Effect of Agrobacterium Infection Time, Co-Cultivation and Cell Density on in vitro Response in Hypocotyl of Eggplant (Solanum melongena L.)

The present study purports to assess the effect of Agrobacterium infection time, co-cultivation and cell density on in vitro response in hypocotyl explants of eggplant (brinjal) cv. Manjarigota. Agrobacterium(OD600 0.3-0.5) infection for 10-15 minutes (24.44±2.34%) was found to be optimum, while, higher or lower infection-time resulted in reduced callus initiation, shoot regeneration and explant survival. Explants with no (only Agrobacterium infection) or short (1 day) co-cultivation, showed reduced callus-initiation response and turned yellow, with no regeneration. Callus-initiation response increased from Day 1 (96.66±03.33%), and reached a maximum on Day 2 and Day 3 (100±00.00%). It decreased on further increase in co-cultivation time. Explants co-cultivated for three days showed highest regeneration response (30.00±02.96%) which thereafter reduced with further increase in co-cultivation time. Explants infected with Agrobacterium culture at 0.05 OD600 showed hardly any regeneration, and turned yellow and necrotic on the selection medium. Highest regeneration response (28.33±02.33%) was obtained in explants infected with 0.1 OD600 culture, and this gradually reduced as celldensity increased (upto 1.0 OD600), becoming zero in explants treated with cultures at 1.5 OD600 or above. Agrobacterium overgrowth was noticed on explants infected with cultures of 0.5 OD600 and above. Exposure of hypocotyl explants to higher cell-density, longer infection-time and prolonged co-cultivation regime resulted in severe necrosis of explants; time taken for development of Agrobacterium overgrowth was less with increase in the level of these factors. Regenerated shoots were healthy, green, elongated and showed root induction on culture medium containing Kanamycin

Factors Affecting in Vitro Shoot Regeneration in Hypocotyls of Brinjal (Solanum melongena L.) in the Early Steps of Agrobacterium-Mediated Transformation

An attempt was made to assess the effect of size, age and position of the explant, pre-culture and high cytokinin concentration in the pre-culture medium on shoot regeneration in brinjal hypocotyls co-cultivated with Agrobacterium. The study was carried out using hypocotyl explants of brinjal cv. Manjarigota, Agrobacterium strain A208 and shoot regeneration medium (full-strength basal MS medium, 2μM BAP + 0.05μM NAA, 3% sucrose and 0.8% agar) containing Cefotaxime (250-500mg l-1) and Kanamycin (100mg l-1). Hypocotyl explants showed callus initiation and shoot regeneration response after 10-12 and 20-22 days of culture, respectively. Five-day-old explants did not survive Agrobacterium infection, and ten-day-old explants showed higher shoot regeneration (29±1.91%) than older explants. Explants of medium size (1cm long; 32±2.62%) from the apical region (38.57±2.61%) showed better shootregeneration ability than explants of any other size or region. A period of four days of pre-culture (33.33±3.76) was optimal best for best shoot-regeneration in hypocotyl explants. No regeneration was seen in hypocotyl explants at shorter or longer pre-culture period. High cytokinin (10μM) in shoot regeneration medium during pre-culture enhanced shoot regeneration response (47.27±2.98%) in explants co-cultivated with Agrobacterium. Effects of various factors documented in this study will be useful in developing an efficient Agrobacterium-mediated transformation protocol in brinjal cv. Manjarigota

Assessment of Pathogenicity in Helminthosporium maydis causing Southern Corn Leaf Blight Disease in the Region of Karnataka

Maize (Zea mays L) is the one of the most beneficial crops, adapted to various ecological and climatic states, it grades third after wheat and rice. Based on the research determinations for the last few years under the leadership of All India Coordinated Maize Improvement Project, 16 out of 61 diseases harmfully affecting this crop. One of the major diseases is Southern corn leaf blight (SCLB). The causative agent of the prevalent was recognized as the fungus Helminthosporium maydis. Research was carried out for pathogenicity assay. Pathogenicity assay was conducted with two methods, by collecting spores (2X105/ml), spraying on one month old maize plant. After 24 – 48 hours, it was found that spores collected from Davanagere (HMS3) and Kodagu (HMS5) region shows more yellow to brown lesion compare to all other regions. Second by extraction of toxin by methanol – chloroform method, purification by adsorption on charcoal and separated by using column chromatography and by thin layer chromatography. The Rf values, FTIR and UV absorption spectrum of purified toxin reveals the production of host specific toxin by H.maydis. Determination minimum toxic concentration required to satisfy the conditions as a host specific toxin. Keywords: Survey, Pathogenicity, Extraction, Host specific Toxin, Southern Corn Leaf Blight

Effect of Age and Size of Hypocotyl Explant on in vitro Shoot Regeneration in Eggplant

In the present study, effect of size and age of hypocotyl explant on in vitro organogenetic responses was assessed in eggplant cv. Manjarigota. Size and age did not affect callus-initiation response, but showed marked influence on shoot regeneration response. Hypocotyl explants 1.5cm long showed highest shoot regeneration response (77.4%); either increase or decrease in size resulted in reduced response. Five to 15 day old hypocotyl explants showed direct shoot regeneration from cut ends, whereas 20-30 day old hypocotyl explants showed indirect shoot regeneration from callus produced on cut ends. Five day old explants were most responsive, with highest (91.23%) and thirty day old explants least responsive with reference to shoot regeneration response (20.85%). Shoot regeneration frequency decreased with increasing age, whereas shoot regeneration efficiency increased with increasing age of hypocotyl explants

Effects of Growth Regulators and Explant-Type on Agrobacterium-Mediated Transformation in Brinjal (Solanum melongena L.) cv. Manjarigota

Effects of growth regulators and type of explants on transformation and in vitro morphogenetic responses of brinjal cv. Manjarigota were studied. Both hypocotyl and cotyledonary explants showed marked influence on in vitro morphogenetic responses after Agrobacterium co-cultivation. Hypocotyl explants showed callus initiation and regeneration responses earlier than cotyledonary leaves. Hypocotyl explants were found to be better than cotyledonary leaf explants in regenerating shoots after Agrobacterium co-cultivation. There was delay and reduction in both callus and regeneration responses in Agrobacterium co-cultivated explants. Hypocotyl explants showed the highest regeneration response on MS medium containing 2 μM BAP and 0.05 μM NAA while cotyledonary leaves did not show regeneration response after Agrobacterium co-cultivation. However, they showed green buds on MS medium containing 10 μM BAP and 1 μM NAA, which could not differentiate into shoots. Overall, hypocotyl explants were found better in regenerating shoots after Agrobacterium co-cultivation

Effect of Antibiotics and Gelling Agents in Transformation of Brinjal (Solanum melongena L.) cv. Manjarigota

A study was conducted to find out the effect of antibiotics and gelling agents on Agrobacterium-mediated transformation using hypocotyl explants of brinjal cv. Manjarigota. Hypocotyl explants of brinjal were found to be sensitive even to the lowest level of kanamycin (25 mg/l) tested. Explants that showed increased callus initiation and regeneration response upon cocultivation with Agrobacterium and on kanamycin at 100 mg/l were selected as this indicated a highly effective selection pressure. Cefotaxime did not affect regeneration response and at 500 mg/l, it effectively inhibited Agrobacterium overgrowth completely on Agrobacterium cocultivated hypocotyl explants. There were marked differences in regeneration response in hypocotyl explants cultured on medium solidified with various gelling agents indicating the influence of gelling agent on the activity of kanamycin in culture medium, which indirectly affects selection and recovery of transformants. Antibiotics and gelling agents could therefore affect, directly or indirectly, transformation of brinjal cv. Manjarigota

Synergistic Use of Hypocotyl Explants and High Bap Preconditioning for Enhanced Transformation Frequency in Brinjal (Solanum melongena L.)

Poor regeneration is one of the limiting factors in the development of transgenic crops since Agrobacterium as a plant pathogen can disturb the fragile in vitro conditions with wounding and infection regimes. We have tried to optimize the transformation system in two important varieties of brinjal after Agrobacterium infection to the explants. The effect of explant was studied and hypocotyls were found to be better than cotyledonary leaves. High BAP during the preconditioning period was found to further enhance the regeneration rate. Therefore, use of hypocotyls and high BAP during preconditioning can improve the regeneration of transformed cells and recovery of transformants in vegetables especially brinjal

Cloning and characterization of microRNAs from rainbow trout (Oncorhynchus mykiss): Their expression during early embryonic development

<p>Abstract</p> <p>Background</p> <p>Current literature and our previous results on expression patterns of oocyte-specific genes and transcription factors suggest a global but highly regulated maternal mRNA degradation at the time of embryonic genome activation (EGA). MicroRNAs (miRNAs) are small, non-coding regulatory RNAs (19–23 nucleotides) that regulate gene expression by guiding target mRNA cleavage or translational inhibition. These regulatory RNAs are potentially involved in the degradation of maternally inherited mRNAs during early embryogenesis.</p> <p>Results</p> <p>To identify miRNAs that might be important for early embryogenesis in rainbow trout, we constructed a miRNA library from a pool of unfertilized eggs and early stage embryos. Sequence analysis of random clones from the library identified 14 miRNAs, 4 of which are novel to rainbow trout. Real-time PCR was used to measure the expression of all cloned miRNAs during embryonic development. Four distinct expression patterns were observed and some miRNAs showed up-regulated expression during EGA. Analysis of tissue distribution of these miRNAs showed that some are present ubiquitously, while others are differentially expressed among different tissues. We also analyzed the expression patterns of Dicer, the enzyme required for the processing of miRNAs and Stat3, a transcription factor involved in activating the transcription of miR-21. Dicer is abundantly expressed during EGA and Stat3 is up-regulated before the onset of EGA.</p> <p>Conclusion</p> <p>This study led to the discovery of 14 rainbow trout miRNAs. Our data support the notion that Dicer processes miRNAs and Stat3 induces expression of miR-21 and possibly other miRNAs during EGA. These miRNAs in turn guide maternal mRNAs for degradation, which is required for normal embryonic development.</p

A graphene-based physiometer array for the analysis of single biological cells

A significant advantage of a graphene biosensor is that it inherently represents a continuum of independent and aligned sensor-units. We demonstrate a nanoscale version of a micro-physiometer – a device that measures cellular metabolic activity from the local acidification rate. Graphene functions as a matrix of independent pH sensors enabling subcellular detection of proton excretion. Raman spectroscopy shows that aqueous protons p-dope graphene – in agreement with established doping trajectories, and that graphene displays two distinct pKa values (2.9 and 14.2), corresponding to dopants physi- and chemisorbing to graphene respectively. The graphene physiometer allows micron spatial resolution and can differentiate immunoglobulin (IgG)-producing human embryonic kidney (HEK) cells from non-IgG-producing control cells. Population-based analyses allow mapping of phenotypic diversity, variances in metabolic activity, and cellular adhesion. Finally we show this platform can be extended to the detection of other analytes, e.g. dopamine. This work motivates the application of graphene as a unique biosensor for (sub)cellular interrogation.National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)U.S. Army Research LaboratoryUnited States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant P41EB015871-27)Skolkovo Institute of Science and Technolog