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Rho/Rho-kinase downstream of G

A Gram-stain-negative, non-motile, rod-shaped, aerobic bacterial strain, designated 1-3-3-8T, was isolated from soil and characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 1-3-3-8T belongs to the family Cytophagaceae of phylum Bacteroidetes and is most closely related to Hymenobacter paludis KBP-30T (96.8% similarity), Hymenobacter ocellatus Myx2105T (96.8%), Hymenobacter coalescens WW84T (95.6%), and Hymenobacter deserti ZLB-3T (95.4%). The G + C content of the genomic DNA of strain 1-3-3-8T was 63.6 mol%. The isolate contained C15:0 iso (28.4%), summed feature 4 (C17:1 anteiso B/C17:1 iso I; 18.9%), and C15:0 anteiso (17.6%) as major fatty acids, MK-7 as the predominant respiratory quinone, and sym-homospermidine as the predominant polyamine. The major polar lipids were phosphatidylethanolamine and an unidentified lipid. The phenotypic and chemotaxonomic data supported the affiliation of strain 1-3-3-8T with the genus Hymenobacter. The DNA-DNA relatedness between strain 1-3-3-8T and H. paludis KCTC 32237T and H. ocellatus DSM 11117T were 24.5 and 27.4% respectively, clearly showing that the isolate is not related to them at the species level. Overall, the novel strain could be differentiated from its phylogenetic neighbors on the basis of several phenotypic, genotypic, and chemotaxonomic features. Therefore, strain 1-3-3-8T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter jeollabukensis sp. nov. has been proposed. The type strain is 1-3-3-8T (= KCTC 52741T = JCM 32192T).

A Gram-stain-negative, non-motile, non-spore-forming, rodshaped, aerobic bacterial strain, designated S1-2-2-5T, was isolated from the Jeollabuk-do province, Republic of Korea, and was characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain S1-2-2-5T belonged to the family Cytophagaceae in phylum Bacteroidetes, and was most closely related to Hymenobacter terrae DG7AT (98.2%), Hymenobacter rubidus DG7BT (98.0%), Hymenobacter soli PB17T (97.7%), Hymenobacter daeguensis 16F3Y-2T (97.2%) and Hymenobacter saemangeumensis GSR0100T (97.0%). The G + C content of the genomic DNA of strain S1-2-2-5T was 59.4 mol%. The detection of menaquinone MK-7 as the predominant respiratory quinone, a fatty acid profile with summed feature 3 (C16:1ω7c/C16:1ω6c; 32.0%), C15:0 iso (19.0%), and C15:0 anteiso (15.0%) as the major components, and a polar lipid profile with phosphatidylethanolamine as the major component supported the affiliation of strain S1-2-2-5T to the genus Hymenobacter. The DNA-DNA relatedness between strain S1-2-2-5T and H. terrae KCTC 32554T, H. rubidus KCTC 32553T, H. soli KCTC 12607T, H. daeguensis KCTC 52537T, and H. saemangeumensis KACC 16452T were 49.5, 48.2, 34.1, 28.1, and 31.8% respectively, clearly showing that the isolate is not related to them at the species level. Strain S1-2-2-5T could be clearly differentiated from its closest neighbors on the basis of its phenotypic, genotypic and chemotaxonomic features. Therefore, strain S1-2-2-5T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter terrigena sp. nov. is proposed. The type strain is S1-2-2-5T (= KCTC 52737T = JCM 32195T).

We evaluated the effects of 1-methylcyclopropene (1-MCP) treatment and polyethylene (PE) film liner application on the fruit quality attributes of cold-stored ‘Gamhong’ apples. Flesh firmness was better retained with 1-MCP treatment than by PE film liner treatment, regardless of 1-MCP treatment time. Titratable acidity was also well-maintained during cold storage. In contrast, the soluble solids contents were not consistent during storage. The 1-MCP treatment greatly reduced the internal ethylene concentration, when compared with the control and PE film liner treatments, irrespective of the 1-MCP treatment time. The weight loss of the fruit was greatly reduced by the PE film liner treatments, as well as by the 1-MCP treatments. The fruit peel color variables were not affected by the 1-MCP or PE film liner treatments during cold storage. Nevertheless, the peel color variables were greatly different in the shaded tissue than in the sunny side during cold storage. Our results indicate that 1-MCP treatment, as well as PE film liner application, should be highly recommended for retaining the fruit quality of cold-stored ‘Gamhong’ apples.

We report an Agrobacterium-mediated transformation procedure optimized for ‘Kyoho’ that is a major table grapevine cultivar in Korea, and its transgenic plants with antifreeze protein gene of carrot (DcAFP). The full length of DcAFP coding region in accordance with the previous report was isolated from young leaves of carrot and recombined into a plant transformation vector. Ethylene inhibitors such as silver nitrate and aminoethoxyvinylglycin (AVG) supplemented in a co-cultivation medium distinctly increased frequency of shoot regeneration when explants were sub-cultured in a selection medium: particularly ten-fold higher in treatment with 0.1 mg/L AVG than one without ethylene inhibitor. Among various antibiotics and their concentrations, the combination of 150 mg/L cefotaxime plus 150 mg/L ClavamoxTM was selected for elimination of Agrobacterium cells in addition to minimization of adverse effect on shoot regeneration, while 50 mg/L kanamycin monosulfate effectively suppressed regeneration of nontransgenic shoots. Applying the elucidated culture condition, we finally obtained a total of 5 transgenic ‘Kyoho’ plantlets with DcAFP, of which integration with the grapevine genome and transcription was confirmed by nucleic acid analyses.

Based on the results in this study, here we propose a systematic micropropagation process for ‘Gisela 5’ that is one of the important dwarfing cherry rootstocks. When the apical tips detached from newly developed shoot in spring season were cultured on the half strength MS media with 0.5 mg/L IBA and 0.5 ~ 1.0 mg/L BA, the cultures scored the highest acquisition rate at 90% for normal shoot with vigorous growth and without hyperhydricity. As next step, the young shoots maintained in vitro well multiplied on the full strength MS medium supplemented with 0.5 mg/L IBA and 0.5 mg/L BA, in which multiplication rate was approximately nine-fold. Given the half strength MS medium containing 2.0 mg/L IBA, each transplanted shoot fu rther developed robust roots. Finally, the plantlets were easily acclimatized in the compost consisted of vermiculite, perlite, and peatmoss in the proportion of 1:1:1. We expect that the results are useful for cherry cultivation and its rootstock production.

We investigated the effects of sucrose and Nano-silver (NAg) on extending the vase life of cut carnation flowers ‘Edun’. Sucrose suppressed ethylene production by downregulating the genes that code for its biosynthesis. Relative to the control, however, sucrose significantly increased bacterial proliferation on cut stem surfaces and reduced relative fresh weight (RFW), 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 bacterial 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, which is partially or totally insensitive to ethylene. 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.

We investigated the role of nano-silver (NAg) and the bacterial strain Enterobacter cloacae in increasing the vase life of cut carnation flowers ‘Omea’. NAg treatment extended vase life of the flowers by increasing relative fresh weight, antioxidant activities, and expression level of the cysteine proteinase inhibitor gene (DcCPi), and by suppressing bacterial blockage in stem segments, ethylene production and expression of ethylene biosynthesis genes and DcCP1 gene, compared with the control. Out of all the treatments, administration of 25 mg L-1 NAg gave the best results for all the analyzed parameters. Interestingly, application of E. cloacae also extended the vase life of cut flowers by three days in comparison with control flowers, and overall, showed better results than the control for all the analyzed parameters. Taken together, these results demonstrate the positive role of NAg and E. cloacae in increasing the longevity of cut carnation flowers, and indicate that this effect is brought about through multiple modes of action.

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.

Bottle gourd (Lagenaria siceraria Standl.) has been used as a source of rootstock for grafting watermelon to improve its fruit quality. We report here the development of a bottle gourd with resistance to drought by ectopic expression of the ArabidopsisAVP1 gene that encodes a vacuolar H+-pyrophosphatase. The drought resistance of AVP1-expressing and wild-type plants was assessed by growing plants under drought conditions. After 12 days of water deprivation, both AVP1-expressing and wild-type plants demonstrated reduced growth. After 10 days of re-watering, wild-type plants showed minimal growth while the AVP1-expressing plants resumed rapid growth. Further, AVP1-expressing plants displayed longer primary roots and more robust root systems than wild-type plants.

Chrysanthemum is one of the most economically valuable ornamental plants worldwide. However, the high temperature is a hindrance to chrysanthemum cultivation, causing delayed flowering, poor quality of ray florets, and reduced shelf life. Here, we report that ectopic overexpression of an Arabidopsis monothiol glutaredoxin AtGRXS17 in chrysanthemum improved response to heat stress, 35/25 °C (day/night temperatures) and 45/25 °C following 35/25 °C, in terms of flowering time and ray floret quality, respectively. Under heat stress conditions, AtGRXS17-overexpressing (AtGRXS17-OE) chrysanthemum plants had reduced H2O2 production, lower oxidative damage of cellular membranes, and greater photosynthetic ability compared to wild-type controls. Ectopic overexpression of AtGRXS17 promoted faster flower bud formation, resulting in earlier flowering, and prevented heat stress-caused loss of ray florets shape and wilting compared to wild-type controls. The findings demonstrate an approach to enhance quality flower production in a valuable ornamental crop.

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.

On the basis of the reported agriculturally valuable phenotypes resulted from ectopic overexpression of Arabidopsis vacuolar W-PPase (AVP 1), we generated the Chinese cabbage lines expressing AVP I which then subjected to salt stress to determine the A V P1 expression if it consistently confers the capability for increasing biomass and enhancing tolerance to salinity in other species. Collectively, here we demonstrate that the transgenic young plants show more vigorous growth and higher tolerance to salt stress than wild-type ones. Increased biomass phenotype by A VP1 expression was supported by comparing fresh and dry weights of transgenic and wild type plants grown under normal condition, while higher salt tolerance trait was confirmed by tracing the kinetics of photosystem II quantum yield and DAB-staining under gradually intensified salt stress induced by MS salt or NaCl, followed by normal condition.

A MYB transcription factor gene, RsMYB1, from radish was introduced into the chrysanthemum cultivars 'Peach ND', 'Peach Red', and 'Vivid Scarlet' under the control of the cauliflower mosaic virus 35S promoter. Presence of RsMYB1 in transgenic lines was confirmed using polymerase chain reaction (PCR). Results of reverse-transcription-PCR analysis revealed that the expression of RsMYB1 was stable in all transgenic lines and could enhance the expression levels of three key biosynthetic genes (F3H, DFR, and ANS) involved in anthocyanin production. Accordingly, anthocyanin content was significantly higher in transgenic lines than in control of all the cultivars, although the increasement was not visually observed in any of the transgenic lines. Therefore, these results demonstrate that RsMYB1 has potential to enhance anthocyanin content in the chrysanthemums.

Deregulated expression of an Arabidopsis H⁺/Ca²⁺ antiporter (sCAX1) in agricultural crops increases total calcium (Ca²⁺) but may result in yield losses due to Ca²⁺ deficiency-like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca²⁺ binding protein located at endoplasmic reticulum) in sCAX1-expressing tobacco and tomato plants mitigated these adverse effects while maintaining enhanced Ca²⁺ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca²⁺ transporters and binding proteins at different intracellular compartments can alter the content and distribution of Ca²⁺ within the plant matrix.

수요가 증가하고 있는 단자엽 구근 화훼작물인 무스카리( $Muscari$ $armeniacum$ Leichtl. Ex Bak.) 'Early Giant' 품종의 캘러스를 재료로 체세포배발생을 통한 기내 대량증식 체계를 확립하기 위하여 본 연구를 수행하였다. 잎 절편을 1-naphthalene acetic acid(NAA), 2,4-dichlorophenoxyacetic acid(2,4-D) 등 오옥신이 0.1~3.0 $mg{\cdot}L^{-1}$ 첨가된 배지에 배양하여 모든 배지에서 부드러운 엷은 연두색 캘러스를 고빈도로 유기하였지만 유기된 캘러스를 동일한 조성의 배지로 이식하였을 때 2,4-D, 4-amino-3,5,6-tri-chloropicolinic acid(picloram) 및 3,6-dichloro-o-anisic acid (dicamba) 첨가배지에서만 증식이 양호하게 이루어졌다. 비록 체세포배발생의 빈도가 캘러스의 기원과 배발생 유도 배지의 식물생장조절제 조성에 따라 차이가 있기는 했지만 식물생장조절제 무첨가 배지를 비롯하여 $N^6$ -Benzylaminopurine (BAP) 등 다양한 시토키닌과 NAA 0.01 $mg{\cdot}L^{-1}$ 가 혼용된 배지에서 체세포배가 유기되었다. 무스카리 잎 절편을 picloram 0.1 $mg{\cdot}L^{-1}$ 배지에 치상하여 캘러스를 유기하고 동일한 배지로 이식하여 증식한 후 NAA 0.01 $mg{\cdot}L^{-1}$ 와 BAP 0.5 $mg{\cdot}L^{-1}$ 혼용배지로 이식했을 때 최고빈도인 캘러스 덩어리당 9.3개의 체세포배를 획득할 수 있었다. 또한 무스카리 체세포배는 구형, 편심장형, 편어뢰형, 초기 자엽형, 중기 자엽형, 후기 자엽형, 초기 맹아형, 중기 맹아형 및 후기 맹아형배 등 총 9단계로 그 외형이 뚜렷이 구분되었다. 【Using calli of $Muscari$ $armeniacum$ cv. 'Early Giant' that is monocotyledonous ornamental bulb crop with increasing demand in Korea, we carried out current studies to establish an in vitro multiple propagation protocol via somatic embryogenesis. We found that soft pale yellow green calli were induced from leaf explants cultured on all media containing 0.1~3.0 $mg{\cdot}L^{-1}$ auxins such as 1-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). However, induced calli showed vigorous growth only when they further transferred on same media containing 2,4-D, 4-amino-3,5,6-tri-chloropicolinic acid (picloram), or 3,6-dichloro-o-anisic acid (dicamba). Although frequency of somatic embryo induction depended on callus source and PGR composition in somatic embryo induction media, somatic embryogenesis was initiated on surface of proliferated calli after transferring on media with no PGR or 0.01 $mg{\cdot}L^{-1}$ NAA co-supplemented with various cytokinins such as $N^6$ -benzylaminopurine (BAP). Highest number of embryo at 9.3 per callus clump was obtained when calli which were grown under 0.1 $mg{\cdot}L^{-1}$ picloram supplementation were sub-cultured on medium with 0.01 $mg{\cdot}L^{-1}$ NAA and 0.5 $mg{\cdot}L^{-1}$ BAP. In addition, morphological characteristics of somatic embryo were categorized into following nine phases: globular, biased heart, biased torpedo, early cotyledonary, middle cotyledonary, late cotyledonary, early sprouting, middle sprouting, and late sprouting embryos.】

To ascertain the effect of over-expressed maize calreticulin in tomato plant on tobamovirus movement in addition to validating potentiality of the gene (ZmCRT) as a means for the virus-resistance resource, four ZmCRT-expressing homozygous lines were generated from the T0 plants as using an Agrobacterium-mediated transformation, nucleic acid analyses, and a conventional breeding method. Of them, a line was subjected to the bioassay for tolerances to tobacco mosaic virus-U1 (TMV-U1) and tomato mosaic virus (ToMV) followed by RT-PCR and a chlorophyll fluorescence quenching analyses. Both transgenic plants transcribing ZmCRT and wild-type plants showed no symptom by 20 days after viruses inoculation, however the photosystem II quantum yield parameter measured from the upper leaves of ToMV-inoculated plants revealed that ZmCRT transgenic plants have higher photosynthetic ability than wild-type ones at that time, which indirectly implies that over-expressed ZmCRT product acts as a barrier to the cell-to-cell and/or systemic movement of ToMV. Moreover, ZmCRT transgenic plants showed remarkably longer shoot length than wild-type ones in 40 days after TMV-U1 or ToMV inoculation each, which might be resulted from higher photosynthetic ability during the phase not yet showing any external symptoms. Collectively, over-expressed ZmCRT protein in tomato plants is able to interrupt the systemic movement of infected TMV-U1 and ToMV even though not perfect.

Here we focused on tip-burn and blossom-end rot (BER) symptoms in the tomato plants expressing the constitutively active form of Ca 2+ /H + antiporter (sCAX1) and/or a Ca-binding protein (calreticulin, CRT) genes during their whole growth period. Conclusively we demonstrated that CRT is able to suppress the tip-burn and BER symptoms that were induced by sCAX1. Under poor nutrition con- dition, tomato plants overexpressing sCAX1 showed severe necrotic collapses in both roots and shoot polar tissues, which are in accordance with Ca 2+ deficient symptoms frequently observed in an agricultural cultivation of tomato. Reciprocal grafting trials using sCAX1 and wild type plants revealed that the tip-burn symptom by sCAX1 overexpression is not caused by hindrance of Ca 2+ uptake from soil. We con- structed CRT overexpressing transgenic tomatoes, and crossed them with sCAX1 transgenic plants to investigate the effects of CRT on the symptoms of sCAX1 transgenic plants. Co- expression of sCAX1 and CRT significantly suppressed the Ca 2+ deficient symptoms of sCAX1 transgenic plants. Those results suggest the model that Ca 2+ homeostasis disturbed by the overexpression of sCAX1 may be suppressed by the co-expression of CRT.

An efficient plant regeneration system has been developed for figleaf gourd (Cucurbita ficifolia Bouche), which is exclusively used as a rootstock for cucumber. The protocol is based on results obtained from a series of culture experiments involving different parts of the cotyledons and various media. The culture of cotyledon explants was critical for the enhancement of shoot regeneration frequency. The lower parts of the cotyledon excised at the plumule base were found to display a markedly enhanced production of adventitious shoots compared to other cotyledon regions. Culture in silver nitrate-supplemented Murashige and Skoog (MS) medium was not beneficial for shoot regeneration and suppressed root regeneration. Efficient shoot regeneration was obtained on MS medium containing 1.0 mg l−1 zeatin and 0.1 mg l−1 indole-3-acetic acid. Regenerated shoots successfully elongated and rooted in medium containing 0.1 mg l−1 1-naphthaleneacetic acid after 10–15 days of subculturing. The plantlets were satisfactorily acclimatized in a greenhouse and grew into normal plants without any morphological alterations.

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.

We generated transgenic tomato plants with Arabidopsis thaliana /cation exchanger gene (C4X4) by Agrobactrium-mediated transformation. We confirmed transgene copy number and transcription by Southern and Northern blot analyses. The intact CAX4-expressing tomato (Lycopersicon esculentum) fruits contained 63-71% more calcium () than wild-type fruits. Moreover, ectopic expression of C4X4 in tomato fruits did not show any significant increase of the four kinds of metallic cations analyzed (, , , and . The C4X4-expressing tomato plants including their fruits did not show any morphological alternations during whole growth period. These results suggest the enhanced Ca-substrate specificity of CAX4 exchanger in tomato. Therefore, intact CAX4 exchanger can be a useful tool for nutrient enrichment of tomato fruits with reduced accumulation of undesirable cations.

Here we describe a procedure for Chinese cabbage protoplast culture and effects of various treatments. Chinese cabbage protoplasts were isolated from different parts of young seedlings as using an enzyme mixture, of which yield was maximized in seven hours around after digestion. The highest rate of initial cell division followed by micro-callus formation was obtained in the medium with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, and 1.0 mg/L BA when the cotyledon-derived protoplasts were cultured. Initiation of cell division and micro-callus proliferation significantly depended upon Chinese cabbage genotype under a same culture circumstances. The micro-calli developed from cotyledon tissue of Norang-Bom cultivar successfully grew toward callus colonies on the solidified medium with 0.2 mg/L zeatin and 0.1 mM spermidine. The callus colonies generated de novo shoots at the maximum frequency of 4.3% on the medium with 5.0 mg/L BA and 1.0 mg/L NM. Our results might be helpful for further studies to enhance the regeneration efficiency in Chinese cabbage protoplast culture.

Bottle gourd (Lagenaria siceraria Standl.) has been used as a rootstock for the watermelon cultivation because of better growth ability at low temperature and avoidance from contamination of the soil disease. Since the genetic source for the elite rootstock is limited in nature, the genetic engineering method is inevitable to develop new lines especially to obtain the functionally important or multi-disease resistant bottle gourd. Recently, our lab has set up a successful system to transform the bottle gourd. in order to monitor the transformation process, GFP gene is used. Cotyledons of the inbred line 9005, 9006 and G5 were used to induce the shoot under the selection media with MS + 30 g/L sucrose + 3.0 mg/L BAP + 100 mg/L kanamycin + 500 mg/L cefotaxime + 0.5 mg/L , pH 5.8. The shoot was developed from the cut side of the explants after 3 weeks on the selection media. The shoot was incubated in the rooting media with 1/2 MS + 30 g/L sucrose + 0.1 mg/L IAA + 50 mg/L kanamycin + 500 mg/L cefotaxime, pH 5.8 and moved to pot for acclimation. Although the shoot development rate was depended on the genotype, the G5 was the best line to be transformed. Monitoring GFP expression from the young shoot under microscope could make the selection much easier to distinguish the transformed shoot from the non-transformed shoots.

Previously, we made a chimeric Arabidopsis thaliana vacuolar transporter CAX2B [a variant of N-terminus truncated form of CAX2 (sCAX2) containing the "B" domain from CAX1] that has enhanced calcium (Ca(2+)) substrate specificity and lost the manganese (Mn(2+)) transport capability of sCAX2. Here, we demonstrate that potato (Solanum tuberosum L.) tubers expressing the CAX2B contain 50-65% more calcium (Ca(2+)) than wild-type tubers. Moreover, expression of CAX2B in potatoes did not show any significant increase of the four metals tested, particularly manganese (Mn(2+)). The CAX2B-expressing potatoes have normally undergone the tuber/plant/tuber cycle for three generations; the trait appeared stable through the successive generations and showed no deleterious alternations on plant growth and development. These results demonstrate the enhanced substrate specificity of CAX2B in potato. Therefore, CAX2B can be a valuable tool for Ca(2+) nutrient enrichment of potatoes with reduced accumulation of undesirable metals.

Increased calcium (Ca) in potatoes may increase the production rate by enhancing tuber quality and storability. Additionally, increased Ca levels in important agricultural crops may help ameliorate the incidence of osteoporosis. However, the capacity to alter Ca levels in potato tubers through genetic manipulations has not been previously addressed. Here we demonstrate that potato tubers expressing the Arabidopsis H+/Ca2+ transporter sCAX1 (N-terminal autoinhibitory domain truncated version of CAtion eXchanger 1) contain up to 3-fold more Ca than wild-type tubers. The increased Ca appears to be distributed throughout the tuber. The sCAX1-expressing potatoes have normally undergone the tuber/plant/tuber cycle for three generations; the trait appeared stable through successive generations. The expression of sCAX1 does not appear to alter potato growth and development. Furthermore, increased Ca levels in sCAX1-expressing tubers do not appear to alter tuber morphology or yield. Given the preponderance of potato consumption worldwide, these transgenic plants may be a means of marginally increasing Ca intake levels in the population. To our knowledge, this study represents the first attempts to use biotechnology to increase the Ca content of potatoes.

A procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 carrying a binary vector pCAMBIA3301, which contains glufosinate ammonium-resistant (bar) and the reporter (gus) genes, is describe. Infection was the most effective (highest infection frequency and index) when explants were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.001-0.1 mg/L L-a-(2-aminoethoxyvinyl) glycine (AVG). Transgenic plants were obtained with frequencies of about 0.2% when the explants were cultured on selection medium (MS medium supplemented with 3.0 mg/L BAP, 0.5 mg/L AgNO3, 500 mg/L cefotaxime, 2.0 mg/L DL-phosphinothricin, 0.3% sucrose and 0.8% Plant Agar. A histochemical gus assay, PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progenies showed that the transgenes were inherited in a Mendelian fashion. To our knowlege, this study represents the first report for Agrobacterium-mediated transformation in bottle gourd, rootstock for watermelon and other cucurbit crops in many countries.

Background Rosea1 ( 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. Results In 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: T 0 -P, red: T 0 -R, and strong red: T 0 -S), resulting from varying levels of biosynthetic gene transcripts. Under salt stress, the T 2 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). Conclusion We 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.