[This corrects the article on p. 703 in vol. 66, PMID: 28163586.].

Abiotic stresses have negative effects on potato growth and production. To enhance the abiotic stress tolerance of the commercial potato cultivar, Desiree, rd29A::AtDREB1A transgenic lines have been developed. The salinity and freeze tolerance of these transgenic lines has previously been demonstrated; however, their dehydration tolerance remains to be elucidated. First of all, we have tackled the reproducible tolerance evaluation methodology, which have been the hurdle for selecting the stable dehydration tolerant phenotypes in potato. A novel in vitro method was developed using the rotary liquid culture combined with PEG. This method enhanced oxygen diffusion into the medium and diminished root damage/injury during plant transfer, thereby reducing the side effects caused by hypoxia and penetration of osmotica. In the present study, we evaluated the dehydration tolerance of twelve transgenic potato lines, and seven of the transgenic lines showed enhanced dehydration tolerance in comparison with the non-transgenic line. However, we observed growth retardation in some dehydration tolerant lines. Therefore, balance between the dehydration tolerance and growth retardation of the transgenic plants should be considered. Four of seven of the transgenic lines displayed enhanced dehydration tolerance without growth retardation, and may represent good candidates for practical application.

Untargeted metabolome analyses play a critical role in understanding possible metabolic fluctuations of crops under varying environmental conditions. This study reports metabolic profiles of transgenic potato tubers expressing the Arabidopsis DREB1A transcription factor gene, which induces expression of genes involved in environmental stress tolerance. A combination of targeted and untargeted metabolomics demonstrated considerable metabolome differences between the transgenic lines and nontransgenic parent cultivars. In the transgenic lines, stimulation of stress responses was suggested by elevated levels of the glutathione metabolite, gamma-aminobutyric acid (GABA), and by the accumulation of beta-cyanoalanine, a byproduct of ethylene biosynthesis. These results suggest that the Arabidopsis DREB1A expression might directly or indirectly enhance endogenous potato stress tolerance systems. The results indicate that transgenesis events could alter the metabolic compositions in food crops, and therefore metabolomics analysis could be a most valuable tool to monitor such changes.

To produce crops that are more tolerant to stresses such as heat, cold, and salt, transgenic plants have been produced those express stress-associated proteins. In this study, we used immunoproteomic and two-dimensional difference gel electrophoresis (2D-DIGE) methods to investigate the allergenicity of transgenic potatoes expressing Arabidopsis DREB1A (dehydration responsive element-binding protein 1A), driven by the rd29A promoter or the 35S promoter. Immunoproteomic analysis using sera from potato-allergic patients revealed several immunoglobulin E (IgE)-binding protein spots. The patterns of protein binding were almost the same between transgenic and non-transgenic potatoes. The IgE-binding proteins in potato were identified as patatin precursors, a segment of serine protease inhibitor 2, and proteinase inhibitor II by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS/MS. 2D-DIGE analysis revealed several differences in protein expression between non-transgenic potato and transgenic potato; those showing increased expression in transgenic potatoes were identified as precursors of patatin, a major potato allergen, and those showing decreased expression in transgenic potatoes were identified as lipoxygenase and glycogen (starch) synthase. These results suggested that transgenic potatoes may express slightly higher levels of allergens, but their IgE-binding patterns were almost the same as those of control potatoes. Further research on changes in protein expressions in response to environmental factors is required to confirm whether the differences observed in this study are due to gene transfection, rather than environmental factors.

A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39 936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5′ and 3′ ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22 674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5′- and 3′-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.

The freezing tolerance of 38 independent transgenic potato lines derived from the cultivar Desiree was tested in vitro using plantlets. The lines were transgenic for the DREB1A gene under control of the rd29A promoter, both of which were derived from Arabidopsis thaliana. The level of damage caused by freezing varied significantly among the transgenic clones and a non-transgenic control (cv. Desiree). Phenotypic evaluation indicated that the variable responses to freezing were attributable to genotypic variation, but freezing tolerance was not dependent on the number of insertions. Northern blot analysis using a DREB1A cDNA probe revealed high levels of DREB1A expression among the transgenic clones during the initial cold exposure at 4 degrees C (after 2 h) and in the early stages of freezing (-20 degrees C, 1-10 min). Furthermore, a linear correlation was detected between the level of expression and the phenotypic response for all lines except D138. Thus, in the case of potato, a significant increase in freezing tolerance was observed in vitro on a small scale following the introduction of rd29A::DREB1A. Additional testing will show whether this strategy can be used for tolerance breeding in potato and to increase the freezing tolerance of other agriculturally important crops.

Transgenic potato lines of cv. Desiree containing the DREB1A gene driven by the rd29A promoter were generated using Agrobacterium-mediated transformation. The morphological appearance of the 120 transgenic lines was classified into three categories as determined by in vitro test-tube evaluation. Southern blot analyses of genomic DNA were conducted using the restriction enzymes HindIII and DraI. There were significant differences between the transgenic lines and DSC in the quantitative salinity-tolerance evaluations at 1 M NaCl. Two transgenic lines were recognized as highly tolerant to salinity based on Duncan multiple range testing. Furthermore, there was a significant correlation between the mean tolerance level of the transgenic lines and the DREB1A copy number estimated from the Southern hybridization experiments. Northern hybridization experiments were subsequently done using a DREB1A cDNA probe and transgenic lines with different levels of salinity tolerance. Salt-tolerant transgenic lines expressed substantially more of the transgene at 2 to 5 h of salt treatment, after which the expression returned to basal levels. These observations suggest that the gene transfer of rd29A::DREB1A can be used to increase the salt tolerance of important agricultural crops, such as tetrasomic polyploid potatoes, as occurs in diploid model species, such as Arabidopsis.

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) 'Fall Color' genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.

【Objective】 This article aimes at breeding of new materials with tolerance to cold conditions in ground-cover chrysanthemum [Dendranthema grandiflorum (Ramat.) Kitamura]. 【Method】 Young leaf explants of the cultivar, Fall color, were used to determine the optimum conditions of both concentration of plant growth regulator and induction time to establish a high frequency regeneration system through somatic embryogenesis. 【Result】 The explants incubated for 15 d on TM containing 0.75 mg•L^(-1) 2,4-D generated not only embryo callus tissue, but also somatic embryos. The somatic embryos emerged shoot with 93% of shoot regeneration rate after further regeneration culture. AtDREB1A, a stress-inducible transcription factor, driven by 35S cauliflower mosaic virus (CaMV) promoter was transferred into Fall Color through Agrobacterium-mediated transformation. Seed germination and seedling growth at low temperature, and plant growth in winter cultivated in open-field were much improved in transgenic plants compared to WT plants. 【Conclusion】 These results indicate that the authors have established successfully the embryo-generation system of ground-cover cv. Fall color, and have obtained successfully the transgenic lines with tolerance to open-field conditions in winter.

将携带有AtDREB1A基因, 并以35S或rd29A启动子驱动的植物表达载体转入地被菊花(Dendranthema grandiflorum)的粉色品种'Fall color'. 与野生型相比, 35S:DREB1A转基因植株表现出严重的生长抑制, 而rd29A:DREB1A植株生长正常. RT-PCR检测表明, 在胁迫条件下, AtDREB1A基因在35S: DREB1A转基因植株中呈现组成型过量表达, 而在rd29A:DREB1A植株中则是受胁迫诱导型过量表达. 这两种启动子驱动的转基因植株对干旱和盐渍胁迫都表现出较强的耐性, 其中rd29A:DREB1A植株耐性显著强于35S:DREB1A植株. rd29A:DREB1A植株中的脯氨酸含量和SOD活性都强烈地被胁迫诱导升高, 且高于35S:DREB1A植株. 这些结果表明, 在地被菊花中表达AtDREB1A基因可以提高植株对干旱和盐渍胁迫的耐性, 同时这些耐性的升高可能与脯氨酸含量和SOD活性的上升有关.

Summary The gene DREB1A, encoding a stress-inducible transcription factor, driven by the cauliflower mosaic virus 35S (CaMV) promoter or by the stress-inducible rd29A promoter, was transferred into chrysanthemum plants [Dendranthema grandiflorum (Ramat.) Kitamura] cv. ‘Fall Color’ by Agrobacterium-mediated transformation. Integration of the target gene, DREB1A was confirmed by PCR and Southern blotting. When exposed to 2°C, expression of DREB1A was enhanced in the roots of young transgenic 35S:DREB1A plant lines, and induced in transgenic rd29A:DREB1A lines. Electrolyte leakage in leaves of rd29A:DREB1A plant lines was significantly lower than in 35S:DREB1A lines. Compared to control plants, superoxide dismutase activities and proline contents increased slowly in transgenic plants at the start of the cold stress treatment and remained at high levels during later periods, especially in rd29A:DREB1A transgenic lines. Young plants of the rd29A:DREB1A line could tolerate –8°C for 12 h, with a survival rate of 37.5%. No survival was observed in 35S:DREB1A lines or in wild-type plants. These results indicate that a combination of the stress-inducible rd29A promoter and the DREB1A gene enhanced the tolerance of ground-cover chrysanthemum plants to cold stress through a transgenic approach.

将携带有AtDREB1A基因, 并以35S或rd29A启动子驱动的植物表达载体转入地被菊花(Dendranthema grandiflorum)的粉色品种‘Fall color’. 与野生型相比, 35S:DREB1A转基因植株表现出严重的生长抑制, 而rd29A:DREB1A植株生长正常. RT-PCR检测表明, 在胁迫条件下, AtDREB1A基因在35S: DREB1A转基因植株中呈现组成型过量表达, 而在rd29A:DREB1A植株中则是受胁迫诱导型过量表达. 这两种启动子驱动的转基因植株对干旱和盐渍胁迫都表现出较强的耐性, 其中rd29A:DREB1A植株耐性显著强于35S:DREB1A植株. rd29A:DREB1A植株中的脯氨酸含量和SOD活性都强烈地被胁迫诱导升高, 且高于35S:DREB1A植株. 这些结果表明, 在地被菊花中表达AtDREB1A基因可以提高植株对干旱和盐渍胁迫的耐性, 同时这些耐性的升高可能与脯氨酸含量和SOD活性的上升有关.

Using in vitro leaves as explants, the adventitious shoots of ground-cover chrysanthemum(Dendranthema grandiflorum cv. White Snow) were derived from the basal MS media supplemented with different plant growth regulators through organogenesis. The results indicated that the regeneration rate of 93.8% was obtained by the combination of 2.0 mg/L 6-BA and 2 mg/L NAA. The binary vector pBI-DREB1A containing transcription factor DREB1A gene from Arabidopsis thaliana was introduced into Agrobacterium tumefaciens LBA4404. The leaf disks of ground-cover chrysanthemum, White Snow, were infected through Agrobacterium-mediated transformations. The DREB1A gene was integrated into the genome of transgenic plants confirmed by PCR and PCR-Southern analysis. The highest transformation efficiency was obtained through the following transformation procedure: after preculture for 2 d, the explants were infected for 10 min with diluted OD600=0.5~0.7 Agrobacterium tumefaciens culture liquid with a ratio of 1/30, and then co-cultivated for 2 d.

Tetraploid potato cv. Desiree was transformed using an Arabidopsis thaliana stress-inducible promoter rd29A and the DKEB1A gene, which confers multiple tolerances to abiotic stresses (e.g., dehydration and elevated soil salinity). Transformed Desiree lines showed a direct correlation between the DREB1A expression levels and the tolerance to salinity. By producing a filial progeny from the transgenic lines which was considered to harbor a single copy of the DREB1A gene, crossed with a non-transgenic cultivar, the genotype at the transgenic locus of these tetrasomic tetraploids was estimated by chi-square test, suggesting the existence of a Simplex mode. The results showed that low-copy heterozygous loci with the DREB1A gene could sustain the expression, and consequently confer a significant tolerance to salinity in tetrasomic tetraploid potatoes.