An efficient protocol for in vitro multiplication of hybrid tea rose cv. Grand Gala was developed using axillary bud segments.Out of different pre-treatments of explants tried, the highest explant survival (82.16%) was obtained with carbendazim (0.2%) + diathane M-45 (0.2%) + 8-HQC (200 mg/l) for 4 h on a horizontal shaker (120 rpm). Sucrose concentration of 40 g/l in the medium seems to be optimal for in vitro shoot multiplication. Murashige and Skoog medium supplemented with 5.0 mg/l BAP + 0.2 mg/l NAA + 0.5 mg/l GA3 was found most effective for culture establishment and shoot proliferation with highest number of micro-shoots (6.49 shoots/explant). Rooting of micro-shoots was induced on half-strength MS basal medium supplemented with NAA (0.5 mg/l) + IBA (0.5 mg/l). The regenerated plantlets were efficiently hardened in glass jars filled with vermiculite + agropeat (1:2) moistened with half-strength MS medium salts and covered with polypropylene lids, thereafter plants were successfully transferred to the glasshouse with good survival.
An efficient protocol for in vitro multiplication of hybrid tea rose cv. Pusa Mohit was developed using axillary bud explant. Out of different pre-treatments of explants with fungicides and bactericides tried, the highest explant survival (62.47%) was obtained with carbendazim (0.2%) + diathane M-45 (0.2%) + 8-HQC (200 mg/l) for 3 h on a horizontal shaker (120 rpm). The effect of different combinations of BAP (2.5, 3.0 and 3.5 mg/l), NAA (0.1 and 0.2 mg/l), and GA3 (0.3 and 0.5 mg/l) on culture establishment and shoot proliferation was studied. MS medium supplemented with 3.5 mg/l BAP + 0.1 mg/l NAA + 0.5 mg/l GA3 was found most effective for culture establishment and shoot proliferation with highest number of micro-shoots (5.30 shoots per explant). Rooting on micro-shoots was induced on half-strength MS basal medium supplemented with NAA (0.5 mg/l) + IBA (0.5 mg/l). The regenerated plantlets were efficiently hardened in glass jars filled with vermiculite + agropeat (1:2) moistened with one-quarter strength MS medium salts and covered with polypropylene lids. After hardening the plants were successfully transferred to the glasshouse with good survival.
An efficient protocol for in vitro multiplication of Rosa × hybrida L. cv. Happiness was standardized using axillary bud segments. Out of different pre treatments for explants, the highest explant survival (80.25%) was obtained with T1 pre-treatment comprising 0.2% Carbendazim + 0.2% Mancozeb-45 +150 mg/l 8-HQC for 4 hragitation on a horizontal shaker (200 rpm). Sucrose concentration of 30g/l in the medium was found to be optimum for in vitro shoot multiplication. Murashige and Skoog (MS) medium supplemented with 2.5 mg/l BAP + 5.0 mg/l kinetin + 0.1 mg/l NAA + 0.5 mg/l GA3 was found most effective for culture establishment, however, MS medium comprising 2.5 mg/l BAP + 2.5 mg/l kinetin + 0.1mg/l NAA+ 0.5mg/l GA3 along with 40 mg/l adenine sulphate was found to be better for shoot proliferation with highest number of micro shoots (7.10 shoots/explant). Rooting of micro shoots was induced on half strength MS basal medium supplemented with NAA (0.5 mg/l) and IBA (0.5 mg/l) rooting growth regulators. The regenerated plantlets were efficiently hardened in glass jars filled with coco peat + vermiculite + perlite (2:1:1) moistened with half strength MS medium salts and covered with polypropylene lids, thereafter, plants were successfully transferred to the glasshouse with good survival.
Deep sequencing of a plant transcriptome Jeff Martin 1 , Stephen Gross 1 , James Schnable 2 , Cindy Choi 1 , Mei Wang 1 , Kanwar Singh 1 , Erika Lindquist 1 , Feng Chen 1 , Chia-Lin Wei 1 , and Zhong Wang 1 , 1 Department of Energy Joint Genome Institute // LBNL - Walnut Creek, CA 2 University of California Berkeley - Berkeley, CA a To whom correspondence may be addressed. E-mail: jamartin@lbl.gov March 20, 2012 ACKNOWLEDGMENTS: The work conducted by the US Department of Energy (DOE) Joint Genome Institute is supported by the Office of Science of the DOE under Contract Number DE-AC02-05CH11231. The views and opinions of the authors expressed herein do not necessarily state or reflect those of the United States Government, or any agency thereof, or the Regents of the University of California. DISCLAIMER: This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California
ABSTRACT Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many SIP studies rely on 16S rRNA sequences to identify active taxa but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes, and their level of isotopic enrichment, were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytic models for identifying active taxa, and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg , an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. Importance Answering the question of ‘ who is eating what?’ within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. This question is often pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism’s taxonomic identity and genome composition, while providing quantitative estimates of the microorganism’s isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.
Author(s): Singh, Kanwar; Zhao, Zhiying Jean; Zvenigorodsky, Natasha; Froula, Jeff; Pennacchio, Len; Chen, Feng | Abstract: Next generation DNA sequencing provides new opportunities to efficiently accomplish a variety of genomic tasks such as the de novo assembly of genomes. The newly released 454 FLX Titanium in October 2008 advanced the previous 454 FLX Standard system with nearly double the sequence reads and read lengths. A thorough assessment and optimization of 454 FLX Titanium sequencing system was done by the Technology Development Group at Joint Genome Institute. Currently applications like de novo whole genome shotgun assembly, transcriptome profiling, pyrotag sequencing and sequence capture technology are being optimized on the new 454 FLX Titanium systems.
An effort was made to develop the protocol of in vitro mass multiplication in marigold cultivar Pusa Narangi Gainda from the sterilized selfed seeds on Murashige and Skoog medium supplemented with different combinations of BAP, NAA and GA3. High frequency of germination (84.67%) was obtained 1.0 mg/l BAP and 0.5 mg/l GA3. The growth of individual shoot tip were significantly enhanced (4.1 cm) when the excised shoot tips were transferred on to MS medium with 2.0 mg/l BAP, 0.1 mg/l NAA and 0.5 mg/l GA3. Low auxin (0.2 mg/l NAA) in the culture medium in the contrary to high cytokinin (3.0 mg/l BAP) concentration referred the highest shoot multiplication (10.15 shoots/explant). Half-strength MS medium supplemented with 1.0 mg/l NAA and 1.0 mg/l IBA was observed to be efficient for rooting (87.33%). The rooted plants could be ideally acclimatized in glasshouse conditions for field transfer.
supplemented with 13.31 µM 6-benzyl aminopurine (BAP) plus 0.54 µM α-napthalene acetic acid (NAA) plus 1.44 µM giberellic acid (GA3) plus 0.8% w/v agar-agar to induce sprouting and shoot proliferation. The 40 Gy-treatment was found to be the LD50 dose. Morphological abnormalities, such as, fused leaves, leaf albinism and variegated leaves, were observed at the higher doses (25, 40 & 55 Gy). In vitro raised mutant plants and non-irradiated (control) plants were transferred to plastic pots 1 month after acclimatisation and examined for their morphological traits. Two types of flower colour mutants with altered or novel flower colour in comparison to original flower colour were isolated. The aim of the present study was to develop a protocol for induction of mutagenesis that could be used successfully to develop novel traits in rose.
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