Genome-wide transcriptional analysis of super-embryogenic Medicago truncatula explant cultures

BACKGROUND: The Medicago truncatula (M. truncatula) line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than its wild type progenitor Jemalong. To understand the molecular basis for the regeneration capacity of this super-embryogenic line 2HA, using Affymetrix GeneChip®, we have compared transcriptomes of explant leaf cultures of these two lines that were grown on media containing the auxin NAA (1-naphthaleneacetic acid) and the cytokinin BAP (6-benzylaminopurine) for two weeks, an early time point for tissue culture proliferation. RESULTS: Using Affymetrix GeneChip®, GCRMA normalisation and statistical analysis, we have shown that more than 196 and 49 probe sets were significantly (p < 0.05) up- or down-regulated respectively more than 2 fold in expression. We have utilised GeneBins, a database for classifying gene expression data to distinguish differentially displayed pathways among these two cultures which showed changes in number of biochemical pathways including carbon and flavonoid biosynthesis, phytohormone biosynthesis and signalling. The up-regulated genes in the embryogenic 2HA culture included nodulins, transporters, regulatory genes, embryogenesis related arabinogalactans and genes involved in redox homeostasis, the transition from vegetative growth to reproductive growth and cytokinin signalling. Down-regulated genes included protease inhibitors, wound-induced proteins, and genes involved in biosynthesis and signalling of phytohormones auxin, gibberellin and ethylene. These changes indicate essential differences between the super-embryogenic line 2HA and Jemalong not only in many aspects of biochemical pathways but also in their response to auxin and cytokinin. To validate the GeneChip results, we used quantitative real-time RT-PCR to examine the expression of the genes up-regulated in 2HA such as transposase, RNA-directed DNA polymerase, glycoside hydrolase, RESPONSE REGULATOR 10, AGAMOUS-LIKE 20, flower promoting factor 1, nodulin 3, fasciclin and lipoxygenase, and a down-regulated gene ETHYLENE INSENSITIVE 3, all of which positively correlated with the microarray data. CONCLUSION: We have described the differences in transcriptomes between the M. truncatula super-embryogenic line 2HA and its non-embryogenic progenitor Jemalong at an early time point. This data will facilitate the mapping of regulatory and metabolic networks involved in the gaining totipotency and regeneration capacity in M. truncatula and provides candidate genes for functional analysis

Genome-wide transcriptional analysis of super-embryogenic Medicago truncatula explant cultures

Background. The Medicago truncatula (M. truncatula) line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than its wild type progenitor Jemalong. To understand the molecular basis for the regeneration capacity of this super-embryogenic line 2HA, using Affymetrix GeneChip, we have compared transcriptomes of explant leaf cultures of these two lines that were grown on media containing the auxin NAA (1-naphthaleneacetic acid) and the cytokinin BAP (6-benzylaminopurine) for two weeks, an early time point for tissue culture proliferation. Results. Using Affymetrix GeneChip, GCRMA normalisation and statistical analysis, we have shown that more than 196 and 49 probe sets were significantly (p < 0.05) up- or down-regulated respectively more than 2 fold in expression. We have utilised GeneBins, a database for classifying gene expression data to distinguish differentially displayed pathways among these two cultures which showed changes in number of biochemical pathways including carbon and flavonoid biosynthesis, phytohormone biosynthesis and signalling. The up-regulated genes in the embryogenic 2HA culture included nodulins, transporters, regulatory genes, embryogenesis related arabinogalactans and genes involved in redox homeostasis, the transition from vegetative growth to reproductive growth and cytokinin signalling. Down-regulated genes included protease inhibitors, wound-induced proteins, and genes involved in biosynthesis and signalling of phytohormones auxin, gibberellin and ethylene. These changes indicate essential differences between the super-embryogenic line 2HA and Jemalong not only in many aspects of biochemical pathways but also in their response to auxin and cytokinin. To validate the GeneChip results, we used quantitative real-time RT-PCR to examine the expression of the genes up-regulated in 2HA such as transposase, RNA-directed DNA polymerase, glycoside hydrolase, RESPONSE REGULATOR 10, AGAMOUS-LIKE 20, flower promoting factor 1, nodulin 3, fasciclin and lipoxygenase, and a down-regulated gene ETHYLENE INSENSITIVE 3, all of which positively correlated with the microarray data. Conclusion. We have described the differences in transcriptomes between the M. truncatula super-embryogenic line 2HA and its non-embryogenic progenitor Jemalong at an early time point. This data will facilitate the mapping of regulatory and metabolic networks involved in the gaining totipotency and regeneration capacity in M. truncatula and provides candidate genes for functional analysis

Transcriptional profiling of Medicago truncatula meristematic root cells

BACKGROUND: The root apical meristem of crop and model legume Medicago truncatula is a significantly different stem cell system to that of the widely studied model plant species Arabidopsis thaliana. In this study we used the Affymetrix Medicago GeneChip® to compare the transcriptomes of meristem and non-meristematic root to identify root meristem specific candidate genes. RESULTS: Using mRNA from root meristem and non-meristem we were able to identify 324 and 363 transcripts differentially expressed from the two regions. With bioinformatics tools developed to functionally annotate the Medicago genome array we could identify significant changes in metabolism, signalling and the differentially expression of 55 transcription factors in meristematic and non-meristematic roots. CONCLUSION: This is the first comprehensive analysis of M. truncatula root meristem cells using this genome array. This data will facilitate the mapping of regulatory and metabolic networks involved in the open root meristem of M. truncatula and provides candidates for functional analysis.This research is supported by a grant from the Australian Research Council Centre of Excellence Program (CE0348212). PH was supported by an Australian Postgraduate Award. We thank Lily Shen from the ANU Electron Microscopy Unit for assistance with microscopy

Transcriptional profiling of Medicago truncatula meristematic root cells

<p>Abstract</p> <p>Background</p> <p>The root apical meristem of crop and model legume <it>Medicago truncatula </it>is a significantly different stem cell system to that of the widely studied model plant species <it>Arabidopsis thaliana</it>. In this study we used the Affymetrix <it>Medicago </it>GeneChip^®to compare the transcriptomes of meristem and non-meristematic root to identify root meristem specific candidate genes.</p> <p>Results</p> <p>Using mRNA from root meristem and non-meristem we were able to identify 324 and 363 transcripts differentially expressed from the two regions. With bioinformatics tools developed to functionally annotate the <it>Medicago </it>genome array we could identify significant changes in metabolism, signalling and the differentially expression of 55 transcription factors in meristematic and non-meristematic roots.</p> <p>Conclusion</p> <p>This is the first comprehensive analysis of <it>M. truncatula </it>root meristem cells using this genome array. This data will facilitate the mapping of regulatory and metabolic networks involved in the open root meristem of <it>M. truncatula </it>and provides candidates for functional analysis.</p

Effects of Forage Type, Storage Method, and Moisture Level in Crop Residues Mixed with Modified Distillers Grains

Two growing experiments compared effects of feeding a diet consisting of cornstalks or wheat straw and modified distillers grains when ensiled or mixed fresh daily. Wheat-straw based diets also were compared at different moisture levels (50% and 70%) when ensiled and mixed daily. In Experiment 1, steers fed ensiled diets had greater DMI compared to diets mixed daily. Moisture level and crop residue type had no effect on steer performance. In Experiment 2, steers were offered the supplements and a hay mix to determine palatability and forage replacement. Moisture level had no effect, cornstalks were consumed better than wheat straw, and steers fed freshly mixed diets gained more and were more efficient than those fed ensiled mixes

Differences in efficacy of monepantel, derquantel and abamectin against multi-resistant nematodes of sheep

Drug resistance has become a global phenomenon in gastrointestinal nematodes of sheep, particularly resistance to macrocyclic lactones. New anthelmintics are urgently needed for both the control of infections with multi-resistant nematodes in areas where classical anthelmintics are no longer effective, and the prevention of the spread of resistance in areas where the problem is not as severe. Recently, two new active ingredients became commercially available for the treatment of nematode infections in sheep, monepantel (Zolvix®) and derquantel, the latter used only in a formulated combination with the macrocyclic lactone, abamectin (Startect®). In order to assess the potential of the new actives for the control and prevention of spread of anthelmintic resistance, two characterized multi-resistant field isolates from Australia were used in a GLP (good laboratory practice) conducted efficacy study in sheep. Eight infected sheep in each group were treated orally according to the product labels with 2.5 mg/kg body weight monepantel, 0.2 mg/kg abamectin, or with the combination of 2.0 mg/kg derquantel and 0.2 mg/kg abamectin. The results demonstrate that monepantel was fully effective against multi-resistant species, Trichostrongylus colubriformis and Haemonchus contortus (99.9%). In contrast, the combination of derquantel and abamectin was effective against T. colubriformis (99.9%), but was not effective against larval stages of the barber's pole worm H. contortus (18.3%)

Perfusion by Arterial Spin Labelling following Single Dose Tadalafil in Small Vessel Disease (PASTIS): study protocol for a randomized controlled trial

Background Cerebral small vessel disease is a common cause of vascular cognitive impairment in older people, with no licensed treatment. Cerebral blood flow is reduced in small vessel disease. Tadalafil is a widely prescribed phosphodiesterase-5 inhibitor that increases blood flow in other vascular territories. The aim of this trial is to test the hypothesis that tadalafil increases cerebral blood flow in older people with small vessel disease. Methods/design Perfusion by Arterial Spin labelling following Single dose Tadalafil In Small vessel disease (PASTIS) is a phase II randomised double-blind crossover trial. In two visits, 7-30 days apart, participants undergo arterial spin labelling to measure cerebral blood flow and a battery of cognitive tests, pre- and post-dosing with oral tadalafil (20 mg) or placebo. Sample size: 54 participants are required to detect a 15% increase in cerebral blood flow in subcortical white matter (p < 0.05, 90% power). Primary outcomes are cerebral blood flow in subcortical white matter and deep grey nuclei. Secondary outcomes are cortical grey matter cerebral blood flow and performance on cognitive tests (reaction time, information processing speed, digit span forwards and backwards, semantic fluency). Discussion Recruitment started on 4th September 2015 and 36 participants have completed to date (19th April 2017). No serious adverse events have occurred. All participants have been recruited from one centre, St George’s University Hospitals NHS Foundation Trust. Trial registration European Union Clinical Trials Register: EudraCT number 2015-001235-20. Registered on 13 May 2015