furthermore, when phosphinothricin (PPT) was added into media for the selection of transgenic plants bearing bar gene as a selective marker, the transformation efficiency of Jowon cultivar was decreased to lower than 20% and most of cultivars bred in South Korea showed very low transformation ratio. Since Si