Untransformed Control Research Articles

Overexpression of EaALDH7, an aldehyde dehydrogenase gene from Erianthus arundinaceus enhances salinity tolerance in transgenic sugarcane (Saccharum spp. Hybrid)

Aldehyde Dehydrogenases (ALDH), a group of enzymes, are associated with the detoxification of aldehydes, produced in plants during abiotic stress conditions. Salinity remains a pivotal abiotic challenge that poses a significant threat to cultivation and yield of sugarcane. In this study, an Aldehyde dehydrogenase gene (EaALDH7) from Erianthus arundinaceus was overexpressed in the commercial sugarcane hybrid cultivar Co 86032. The transgenic lines were evaluated at different NaCl concentrations ranging from 0 mM to 200 mM for various morpho-physiological and biochemical parameters. The control plants, subjected to salinity stress condition, exhibited morphological changes in protoxylem, metaxylem, pericycle and pith whereas the transgenic events were on par with plants under regular irrigation. The overexpressing (OE) lines showed less cell membrane injury and improved photosynthetic rate, transpiration rate, and stomatal conductance than the untransformed control plants under stress conditions. Elevated proline content, higher activity of enzymatic antioxidants such as sodium dismutase (SOD), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX) and low level of malondialdehyde MDA and hydrogen peroxide (H2O2) in the transgenic lines. The analysis of EaALDH7 expression revealed a significant upregulation in the transgenic lines compared to that of the untransformed control during salt stress conditions. The current study highlights the potentials of EaALDH7 gene in producing salinity-tolerant sugarcane cultivars.

Engineered Chlorella vulgaris improves bioethanol production and promises prebiotic application.

Microalgal biomass for biofuel production, integration into functional food, and feed supplementation has generated substantial interest worldwide due to its high growth rate, non-competitiveness for agronomic land, ease of cultivation in containments, and presence of several bioactive molecules. In this study, genetic engineering tools were employed to develop transgenic lines of freshwater microalga Chlorella vulgaris with a higher starch content, by up-regulating ADP-glucose pyrophosphorylase (AGPase), which is a rate-limiting enzyme in starch biosynthesis. Expression of the Escherichia coli glgC (AGPase homolog) gene in C. vulgaris led to an increase in total carbohydrate content up to 45.1% (dry cell weight, DCW) in the transgenic line as compared to 34.2% (DCW) in the untransformed control. The starch content improved up to 16% (DCW) in the transgenic alga compared to 10% (DCW) in the control. However, the content of total lipid, carotenoid, and chlorophyll decreased differentially in the transgenic lines. The carbohydrate-rich biomass from the transgenic algal line was used to produce bioethanol via yeast fermentation, which resulted in a higher ethanol yield of 82.82mg/L as compared to 54.41mg/L from the untransformed control. The in vitro digestibility of the transgenic algal starch revealed a resistant starch content of up to 7% of total starch. Faster growth of four probiotic bacterial species along with a lowering of the pH of the growth medium indicated transgenic alga to exert a positive prebiotic effect. Taken together, the study documents the utilization of genetically engineered C. vulgaris with enriched carbohydrates as bioethanol feedstock and functional food ingredients.

Enhancing drought tolerance in chilli pepper through AdDjSKI‐mediated modulation of ABA sensitivity, photosynthetic preservation, and ROS scavenging

AbstractDrought stress threatens the productivity of numerous crops, including chilli pepper (Capsicum annuum). DnaJ proteins are known to play a protective role against a wide range of abiotic stresses. This study investigates the regulatory mechanism of the chloroplast‐targeted chaperone protein AdDjSKI, derived from wild peanut (Arachis diogoi), in enhancing drought tolerance in chilli peppers. Overexpressing AdDjSKI in chilli plants increased chlorophyll content, reflected in the maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm) compared with untransformed control (UC) plants. This enhancement coincided with the upregulated expression of PSII‐related genes. Our subsequent investigations revealed that transgenic chilli pepper plants expressing AdDjSKI showed reduced accumulation of superoxide and hydrogen peroxide and, consequently, lower malondialdehyde levels and decreased relative electrolyte leakage percentage compared with UC plants. The mitigation of ROS‐mediated oxidative damage was facilitated by heightened activities of antioxidant enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, coinciding with the upregulation of the expression of associated antioxidant genes. Additionally, our observations revealed that the ectopic expression of the AdDjSKI protein in chilli pepper plants resulted in diminished ABA sensitivity, consequently promoting seed germination in comparison with UC plants under different concentrations of ABA. All of these collectively contributed to enhancing drought tolerance in transgenic chilli plants with improved root systems when compared with UC plants. Overall, our study highlights AdDjSKI as a promising biotechnological solution for enhancing drought tolerance in chilli peppers, addressing the growing global demand for this economically valuable crop.

Response of sugarcane plants with modified cytokinin homeostasis under water deficit conditions

Cytokinins (CK) are one of the main phytohormones involved in plant growth and development and play a role in plant adaptation to stress. In this study, the response to water deficit stress was investigated in sugarcane (Saccharum spp. hybrids) plants after exogenous application of CK and partial silencing of endogenous cytokinin oxidase/dehydrogenase (CKX) gene expression in transgenic plants. The CKX enzyme is responsible for CK breakdown in plant cells. A foliar drench of N6-benzyladenin on leaves (100 μM) resulted in plants that survived more than 10 days of water deficit stress compared with the untreated control plants, which displayed senescence and leaf wilting after 10 days without water. Furthermore, after 10 days without water, the treated plants maintained a high efficiency of the photosystem II (PSII) and displayed higher levels of chlorophyll and relative water content, and after 20 days without water, high catalase activity and proline content when compared with the untreated controls. In addition, sugarcane calli were transformed with a recombinant vector containing a 237 bp sugarcane ShCKX target sequence which included a conserved CK binding domain cloned in sense and antisense orientations. Partial silencing of CKX expression in the transgenic plants resulted in significantly lower salicylic acid levels after 21 days of deficit stress compared with untransformed controls. Stressed transgenic plants accumulated less H2O2, more antioxidants (superoxide dismutase and catalase) and proline when compared with the wild type (WT) plants. Moreover, the transgenic plants were able to retain high levels of chlorophyll and most maintained the efficiency of PSII after watering was stopped. The transgenic genotype with the highest level of CKX silencing displayed delayed leaf senescence and maintained high leaf water content under water deficit stress compared with the WT plants. Both the exogenous application of CK and the partial silencing of CKX expression enabled the plants to resprout after re-watering following the stress period. The results indicate that the manipulation of CK levels, either by increasing CK exogenously or deceasing their break down by partial silencing of the CKX gene, in sugarcane positively affects the response to water deficit stress through a variety of physiological mechanisms.

The Maize ZmBES1/BZR1-9 Transcription Factor Accelerates Flowering in Transgenic Arabidopsis and Rice.

In model plants, the BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) transcription factors play vital roles in regulating growth, development, and stimuli response. However, the roles of maize ZmBES1/BZR1 members are largely unknown. In this research, the ZmBES1/BZR1-9 gene was ectopically expressed in Arabidopsis and rice for the phenotyping of flowering. We found that the complementation and overexpression of ZmBES1/BZR1-9 in bes1-D mutant and wild type Arabidopsis both resulted in early flowering that was about 10 days shorter than in the untransformed control under long-day conditions. In addition, there was no difference in the rosette leaf number between all transgenic lines and the control. Subsequently, the ZmBES1/BZR1-9 gene was overexpressed in rice. It was found that overexpression lines of rice exhibited early flowering with heading dates that were 8 days shorter compared with untransformed plants. Moreover, the results of RNA-seq and qRT-PCR showed that five flowering-regulated genes, namely At2-MMP, AtPCC1, AtMYB56, AtPELPK1, and AtPRP10, were significantly up-regulated in all complementary and overexpressing lines of Arabidopsis. Meanwhile, the results of RNA-seq showed that 69 and 33 differentially expressed genes (DEGs) were up- and down-regulated in transgenic rice, respectively. Four flowering-related genes, namely OsGA20OX1, OsCCR19, OsBTBN19, and OsRNS4 were significantly up-regulated in transgenic lines. To sum up, our findings demonstrate that ZmBES1/BZR1-9 is involved in controlling flowering and provide insights into further underlying roles of BES1/BZR1s in regulating growth and development in crops.

Overexpression of MdFRK2 enhances apple drought resistance by promoting carbohydrate metabolism and root growth under drought stress

Soluble sugars function not only as the energy and structural blocks supporting plants, but also as osmoregulators and signal molecules during plant adaptation to water deficit. Here, we investigated drought resistance in transgenic apple (Malus × domestica) overexpressing MdFRK2, a key gene regulating fructose content and sugar metabolism. There is no obvious phenotypic difference between MdFRK2-overexpressing transgenic plants and WT plants under the well-watered condition. However, the transgenic plants and the grafted plants using MdFRK2-overexpressing rootstock exhibited improved tolerance to drought stress. Overexpression of MdFRK2 significantly promoted the growth of root system under drought stress. RNA sequencing showed that under drought stress, genes involved in sugar metabolism, transcription regulation, signal transduction or hormone metabolism were differentially expressed in MdFRK2 transgenic plants. Consistent with the gene expression profile, the activities of enzyme (SDH, FRK and NI) involved in sugar metabolism in the roots of MdFRK2 transgenic plants were significantly higher than those of untransformed control plants after drought stress. Under drought stress, overexpression of MdFRK2 promoted the accumulation of IAA, and decreased the contents of ABA and CK in apple root system. In conclusion, these results suggest that MdFRK2 can promote the growth of apple roots under drought stress by regulating sugar metabolism and accumulation, hormone metabolism and signal transduction, and then resist drought stress.

Field performance of switchgrass plants engineered for reduced recalcitrance.

Switchgrass (Panicum virgatum L.) is a promising perennial bioenergy crop that achieves high yields with relatively low nutrient and energy inputs. Modification of cell wall composition for reduced recalcitrance can lower the costs of deconstructing biomass to fermentable sugars and other intermediates. We have engineered overexpression of OsAT10, encoding a rice BAHD acyltransferase and QsuB, encoding dehydroshikimate dehydratase from Corynebacterium glutamicum, to enhance saccharification efficiency in switchgrass. These engineering strategies demonstrated low lignin content, low ferulic acid esters, and increased saccharification yield during greenhouse studies in switchgrass and other plant species. In this work, transgenic switchgrass plants overexpressing either OsAT10 or QsuB were tested in the field in Davis, California, USA for three growing seasons. No significant differences in the content of lignin and cell wall-bound p-coumaric acid or ferulic acid were detected in transgenic OsAT10 lines compared with the untransformed Alamo control variety. However, the transgenic overexpressing QsuB lines had increased biomass yield and slightly increased biomass saccharification properties compared to the control plants. This work demonstrates good performance of engineered plants in the field, and also shows that the cell wall changes in the greenhouse were not replicated in the field, emphasizing the need to validate engineered plants under relevant field conditions.

S‐adenosylmethionine synthase (BtSAMS) from Balsas teosinte confers resistance to peach aphids in Arabidopsis

AbstractThe purpose of this study was to clone S‐adenosylmethionine synthase (BtSAMS) from Balsas teosinte (Zea mays ssp. parviglumis) and investigate its function via heterologous expression in Arabidopsis plants. The BtSAMS coding sequence was cloned and its length is 1185 bp. The expression of BtSAMS in Z. mays ssp. parviglumis was a 12.2‐fold increase after aphid infestation. Transgenic Arabidopsis plants heterologously expressing BtSAMS were generated and their morphological characters were observed. A prolonged vegetative growth period was displayed by BtSAMS‐OX plants. The more and longer trichomes were observed on the upper surface of BtSAMS seedlings. The ethylene content in BtSAMS‐OX plants was not significantly different compared with the untransformed controls. These BtSAMS‐OX transgenic plants showed greater resistance to peach aphids compared with the untransformed control plants. Peach aphids appeared to prefer the untransformed controls in that aphids number reduced by more than 50% on BtSAMS plants compared to controls. When sprayed with the insecticide avi‐imidacloprid, the number of peach aphids decreased on BtSAMS‐OX plants and untransformed controls by 91.6% and 80.5%, respectively. Peach aphids also showed lower reproduction ability when forced to feed on the transgenic plants. The number of newly born nymphs on BtSAMS plants was less than 50% of that on the untransformed control. We therefore propose that BtSAMS may be a suitable candidate gene to confer resistance to peach aphids in plants.

Transcriptome Analysis of Heat Shock Factor C2a Over-Expressing Wheat Roots Reveals Ferroptosis-like Cell Death in Heat Stress Recovery.

Wheat (Triticum aestivum L.) growing areas in many regions of the world are subject to heat waves which are predicted to increase in frequency because of climate change. The engineering of crop plants can be a useful strategy to mitigate heat stress-caused yield losses. Previously, we have shown that heat shock factor subclass C (TaHsfC2a-B)-overexpression significantly increased the survival of heat-stressed wheat seedlings. Although previous studies have shown that the overexpression of Hsf genes enhanced the survival of plants under heat stress, the molecular mechanisms are largely unknown. To understand the underlying molecular mechanisms involved in this response, a comparative analysis of the root transcriptomes of untransformed control and TaHsfC2a-overexpressing wheat lines by RNA-sequencing have been performed. The results of RNA-sequencing indicated that the roots of TaHsfC2a-overexpressing wheat seedlings showed lower transcripts of hydrogen peroxide-producing peroxidases, which corresponds to the reduced accumulation of hydrogen peroxide along the roots. In addition, suites of genes from iron transport and nicotianamine-related gene ontology categories showed lower transcript abundance in the roots of TaHsfC2a-overexpressing wheat roots than in the untransformed control line following heat stress, which are in accordance with the reduction in iron accumulation in the roots of transgenic plants under heat stress. Overall, these results suggested the existence of ferroptosis-like cell death under heat stress in wheat roots, and that TaHsfC2a is a key player in this mechanism. To date, this is the first evidence to show that a Hsf gene plays a key role in ferroptosis under heat stress in plants. In future, the role of Hsf genes could be further studied on ferroptosis in plants to identify root-based marker genes to screen for heat-tolerant genotypes.

The coexpression of two desaturases provides an optimized reduction of saturates in camelina oil.

Reducing the saturate content of vegetable oils is key to increasing their utility and adoption as a feedstock for the production of biofuels. Expression of either the FAT5 16:0-CoA desaturase from Caenorhabditis elegans, or an engineered cyanobacterial 16:0/18:0-glycerolipid desaturase, DES9*, in seeds of Arabidopsis (Arabidopsis thaliana) substantially lowered oil saturates. However, because pathway fluxes and regulation of oil synthesis are known to differ across species, translating this transgene technology from the model plant to crop species requires additional investigation. In the work reported here, we found that high expression of FAT5 in seeds of camelina (Camelina sativa) provided only a moderate decrease in saturates, from 12.9% of total oil fatty acids in untransformed controls to 8.6%. Expression of DES9* reduced saturates to 4.6%, but compromised seed physiology and oil content. However, the coexpression of the two desaturases together cooperatively reduced saturates to only 4.0%, less than one-third of the level in the parental line, without compromising oil yield or seedling germination and establishment. Our successful lowering of oil saturates in camelina identifies strategies that can now be integrated with genetic engineering approaches that reduce polyunsaturates to provide optimized oil composition for biofuels in camelina and other oil seed crops.

Hypertension control rate in India: systematic review and meta-analysis of population-level non-interventional studies, 2001–2022

Hypertension is a significant contributor to mortality in India. Achieving better hypertension control rate at the population level is critical in reducing cardiovascular morbidity and mortality. Hypertension control rate was defined as the proportion of patients with their blood pressure under control (systolic blood pressure <140mmHg and diastolic blood pressure <90mmHg). We conducted a systematic review and meta-analysis of community-based, non-interventional studies published after 2001 that reported hypertension control rates. We searched PubMed, Embase, and Web of Science databases, and grey literature, and extracted data using a common framework, and summarized the study characteristics. We conducted random-effects meta-analysis using untransformed hypertension control rates and reported the overall summary estimates and subgroup estimates of control rates as percentages and 95% confidence intervals. We also conducted mixed-effects meta-regression with sex, region, and study period as covariates. The risk of bias was assessed, and level of evidence was summarized using SIGN-50 methodology. The protocol was pre-registered with PROSPERO, CRD42021267973. The systematic review included 51 studies (n=338,313 hypertensive patients). 21 studies (41%) reported poorer control rates among males than females, and six studies (12%) reported poorer control rates among rural patients. The pooled hypertension control rate in India during 2001-2020 was 17.5% (95% CI: 14.3%-20.6%)-with significant increase over the years, reaching 22.5% (CI: 16.9-28.0%) in 2016-2020. Sub-group analysis showed significantly better control rates in the South and West regions, and significantly poorer control rates among males. Very few studies reported data on social determinants or lifestyle risk factors. Less than one-fourth of hypertensive patients in India had their blood pressure under control during 2016-2020. Although the control rate has improved compared to previous years, substantial differences exist across regions. Very few studies have examined the lifestyle risk factors and social determinants relevant to hypertension control in India. The country needs to develop and evaluate sustainable, community-based strategies and programs to improve hypertension control rates. Not applicable.

Enhancement of sesame omega-3 fatty acid content using Fusarium moniliforme bifunctional desaturase gene

Alpha-linolenic acid (ALA) is the precursor for the synthesis of essential fatty acids such as EPA and DHA. The dietary requirement of omega 3 fatty acid is 1.59 g for females and 2.06 g for males. There is an urgent need for enhancing omega 3 fatty acid in vegetative sources. Sesame oil is unique in its flavor and medicinal properties but it contains only traces of ALA. In this study attempts were made to increase the omega 3 fatty acid content in sesame seeds using a bi-functional ∆12/ω3 fatty acid desaturase gene from Fusarium moniliforme . Sesame seed-specific promoter isolated from oleic acid desaturase was used for high and tissue-specific expression. The fungal bi-functional ∆12/ω3 fatty acid desaturase gene under the control of sesame seed-specific promoter was transferred to sesame the ALA content in transgenic sesame was evaluated. Expression of fungal ∆12/ω3 fatty acid desaturase gene resulted in increased ALA content up to 12 fold in transformed sesame plants compared to untransformed control. • Transgenic sesame plants were generated using fungal bi-functional desaturase gene to enhance omega 3 fatty acid. • This opens the possibility of using sesame leaves for omega 3 fatty acid production. • Constitutive promoters produce more of ALA throughout the plant than the strong oleic acid promoter in seeds.

A two-gene strategy increases iron and zinc concentrations in wheat flour, improving mineral bioaccessibility.

Dietary deficiencies of iron and zinc cause human malnutrition that can be mitigated by biofortified staple crops. Conventional breeding approaches to increase grain mineral concentrations in wheat (Triticum aestivum L.) have had only limited success, and our understanding of the genetic and physiological barriers to altering this trait is incomplete. Here we demonstrate that a transgenic approach combining endosperm-specific expression of the wheat VACUOLAR IRON TRANSPORTER gene TaVIT2-D with constitutive expression of the rice (Oryza sativa) NICOTIANAMINE SYNTHASE gene OsNAS2 significantly increases the total concentration of zinc and relocates iron to white-flour fractions. In two distinct bread wheat cultivars, we show that the so called VIT-NAS construct led to a two-fold increase in zinc in wholemeal flour, to ∼50 µg g-1. Total iron was not significantly increased, but redistribution within the grain resulted in a three-fold increase in iron in highly pure, roller-milled white flour, to ∼25 µg g-1. Interestingly, expression of OsNAS2 partially restored iron translocation to the aleurone, which is iron depleted in grain overexpressing TaVIT2 alone. A greater than three-fold increase in the level of the natural plant metal chelator nicotianamine in the grain of VIT-NAS lines corresponded with improved iron and zinc bioaccessibility in white flour. The growth of VIT-NAS plants in the greenhouse was indistinguishable from untransformed controls. Our results provide insights into mineral translocation and distribution in wheat grain and demonstrate that the individual and combined effects of the two transgenes can enhance the nutritional quality of wheat beyond what is possible by conventional breeding.

Silencing P25, HC-Pro and Brp1 of Potato Virus (Viroid) Using Artificial microRNA Confers Resistance to PVX, PVY and PSTVd in Transgenic Potato.

Virus infection is the key constraint to potato cultivation worldwide. Especially, coinfection by multiple viruses could exacerbate the yield loss. Transgenic plants expressing artificial microRNAs (amiRNAs) have been shown to confer specific resistance to viruses. In this study, three amiRNAs containing Arabidopsis miR159 as a backbone, expressing genes targeting P25, HC-Pro and Brp1 of potato virus X (PVX), potato virus Y (PVY) and potato spindle tuber viroid (PSTVd), were constructed. amiR-159P25, amiR-159HCPro and amiR-159Brp1 were cloned into the plant expression vector pCAMBIA1301 with a CaMV35S promoter, producing the p1301-pre-amiRP25−HCPro−Brp1 vector. Twenty-three transgenic plants (Solanum tuberosum cv. ‘Youjin’) were obtained by Agrobacterium tumefaciens–mediated transformation, and ten PCR-positive transplants were chosen for further analysis. Quantitative real-time PCR results indicated that 10 transgenic plants could express amiRNAs successfully. Southern blotting hybridization proved that amiR-159P25−HCPro−Brp1 had integrated into potato genome in transgenic lines. Viral (viroid) challenge assays revealed that these transgenic plants demonstrated resistance against PVX, PVY and PSTVd coinfection simultaneously, whereas the untransformed controls developed severe symptoms. This study demonstrates a novel amiRNA-based mechanism that may have the potential to develop multiple viral resistance strategies in potato.

Identification of Candidate Genes Responsible for Flower Colour Intensity in Gentiana triflora.

Gentians cultivated in Japan (Gentiana triflora and Gentiana scabra and hybrids) have blue flowers, but flower colour intensity differs among cultivars. The molecular mechanism underlying the variation in flower colour intensity is unclear. Here, we produced F2 progeny derived from an F1 cross of intense- and faint-blue lines and attempted to identify the genes responsible for flower colour intensity using RNA-sequencing analyses. Comparative analysis of flower colour intensity and transcriptome data revealed differentially expressed genes (DEGs), although known flavonoid biosynthesis-related genes showed similar expression patterns. From quantitative RT-PCR (qRT-PCR) analysis, we identified two and four genes with significantly different expression levels in the intense- and faint-blue flower lines, respectively. We conducted further analyses on one of the DEGs, termed GtMIF1, which encodes a putative mini zinc-finger protein homolog, which was most differently expressed in faint-blue individuals. Functional analysis of GtMIF1 was performed by producing stable tobacco transformants. GtMIF1-overexpressing tobacco plants showed reduced flower colour intensity compared with untransformed control plants. DNA-marker analysis also confirmed that the GtMIF1 allele of the faint-blue flower line correlated well with faint flower colour in F2 progeny. These results suggest that GtMIF1 is one of the key genes involved in determining the flower colour intensity of gentian.

Field Performance of Resistant Potato Genotypes Transformed with the EFR Receptor from Arabidopsis thaliana in the Absence of Bacterial Wilt (Ralstonia solanacearum).

Bacterial wilt caused by the pathogen Ralstonia solanacearum is a devastating disease of potato crops. Harmonizing immunity to pathogens and crop yield is a balance between productive, economic, and environmental interests. In this work, the agronomic performance of two events of potato cultivar INIA Iporá expressing the Arabidopsis thaliana EFR gene (Iporá EFR 3 and Iporá EFR 12) previously selected for their high resistance to bacterial wilt was evaluated under pathogen-free conditions. During two cultivation cycles, the evaluated phenotypic characteristics were emergence, beginning of flowering, vigor, growth, leaf morphology, yield, number and size of tubers, analyzed under biosecurity standards. The phenotypic characteristics evaluated did not show differences, except in the morphology of the leaf with a more globose appearance and a shortening of the rachis in the transformation events with respect to untransformed Iporá. The Iporá EFR 3 genotype showed a ~40% yield decrease in reference to untransformed Iporá in the two trials, while Iporá EFR 12 did not differ statistically from untransformed Iporá. Iporá EFR 12 shows performance stability in the absence of the pathogen, compared to the untransformed control, positioning it as an interesting candidate for regions where the presence of the pathogen is endemic and bacterial wilt has a high economic impact.

CRISPR/Cas9-mediated genome editing of phytoene desaturase in Carica papaya L

ABSTRACT Despite a long history of genetic engineering in Carica papaya L., genome editing protocols have not been previously validated for this species. To address this gap in the papaya improvement toolkit, we deployed CRIPSR/Cas9 to target the candidate Carica papaya L. phytoene desaturase (CpPDS) gene with the goal of inducing a visually scorable albino phenotype in edited tissue. A total of 73 plant lines stably transformed with pAC0025 (CRISPR construct targeting CpPDS) were obtained. Of these, 59 (81%) were fully albino. Genotyping of the targeted region within CpPDS was carried out for ten pAC0025 (CpPDS construct) lines, one untransformed control, and one line transformed with a negative control construct pAC0026 (no gRNA construct). Alignment of Sanger sequencing results revealed diverse edits in pAC0025-transformed plants including insertions of up to 415 bp, inversions and large deletions between gRNA target sites, and many smaller deletions. Mutations were observed at all three gRNA target sites. Overall, the high percentage of albino plants recovered in combination with the abundance of mutations observed across CpPDS gRNA target sites suggests an efficient genome editing protocol that could be used to improve papaya at the genetic level.

Host-induced gene silencing reveals the role of Sclerotinia sclerotiorum oxaloacetate acetylhydrolase gene in fungal oxalic acid accumulation and virulence

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating necrotrophic pathogen which causes severe yield losses to oilseed production worldwide. Most of efforts at the genetic mitigation of the disease have not been successful. Present investigation was conducted to functionally characterize the effect of down-regulating Ssoah1 during host infection and explore the possibility of boosting host resistance by silencing this gene. We utilized host-induced gene silencing (HIGS) to silence Ssoah1 gene in the S. sclerotiorum fungus. A HIGS based vector was constructed and transformed into Arabidopsis thaliana. The pathogenicity assays in the transgenic A. thaliana lines revealed three T3 transformants with significantly higher resistance to S. sclerotiorum in comparison to untransformed controls. There was a concomitant reduction in expression of Ssoah1 and accumulation of oxalic acid in the necrotic regions of transgenic lines as compared to the non-transgenic controls. Specific Ssoah1-siRNA was highly expressed in HIGS Ssoah1 transgenic lines, as compared with WT and EV plants. The outcomes of oxalic acid estimation revealed that silencing of Ssoah1 results in decreased OA accumulation. The recovered mycelium plugs from HIGS Ssoah1 transgenic lines showed decreased Ssoah1 expression and pathogenesis. These results provide the possibility of using HIGS of Ssoah1 for engineering resistance against S. sclerotiorum.

Transcriptional loss of domestication-driven cytoskeletal GhPRF1 gene causes defective floral and fiber development in cotton (Gossypium)

Constitutive- and fiber-specific RNAi of GhPRF1 gene illustrated strong correlation between domestication-driven profilin genes and floral/fiber architecture in cotton. During morpho-transformation of short-fuzz of wild cotton into the elongating spinnable fibers under the millennia of human selection, actin-polymerizing cytoskeletal profilin genes had undergone significant sequence alterations and spatiotemporal shift in their transcription levels. To comprehend the expression dynamics of profilin genes with their phenotypic implications, transgenic expression modulation of cotton profilin 1 (GhPRF1) gene was performed in the constitutive- and fiber-specific manner in Coker 310FR cottoncultivar. The constitutive GhPRF1-RNAi lines (35S:GhPRF1-RNAi) exhibited distorted 'monadelphous' staminal-tube, reduced pollen-viability and poorly developed fibers, whereas floral and fiber development of fiber-specific GhPRF1-RNAi lines showed no abnormalities. Moreover, the fiber-specific GhPRF1 overexpression lines (FBP7:GhPRF1-Ox) showed increased emergence of fiber-initials on the ovule surface, on thecontrary to no fiber-initials in fiber-specific RNAi lines (FBP7:GhPRF1-RNAi). Interestingly, theaverage seed weight and fiber weight of FBP7:GhPRF1-Ox lines increased > 60% and > 38%, respectively, compared with FBP7:GhPRF1-RNAi lines and untransformed control seeds. On a molecular basis, the aberrant floral and fiber development of 35S:GhPRF1-RNAi lines was largely associated with sugar metabolism and hormone-signaling mechanisms. These observations illustrated the strong correlation between domestication-driven GhPRF genes, and floral/fiber development in cotton. Also, the enhanced agronomic traits in GhPRF1-Oxlines of cotton empowered us to recognize their imperative roles, and theirfuture deployment for the sustainable cotton crop improvement.

Alteration of Flower Color in Viola cornuta cv. “Lutea Splendens” through Metabolic Engineering of Capsanthin/Capsorubin Synthesis

Flower color is an important characteristic that determines the commercial value of ornamental plants. The development of modern biotechnology methods such as genetic engineering enables the creation of new flower colors that cannot be achieved with classical methods of hybridization or mutational breeding. This is the first report on the successful Agrobacterium-mediated genetic transformation of Viola cornuta L. The hypocotyl explants of cv. “Lutea Splendens” variety with yellow flowers were transformed with A. tumefaciens carrying empty pWBVec10a vector (Llccs−) or pWBVec10a/CaMV 35S::Llccs::TNos vector (Llccs+) for capsanthin/capsorubin synthase gene (Llccs) from tiger lily (Lilium lancifolium). A comparative study of shoot multiplication, rooting ability during culture in vitro, as well as phenotypic characteristics of untransformed (control) and transgenic Llccs− and Llccs+ plants during ex vitro growth and flowering is presented. Successful integration of Llccs transgene allows the synthesis of red pigment capsanthin in petal cells that gives flowers different shades of an orange/reddish color. We demonstrate that the ectopic expression of Llccs gene in ornamental plants, such as V. cornuta “Lutea Splendens” could successfully be used to change flower color from yellow to different shades of orange.