Plant Biotechnology Institute

Abstract Ocimum sanctum is a multipurpose herb with highly significant medicinal properties. An in vitro direct regeneration protocol for propagation of a valuable medicinal plant Ocimum sanctum, using petiole explants has been successfully developed. The protocol employed regeneration of shoots directly, without any intervening callus using Murashige and Skoog (MS) medium fortified with 3 mg L-1 BAP + 1 mg L-1 NAA. The maximum regeneration frequency of 98% with 9.6 shoots per explants was achieved. Agrobacterium tumefaciens mediated genetic transformation (ATMT) protocol (transient and stable) was established using LBA4404 strain harboring pBI121 with uid-A reporter gene and neomycin phosphotransferase (npt-II) as selection marker. The putative transformants were screened on MS with 50 mg L-1 kanamycin and subsequently rooted on the half-strength MS medium. The confirmation was done via polymerase chain reaction (PCR) using npt-II and gus-A gene-specific primers. The maximum stable transformation frequency 70% ± 0.35. Thus, it is apparent that the established in vitro direct regeneration and ATMT method was suitable for integrating novel genes and modulating the metabolic flux for obtaining desired agronomic trait in planta.

Studies on the model plant Arabidopsis thaliana have uncovered the identities of most enzymatic components involved in seed storage lipid biosynthesis. However, much remains to be learned on how pathway interactions operate in the seed metabolic network. In this study, we dissected seed glycerolipid molecular compositional changes in the Arabidopsis mutant deficient in diacylglycerol acyltransferase 1 (DGAT1). Our results indicate that metabolic adjustments occurred in both phosphatidylcholine synthesis and deacylation in developing seeds. Ultrastructural changes of perturbed oil and protein bodies were also evident in cotyledon parenchyma cells. To unmask the physiological and developmental role associated with DGAT1-mediated neutral lipid biosynthesis, we attempted to combine dgat1 mutation with lpcat2 that harbors a defect in lysophosphatidylcholine acyltransferase 2 (LPCAT2). Disruption in both DGAT1 and LPCAT2 led to an apparent defect in pollen development that manifested as pollen sterility. Collectively, our results highlight a role of DGAT1 in both storage lipid synthesis and plant development.

Core Ideas Carinata growth and yield was influenced more by row spacing than seeding rate. Rows spaced at 36 cm maximized carinata yield. Carinata branching was favored by wider row spacing. Carinata (Brassica carinata A. Braun) is an oilseed crop with potential as a winter crop in the southeastern United States to diversify crop rotations and provide biofuel production and livestock feed. The objective was to evaluate the effects of row spacing and seeding rate on carinata yield and oil composition. Field experiments were conducted in Jay and Quincy, FL, from 2013 to 2016 evaluating carinata growth, seed and oil yield, and oil composition grown in a factorial arrangement of four seeding rates (3, 6, 9, and 12 kg ha−1) and four row spacings (18, 36, 53, and 89 cm). No interactions between seeding rate and row spacing were detected. Seeding rate did not influence any of the variables studied. In contrast, row spacing affected seed and oil yield, branch production, and pods per plant. Seed yield (ranked from highest to lowest) was 2761, 2286, 1851, and 1572 kg ha−1 for rows spaced at 36, 18, 53, and 89 cm, respectively. Branching and pods per plant increased with row spacing. Neither seeding rate nor row spacing affected oil concentration and quality. Oil concentration averaged 40%, of which more than a third was erucic acid (C22:1). Protein concentration was 31%, and glucosinolate concentration was 93 μmol g−1. The results of the present study demonstrated that carinata can be successfully grown in the southeastern United States, reaching yields and oil quality similar to those reported at other latitudes, and can be a source of biofuel, protein for animal feed, and cropping system diversification for growers.

The present study investigated the influence of chia consumption on inflammation, oxidative stress, and lipid profiles in adult female ovariectomized rats fed a high-fat diet. Forty ovariectomized and 40 intact (SHAM) rats were allocated into 8 groups (n = 10), and each rat received one of the following four diets: standard diet (ST); standard diet + chia (STC); high-fat diet (HF); and high-fat diet + chia (HFC) for 126 days. Biochemical parameters and biomarkers of lipid peroxidation, inflammation, and oxidative stress were evaluated. The mRNA expression levels of PPAR-α, NFκB, TNF-α and Zn-SOD1 were analyzed, as well as those of TNF-α and IL-1β. Chia intake increased HDL cholesterol (HDL-c) and reduced LDL cholesterol (LDL-c) levels. Plasma catalase activity was elevated in the STC group. Concentrations of TBARS were higher in all groups fed HF. PPAR-α mRNA expression was elevated, and levels of NFκB mRNA expression were reduced in the STC group. mRNA expression and protein levels of TNF-α were lower in rats fed the standard diet. Protein levels of IL-1β were reduced in rats fed the standard diet, and the high fat diet with chia. In general, ovariectomy did not influence the inflammatory and oxidative stress parameters. Chia intake improved antioxidant activity by increasing SOD expression, PPAR-α expression, catalase activity, and HDL-c levels. In addition, chia consumption decreased the concentrations of the inflammatory markers IL-1β and LDL-c.

Phytophthora infestans (P. infestans) is the causal agent of potato late blight, which caused the devastating Irish Potato Famine during 1845-1852. Until now, potato late blight is still the most serious threat to potato growth and has caused significant economic losses worldwide. Melatonin can induce plant innate immunity against pathogen infection, but the direct effects of melatonin on plant pathogens are poorly understood. In this study, we investigated the direct effects of melatonin on P. infestans. Exogenous melatonin significantly attenuated the potato late blight by inhibiting mycelial growth, changing cell ultrastructure, and reducing stress tolerance of P. infestans. Notably, synergistic anti-fungal effects of melatonin with fungicides on P. infestans suggest that melatonin could reduce the dose levels and enhance the efficacy of fungicide against potato late blight. A transcriptome analysis was carried out to mine downstream genes whose expression levels were affected by melatonin. The analysis of the transcriptome suggests that 66 differentially expressed genes involved in amino acid metabolic processes were significantly affected by melatonin. Moreover, the differentially expressed genes associated with stress tolerance, fungicide resistance, and virulence were also affected. These findings contribute to a new understanding of the direct functions of the melatonin on P. infestans and provide a potential ecofriendly biocontrol approach using a melatonin-based paradigm and application to prevent potato late blight.

Fatty acid biosynthesis is a primary metabolic pathway that occurs in plastids, whereas the formation of glycerolipid molecules for the majority of cellular membrane systems and the deposition of storage lipid in seeds takes place in the cytosolic compartment. In this report, we present a study of an Arabidopsis mutant, ar21, with a novel seed fatty acid phenotype showing higher contents of eicosanoic acid (20:1) and oleic acid (18:1) and a reduced level of α-linolenic acid (18:3). A combination of map-based cloning and whole-genome sequencing identified the genetic basis underlying the fatty acid phenotype as a lesion in the plant-specific eukaryotic translation initiation factor eIFiso4G1. Transcriptome analysis on developing seeds revealed a reduced level of plastid-encoded genes. Specifically, decreases in both transcript and protein levels of an enzyme involved in fatty acid biosynthesis, the β-subunit of the plastidic heteromeric acetyl-CoA carboxylase (htACCase) encoded by accD, were evident in the mutant. Biochemical assays showed that the developing seeds of the mutant possessed a decreased htACCase activity in the plastid but an elevated activity of homomeric acetyl-CoA carboxylase (hmACCase). These results suggested that the increased 20:1 was attributable at least in part to the enhanced cytosolic hmACCase activity. We also detected a significant repression of FATTY ACID DESATURASE 3 (FAD3) during seed development, which correlated with a decreased 18:3 level in seed oil. Together, our study on a mutant of eIFiso4G1 uncovered multifaceted interactions between the cytosolic and plastidic compartments in seed lipid biosynthesis that impact major seed oil traits.

Allelic diversity of the allied species of Brassica napus L. as well as of the winter form of this species has been demonstrated to be related with increasing productivity of hybrid spring B. napus cultivars. To compare potential value of the different gene pools of Brassica species three spring B. napus inbred populations were developed by use of a B. oleracea L. line, a spring B. napus breeding line, and a winter B. napus cultivar crossed to a spring B. napus ‘Hi‐Q’; and test hybrids of these inbred lines were produced by crossing with Hi‐Q as the common tester. Mid‐parent heterosis (MPH) showed a negative correlation with seed yield of the inbred lines in all three populations; however, a positive correlation existed between seed yield of the inbred lines and heterosis over Hi‐Q (HiQH) (or, inbred vs. hybrid yield). On average, the level of MPH in hybrid of the inbred lines derived from B. napus × B. oleracea cross was twice greater than the level of heterosis found for the inbred lines derived from spring × spring or winter × spring B. napus crosses. The inbred population derived from winter × spring cross gave highest seed yield, and this population also gave highest HiQH. The results suggested that B. oleracea and winter canola could be used in spring B. napus canola breeding for accumulating additive and non‐additive effect genes for increased seed yield in hybrid cultivars.