Biotechnological Tools to Achieve 21st Century Agriculture and Forestry

The complete netting of orchards is one strategy to protect fruit trees from pest and pathogen damage by reducing insect movement. When the cultivated trees were derived from genetic engineering (GE), reduced pollinator movement may also reduce outcrossing to cultivated or wild non-GE trees. We report on a field study over four years in a plot of apple tress supplied with insect side nets and covered with hail nets that were closed from shortly before flowering to harvest. A reduced number of arthropods in general, and large bees in particular, were recorded inside the netted plot compared with outside. However, wild bees colonized the plot before the net was closed and built up populations inside. An additional experiment demonstrated that small bees were able to cross the hail net. While the nets were effective in excluding large bees as active pollen vectors, the proportion of small bees acting as such remained unquantified. Furthermore, a companion study showed occasional cross-pollination events through the netting. For the field release of GE apple trees, acceptable levels of outcrossing thus need to be defined. Full article

This paper is the first to report the soluble carbohydrate content at harvest for eight Transylvanian potato cultivars: Christian, Cumidava, Kronstadt, Riviera, Roclas, Rustic, Tampa and Zamolxis. The aim of this study is to explore the soluble carbohydrate composition of the above-mentioned cultivars, since such quantitative information is important for breeding programs, consumers and processing units. High performance liquid chromatography was used for analysis, separations being achieved using a Prominence Shimadzu system with a refractive index detector, under isocratic conditions with a mobile phase consisting of acetonitrile: water (80:20%) delivered at 1 mL/min; baseline separations of the target analytes were accomplished with an EC 250/4 Nucleodur 100–5 NH₂ RP column in less than 10 min. The carbohydrate concentrations were found to range from 24.03 mg/100 g (Zamolxis) to 76.58 mg/100 g (Riviera) for fructose, while the corresponding range was from 52.78 mg/100 g (Zamolxis) to 232.97 mg/100 g (Riviera) for glucose and from 238.41 mg/100 g (Zamolxis) to 378.45 (Cumidava) for sucrose. Chromatographic data were then subjected to chemometric analysis; the association of these complementary techniques allowed a fast selection of cultivars with low-reducing carbohydrate content for food processing purposes—the cultivars Zamolxis, Kronstadt, Christian and Roclas were outlined exhibiting both the lowest reducing carbohydrate content and the lowest sucrose content. Full article

Selection of reference genes (RGs) for normalization of PCR-gene expression data includes two crucial steps: determination of the between-sample transcriptionally more stable genes, and subsequent choosing of the most suitable genes as internal controls. Both steps can be carried out through generally accepted strategies, each having different strengths and weaknesses. The present study proposes reinforcement of the normalization of gene expression data by integrating analytical revision at critical steps of those accepted procedures. In vitro olive adventitious rooting was used as an experimental system. Candidate RGs were ranked according to transcriptional stability according to several methods. An algorithm of one of these programs (GeNorm) was adapted to allow for partial automatization of RG selection for any strategy of transcriptional-gene stability ordering. In order to choose the more appropriate set of RGs, the achieved results were analytically revised, with special emphasis on biasing effects such as co-regulation. The obtained putative RG sets were also tested for cases restricted to fewer variables. The set formed by the genes H2B, OUB and ACT is valid for normalization in transcriptional studies on olive microshoot rooting when comparing treatments, time points and assays. Such internal reference is now available for wider expression studies on any target gene in similar biological systems. The overall methodology aims to constitute a guide for general application. Full article

Sucrose is the primary form of mobile photoassimilates, and its level is regulated by sucrose-phosphate synthase (SPS) in plants. Increasing in the SPS activity was accompanied by an increase in sucrose accumulation. This study was designed to examine the effect of the overexpression sugarcane SoSPS1 gene on sucrose metabolizing enzymes, growth, and grain yield of indica rice. The SoSPS1 gene was constructed in a binary vector under the control of a rice ubiquitin promoter and transformed into indica rice using an Agrobacterium vector. Five lines of transgenic rice were selected to develop homozygous transgenic lines and used for analysis. The overexpression of the SoSPS1 gene significantly increased the transcript and protein levels, followed by increasing in SPS activity and sucrose content in the leaves of the transgenic rice lines. Moreover, the activity of soluble acid invertase (SAI) was elevated rather than sucrose synthase (SuSy) in the transgenic lines. The increase in the sucrose-degrading enzymes leads to an increase in plant growth and development. The plant height and number of tillers were significantly higher in the transgenic line compared to non-transgenic (NT) rice. In addition, the amylose content, the number of seeds per panicle, and the weight of 1000 grains of seed, including dry biomass weight, were increased in the transgenic lines. The results indicated that enhancement of SPS activity, as well as sucrose content, provides a higher carbon partitioning for higher growth and productivity of the transgenic rice lines. Full article

With the intensification of coastal erosion, damage to coastal shelterbelts has gradually increased. Arbuscular mycorrhizal fungi (AMF) can improve the salinity tolerance and productivity of plants in saline–alkali soils using various strategies including nutrient uptake, osmotic regulation, soil shaping, etc. Thus, the application of AMF to alleviate the impacts of salinization for these shelterbelts has become a research hotspot. For this study, we investigated the effects of inoculation with different AMF strains on the growth and nitrogen (N) utilization of Gleditsia sinensis Lam. and Zelkova serrata (Thunb.) Makino leaves under different salt concentrations. As the salt concentration increased, the growth rates and leaf areas of the autoclaved AMF inoculant (CK) treatment exhibited a decreasing trend for both G. sinensis and Z. serrata, while Funneliformis mosseae (FM) and Corymbiglomus tortuosum (CT) treatments weakened this trend. Between them, on average, FM increased the G. sinensis height growth rate by 396.9%, ground diameter growth rate by 99.0%, and Z. serrata leaf area by 29.1%. At a salt concentration of 150 mM, the chlorophyll content and nitrate reductase activities of leaves under the FM treatment for both tree species were significantly higher than for CK, with an average increase in chlorophyll content of 106.1% and nitrate reductase activities by 74.6%. Moreover, the AMF inoculation significantly reduced the leaf N content and photosynthetic N-use efficiency of G. sinensis in contrast to Z. serrata. Further, in contrast to G. sinensis, the photosynthetic N-use efficiency was significantly positively correlated with the growth rate and leaf area of Z. serrata. Meanwhile, the nitrate reductase activity contributed most to the growth rate and leaf area of Z. serrata. Our results suggest that the issues with coastal shelterbelts might be effectively alleviated through appropriate AMF–plant combinations, which is of great significance for the optimization of forestry production. Full article

Towards the improvement of plant productivity in saline–alkali soils, the application of arbuscular mycorrhizal fungi (AMF) is an intensive topic of research. For this study, three inoculation treatments, namely, autoclaved AMF inocula (CK), Funneliformis mosseae (FM), and Corymbiglomus tortuosum (CT), and four NaCl levels, namely, 0, 50, 100, and 150 mM were established to investigate the growth and physiological responses of mycorrhizal Gleditsia sinensis Lam. root systems to increase salinity through root dry weight, morphology, nutrient content, and physiology, and soil nutrient content. As NaCl levels increased, root dry weight, morphology, and nutrient content under the CK treatment exhibited a downward trend, while FM and CT treatments weakened this trend and significantly improved root dry weight and morphology, which increased by more than 200%. Under high NaCl levels, root activity under the FM treatment was significantly higher than that under the CK, with an average increase of 120.86%. In contrast to the activity of nitrate reductase, niacinamide adenine dinucleotide oxidase activity under CK was significantly less than that in FM and CT treatments. Moreover, inoculation with AMF significantly affected soil alkali-hydrolyzable nitrogen (AN), total nitrogen (TN), and phosphorus (TP), while NaCl had no significant impact on soil nutrients. Further, both soil salinity and mycorrhizal colonization rate had significant direct effects on root growth. However, soil salinity primarily influenced root growth through indirect effects on root nitrogen content, while mycorrhizal colonization rate indirectly impacted root nitrate reductase activity, and root nitrogen and phosphorus content. Our results suggested that the use of suitable AMF (e.g., Funneliformis mosseae) might effectively improve the currently unfavorable situation of economic tree species production on land with saline soils, which may greatly optimize the utility of these areas. Full article

In this study, we compare two rapid cryopreservation (−196 °C) procedures, droplet-vitrification and encapsulation-dehydration for rose (Rosa × hybrida L., cultivars ‘Ioana’, ‘Mariana’, ‘Vulcan’). Significant factors for cryopreservation, such as sucrose concentration during osmoprotection, treatment duration with plant vitrification solution 2 (PVS2) in droplet-vitrification, duration of air desiccation and moisture content of alginate beads in encapsulation-dehydration, were investigated. In addition, the morphogenetic response to in vitro culture and to liquid nitrogen storage and the content in photosynthetic pigments have been assessed. The in vitro cultures were initiated from plant material originating from field collection. The highest regeneration frequencies were obtained for cv. ‘Vulcan’ in both of the cryopreservation procedures tested, 72% in droplet-vitrification and 65% following encapsulation-dehydration. The morphogenetic response (multiplication index and height of shoots) to liquid nitrogen storage was direct multiple shoot formation per initial shoot tip for all genotypes. The content in chlorophyll a and b was statistically comparable in plant material resulting from cryopreserved and non-cryopreserved shoot tips in all cultivars. The findings expand the information on Rosa‘s response to in vitro culture conditions and cryopreservation, providing protocols with a high regeneration capacity for the storage of genotypes with high ornamental value. Full article

The effects of soilless substrate-based versus soil cultivation on overall fruit quality and yield in tomato (Solanum lycopersicum) were studied using the tomato cv. Zhonghua Lvbao. Experiments for tomato soilless cultivation were carried out under greenhouse conditions. Plant growth, fruit quality and yield, and physiologic traits were observed. RNA-seq and RT-PCR, as well as metabolomic analyses were performed to examine the expressed genes and metabolites under soilless substrate cultivation. The results showed that the plant height, stem diameter, and chlorophyll contents of tomato under substrate-based cultivation were increased by 37.3%, 19.8%, and 15.3%, respectively, compared with soil cultivation system. Leaf photosynthetic and transpiration rates, stomatal conductance, and root vitality of tomato, under substrate-based cultivation, increased by 29.0%, 21.2%, 43.9%, and 84.5%, respectively, compared with soil cultivation. The yield reached 7177.5 kg/667 m², and the relative yield increased by 10.1%, compared with soil cultivation. The contents of total soluble sugar, soluble solids, and vitamin C increased by 35.7%, 19.7%, and 18.2%, respectively, higher than those of soil cultivation in tomato fruits, while nitrate content and titratable acid decreased by 29.4% and 11.8%, respectively. Therefore, substrate-based-cultivation can increase production and improve tomato fruit quality and taste. We examined the expressed genes and metabolites to explore the molecular mechanism of plant growth and overall fruit quality improvement in substrate-based cultivation. A total of 476 differentially expressed genes were identified by transcriptomes profiling, of which 321 and 155 were significantly up- and down-regulated, respectively. The results of metabolomics analysis showed that 441 metabolites were detected, where 24 and 36 metabolites were up- and down-regulated, respectively. By combining analyses of transcriptomic and metabolic groups, genes and metabolites related to the fruit quality were mainly concentrated in the vitamin B6/ascorbic acid/aldonic acidmetabolism, and glycerophospholipid metabolic pathways. Therefore, substrate-based cultivation can elevate vitamin and soluble sugar contents and the expression of fruit flavor related genes, which lays an initial background for exploring the mechanism of substrate-based cultivation, in order to improve the quality of tomato in the future. Full article

Sunflower is an important oilseed crop in which the biochemical pathways leading to seed oil synthesis and accumulation have been widely studied. However, how these pathways are regulated is less well understood. The WRINKLED1 (WRI1) transcription factor is considered a key regulator in the control of triacylglycerol biosynthesis, acting through the AW box binding element (CNTNG(N)₇CG). Here, we identified the sunflower WRI1 gene and characterized its activity in electrophoretic mobility shift assays. We studied its role as a co-regulator of sunflower genes involved in plastidial fatty acid synthesis. Sunflower WRI1-targets included genes encoding the pyruvate dehydrogenase complex, the α-CT and BCCP genes, genes encoding ACPs and the fatty acid synthase complex, together with the FATA1 gene. As such, sunflower WRI1 regulates genes involved in seed plastidial fatty acid biosynthesis in a coordinated manner, establishing a WRI1 push and pull strategy that drives oleic acid synthesis for its export into the cytosol. We also determined the base bias at the N positions in the active sunflower AW box motif. The sunflower AW box is sequence-sensitive at the non-conserved positions, enabling WRI1-binding. Moreover, sunflower WRI1 could bind to a non-canonical AW-box motif, opening the possibility of searching for new target genes. Full article

Myelocytomatosis (MYC) transcription factors play a core regulator in the jasmonic acid signaling pathway, which regulates the secondary laticifer differentiation and rubber biosynthesis in rubber tree (Hevea brasiliensis). However, there are currently no reports on the MYC gene family in rubber trees, an important industrial raw material crop worldwide. In the present study, 32 HblMYCs were isolated and identified. The diversity in gene structure and presence of various cis-regulatory elements in promotors suggest that HblMYCs participate in various biological processes. Based on the expression patterns in the cambium region and laticifer in, respectively, response to coronatine (COR) and tapping, and the phylogenetic relationship with the MYCs that have been functionally identified in other plants, the HblMYC24 and HblMYC30 may be related to laticifer differentiation while the HblMYC6, HblMYC11 and HblMYC15, as well as HblMYC16 and HblMYC21, may positively regulate rubber biosynthesis. The results provide a foundation for understanding the molecular mechanism of jasmonate signaling in regulating laticifer differentiation and rubber biosynthesis in rubber tree. Full article