Characterization and functional analysis of eugenol O-methyltransferase gene reveal metabolite shifts, chemotype specific differential expression and developmental regulation in Ocimum tenuiflorum L.

Eugenol-O-methyltransferase (EOMT) catalyzes the conversion of eugenol to methyleugenol in one of the final steps of phenylpropanoid pathway. There are no comprehensive reports on comparative EOMT gene expression and developmental stage specific accumulation of phenylpropenes in Ocimum tenuiflorum. Seven chemotypes, rich in eugenol and methyleugenol, were selected by assessment of volatile metabolites through multivariate data analysis. Isoeugenol accumulated in higher levels during juvenile stage (36.86 ng g(-1)), but reduced sharply during preflowering (8.04 ng g(-1)), flowering (2.29 ng g(-1)) and postflowering stages (0.17 ng g(-1)), whereas methyleugenol content gradually increased from juvenile (12.25 ng g(-1)) up to preflowering (16.35 ng g(-1)) and then decreased at flowering (7.13 ng g(-1)) and post flowering (5.95 ng g(-1)) from fresh tissue. Extreme variations of free intracellular and alkali hydrolysable cell wall released phenylpropanoid compounds were observed at different developmental stages. Analyses of EOMT genomic and cDNA sequences revealed a 843 bp open reading frame and the presence of a 90 bp intron. The translated proteins had eight catalytic domains, the major two being dimerisation superfamily and methyltransferase_2 superfamily. A validated 3D structure of EOMT protein was also determined. The chemotype Ot7 had a reduced reading frame that lacked both dimerisation domains and one of the two protein-kinase-phosphorylation sites; this was also reflected in reduced accumulation of methyleugenol compared to other chemotypes. EOMT transcripts showed enhanced expression in juvenile stage that increased further during preflowering but decreased at flowering and further at postflowering. The expression patterns may possibly be compared and correlated to the amounts of eugenol/isoeugenol and methyleugenol in different developmental stages of all chemotypes.

The relationship between growth stages and aroma composition of lemon basil Ocimum citriodorum Vis.

Volatiles from infusions of lemon basil Ocimum citriodorum Vis were evaluated by SPME-GC/MS. Citral, linalool and estragole were the major constituents. Citral, the major contributor to the lemony flavour, was significantly higher in post-flowering (79%) and full-flowering (65%), it was reduced to 42% at pre-flowering. Linalool was consistent throughout the growth cycle (2-3%). Estragole was higher in pre-flowering representing 15% of the total volatiles present. Linalool levels dropped sharply during the full-flowering and post-flowering stages to 2% and 0.4%, respectively. Volatiles from different parts of lemon basil were evaluated to determine the parts that influence the flavour. The percentage composition of citral for leaves and flowers was 64.5% and 58% for the branches. Linalool was much higher in flowers (13%), followed by 11% in branches and 3% in leaves. The flavour attributes of lemon basil infusions can be improved by incorporating aerial branches and flowers in the tea.

Armazenamento de sementes de Alfavaca-cravo (Ocimum gratissimum L. ) / Storage of Clove Basil (Ocimum gratissimum L. ) seeds

A espécie Ocimum gratissimum L. é um subarbusto aromático, pertencente à família Lamiaceae, considerado como importante produtor de óleo essencial e largamente utilizado na medicina popular. Devido à escassez de informações sobre a conservação das sementes dessa espécie o presente estudo objetivou analisar a longevidade das sementes submetidas a diferentes condições de armazenamento, por 12 meses, através de avaliações do potencial germinativo e da capacidade de formação de plântulas normais. As sementes foram armazenadas em três tipos de embalagens: sacos de papel, sacos de papel aluminizado, e frascos de vidro, e nas seguintes condições ambientais: balcão de laboratório, geladeira, e câmara fria. Aos 6, 8, 10 e 12 meses de armazenamento foram avaliados os seguintes parâmetros: umidade, porcentagem de germinação, índice de velocidade de germinação e porcentagem de plântulas normais. Durante o período de armazenamento a manutenção da umidade nas sementes foi mantida, bem como a porcentagem de germinação e de plântulas normais em todos os tratamentos avaliados. As sementes armazenadas apresentaram maiores valores de IVG quando comparadas com as recém-colhidas.

The Ocimum gratissimum L. species is an aromatic subshrub that belongs to the Lamiaceae family and is considered as an important producer of essential oil, widely used in folk medicine. Because of the lack of information about the conservation of O. gratissimum seeds, we aimed to evaluate the longevity of the seeds on different storage conditions for 12 months through the germination and seedling formation percentage in this study. The seeds were stored in three types of packages: paper bags (permeable), aluminized paper bags (semipermeable) and glass flasks (impermeable), in the settings of laboratory bench, refrigerator and cold room over the 12 month period. The germination tests were performed on the 6th, 8th, 10th and 12th month, when moisture, germinability, germination velocity index and percentage of normal seedlings were evaluated. During the storage period, moisture, germinability and percentage of normal seedling were kept in each storage condition. The stored seeds showed increased values of GSI compared to the newly- harvested ones.

Influence of Npk inorganic fertilizer treatment on the proximate composition of the leaves of Ocimum gratissimum (L.) and Gongronema latifolium (benth).

The influence of NPK inorganic fertilizer treatment on the proximate composition of the leaves of Ocimum gratissimum (L.) and Gongronema latifolium (Benth) was investigated. Cultivated O. gratissimum and G. latifolium were treated with NPK (15:15:15) fertilizer at 100, 200, 300, 400 and 500 kg h(-1) treatment levels in planting buckets derived using the furrow slice method two months after seedling emergence. No fertilizer treatment served as control. The leaves of the plants were harvested for analysis one month after treatment. The leaf was used for the analysis because it the most eaten part. Fertilizer treatment significantly (p < 0.05) increased the dry matter, moisture content, ash, crude protein, crude fibre, crude fat contents of the leaves of both plants. On the other hand, fertilizer treatment significantly, (p < 0.05) decreased the carbohydrate and the calorific value of the leaves of the plants. The increase in the concentrations of these substances as a result of fertilizer of fertilizer treatment might be due to the role of fertilizer in chlorophyll content of plant's leaves, which in turn enhanced the process of photosynthesis leading to increased synthesis of these substances. The decrease in the carbohydrate content might be due to its conversion to other materials in the plants. The results obtained were discussed in line with current literatures.

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation.

Ocimum sanctum grown as rain-fed crop, is known to be poorly adapted to waterlogged conditions. Many a times the crop suffers extreme damages because of anoxia and excessive ethylene generation due to waterlogging conditions present under heavy rain. The usefulness of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth promoting rhizobacteria was investigated under waterlogging stress. The comparison of herb yield and stress induced biochemical changes of waterlogged and non-waterlogged plants with and without ACC deaminase-containing microbiological treatments were monitored in this study. Ten plant growth promoting rhizobacteria strains containing ACC-deaminase were isolated and characterized. Four selected isolates Fd2 (Achromobacter xylosoxidans), Bac5 (Serratia ureilytica), Oci9 (Herbaspirillum seropedicae) and Oci13 (Ochrobactrum rhizosphaerae) had the potential to protect Ocimum plants from flood induced damage under waterlogged glass house conditions. Pot experiments were conducted to evaluate the potential of these ACC deaminase-containing selected strains for reducing the yield losses caused by waterlogging conditions. Bacterial treatments protected plants from waterlogging induced detrimental changes like stress ethylene production, reduced chlorophyll concentration, higher lipid peroxidation, proline concentration and reduced foliar nutrient uptake. Fd2 (A. xylosoxidans) induced maximum waterlogging tolerance as treated waterlogged plants recorded maximum growth and herb yield (46.5% higher than uninoculated waterlogged plants) with minimum stress ethylene levels (53% lower ACC concentration as compared to waterlogged plants without bacterial inoculation) whereas under normal non-waterlogged conditions O. rhizosphaerae was most effective in plant growth promotion.

Efeito do BAP no cultivo in vitro de Ocimum selloi Benth / Effect of BAP on in vitro culture of Ocimum selloi Benth

Visando promover a proliferação de brotações em segmentos apicais e nodais de Ocimum selloi em diferentes concentrações de BAP, plantas jovens de 60 dias serviram de doadoras de segmentos apicais e nodais. Os segmentos foram inoculados em meio MS preparado com a metade da concentração dos sais, e acrescido de 1,5% de sacarose e diferentes concentrações de BAP. O experimento foi conduzido no delineamento fatorial, 3 x 4, sendo 3 as posições dos segmentos de O. selloi (segmento apical, primeiro e segundo segmentos nodais) e 4 concentrações de BAP (0 - controle; 2; 4 e 6 mg L-1). Aos 30 dias, foram avaliados o número, comprimento e biomassa fresca e seca de brotos e raízes. Os primeiros e segundos segmentos apresentaram melhores resultados na indução de brotos de O. selloi, 7 e 8 brotos/explante, nas diferentes concentrações de BAP; porém, não houve formação de raízes na presença da citocinina. Nas condições testadas, recomenda-se o uso do primeiro e segundo segmento nodal suplementando o meio de cultivo com BAP para a proliferação in vitro de brotações de O. selloi.

The present study was undertaken to develop the proliferation of sprouts in apical and nodal segments of Ocimum selloi with different BAP levels. Young plants aged 60 days were used as donors of nodal and apical segments. The segments were inoculated in MS medium at half the concentration of salts supplemented with 1.5% of sucrose and different BAP levels. The experiment was in 3 x 4 factorial arrangement, 3 positions of O. selloi segments (apical segment; first and second nodal segment) and 3 BAP levels (0 - control; 2; 4 and 6 mg L-1). After 30 days, the number, the length, and the fresh and dry biomass of sprouts and roots were evaluated. The first and the second segments showed better results in inducing O. selloi sprouts, 7 and 8 sprouts/explant, at the different BAP levels, but there was not root formation in the presence of the cytokinin. Under the tested conditions, use of the first and the second nodal segments is recommended in addition to supplementing the culture medium with BAP for in vitro proliferation of O. selloi sprouts.

Phytoremediatory effect and growth of two species of Ocimum in endosulfan polluted soil.

Endosulfan is a hazardous organochlorine pesticide banned or restricted in several countries. However, it has been found in the environment and in animal samples. To study a potential way to bioremediate soils contaminated with this pesticide, two plant species of the genus Ocimum were studied: Ocimum basilicum L. and Ocimum minimum L., since they are economically feasible and well adapted to the climatic conditions of the Nayarit zone (Mexican pacific coast). Young plants were transplanted into soil experimentally polluted with endosulfan. Growth of both species was not affected by endosulfan, the plants grew, flourished, and produced seeds; 30 days later, endosulfan concentration was lower in the soil with O. basilicum than in the soil without plants. On day 90, no differences in endosulfan concentrations were found between soil with or without O. minimum. At day 1, plants in the polluted soil showed lipoperoxidation, as measured by thiobarbituric acid-reactive species (TBARS). Interestingly, a higher TBARS value was observed at day 3 in transplanted plants as compared to non-transplanted plants. In conclusion, both species can endure endosulfan pollution (as high as 1 g kg(-1)) in soils. O. basilicum seems to be an adequate candidate for bioremediation of soils polluted with endosulfan.

Detecção de Aethalion reticulatum (L., 1767) (Hemiptera: Aethalionidae) em alfavaca-cravo (Ocimum gratissimum L.) e observações sobre sua ocorrência / Detection of Aethalion reticulatum (L., 1767) (Hemiptera: Aethalionidae) in clove basil (Ocimum gratissimum L.) and observations of its occurrence

O objetivo deste trabalho foi relatar a ocorrência da cigarrinha Aethalionidae em alfavaca-cravo (Ocimum gratissimum L.) no município de Bandeirantes-PR. Plantas com cerca de dois anos, na fase reprodutiva, com inflorescências, flores, frutos e sementes, serviram como fonte para a coleta de ninfas e adultos das cigarrinhas. Registrou-se em alfavaca-cravo a espécie Aethalion reticulatum e a interação com as formigas Camponotus rufipes e C. crassus.

The aim of this work was to report the occurrence of the leafhopper Aethalionidae in clove basil (Ocimum gratissimum) in Bandeirantes Municipality, Paraná State, Brazil. Plants around two years old, in the reproductive phase, presenting inflorescences, flowers, fruits and seeds, were used as source for collection of leafhopper nymphs and adults. In clove basil, the species Aethalion reticulatum was recorded, in addition to its interaction with the ants Camponotus rufipes and C. crassus.

Endophytic bacteria from Ocimum sanctum and their yield enhancing capabilities.

Endophytes are beneficial microbes that reside intercellularly inside the plants. Interaction of endophytes with the host plants and their function within their host are important to address ecological relevance of endophyte. Four endophytic bacteria OS-9, OS-10, OS-11, and OS-12 were isolated from healthy leaves of Ocimum sanctum. These isolated microbes were screened in dual culture against various phytopathogenic fungi viz. Rhizoctonia solani, Sclerotium rolfsii, Fusarium solani, Alternaria solani, and Colletotrichum lindemuthianum. Of these, strain OS-9 was found to be antagonistic to R. solani, A. solani, F. solani, and C. lindemuthianum while OS-11 was found antagonistic against A. solani only. The growth-promoting benefits of the endophytes were initially evaluated in the glasshouse by inoculated seeds of O. sanctum. Treatment with endophytes OS-10 and OS-11 resulted in significant enhancement of growth as revealed by increase in fresh as well as dry weight. Further, field trials involving two genotypes OS Purple and CIM-Angana were conducted with strains OS-10 and OS-11. The growth-promoting effect was visible on both the genotypes tested as the endophytes significantly enhanced fresh herbage yield (t/ha). Interestingly, these endophytes increased the content of essential oil particularly in cultivar OS Purple and thereby increasing the total oil yields. Molecular characterization of strain OS-11 indicated the strain to be highly related to the type strain of Bacillus subtilis.

Rosmarinic acid content and RAPD analysis of in vitro regenerated basil (Ocimum americanum) plants.

Various in vitro cultures were established from shoot tips of Ocimum americanum seedlings. Rosmarinic acid content of the in vitro produced plants as well as parent plant were determined by HPLC analysis and subjected to RAPD analysis. MS medium with BA at a concentration of 1 mg/l and 0.25 mg/l IAA supports maximum rosmarinic acid production in plants produced from cultures grown on that medium. RAPD analysis revealed 64 scorable bands from four primers, including six polymorphic bands. The band pattern revealed differences between the parent plant and the in vitro regenerated plants. Certain band changes were found in O. americanum plants regenerated in vitro, suggesting the existence of genetic variation that might affect the biochemical synthesis of plants derived from tissue culture.

Essential oil yield and quality of methyl eugenol rich Ocimum tenuiflorum L.f. (syn. O. sanctum L.) grown in south India as influenced by method of harvest.

A field experiment carried out during 2001--2002 under semi-arid conditions of Hyderabad, India investigated the effect of three different methods of harvesting at full bloom stage, on essential oil yield and quality of methyl eugenol rich sacred/holy basil (Ocimum tenuiflorum L.f.; Lamiaceae). The harvest methods were: harvesting of primary branches, secondary branches and shoot biomass cut at 30 cm above ground level. Four harvests at 102, 192, 287 and 360 days after transplanting of the crop were taken in 1 year in each method of harvest. Harvesting of secondary branches led to maximum plant height and number of secondary branches per plant compared to harvesting of primary branches or shoot biomass cut at 30 cm above ground during second, third and fourth harvests. On the contrary, secondary branch harvest gave least biomass yield in all the four harvests. But due to higher essential oil content, secondary branch harvest gave 25.2 and 15.4% higher total (sum total of all four harvests) essential oil yield (kg/ha per year) over primary branches and shoot biomass cut at 30 cm above ground methods of harvesting, respectively. A similar treatment difference was observed in respect of oil composition studied in the first harvest. Harvesting shoot biomass at 30 cm above ground produced oil containing highest amount of methyl eugenol. The content of methyl eugenol decreased in the order of shoot biomass cut at 30 cm above ground > primary branch > secondary branch treatments. A reverse trend was observed, however, in respect of (E)-cinnamyl acetate, eugenol and beta-elemene constituents of the oil. Little variability was, however, observed among the treatments in respect of 24 other constituents of the oils.