Non-transgenic Lines Research Articles

Expression of a sphingomyelinase‐coding gene from Trichoderma harzianum conferred bacterial tolerance in tobacco

AbstractDiseases caused by bacteria are an important and widespread constraint, occurring in almost all crops, including vegetables, pulses, cereals, ornamentals, fruit and forages. Although several strategies have been developed to obtain disease resistance in plants using genetic engineering, few studies have effectively demonstrated the control of bacterial diseases. It has previously been reported that a gene encoding a sphingomyelinase (ThSMase), identified in Trichoderma harzianum, is up‐regulated during biocontrol against phytopathogens, and this may have biotechnological applications. SMases are involved in multiple cellular functions, including the immune response against pathogens. We hypothesized that the expression of this gene from fungi in transgenic tobacco could generate resistance to plant pathogens. ThSMase was cloned under control of the double CaMV 35S promoter plus a leader sequence from alfalfa mosaic virus (AMV), and stably introduced and expressed in tobacco. Reverse transcription‐quantitative PCR analysis revealed that the ThSMase gene was expressed at similar levels in all transgenic lines tested. Our results showed no statistically significant difference in susceptibility after challenging transgenic and nontransgenic lines with the fungus Sclerotinia sclerotiorum. However, transgenic tobacco plants revealed a significant resistance to the bacterium Pseudomonas syringae pv. tabaci, and a tolerance to Xylella fastidiosa. Our results demonstrated the strong potential of ThSMase in biotechnological processes such as molecular breeding of the plants.

T-DNA integration and its effect on gene expression in dual Bt gene transgenic Populus ×euramericana cv. Neva

The integration of Bacillus thuringiensis (Bt) genes is often accompanied by unintended effects along with the improved resistance to targeted pests. The insertion information of transfer DNA (T-DNA) and the expression information of upstream and downstream genes are of great significance for related research on unexpected effects and molecular-level mechanisms. In this study, six dual Bt transgenic Populus × euramericana cv. Neva (poplar 107) lines were used as research objects. We determined growth and physiological indices, and characterized the T-DNA integration using next-generation sequencing technology. The transgenic and non-transgenic lines showed no significant difference in growth index. However, while insect resistance was enhanced, effects related to the integration sites of the Bt gene occurred. A total of 15 insertion sites were detected among the six transgenic lines, and T-DNA preferentially inserted into AT-rich regions of the poplar genome. RNA sequencing and weighted gene co-expression network analysis were used to explore the effects of T-DNA insertion on the gene expression of poplar 107. As a result, the metabolic pathways most affected by the Bt gene were starch and sucrose metabolism and pentose and gluconate conversion. The expression of exogenous Bt had a greater impact than the insertion position or copy number on poplar 107 gene expression, and the Cry1Ac toxin protein also played a major role. This study provides theoretical support for the cultivation and management of poplar 107 new varieties, and provides reference for Poplar insect resistance breeding and its molecular response to Bt gene.

TmNAS3 from Triticum monococum directly regulated by TmbHLH47 increases Fe content of wheat grain.

Biofortification is an effective way to enhance wheat grain Fe content. However, Fe overload inhibits the growth and development of wheat. In this work, the impact of Triticum monococcum nicotianamine synthase 3 (TmNAS3) on Fe accumulation in wheat grain was analyzed. Transgenic wheat expressing TmNAS3 was obtained via Agrobacterium-mediated transformation. The concentrations of Fe in the grains of two transgenic wheat lines were 62.42μg/g and 68.75μg/g, while that in the non-transgenic line (NT) was only 29.51μg/g. Exogenous Fe application induced the expression of natural resistance-associated macrophage protein 3 (NRAMP3), NRAMP6, yellow stripe-like protein 3 (YSL3), YSL6, and vacuolar iron transporter 2 in transgenic wheat. The transcription factor that bound to the TmNAS3 promoter was identified, and the findings suggested that TmbHLH47 directly interacted and promoted the transcription of TmNAS3. Moreover, TmbHLH47 was observed to bind directly to the G-box in TmNAS3 promoter and regulated the transcriptional level of TmNAS3. Our findings contribute a TmbHLH47/TmNAS3 transcriptional pathway and thereby provide a potential strategy for improving the Fe concentration of wheat through genetic engineering.

Identification Transgenic DNA of Transformed <i>Arabidopsis</i> Using PCR and Southern Blot

For identification of transgenic DNA of Arabidopsis plant material, the followed methods are transformation and growing of seeds after bleaching them for sterilization should be performed then using polymerase chain reaction (PCR) for amplification the gene before making gel electrophoresis to be ready for southern blot analysis, this method is possible because the length of the given primers, there are slightly modifications in the methods as ligating PCR technique developed for amplification of the unknown DNA after extracting it to determine its transgenicity. The methods were specific and reproducible for many different plants from transgenic and non-transgenic cell lines. Furthermore, the number of products of PCR result can be considered as a good estimation of transgenic DNA, during comparison to southern blot analysis, the results obtained by the PCR give information about DNA of Arabidopsis, applications of the approach to Arabidopsis plants after transformation with an Agrobacterium-mediated transformation will be described.

Molecular and morphological evaluation of transgenic Persian walnut plants harboring Fld gene under osmotic stress condition.

Soil drought stress is a limiting factor of productivity in walnut (Juglans regia L). Ferredoxin (Fd) level decreases under adverse environmental stress. Functional replacement of decreased Fd by Fld (Flavodoxin) had been shown to have protective effect under abiotic stress condition. This study aimed to evaluate four transgenic lines (L3, L4, L13 and L17) along with non-transgenic line under three osmotic stresses levels (0, 10 and 12% PEG). This experiment carried out based on a completely randomized design with four replications. To confirm that the Fld gene is successfully integrated into the walnut genome, PCR and dot blot analysis were carried out. The transgenic lines of walnut expressing Fld displayed increased tolerance to osmotic stress at 10 and 12% PEG condition. Lines expressing Fld exhibited increasing tolerance to drought stress and maintained health of plants under osmotic conditions. Results of real time PCR showed that expression level of Fld gene in L4 was higher than the others. Among transgenic lines, L4 was more tolerant than other lines under osmotic stress. These findings indicate that expression of Fld gene can increase tolerance to osmotic stress in Persian walnut and is useful tool for walnut production in arid and semi-arid regions.

DNA Methylation Level Changes in Transgenic Chinese Cabbage (Brassica rapa ssp. pekinensis) Plants and Their Effects on Corresponding Gene Expression Patterns.

Plant tissue culture is an in vitro technique used to manipulate cells, tissues, or organs, and plays an important role in genetic transformation. However, plants cultured in vitro often exhibit unintended genetic and epigenetic variations. Since it is important to secure the stability of endogenous and exogenous gene expressions in transgenic plants, it is preferable to avoid the occurrence of such variations. In this study, we focused on epigenetic variations, exclusively on methylation level changes of DNA, in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) plants. To detect these methylation level changes of DNA, bisulfite sequencing was performed and the obtained sequences were compared with the ‘CT001’ reference genome. Differentially methylated regions (DMRs) of DNA between the non-transgenic and transgenic lines were detected by bisulfite sequencing, and ten DMRs located in exonic regions were identified. The regions with methylation variations that were inherited and consistently maintained in the next generation lines were selected and validated. We also analyzed the relationship between methylation status and expression levels of transformant-conserved DMR (TCD) genes by quantitative reverse transcription-PCR. These results suggested that the changes in methylation levels of these DMRs might have been related to the plant transformation process, affecting subsequent gene expression. Our findings can be used in fundamental research on methylation variations in transgenic plants and suggest that these variations affect the expression of the associated genes.

Gene Transformation by Chitosan Nanoparticle to Enhance Fatty Acid Production in Zea mays (L.)

Zea mays is an important crop and an essential source of fatty acids. Hence, increasing and adding new fatty acids led to the enhancement of these properties. Transformation of external Acetyl-CoA gene (Aco) can enhance fatty acid components, as ACo is expressed into Acetyl-CoA carboxylase (ACCase) enzyme, which is the first essential step in the fatty acid production process. Chitosan nanoparticles are safe and fast polymer nanoparticles that are applied for gene transformation. Conventional PCR was performed for the detection of the ACo gene in both transgenic and nontransgenic maize lines. The results confirm the presence of the gene in the transgenic lines and absence in non-transgenic lines. The Gas chromatography-mass spectrometry (GC-MS) analysis for fatty acid contents in transgenic and non-transgenic maize lines showed an increase in fatty acid contents in transgenic lines compared to non-transgenic ones. Besides, the transgenic maize’s lines produced extra new fatty acids not found in non-transgenic ones.

Tolerance of Pima and Upland cotton to trifloxysulfuron (Envoke) herbicide under field conditions

Trifloxysulfuron (Envoke) is an acetolactate synthase-inhibitor herbicide and can be used to control many broadleaf weeds and nutsedges in cotton production. However, there is a lack of information on genotypic variation in response to the herbicide. In this field study, 60 Pima (Gossypium barbadense L.) lines, 122 Upland (G. hirsutum L.) lines, and 9 Upland × Pima segregating populations were divided into five tests (18A, 18B, 18G, 18RB, and 18HQ) to evaluate trifloxysulfuron tolerance at the 7-true leaf stage (42 days after planting) under the same field conditions in 2018. Across the five tests, Pima cotton genotypes tested in this study did not show any visual crop injury based on percentage of plants with chlorosis at 6 days after treatment (DAT), indicating consistent and high levels of trifloxysulfuron tolerance. However, the response to trifloxysulfuron within Upland cotton is highly variable. While Upland cotton is overall more sensitive to trifloxysulfuron with crop injury up to 80% than Pima cotton, 19 lines had injury below 5% including one line with no visual injury, and 19 lines had injury between 5% and 10%. In test 18HQ with 15 transgenic Upland cultivars and 17 non-transgenic Upland lines, the analysis of variance detected a significant genotypic difference. The broad-sense heritability estimates for trifloxysulfuron tolerance based on crop injury at 6 DAT was 0.555, suggesting that trifloxysulfuron tolerance in Upland cotton is moderately heritable. This study represents the first report that Pima cotton and many Upland cotton lines are highly tolerant to trifloxysulfuron with no or little crop injury.

Overexpression of antisense phosphatase 2C affords cold resistance in hybrid Populus davidiana × Populus bolleana.

Overexpression of the abiotic and biotic stress-resistance genes of the plant signaling pathway is well known for its significant role in the regulation of plant growth and enhancement of the productivity of agricultural land under changing climatic conditions. This research aimed to clone Populus davidiana × Populus bolleana PP2C (PdPP2C) gene and analyze its structure and function, and downregulate PdPP2C by overexpression of its antisense PdPP2C (AS-PdPP2C) gene for enhancing cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. PdPP2C was cloned and transformed to identify its function, and its antisense was overexpressed via downregulation to increase the cold resistance in transgenic lines of hybrid P. davidiana × P. bolleana. Antisense inhibition of protein phosphatase 2C accelerates the cold acclimation of Poplar (P. davidiana × P. bolleana) gene in terms of antifreeze. PdPP2C was expressed in the roots, stems, and leaves of P. davidiana × P. bolleana, and the expression was higher in the leaves. The expression of PdPP2C was also significantly downregulated at low-temperature (0°C and 4°C) stress. The relative conductivity and malondialdehyde content of non-transgenic lines were higher than those of AS-PdPP2C lines after 2days of cold treatment at - 1°C. The leaves of the transgenic lines were not wilted and showed no chlorosis compared with those of the non-transgenic lines. The AS-PdPP2C transgenic lines also showed higher freezing resistance than the non-transgenic lines. AS-PdPP2C participated in the regulation of freezing resistance.

Successful use of Nicotiana tabacum hairy roots for the recombinant production of Cecropin A peptide.

Cecropin A, as an antimicrobial peptide (AMP), is possible to use in medical and agricultural fields as a new and safe biocontrol agent. Therefore, it is highly necessary to find a cost-effective and scalable approach to generate a large scale of it. In this research, the Agrobacterium rhizogenes strain ATCC 15834 was used to transfer the Cecropin A gene to the Nicotiana tabacum. After confirmation of transgenic hairy roots, the antibacterial activity of purified Cecropin A peptide was measured using the agar gel diffusion method. Successful transforming of Cecropin A was confirmed at the RNA and protein levels in hairy root cells using RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The highest Cecropin A amount was detected in line 4 of the transgenic lines using ELISA in comparison with the nontransgenic line. Subsequently, the antimicrobial activity of Cecropin A extracted from line 4 showed the highest inhibition activity against Aspergillus niger. Besides, this activity was stable against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans pathogens after 7 days. The recombinant production of Cecropin A AMP had a yield of 63.81μg/g of fresh weight. According to a significant yield, this system can be used to produce the Cecropin A peptide for pharmacological and food science applications.

Expression of a DREB 5-A subgroup transcription factor gene from Ricinus communis (RcDREB1) enhanced growth, drought tolerance and pollen viability in tobacco

Climate change has increased the frequency of long periods of drought, affecting crop cultivation worldwide. Losses due to water stress exceed 10% of world production of major crops, reaching three-quarters of production areas, with severe economic losses. Therefore, the generation of environmental stress-tolerant genotypes that are more efficient in water use is extremely important. We have previously isolated and characterized a DREB transcription factor coding gene, named RcDREB1, from castor bean (Ricinus communis L.), which probably belongs to the CBF/DREB subfamily subgroup A-5. Aiming to develop drought-tolerant lines, we have stably introduced and expressed the RcDREB1 transgene into tobacco. Transgenic lines have revealed an enhanced drought tolerance. Genetically modified lines cultivated under water deficit presented a higher photosynthetic rate, stomatal conductance, leaf water potential and leaf water content when compared to the control. Transgenic lines revealed lower transpiration rates. In addition, biometric analyses showed that transgenic lines cultivated under water stress presented higher biomass, higher fresh and dry weight and higher plant height than the non-transgenic lines. After re-watering, transgenic lines recovered faster than non-transgenic plants. Moreover, pollen grains from transgenic plants revealed a remarkable increase in viability after exposure to heat (38 °C) and desiccation stresses. The results presented here will be the foundation for production of commercial crops that are more tolerant to environmental stresses and long-life pollen grains, increasing pollination and in consequence, productivity. A DREB gene isolated from castor bean was expressed in tobacco plants. Genically modified plants presented a remarkable tolerance to water-deific stress and pollens were more tolerance to high temperatures and dehydration. These results paved the way for the development of drought tolerant and high yield crops.

Pollen Flow of Winter Triticale (x Triticosecale Wittmack) Investigated with Transgenic Line Expressing β-Glucuronidase Gene

A transgenic winter triticale line expressing the uidA gene, encoding β-glucuronidase, was used to assess the pollen flow in field experiments over two consecutive vegetation seasons in central Poland. The experimental design included two variants of mixed transgenic and non-transgenic lines. Pollen grains were collected using passive traps located at 0, 10, 30, 60 and 85 m from the transgenic line. GM pollen grains were detected histochemically by staining with x-Gluc. A positive effect of temperature increase, as well as the strength and direction of the wind on the number and spread of pollen grains was observed. Regardless of the experiment year and variant, only few pollen grains were observed at a distance of 85 m. In the first year of the study the amount of pollen grains at 85 m was 300-fold lower than at the source and 140-fold lower in the second year. The number of transgenic pollen grains was two times lower when the field with the transgenic triticale was surrounded by a non-transgenic line, compared to an empty field. On the basis of the obtained results, we suggest 100 m as the distance for triticale pollen migration, although longer flight incidents are possible in extreme atmospheric conditions.

Development of broad-spectrum and sustainable resistance in cotton against major insects through the combination of Bt and plant lectin genes.

Second generation Btinsecticidaltoxinin comibination with Allium sativum leaf agglutinin gene has been successfully expressed in cotton to develop sustainable resistance against major chewing and sucking insects. The first evidence of using the Second-generationBt gene in combination with Allium sativumplant lectin to develop sustainable resistance against chewing and sucking insects has been successfully addressed in the current study. Excessive use of Bt δ-endotoxins in the field is delimiting its insecticidal potential. Second-generation Bt Vip3Aa could be the possible alternative because it does not share midgut receptor sites with any known cry proteins. Insecticidal potential of plant lectins against whitefly remains to be evaluated. In this study, codon-optimized synthetic Bt Vip3Aa gene under CaMV35S promoter and Allium sativum leaf agglutinin gene under phloem-specific promoter were transformed in a local cotton variety. Initial screening of putative transgenic cotton plants was done through amplification, histochemical staining and immunostrip assay. The mRNA expression of Vip3Aa gene was increased to be ninefold in transgenic cotton line L6P3 than non-transgenic control while ASAL expression was found to be fivefold higher in transgenic line L34P2 as compared to non-transgenic control. The maximum Vip3Aa concentration was observed in transgenic line L6P3. Two copy numbers in homozygous form at chromosome number 9 and one copy number in hemizygous form at chromosome number 10 was observed in transgenic line L6P3 through fluorescent in situ hybridization. Significant variation was observed in transgenic cotton lines for morphological characteristics, whereas physiological parameters of plants and fiber characteristics (as assessed by scanning electron microscopic) remained comparable in transgenic and non-transgenic cotton lines. Leaf-detach bioassay showed that all the transgenic lines were significantly resistant to Helicoverpa armigera showing mortality rates between 78% and 100%. Similarly, up to 95% mortality of whiteflies was observed in transgenic cotton lines when compared with non-transgenic control lines.

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.

Expression of ice recrystallization inhibition protein in transgenic potato lines associated with reduced electrolyte leakage and efficient recovery post freezing injury

Potato (Solanum tuberosum L.) is considered to be frost-susceptible as short spells of frost can reduce the tuber yield and quality. Ice recrystallization inhibition (IRI) protein helps prevent growth of ice crystals in the cell apoplast during frost and help prevent damage associated with freezing stress. In this study, we investigated the in planta potential of Lolium perenne derived IRI3 transgene in improving the tolerance of transgenic potato lines for freezing stress. The codon optimized IRI3 transgene was introduced into potato cultivar Diamant through Agrobacterium mediated transformation. Three transgenic potato lines were successfully generated which were confirmed for transgene insertion and genomic integration by polymerase chain reaction and Southern blot. It was evident that the IRI3 transcript decreased in initial 24 h of cold stress treatment while the IRI3 mRNA expression up regulated in subsequent hours of cold treatment with maximum increase to 20 folds at 96 h post stress. A similar trend was also revealed in ion-leakage assay which showed that during cold stress, the transgenic potato lines depicted reduced ion leakage of 14–22% as compared to non-transgenic control plants. Further, the generated transgenic potato lines were tolerant to the frost spell in quarantine field conditions as compared to the non-transgenic potato lines. Additionally, the transgenic lines exhibited efficient recovery post frost injury in field conditions. The biochemical profiles of chlorophyll, proline and higher levels of antioxidant enzyme (superoxide dismutase, Catalase) activity and malondialdehyde levels showed that despite the phenotypic impact of low temperature, the transgenic potato lines quickly adjusted to maintain their cellular homeostasis post freezing stress by increasing the antioxidant defenses. This study suggests that up regulation of IRI3 transcript and regulatory network of cold stress response in transgenic potato lines improve frost tolerance and help stabilize yield in cultivated potato.

Genetic bioengineering of overexpressed guanylate binding protein family BmAtlastin-n enhances silkworm resistance to Nosema bombycis

Microsporidia are obligate single-celled eukaryote parasites. Microsporidian infection can cause large economic losses to beneficial insects such as silkworms and honey bees. Identification of resistance biomacromolecules and breeding of transgenic lines resistant to the microsporidian Nosema bombycis are important for disease management. We previously used transcriptome analysis to identify a guanylate binding protein family BmAtlastin-n gene that was significantly upregulated after Nosema bombycis infection, and we determined that the molecule was highly expressed in resistance-related tissues such as the midgut, fat body and the epidermis. The transgenic silkworm line overexpressing BmAtlastin-n biomolecules had economic characters similar to those of non-transgenic lines. The transgenic OE-BmAtlastin-n lines had significantly improved survival after microspore infection. We used RT-PCR and H&E staining to show that the number of spores in the transgenic lines was significantly lower than in the control lines. In this study, we identified a BmAtlastin-n macromolecule with resistance to N. bombycis and developed a transgenic line. The results improved understanding of the GBP protein family and provided biomacromolecule material for the treatment and prevention of microsporidia.

Generation and Starch Characterization of Non-Transgenic BEI and BEIIb Double Mutant Rice (Oryza sativa) with Ultra-High Level of Resistant Starch

BackgroundCereals high in resistant starch (RS) are gaining popularity, as their intake is thought to help manage diabetes and prediabetes. Number of patients suffering from diabetes is also increasing in Asian countries where people consume rice as a staple food, hence generation of practically growable high RS rice line has been anticipated. It is known that suppression of starch branching enzyme (BE) IIb increases RS content in cereals. To further increase RS content and for more practical use, we generated a non-transgenic be1 be2b double mutant rice (Oryza sativa) line, which completely lacked both proteins, by crossing a be1 mutant with a be2b mutant.ResultsThe be1 be2b mutant showed a decrease in intermediate amylopectin chains and an increase in long amylopectin chains compared with be2b. The amylose content of be1 be2b mutant (51.7%) was the highest among all pre-existing non-transgenic rice lines. To understand the effects of chewing cooked rice and cooking rice flour on RS content, RS content of mashed and un-mashed cooked rice as well as raw and gelatinized rice flour were measured using be1 be2b and its parent mutant lines. The RS contents of mashed cooked rice and raw rice flour of be1 be2b mutant (28.4% and 35.1%, respectively) were 3-fold higher than those of be2b mutant. Gel-filtration analyses of starch treated with digestive enzymes showed that the RS in be1 be2b mutant was composed of the degradation products of amylose and long amylopectin chains. Seed weight of be1 be2b mutant was approximately 60% of the wild type and rather heavier than that of be2b mutant.ConclusionsThe endosperm starch in be1 be2b double mutant rice were enriched with long amylopectin chains. This led to a great increase in RS content in cooked rice grains and rice flour in be1 be2b compared with be2b single mutant. be1 be2b generated in this study must serve as a good material for an ultra-high RS rice cultivar.

Spatial and temporal distribution of Bt proteins in Bt maize leaves

ABSTRACTTo learn the spatial and temporal distribution of Bt proteins in Bt maize leaves, Bt protein levels of Bt maize leaves and their corresponding non-transgenic lines were detected and analysed by the ELISA method. The results showed that Bt proteins were expressed in the leaves of Bt maize varieties during all periods of their growth, while no such Bt proteins were detected in the leaves of non-transgenic maize varieties. The range of leaf Bt proteins was 1.08∼5.81 µg.g−1 FW, which could effectively protect Bt maizes from the corn borer pest during their growth periods. Furthermore, the younger leaves of Bt maizes had significantly lower levels of Bt proteins than their older leaves. In most periods, Bt protein content of Bt maize leaves under drought stress was lower than that under normal growth conditions, but Bt protein content under nutrition stress was higher than that under normal growth conditions. The results provided a full pattern of the spatial and temporal distribution of Bt proteins in Bt maize leaves.

Engineered Ripening-Specific Accumulation of Polyamines Spermidine and Spermine in Tomato Fruit Upregulates Clustered C/D Box snoRNA Gene Transcripts in Concert with Ribosomal RNA Biogenesis in the Red Ripe Fruit.

Ripening of tomato fruit leads, in general, to a sequential decrease in the endogenous levels of polyamines spermidine (SPD) and spermine (SPM), while the trend for the diamine putrescine (PUT) levels is generally an initial decrease, followed by a substantial increase, and thereafter reaching high levels at the red ripe fruit stage. However, genetic engineering fruit-specific expression of heterologous yeast S-adenosylmethionine (SAM) decarboxylase in tomato has been found to result in a high accumulation of SPD and SPM at the cost of PUT. This system enabled a genetic approach to determine the impact of increased endogenous levels of biogenic amines SPD and SPM in tomato (579HO transgenic line) and on the biogenesis, transcription, processing, and stability of ribosomal RNA (rRNA) genes in tomato fruit as compared with the non-transgenic 556AZ line. One major biogenetic process regulating transcription and processing of pre-mRNA complexes in the nucleus involves small nucleolar RNAs (snoRNAs). To determine the effect of high levels of SPD and SPM on these latter processes, we cloned, sequenced, and identified a box C/D snoRNA cluster in tomato, namely, SlSnoR12, SlU24a, Slz44a, and Slz132b. Similar to this snoRNA cluster housed on chromosome (Chr.) 6, two other noncoding C/D box genes, SlsnoR12.2 and SlU24b, with a 94% identity to those on Chr. 6 were found located on Chr. 3. We also found that other snoRNAs divisible into snoRNA subclusters A and B, separated by a uridine rich spacer, were decorated with other C/D box snoRNAs, namely, J10.3, Z131a/b, J10.1, and Z44a, followed by z132a, J11.3, z132b, U24, Z20, U24a, and J11. Several of these, for example, SlZ44a, Slz132b, and SlU24a share conserved sequences similar to those in Arabidopsis and rice. RNAseq analysis of high SPD/SPM transgenic tomatoes (579HO line) showed significant enrichment of RNA polymerases, ribosomal, and translational protein genes at the breaker+8 ripening stage as compared with the 556AZ control. Thus, these results indicate that SPD/SPM regulates snoRNA and rRNA expression directly or indirectly, in turn, affecting protein synthesis, metabolism, and other cellular activities in a positive manner.

Physiological responses and P5CS gene expression of transgenic oil palm plantlet induced by drought stress

Drought is one of the limiting factors in crop cultivation, such as in oil palm (Elaeis guineensis Jacq.). The transgenic approaches are expected to increase plant tolerance to drought stress and minimize low productivity when drought occurs. Proline is an osmoprotectant compound in plants which its biosynthesis involved the P5CS gene. The objective of this study was to evaluate the tolerance level of P5CS-transgenic oil palm to drought stress induced by polyethylene glycol 6000 (PEG-6000). In this present study, the transgenic and non-transgenic oil palms were treated by 0, 2, and 4% PEG-6000 under in vitro conditions. The experiment was arranged as a factorial completely randomized design with three replications. The drought level score, total chlorophyll content, carotenoids, and proline content, as well as P5CS gene expression in leaf tissues were observed at 7 and 14 days after stress treatments. The result showed that transgenic plantlets had a lower drought level score than those of non-transgenic lines. A concentration of 4% PEG-6000 treatment reduced the total chlorophyll and carotenoids contents than that of 2% concentration in non-transgenic plantlets at 7 and 14 day after treatments (DAT). In addition, proline content and P5CS gene expression level in transgenic had been significantly increased during stress treatment. Based on these results, it can be concluded that the P5CS transgene increased the drought stress tolerance of oil palm.