Variations in photoperiods and their impact on yield, photosynthesis and secondary metabolite production in basil microgreens.

BACKGROUND: The effects of different photoperiods on plant phytochemical synthesis can be improved by adjusting the daily light integral. Photoperiod is one of the most important environmental factors that control growth, plant's internal rhythm and the synthesis of secondary metabolites. Information about the appropriate standard in terms of photoperiod for growing basil microgreens as one of the most important medicinal plants is limited. In this study, the effects of five different photoperiods, 6 (6 h × 3 cycles), 8 (8 h × 2 cycles), 16, 18, and 24 h day- 1 on the yield, photosynthesis and synthesis of secondary metabolites of three cultivars and one genotype of basil microgreens in floating system were evaluated. The purpose of this research was to determine the feasibility of using permanent light in growing basil microgreens and to create the best balance between beneficial secondary metabolites and performance. RESULTS: The results showed that the effects of photoperiod and cultivar on all investigated traits and their interaction on photosynthetic pigments, antioxidant capacity, total phenolic compounds, proline content and net photosynthesis rate were significantly different at the 1% level. The highest levels of vitamin C, flavonoids, anthocyanins, yield and antioxidant potential composite index (APCI) were obtained under the 24-h photoperiod. The highest antioxidant capacity was obtained for the Kapoor cultivar, and the highest total phenolic compound and proline contents were measured for the Ablagh genotype under a 24-h photoperiod. The highest yield (4.36 kg m- 2) and APCI (70.44) were obtained for the Ablagh genotype. The highest nitrate content was obtained with a photoperiod of 18 h for the Kapoor cultivar. The highest net photosynthesis rate was related to the Violeto cultivar under a 24-hour photoperiod (7.89 µmol CO2 m- 2 s- 1). Antioxidant capacity and flavonoids had a positive correlation with phenolic compounds and vitamin C. Yield had a positive correlation with antioxidant capacity, flavonoids, vitamin C, APCI, and proline. CONCLUSIONS: Under continuous light conditions, basil microgreens resistance to light stress by increasing the synthesis of secondary metabolites and the increase of these biochemical compounds made basil microgreens increase their performance along with the increase of these health-promoting compounds. The best balance between antioxidant compounds and performance was achieved in continuous red + blue light. Based on these results, the use of continuous artificial LED lighting, due to the increase in plant biochemical with antioxidant properties and yield, can be a suitable strategy for growing basil microgreens in floating systems.

Companion basil plants prime the tomato wound response through volatile signaling in a mixed planting system.

KEY MESSAGE: Volatile compounds released from basil prime the tomato wound response by promoting jasmonic acid, mitogen-activated protein kinase, and reactive oxygen species signaling. Within mixed planting systems, companion plants can promote growth or enhance stress responses in target plants. However, the mechanisms underlying these effects remain poorly understood. To gain insight into the molecular nature of the effects of companion plants, we investigated the effects of basil plants (Ocimum basilicum var. minimum) on the wound response in tomato plants (Solanum lycopersicum cv. 'Micro-Tom') within a mixed planting system under environmentally controlled chamber. The results showed that the expression of Pin2, which specifically responds to mechanical wounding, was induced more rapidly and more strongly in the leaves of tomato plants cultivated with companion basil plants. This wound response priming effect was replicated through the exposure of tomato plants to an essential oil (EO) prepared from basil leaves. Tomato leaves pre-exposed to basil EO showed enhanced expression of genes related to jasmonic acid, mitogen-activated protein kinase (MAPK), and reactive oxygen species (ROS) signaling after wounding stress. Basil EO also enhanced ROS accumulation in wounded tomato leaves. The wound response priming effect of basil EO was confirmed in wounded Arabidopsis plants. Loss-of-function analysis of target genes revealed that MAPK genes play pivotal roles in controlling the observed priming effects. Spodoptera litura larvae-fed tomato leaves pre-exposed to basil EO showed reduced growth compared with larvae-fed control leaves. Thus, mixed planting with basil may enhance defense priming in both tomato and Arabidopsis plants through the activation of volatile signaling.

Suppression of essential oil biosynthesis in sweet basil cotyledons under hypergravity conditions.

The mechanism through which gravity influences the biosynthesis of essential oils in herbs is an important issue for plant and space biology. Sweet basil (Ocimum basilicum L.) seedlings were cultivated under centrifugal hypergravity conditions at 100 g in the light, and the growth of cotyledons, development of glandular hairs, and biosynthesis of essential oils were analyzed. The area and fresh weight of the cotyledons increased by similar amounts irrespective of the gravitational conditions. On the abaxial surface of the cotyledons, glandular hairs, where essential oils are synthesized and stored, developed from those with single-cell heads to those with four-cell heads; however, hypergravity did not affect this development. The main components, methyl eugenol and 1,8-cineole, in the essential oils of cotyledons were lower in cotyledons grown under hypergravity conditions. The gene expression of enzymes in the phenylpropanoid pathway involved in the synthesis of methyl eugenol, such as phenylalanine ammonia lyase (PAL) and eugenol O-methyltransferase (EOMT), was downregulated by hypergravity. Hypergravity also decreased the gene expression of enzymes in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway involved in the synthesis of 1,8-cineole, such as 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 1,8-cineole synthase (CINS). These results indicate that hypergravity without affecting the development of glandular hairs, decreases the expression of genes related to the biosynthesis of methyl eugenol and 1,8-cineole, which may cause a decrease in the amounts of both essential oils in sweet basil cotyledons.

Increase of secondary metabolites in sweet basil (Ocimum basilicum L.) leaves by exposure to N2O5 with plasma technology.

Exposure to N2O5 generated by plasma technology activates immunity in Arabidopsis through tryptophan metabolites. However, little is known about the effects of N2O5 exposure on other plant species. Sweet basil synthesizes many valuable secondary metabolites in its leaves. Therefore, metabolomic analyses were performed at three different exposure levels [9.7 (Ex1), 19.4 (Ex2) and 29.1 (Ex3) µmol] to assess the effects of N2O5 on basil leaves. As a result, cinnamaldehyde and phenolic acids increased with increasing doses. Certain flavonoids, columbianetin, and caryophyllene oxide increased with lower Ex1 exposure, cineole and methyl eugenol increased with moderate Ex2 exposure and L-glutathione GSH also increased with higher Ex3 exposure. Furthermore, gene expression analysis by quantitative RT-PCR showed that certain genes involved in the syntheses of secondary metabolites and jasmonic acid were significantly up-regulated early after N2O5 exposure. These results suggest that N2O5 exposure increases several valuable secondary metabolites in sweet basil leaves via plant defense responses in a controllable system.

Genetic linkage mapping and quantitative trait locus (QTL) analysis of sweet basil (Ocimum basilicum L.) to identify genomic regions associated with cold tolerance and major volatiles.

Chilling sensitivity is one of the greatest challenges affecting the marketability and profitability of sweet basil (Ocimum basilicum L.) in the US and worldwide. Currently, there are no sweet basils commercially available with significant chilling tolerance and traditional aroma profiles. This study was conducted to identify quantitative trait loci (QTLs) responsible for chilling tolerance and aroma compounds in a biparental mapping population, including the Rutgers advanced breeding line that served as a chilling tolerant parent, 'CB15', the chilling sensitive parent, 'Rutgers Obsession DMR' and 200 F2 individuals. Chilling tolerance was assessed by percent necrosis using machine learning and aroma profiling was evaluated using gas chromatography (GC) mass spectrometry (MS). Single nucleotide polymorphism (SNP) markers were generated from genomic sequences derived from double digestion restriction-site associated DNA sequencing (ddRADseq) and converted to genotype data using a reference genome alignment. A genetic linkage map was constructed and five statistically significant QTLs were identified in response to chilling temperatures with possible interactions between QTLs. The QTL on LG24 (qCH24) demonstrated the largest effect for chilling response and was significant in all three replicates. No QTLs were identified for linalool, as the population did not segregate sufficiently to detect this trait. Two significant QTLs were identified for estragole (also known as methyl chavicol) with only qEST1 on LG1 being significant in the multiple-QTL model (MQM). QEUC26 was identified as a significant QTL for eucalyptol (also known as 1,8-cineole) on LG26. These QTLs may represent key mechanisms for chilling tolerance and aroma in basil, providing critical knowledge for future investigation of these phenotypic traits and molecular breeding.

Tobacco introduced Perilla frutescens and Ocimum basilicum genes attenuates neutrophilic inflammation in lung tissues of COPD rats.

The new-type tobacco varieties "Zisu" and "Luole" were obtained by distant hybridization between N. tabacum L. var. HHY and Perilla frutescens and Ocimum basilicum, with obviously different chemical composition. Smoking is the major risk factor for COPD, characterized by neutrophil-dominant inflammation. In the present study, rat COPD model was established by cigarette exposure, and the health hazard of three varieties was compared by general condition observation, pathological and morphological evaluation, total and differential cell numeration, and characterization of major inflammatory mediators and MAPK/NF-κB pathway, etc. Rats in "HHY" group developed obvious symptoms such as cough, dyspnea, mental fatigue, etc., but these symptoms were obviously mitigated in "Zisu" and "Luole" groups. H&E staining analysis, including score, MLI, MAN, wt% and WA%, showed that "Zisu" and "Luole" significantly alleviated lung injury and the degree of airway remodeling and emphysema compared to "HHY". In BALF, the number of total leukocyte and the percent neutrophils in "Zisu" and "Luole" groups were evidently lower than "HHY" group. The levels of inflammatory mediators, such as IL-8, MPO, MIP-2, LTB4, TNF-α and neutrophil elastase, in "HHY" group were obviously higher than "Zisu" and "Luole" groups. The ROS-mediated NF-κB p65 and p38MAPK pathways may play an important role. Results indicated that tobacco introduced perilla and basil genes could remarkably attenuate recruitment, infiltration and activation of neutrophils and intervene in airway inflammation, retarding disease progression, especially "Zisu". Changes in chemical composition via breeding techniques may be a novel way for tobacco harm reduction.

Low-temperature perception and modulations in Ocimum basilicum commercial cultivar CIM-Shishir: Biosynthetic potential with insight towards climate-smart resilience.

The newly released interspecific hybrid variety CIM-Shishir, resulting from a cross between Ocimum basilicum and Ocimum kilimandscharicum claims to be a multicut, lodging resistant, cold tolerant, high essential oil yielding with linalool rich variety. It has a purple-green stem and has a unique feature and advantage of better survival in the winter season than other O. basilicum varieties, illustrating its physiological mechanisms for cold tolerance. In this study, we subjected both the CIM-Shishir variety and a control plant to cold stress to investigate the impact of low temperatures on various physiological, trichome developments, secondary metabolite constitution aspects related to essential oil production, and gene expression. The analysis revealed a significantly higher density and altered morphology of trichomes on the leaf surface of the variety subjected to low temperatures, indicating its adaptation to cold conditions. Furthermore, when comparing the treated plants under low-temperature stress, it was observed that the relative electrolyte leakage and Malondialdehyde (MDA) contents substantially increased in the control in contrast to the CIM-Shishir variety. This finding suggests that CIM-Shishir exhibits superior cold tolerance. Additionally, an increase in proline content was noted in the variety exposed to low temperatures compared to the control. Moreover, the chlorophyll and anthocyanin content gradually increased with prolonged exposure to low-temperature stress in the newly developed variety, indicating its ability to maintain photosynthetic capacity and adapt to cold conditions. The activities of superoxide dismutase (SOD) also increased under low-temperature conditions in the CIM-Shishir variety, further highlighting its cold tolerance behaviour. In our research, we investigated the comprehensive molecular mechanisms of cold response in Ocimum. We analyzed the expression of key genes associated with cold tolerance in two plant groups: the newly developed hybrid variety known as CIM-Shishir Ocimum, which exhibits cold tolerance, and the control plants susceptible to cold climates that include WRKY53, ICE1, HOS1, COR47, LOS15, DREB5, CBF4, LTI6, KIN, and ERD2. These genes exhibited significantly higher expression levels in the CIM-Shishir variety compared to the control, shedding light on the genetic basis of its cold tolerance. The need for climate-smart, resilient high-yielding genotype is of high importance due to varied climatic conditions as this will hit the yield drastically and further to the economic sectors including farmers and many industries that are dependent on the bioactive constituents of Ocimum.

The variations in gene expression of GAPDH in Ocimum basilicum cultivars under drought-induced stress conditions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) holds a pivotal role within the glycolytic pathway of higher plants. It has garnered attention as a significant target protein in instances of oxidative stress, where it can engage in thiolation reactions within its active site. Numerous genes encoding cytosolic iterations of GAPDH have been identified and analyzed in specific plant species. This investigation was conducted to gain insights into GAPDH's function amidst drought-induced stress. Within this framework, the basil plant (Ocimum basilicum) was chosen for focused exploration, encompassing the cloning of the comprehensive cDNA of basil GAPDH (ObGAPDH) and scrutinizing its patterns of expression. The complete sequence of Ob-GAPDH spanned 1315 base pairs. The resultant protein derived from this sequence comprised 399 amino acids, projecting a molecular weight of approximately 42.54 kDa and an isoelectric point (pI) of 6.01. An examination of the evolutionary connections among various GAPDH proteins unveiled ObGAPDH's shared lineage with GAPDH proteins sourced from other plants, such as Salvia splendens and Sesamum indicum. Furthermore, computational methodologies were harnessed to predict the potential oxidative role of ObGAPDH in response to external signals. Molecular docking simulations illuminated the interaction between ObGAPDH and hydrogen peroxide (H2O2) as a ligand. Scrutinizing the expression patterns of the ObGAPDH gene under conditions of water scarcity stress brought to light diverse levels of transcriptional activity. Collectively, these findings underscore the notion that the regulation of ObGAPDH expression is contingent upon both the specific plant cultivar and the presence of stress stemming from drought conditions.

Evaluation of basil (Ocimum basilicum) accessions under different drought conditions based on yield and physio-biochemical traits.

BACKGROUND: Basil is one of the most famous herbs, which has broad usage as a fresh vegetable and therapeutic and pharmaceutical services. The main abiotic stress limiting basil production globally is drought. As a result, appropriate drought screening-which effectively separates high-yielding but drought-sensitive genotypes from drought-tolerant genotypes-is necessary for the optimal selection of high-yielding basil cultivars under drought stress conditions. So, a split plot experiment with three replications based on a completely randomized design were carried out in a pot under field conditions for this investigation. Water levels (full irrigation or control, moderate stress, and severe stress) were assigned as main plots, while 22 basil accessions were given as sub-plots. In this study, leaf yield as well as physio-biochemical traits had measured on accessions. RESULTS: Our results revealed large variation in yield, essential oil (%), protein, proline, chlorophyll, total phenol and flavonoids traits across the 22 accessions. The percentage of leaf yield reduction in moderate drought stress than normal conditions showed that G1 (-6.5%), G17 (-7.05%), G20 (-9.01%), and G12 (-10.9%) accessions had the least changes, respectively. Although in severe drought stress than normal conditions, the G1 (-32.01%), G12 (-33.12%), G4 (-33.24%), G7 (-34.11%), and G17 (-34.93%) accessions had the least amount of change in plant leaf yield, respectively. Furthermore, the highest yield reduction occurred in moderate and severe stress conditions in G18 (-25.36%) and G8 (-42.98%) accessions, respectively. Cluster analysis based on the ward method in both conditions (moderate and severe drought conditions) placed the accessions in three groups, and accessions were identified as tolerant, whose average traits in that group were higher than the total average. The principal component analysis also showed that in moderate drought conditions, the first two components explained about 95.28% of the total variation, while in severe drought conditions, these two components explained about 96.37% of the total variation. CONCLUSIONS: The different multivariate analyses (cluster analysis, PCA, mean comparison) were used to identify tolerant and sensitive accessions based on all traits. The accessions G3, G4, G6, and G7 were found to be tolerant to stress, while G10, G15, G16, and G20 were found to be sensitive to drought. These accessions are a useful step in producing drought-tolerant, high-yielding accessions and can be utilized in breeding programs for basil.

Supplementary UV-A and UV-B radiation differentially regulate morphology in Ocimum basilicum.

UV-A- or UV-B-enriched growth light was given to basil plants at non-stress-inducing intensities. UV-A-enriched growth light gave rise to a sharp rise in the expression of PAL and CHS genes in leaves, an effect that rapidly declined after 1-2 days of exposure. On the other hand, leaves of plants grown in UV-B-enriched light had a more stable and long-lasting increase in the expression of these genes and also showed a stronger increase in leaf epidermal flavonol content. UV supplementation of growth light also led to shorter more compact plants with a stronger UV effect the younger the tissue. The effect was more prominent in plants grown under UV-B-enriched light than in those grown under UV-A. Parameters particularly affected were internode lengths, petiole lengths and stem stiffness. In fact, the bending angle of the 2nd internode was found to increase as much as 67% and 162% for plants grown in the UV-A- and UV-B-enriched treatments, respectively. The decreased stem stiffness was probably caused by both an observed smaller internode diameter and a lower specific stem weight, as well as a possible decline in lignin biosynthesis due to competition for precursors by the increased flavonoid biosynthesis. Overall, at the intensities used, UV-B wavelengths are stronger regulators of morphology, gene expression and flavonoid biosynthesis than UV-A wavelengths.

Morphological and molecular characterization of downy mildew on sweet basil (Ocimum basilicum) caused by Peronospora belbahrii in Turkiye.

BACKGROUND: Sweet basil (Ocimum basilicum) is one of the most significant aromatic plants in Turkiye. Recently, a new pathogen induced symptoms were discovered and identified as basil downy mildew caused by Peronospora belbahrii Thines. The pathogen has been introduced into the country and it has quickly become the most damaging disease in basil cultivation. The purpose of this study was to investigate the molecular and morphological properties of the causal organism of downy mildew observed on sweet basil and determine the disease incidence and prevalence in Antalya province. METHODS AND RESULTS: According to morphological characteristics (conidia, conidiophores) disease was determined as downy mildew caused by P. belbahrii. Pathogenicity tests were performed by spraying with a sporangial suspension of P. belbahrii (1 × 105 sporangia/mL). After 1 week, all inoculated plants exhibited characteristic downy mildew symptoms on their leaves, whereas non-inoculated control plants remained disease-free. All molecular analyses involving the internal transcribed spacer region were amplified using Nested PCR with primer pairs ITS4 and ITS6 for the first round and ITS4 and DC6 for the second round. Resulting sequences of all the nested PCR products had 99% similarity with P. belbahrii isolates. Disease incidence was 22.4-70.2% of sweet basil cultivation area in Antalya province. CONCLUSIONS: Based on the molecular analysis, morphological characteristics and pathogenicity tests the pathogen was identified as P. belbahrii. To our knowledge, this is the first report of downy mildew caused by P. belbahrii on sweet basil in Turkiye.

Genome-based high-resolution mapping of fusarium wilt resistance in sweet basil.

Fusarium wilt of basil is a disease of sweet basil (Ocimum basilicum L.) plants caused by the fungus Fusarium oxysporum f. sp. basilici (FOB). Although resistant cultivars were released > 20 years ago, the underlying mechanism and the genes controlling the resistance remain unknown. We used genetic mapping to elucidate FOB resistance in an F2 population derived from a cross between resistant and susceptible cultivars. We performed genotyping by sequencing of 173 offspring and aligning the data to the sweet basil reference genome. In total, 23,411 polymorphic sites were detected, and a single quantitative trait locus (QTL) for FOB resistance was found. The confidence interval was < 600 kbp, harboring only 60 genes, including a cluster of putative disease-resistance genes. Based on homology to a fusarium resistance protein from wild tomato, we also investigated a candidate resistance gene that encodes a transmembrane leucine-rich repeat - receptor-like kinase - ubiquitin-like protease (LRR-RLK-ULP). Sequence analysis of that gene in the susceptible parent vs. the resistant parent revealed multiple indels, including an insertion of 20 amino acids next to the transmembrane domain, which might alter its functionality. Our findings suggest that this LRR-RLK-ULP might be responsible for FOB resistance in sweet basil and demonstrate the usefulness of the recently sequenced basil genome for QTL mapping and gene mining.

Pretreatment with LEDs regulates antioxidant capacity and polyphenolic profile in two genotypes of basil under salinity stress.

In the present study, we evaluated a pretreatment with four LED light sources (red, blue, red + blue, and white) in two genotypes (green and purple) of basil on the growth parameters, stress oxidative markers, non-enzymatic antioxidants, osmoprotectant compounds, ion content, and polyphenolic profile under both control and salinity stress conditions. The results indicated that 150 mM of NaCl decreased biomass, RWC, and K+/Na+ ratio but increased the content of proline and antioxidant capacity in the leaves of both genotypes of basil grown under GH (greenhouse) conditions. The results suggested that RB LED-exposed plants in the green genotype and R LED-exposed plants in the purple genotype improved accumulation of shoot biomass, K+/Na+ ratio, proline and soluble sugars, glutathione and ascorbate, polyphenolic profile, and thioredoxin reductase activity in the leaves of basil under both control and salinity stress conditions. NaCl stress (150 mM) increased oxidative markers, which are responsible for disturbance of routine functions of various plant cellular modules. LED light pretreatments diminished these markers under both control and salinity stress conditions. It could be concluded that intensification of non-enzymatic antioxidant systems during light-mediated priming can diminish the deleterious effects of ROS induced by NaCl stress (150 mM) through preventing the lipid peroxidation, scavenging cytotoxic H2O2, and enhancement of antioxidant potentials. Therefore, usage of LED lighting systems as a pretreatment or to supplement natural photoperiods under both control and salinity stress conditions may be advantageous for increasing biomass and phytochemical accumulation in basil.

Joint Action of Pb1 and Pb2 Provides Dominant Complementary Resistance Against New Races of Peronospora belbahrii (Basil Downy Mildew).

Sweet basil (Ocimum basilicum, 2n = 4x = 48) is susceptible to downy mildew caused by Peronospora belbahrii. The Pb1 gene exhibits complete resistance to the disease. However, Pb1 became prone to disease because of occurrence of new virulent races. Here, we show that Zambian accession PI 500950 (Ocimum americanum var. pilosum) is highly resistant to the new races. From an interspecies backcross between PI 500950 and the susceptible 'Sweet basil' we obtained, by embryo rescue, a population of 131 BC1F1 plants. This population segregated 73 resistant (58) and susceptible (1:1; P = 0.22) plants, suggesting that resistance is controlled by one incompletely dominant gene called Pb2. To determine whether allelic relationship exists between Pb1 and Pb2, we used two differential races: race 0, which is avirulent to both PI 500945 (Pb1) and PI 500950 (Pb2), and race 1, which is virulent to PI 500945 but avirulent to PI 500950. F1 plants obtained from '12-4-6' (BC6F3 derived from PI 500945) and '56' (BC3F3 derived from PI 500950) showed resistant superiority to both races through dominant complementary interaction. F2 plants segregated to race 0 as follows: 12:3:1 (immune/incomplete resistant/susceptible) as opposed to 9:3:4 to race 1, indicating that Pb1 and Pb2 are not alleles. Because joint action is contributed in F1 plants and in advanced [BC3F3(56) × BC6F3(12-4-6) F4] populations that carry both genes, it can be assumed that both accessions carry two unlinked genes but share a common signal transduction pathway, which leads to dominant complementation superiority of the resistance against different races of basil downy mildew.

Water deficit stress responses of monoterpenes and sesquiterpenes in different Iranian cultivars of basil.

Ocimum basilicum, a popular aromatic plant, contains aromatic terpenes of terpenoids with in vivo and in vitro verified cytotoxicity. Considering the characteristics and potential of its utilization, it would be attractive to reveal its regulation and biosynthesis, originally at the molecular level under water deficit stress. For this aim, for the first time, the gene encoding the enzyme involved in the end step of the MEP biosynthetic pathways (HDR) was cloned, and the accumulation ratio of linalool, germacrene D and γ-cadinene compounds as well as the expression trait of four critical genes (i.e., HDR, LinS, GerS, and GadS) was assessed under water deficit stress in three Iranian cultivars of basil. The highest value of linalool and γ-cadinene were detected for Cultivar 1 (Cult. 1) under mild stress (W1; 52.6 and 21.1%), while insignificant amounts were obtained for Cultivar 3 (Cult. 3). The germacrene D level of Cultivar 2 (Cult. 2) increased under severe and moderate water stresses as compared with mild water deficit stress. Apart from some expectation, all the studied genes demonstrated divergent transcription ratios under water deficit stress. Principal component analyses (PCA) showed that the relative water content (RWC) and HDR gene expression correlated significantly with essential oil components and gene expression in Cult. 1 and 2, which could represent an elevated demand for corresponding metabolites in the plant tissues. The present work elaborates on the regulation of the mentioned genes, and the results indicate that the production of terpenoids might be a drought stress-dependent and cultivar-dependent procedure.

CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.

Sweet basil (Ocimum basilicum) is an economically important allotetraploid (2n = 4x = 48) herb whose global production is threatened by downy mildew disease caused by the obligate biotrophic oomycete, Peronospora belbahrii. Generation of disease resistant cultivars by mutagenesis of susceptibility (S) genes via CRISPR/Cas9 is currently one of the most promising strategies to maintain favored traits while improving disease resistance. Previous studies have identified Arabidopsis DMR6 (Downy Mildew Resistance 6) as an S gene required for pathogenesis of the downy mildew-causing oomycete pathogen Hyaloperonospora arabidopsidis. In this study, a sweet basil homolog of DMR6, designated ObDMR6, was identified in the popular sweet basil cultivar Genoveser and found to exist with a high copy number in the genome with polymorphisms among the variants. Two CRISPR/Cas9 constructs expressing one or two single guide RNAs (sgRNAs) targeting the conserved regions of ObDMR6 variants were generated and used to transform Genoveser via Agrobacterium-mediated transformation. 56 T0 lines were generated, and mutations of ObDMR6 were detected by analyzing the Sanger sequencing chromatograms of an ObDMR6 fragment using the Interference of CRISPR Edits (ICE) software. Among 54 lines containing mutations in the targeted sites, 13 had an indel percentage greater than 96% suggesting a near-complete knockout (KO) of ObDMR6. Three representative transgene-free lines with near-complete KO of ObDMR6 determined by ICE were identified in the T1 segregating populations derived from three independent T0 lines. The mutations were further confirmed using amplicon deep sequencing. Disease assays conducted on T2 seedlings of the above T1 lines showed a reduction in production of sporangia by 61-68% compared to the wild-type plants and 69-93% reduction in relative pathogen biomass determined by quantitative PCR (qPCR). This study not only has generated transgene-free sweet basil varieties with improved downy mildew resistance, but also contributed to our understanding of the molecular interactions of sweet basil-P. belbahrii.

Genetically Engineered Oleaginous Yeast Lipomyces starkeyi for Sesquiterpene α-Zingiberene Production.

Oleaginous yeast, such as Lipomyces starkeyi, are logical organisms for production of higher energy density molecules like lipids and terpenes. We demonstrate that transgenic L. starkeyi strains expressing an α-zingiberene synthase gene from lemon basil or Hall's panicgrass can produce up to 17 mg/L α-zingiberene in yeast extract peptone dextrose (YPD) medium containing 4% glucose. The transgenic strain was further examined in 8% glucose media with C/N ratios of 20 or 100, and YPD. YPD medium resulted in 59 mg/L α-zingiberene accumulation. Overexpression of selected genes from the mevalonate pathway achieved 145% improvement in α-zingiberene synthesis. Optimization of the growth medium for α-zingiberene production led to 15% higher titer than YPD medium. The final transgenic strain produced 700 mg/L α-zingiberene in fed-batch bioreactor culture. This study opens a new synthetic route to produce α-zingiberene or other terpenoids in L. starkeyi and establishes this yeast as a platform for jet fuel biosynthesis.

Production of rosmarinic acid and correlated gene expression in hairy root cultures of green and purple basil (Ocimum basilicum L.).

Rosmarinic acid (RA) is an active constituent of Ocimum basilicum. It has been shown that hairy root production (measured as dry weight) improves when green basil (O. basilicum "Cinnamon") is cultured under the light. In contrast, purple basil (O. basilicum "Purpurascens") shows greater hairy root production when cultured under dark conditions. The level of gene expression was highest in hairy roots of green basil under dark conditions for up to 1 week. Transcript levels were highest in hairy roots of purple basil under both dark and light conditions after 2 weeks of culturing. After 3 weeks of culture under light conditions, green basil had accumulated 1.9-fold higher RA content than that of purple basil, which in turn was fivefold higher than that of the natural roots (42.86 µg/mg). Tyrosine aminotransferase showed a higher transcript level when compared to the other phenylpropanoid pathway genes (phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and coenzyme-A ligase) in both dark and light conditions and in all-time regimens. RA accumulation was higher in the cultured hairy roots of green basil than those of purple basil under both light and dark conditions.

The genome sequence of tetraploid sweet basil, Ocimum basilicum L., provides tools for advanced genome editing and molecular breeding.

Sweet basil, Ocimum basilicum L., is a well-known culinary herb grown worldwide, but its uses go beyond the kitchen to traditional medicine, cosmetics and gardening. To date, the lack of an available reference genome has limited the utilization of advanced molecular breeding methods. We present a draft version of the sweet basil genome of the cultivar 'Perrie', a fresh-cut Genovese-type basil. Genome sequencing showed basil to be a tetraploid organism with a genome size of 2.13 Gbp, assembled in 12,212 scaffolds, with > 90% of the assembly being composed of 107 scaffolds. About 76% of the genome is composed of repetitive elements, with the majority being long-terminal repeats. We constructed and annotated 62,067 protein-coding genes and determined their expression in different plant tissues. We analysed the currently known phenylpropanoid volatiles biosynthesis genes. We demonstrated the necessity of the reference genome for a comprehensive understanding of this important pathway in the context of tetraploidy and gene redundancy. A complete reference genome is essential to overcome this redundancy and to avoid off-targeting when designing a CRISPR: Cas9-based genome editing research. This work bears promise for developing fast and accurate breeding tools to provide better cultivars for farmers and improved products for consumers.

Virus-Induced Gene Silencing in Sweet Basil (Ocimum basilicum).

Virus-induced gene silencing (VIGS) is a powerful reverse genetic tool for rapid functional analysis of plant genes. Over the last decade, VIGS has been widely used for conducting rapid gene knockdown experiment in plants and played a crucial role in advancing applied and basic research in plant science. VIGS was studied extensively in model plants Arabidopsis and tobacco. Moreover, several non-model plants such as Papaver (Hileman et al., Plant J 44:334-341, 2005), Aquilegia (Gould and Kramer, Plant Methods 3:6, 2007), Catharanthus (Liscombe and O'Connor, Phytochemistry 72:1969-1977, 2011), Withania (Singh et al., Plant Biol J 13:1287-1299, 2015), and Ocimum (Misra et al., New Phytol 214:706-720, 2017) were also successfully explored. We have recently developed a robust protocol for VIGS in sweet basil (Ocimum basilicum). Sweet basil, a popular medicinal/aromatic herb, is being studied for the diversity of specialized metabolites produced in it.