Transgenic poplar trees overexpressing AtGolS2, a stress-responsive galactinol synthase gene derived from Arabidopsis thaliana, improved drought tolerance in a confined field.

Drought is an abiotic stress that limits plant growth and productivity, and the development of trees with improved drought tolerance is expected to expand potential plantation areas and to promote sustainable development. Previously we reported that transgenic poplars (Populus tremula × P. tremuloides, T89) harboring the stress-responsive galactinol synthase gene, AtGolS2, derived from Arabidopsis thaliana were developed and showed improved drought stress tolerance in laboratory conditions. Herein we report a field trial evaluation of the AtGolS2-transgenic poplars. The rainfall-restricted treatments on the poplars started in late May 2020, 18 months after transplanting to the field, and were performed for 100 days. During these treatments, the leaf injury levels were observed by measuring photosynthetic quantum yields twice a week. Observed leaf injury levels varied in response to soil moisture fluctuation and showed a large difference between transgenic and non-transgenic poplars during the last month. Comparison of the leaf injury levels against three stress classes clustered by the machine learning approach revealed that the transgenic poplars exhibited significant alleviation of leaf injuries in the most severe stress class. The transgenes and transcript levels were stable in the transgenic poplars cultivated in the field conditions. These results indicated that the overexpression of AtGolS2 significantly improved the drought stress tolerance of transgenic poplars not only in the laboratory but also in the field. In future studies, molecular breeding using AtGolS2 will be an effective method for developing practical drought-tolerant forest trees.

Evaluation of potential impacts on biodiversity of the salt-tolerant transgenic Eucalyptus camaldulensis harboring an RNA chaperonic RNA-Binding-Protein gene derived from common ice plant.

We recently reported that a genetic transformation of the RNA-Binding-Protein (McRBP), an RNA chaperone gene derived from common ice plant (Mesembryanthemum crystallinum), alleviated injury and loss of biomass production by salt stress in Eucalyptus camaldulensis in a semi-confined screen house trial. In this study, we assessed the potential environmental impact of the transgenic Eucalyptus in a manner complying with Japanese biosafety regulatory framework required for getting permission for experimental confined field trials. Two kinds of bioassays for the effects of allelopathic activity on the growth of other plants, i.e., the sandwich assay and the succeeding crop assay, were performed for three transgenic lines and three non-transgenic lines. No significant differences were observed between transgenic and non-transgenic plants. No significant difference in the numbers of cultivable microorganisms analyzed by the spread plate method were observed among the six transgenic and non-transgenic lines. These results suggested that there is no significant difference in the potential impact on biodiversity between the transgenic McRBP-E. camaldulensis lines and their non-transgenic comparators.

Development and evaluation of novel salt-tolerant Eucalyptus trees by molecular breeding using an RNA-Binding-Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.).

The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA-Binding-Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened-house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants' acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high-stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non-transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate-stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non-transgenic status survived the 70 mM NaCl treatment, but after 6-month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non-transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non-transgenic plants after 15-week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.

Climatic changes and potatoes: How can we cope with the abiotic stresses?

Climate change triggers increases in temperature, drought, and/or salinity that threaten potato production, because they necessitate specific amounts and quality of water, meanwhile lower temperatures generally support stable crop yields. Various cultivation techniques have been developed to reduce the negative effects of drought, heat and/or salinity stresses on potato. Developing innovative varieties with relevant tolerance to abiotic stress is absolutely necessary to guarantee competitive production under sub-optimal environments. Commercial varieties are sensitive to abiotic stresses, and substantial changes to their higher tolerance levels are not easily achieved because their genetic base is narrow. Nonetheless, there are several other possibilities for genetic enhancement using landraces and wild relatives. The complexity of polysomic genetics and heterozygosity in potato hamper the phenotype evaluation over abiotic stresses and consequent conventional introgression of tolerance traits, which are more challenging than previous successes shown over diseases and insects resistances. Today, potatoes face more challenges with severe abiotic stresses. Potato wild relatives can be explored further using innovative genomic, transcriptomic, proteomic, and metabolomic approaches. At the field level, appropriate cultivation techniques must be applied along with precision farming technology and tolerant varieties developed from various breeding techniques, in order to realize high yield under multiple stresses.

Erratum: Evaluation of the yield of abiotic-stress-tolerant AtDREB1A transgenic potato under saline conditions in advance of field trials.

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

Genetic diversity and population structure of 'Khao Kai Noi', a Lao rice (Oryza sativa L.) landrace, revealed by microsatellite DNA markers.

Rice (Oryza sativa L.) is the main food for people in Laos, where it has been grown and eaten since prehistory. Diverse landraces are grown in Laos. 'Khao Kai Noi', a landrace favored for its eating quality, is held in the nationwide collection of traditional landraces in the Lao national genebank. Genetic diversity is crucial for sustainable use of genetic resources and conservation. To investigate the genetic diversity of 'Khao Kai Noi' for conservation, we genotyped 70 accessions by using 23 polymorphic simple sequence repeat markers. The markers generated 2 to 17 alleles (132 in total), with an average of 5.7 per locus. The total expected heterozygosity over all 'Khao Kai Noi' accessions was 0.271. Genetic variation was largest among accessions and smallest within accessions. Khao Kai Noi accessions were classified into three different genetic backgrounds, but there was unclear association between the three inferred population and name subgroups and geographical distribution. Most of the accessions were clustered with temperate japonica and showed genetic relatedness to rice from neighboring provinces of Vietnam, suggesting a Vietnamese origin. The results of this study will contribute to the conservation, core collection and future breeding of the Khao Kai Noi population.

Evaluation of the yield of abiotic-stress-tolerant AtDREB1A transgenic potato under saline conditions in advance of field trials.

Cultivated potato is a drought-, salinity-, and frost-sensitive species. The transgenic approach is one of the methods used to mitigate abiotic stress. The utility of transgenic potatoes that have abiotic stress tolerance should be judged from their yield under stress conditions. In order to establish transgenic potato lines with the AtDREB1A gene that could be used in practical applications, we screened candidate lines in a growth room with growth profiles under non-stress conditions rather than the expression level of transgene. After identifying better transgenic lines (D163 and D164), yield of those lines under stress conditions was evaluated in the special netted-house. Although the yield was lower than the yield under non-stress conditions, two selected transgenic lines were able to maintain their yield under high saline conditions (EC > 10 mS/cm). In this study, fertilizer was not added beyond what was already contained in the soil mix in order to evaluate the yield of the transgenic lines under saline conditions in as simple a manner as possible. In future studies, it will be necessary to evaluate their yield in a farming context in an isolated field after assessing the environmental biosafety of these transgenic potato lines.

Genetic variation of rice (Oryza sativa L.) germplasm in Myanmar based on genomic compositions of DNA markers.

The genetic diversity of 175 rice accessions from Myanmar, including landraces and improved types from upland and lowland ecosystems in five different areas-Western (hilly), Northern (mountainous), North and South-eastern (plateau), and Southern (plain)-was evaluated on the basis of polymorphism data for 65 DNA markers and phenol reactions. On the basis of the DNA polymorphism data, high genetic diversity was confirmed to conserve in the accessions from each ecosystem and area. And the accessions were classified into two cluster groups I and II, which corresponded to Indica Group and Japonica Group, respectively. Cluster group I accessions were distributed mainly in upland ecosystems; group II were distributed in lowland in the Southern area, and the distributions of dominant groups differed among areas. Rice germplasm in Myanmar has maintained high genetic diversity among ecosystems and areas. This information will be used for advanced studies in germplasm and rice breeding in Myanmar.

A multi-year assessment of the environmental impact of transgenic Eucalyptus trees harboring a bacterial choline oxidase gene on biomass, precinct vegetation and the microbial community.

A 4-year field trial for the salt tolerant Eucalyptus globulus Labill. harboring the choline oxidase (codA) gene derived from the halobacterium Arthrobacter globiformis was conducted to assess the impact of transgenic versus non-transgenic trees on biomass production, the adjacent soil microbial communities and vegetation by monitoring growth parameters, seasonal changes in soil microbes and the allelopathic activity of leaves. Three independently-derived lines of transgenic E. globulus were compared with three independent non-transgenic lines including two elite clones. No significant differences in biomass production were detected between transgenic lines and non-transgenic controls derived from same seed bulk, while differences were seen compared to two elite clones. Significant differences in the number of soil microbes present were also detected at different sampling times but not between transgenic and non-transgenic lines. The allelopathic activity of leaves from both transgenic and non-transgenic lines also varied significantly with sampling time, but the allelopathic activity of leaves from transgenic lines did not differ significantly from those from non-transgenic lines. These results indicate that, for the observed variables, the impact on the environment of codA-transgenic E. globulus did not differ significantly from that of the non-transformed controls on this field trial.

Heterogeneous Expression of Arabidopsis Subclass II of SNF1-Related Kinase 2 Improves Drought Tolerance via Stomatal Regulation in Poplar.

Abscisic acid (ABA) is the most important phytohormone involved in the response to drought stress. Subclass II of SNF1-related kinase 2 (SnRK2) is an important signaling kinase related to ABA signal transduction. It regulates the phosphorylation of the target transcription factors controlling the transcription of a wide range of ABA-responsive genes in Arabidopsis thaliana. The transgenic poplars (Populus tremula × P. tremuloides, clone T89) ectopically overexpressing AtSnRK2.8, encoding a subclass II SnRK2 kinase of A. thaliana, have been engineered but almost no change in its transcriptome was observed. In this study, we evaluated osmotic stress tolerance and stomatal behavior of the transgenic poplars maintained in the netted greenhouse. The transgenic poplars, line S22, showed a significantly higher tolerance to 20% PEG treatment than non-transgenic controls. The stomatal conductance of the transgenic poplars tended to be lower than the non-transgenic control. Microscopic observations of leaf imprints revealed that the transgenic poplars had significantly higher stomatal closures under the stress treatment than the non-transgenic control. In addition, the stomatal index was lower in the transgenic poplars than in the non-transgenic controls regardless of the stress treatment. These results suggested that AtSnRK2.8 is involved in the regulation of stomatal behavior. Furthermore, the transgenic poplars overexpressing AtSnRK2.8 might have improved abiotic stress tolerance through this stomatal regulation.

Assessment of the salt tolerance and environmental biosafety of Eucalyptus camaldulensis harboring a mangrin transgene.

Increasing soil salinization of arable land has a major impact on the global ecosystem. One approach to increase the usable global forest area is to develop transgenic trees with higher tolerance to conditions of salt stress. An allene oxide cyclase homolog, mangrin, contains a core protein domain that enhances the salt tolerance of its host. We utilized this feature to develop improved salt-tolerant eucalyptus trees, by using transgenic Eucalyptus camaldulensis carrying the mangrin gene as a model. Since the Japanese government requires an environmental biosafety assessment for the surrounding biosphere, we performed experiments on trees grown in a special netted-house. This study examined the transgenic E. camaldulensis carrying the mangrin gene to assess the feasibility of using these transformants, and assessed their salt tolerance and environmental biosafety. We found that seven of 36 transgenic genotypes had significantly higher salt tolerance than non-transformants, and more importantly, that these plants had no significant impact on environmental biosafety. These results suggest that introduction of the mangrin gene may be one approach to safely enhance salt tolerance in genetically modified Eucalyptus species, and that the transformants have no apparent risks in terms of environmental biosafety. Thus, this study provides valuable information regarding the use of transgenic trees in situ.

A statistical modeling approach based on the small-scale field trial and meteorological data for preliminary prediction of the impact of low temperature on Eucalyptus globulus trees.

Eucalyptus trees are important for industrial forestry plantations because of their high potential for biomass production, but their susceptibility to damage at low temperatures restricts their plantation areas. In this study, a 6-year field trial of Eucalyptus globulus was conducted in Tsukuba, Japan, which is the northernmost reach of Eucalyptus plantations, and leaf damage was quantitatively monitored over four of six winters. Leaf photosynthetic quantum yield (QY) levels, an indicator of cold stress-induced damage, fluctuated synchronously with temperature in the winters. We performed a maximum likelihood estimation of the regression model explaining leaf QY using training data subsets for the first 3 years. The resulting model explained QY by the number of days when the daily maximum temperature was below 9.5 °C over approximately the last 7 weeks as an explanatory variable. The correlation coefficient and coefficient of determination of prediction by the model between the predicted and observed values were 0.84 and 0.70, respectively. The model was then used to perform two kinds of simulations. Geographical simulations of potential Eucalyptus plantation areas using global meteorological data from more than 5,000 locations around the world successfully predicted an area that generally agreed with the global Eucalyptus plantation distribution reported previously. Another simulation based on meteorological data of the past 70 years suggested that global warming will increase the potential E. globulus plantation area in Japan approximately 1.5-fold over the next 70 years. These results suggest that the model developed herein would be applicable to preliminary predictions of E. globulus cold damage in the field.

Exploring Volatile Organic Compounds in Rhizomes and Leaves of Kaempferia parviflora Wall. Ex Baker Using HS-SPME and GC-TOF/MS Combined with Multivariate Analysis.

Volatile organic compounds (VOCs) play an important role in the biological activities of the medicinal Zingiberaceae species. In commercial preparations of VOCs from Kaempferia parviflora rhizomes, its leaves are wasted as by-products. The foliage could be an alternative source to rhizome, but its VOCs composition has not been explored previously. In this study, the VOCs in the leaves and rhizomes of K. parviflora plants grown in a growth room and in the field were analyzed using the headspace solid-phase microextraction (HS-SPME) method coupled with gas chromatography and time-of-flight mass spectrometry (GC-TOF-MS). The results showed a total of 75 and 78 VOCs identified from the leaves and rhizomes, respectively, of plants grown in the growth room. In the field samples, 96 VOCs were detected from the leaves and 98 from the rhizomes. These numbers are higher compared to the previous reports, which can be attributed to the analytical techniques used. It was also observed that monoterpenes were dominant in leaves, whereas sesquiterpenes were more abundant in rhizomes. Principal component analysis (PCA) revealed significantly higher abundance and diversity of VOCs in plants grown in the field than in the growth room. A high level of similarity of identified VOCs between the two tissues was also observed, as they shared 68 and 94 VOCs in the growth room and field samples, respectively. The difference lies in the relative abundance of VOCs, as most of them are abundant in rhizomes. Overall, the current study showed that the leaves of K. parviflora, grown in any growth conditions, can be further utilized as an alternative source of VOCs for rhizomes.

Comparison of Chayote (Sechium edule (Jacq.) Sw.) Accessions from Mexico, Japan, and Myanmar Using Reproductive Characters and Microsatellite Markers.

Promoting neglected and underutilized crop species is a possible solution to deal with the complex challenges of global food security. Chayote is a Neglected and Underutilized Cucurbit Species (NUCuS), which is recognized as a fruit vegetable in Latin America and is widely grown in Asia and Africa. However, basic biological knowledge about the crop is insufficient in scientific sources, especially outside of its center of origin. In this study, limited observations on reproductive characters were conducted, differentiating accessions from Mexico, Japan, and Myanmar. Cytological evaluation among Mexican and Japanese accessions showed that the relative nuclear DNA content is 1.55 ± 0.05 pg, the estimated genome size is 1511 at 2C/Mbp, and the observed mitotic chromosomal number is 2n = 28. The genetic diversity of 21 chayote accessions was also examined using six microsatellite markers. A global low genetic heterozygosity (Ho = 0.286 and He = 0.408) and three genetic groups were detected. The results established the basis to provide insights into chayote arrival history in Asia by looking at the crop's reproductive morphology, cytology, and genetic diversity status outside its origin center. This could help in developing sustainable utilization and conservation programs for chayote.

Genetic structure of landraces in foxtail millet (Setaria italica (L.) P. Beauv.) revealed with transposon display and interpretation to crop evolution of foxtail millet.

Although the origin and domestication process of foxtail millet (Setaria italica subsp. italica (L.) P. Beauv.) has been studied by several groups, the issue is still ambiguous. It is essential to resolve this issue by studying a large number of accessions with sufficient markers covering the entire genome. Genetic structures were analyzed by transposon display (TD) using 425 accessions of foxtail millet and 12 of the wild ancestor green foxtail (Setaria italica subsp. viridis (L.) P. Beauv.). We used three recently active transposons (TSI-1, TSI-7, and TSI-10) as genome-wide markers and succeeded in demonstrating geographical structures of the foxtail millet. A neighbor-joining dendrogram based on TD grouped the foxtail millet accessions into eight major clusters, each of which consisted of accessions collected from adjacent geographical areas. Eleven out of 12 green foxtail accessions were grouped separately from the clusters of foxtail millet. These results indicated strong regional differentiations and a long history of cultivation in each region. Furthermore, we discuss the relationship between foxtail millet and green foxtail and suggest a monophyletic origin of foxtail millet domestication.

Natural Bioactive Compounds of Sechium spp. for Therapeutic and Nutraceutical Supplements.

Natural products are in great demand because certain secondary metabolites (SMs) are sources of antioxidants, flavorings, active substances, or anticancer agents with less aggressiveness and selectivity, among which triterpenes and flavonoids are of importance because they inhibit carcinogenesis. For Sechium spp. P. Br. (chayotes), there is scientific evidence of antiproliferative activity that has occurred when cancer cell lines have been treated with this fruit. In order to compare future therapeutic designs and identify new and ancestral characteristics, triterpenes and flavonoids were determined in contrasting Sechium genotypes. The obtained data were analyzed via a cladistics approach, with the aim of identifying the characteristics and state of phytochemicals and genetic variables. The concentrations of flavonoids and triterpenes were determined, and a more complex composition of secondary metabolites was found in the wild types as compared to their domesticated genotypes. Bitter fruits contained a higher number of SMs, followed by those with a neutral and sweet flavor. A cladogram showed the differentiation of the three groups based on the flavor of the fruits. The diversity of SMs decreases in evolutionary terms, in response to domestication and environmental adaptation. Therefore, genotypes can be feasibly selected based on fruit flavor for gross-breeding, and cytotoxicity can be reduced without losing possible therapeutic effects.

Bioprospecting of Sechium spp. varieties for the selection of characters with pharmacological activity.

Bioprospecting identifies new sources of compounds with actual or potential economic value that come from biodiversity. An analysis was performed regarding bioprospecting purposes in ten genotypes of Sechium spp., through a meta-analysis of 20 information sources considering different variables: five morphological, 19 biochemical, anti-proliferative activity of extracts on five malignant cell lines, and 188 polymorphic bands of amplified fragment length polymorphisms, were used in order to identify the most relevant variables for the design of genetic interbreeding. Significant relationships between morphological and biochemical characters and anti-proliferative activity in cell lines were obtained, with five principal components for principal component analysis (SAS/ETS); variables were identified with a statistical significance (< 0.7 and Pearson values ≥ 0.7), with 80.81% of the accumulation of genetic variation and 110 genetic bands. Thirty-nine (39) variables were recovered using NTSYSpc software where 30 showed a Pearson correlation (> 0.5) and nine variables (< 0.05), Finally, using a cladistics analysis approach highlighted 65 genetic bands, in addition to color of the fruit, presence of thorns, bitter flavor, piriform and oblong shape, and also content of chlorophylls a and b, presence of cucurbitacins, and the IC50 effect of chayote extracts on the four cell lines.

Immunoproteomic and two-dimensional difference gel electrophoresis analysis of Arabidopsis dehydration response element-binding protein 1A (DREB1A)-transgenic potato.

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.

Allelochemical, eudesmane-type sesquiterpenoids from Inula falconeri.

We have identified through bioassay guided isolation an allelochemical, eudesmane-type sesquiterpeniod, 3beta-caffeoxyl-beta1,8alpha-dihydroxyeudesm-4(15)-ene (1), from an endemic plant species growing in the Himalayas. In our search for the bioactive subfraction, the hexane one was highly significant, showing 100% inhibition of lettuce seed growth at 100 ppm while other subfractions (chloroform, ethyl acetate, butanol and water) exhibited inhibitory to stimulatory allelopathic effects. The bioactive hexane subfraction was subjected to chromatographic techniques, using lettuce seeds (Lactuca sativa) as indicator species to reveal the bioactive allelopathic fraction. This resulted in the isolation of compound 1, whose structure was elucidated through NMR techniques. The compound presented 92.34% inhibitory effect on the growth of lettuce at 500 ppm. Further field level experiments may help develop an environmentally friendly herbicide from this lead.

Interaction of Scientific Knowledge and Implementation of the Multilateral Environment Agreements in Relation to Digital Sequence Information on Genetic Resources.

Integration of scientific knowledge into negotiations of the Multilateral Environment Agreements (MEAs) is crucial to effective implementation of those MEAs by ensuring uniformity in their terminology. Recent innovations in the field of biotechnology provoked a discussion over "Digital Sequence Information" (DSI) in fora of several MEAs. In the context of this discussion, the term DSI remains ambiguous and encompasses a wide range of concepts, including, at least, DNA/RNA base sequence data. We focused on how the term "DSI" was regarded in negotiations of the Convention on Biological Diversity and the International Treaty on Plant Genetic Resources for Food and Agriculture, analyzed the changes of terminology for DSI in the opinions or views of the Parties in the supreme decision-making bodies of these agreements from the perspective of the MEAs implementation. Based on these efforts we suggest the ways and means to support challenges regarding integration of scientific knowledge into MEAs.