Project description:There are several issues complicating somatic embryogenesis in conifers, for instance the low initiation frequency, the weak maturation capacity, and the loss of an embryogenic potential after a prolonged cultivation. The aim of our study was to clarify molecular details of the this process in the tree Pinus nigra Arn. We performed comparative proteomic analysis based on the two-dimensional gel electrophoresis in 2 genotypes coded 362 and 366 of: 1) proliferating embryogenic tissues (E) initiated from immature zygotic embryos, 2) non-embryogenic calli (NEC) started from cotyledons of somatic seedlings, 3) embryogenic tissues that lost the maturation capacity (E-L). Pine tissues showed distinct morphologic features: a) E362 and E366 were characterized by the presence of early bipolar structures capable of maturation and plantlet regeneration; b) NEC362 and NEC366 were composed of round shaped cells without any organisation; c) for E-L362 and E-L366 the long-term culture of initially embryogenic tissues resulted in the disorganisation of early bipolar structures. We found 24 differentially accumulated protein spots common for both genotypes after comparison of E versus NEC.
Project description:6 timepoints: Day 0 (normal controls), progressively developing neointimal vascular proliferation and pulmonary hypertension in vehicle treated animals (Days 14, 21, 28 and 35) and triptolide-treated animals at Day 35. Replicates: 6 for Day 0 (normal) 2 for Daty 14 3 each for Days 21, 28, 35 and Triptolide -treated at day 35 (T) Keywords: time-course
Project description:Transcript accumulation was measured using the Affymetrix Arabidopsis ATH1 Genome Array [ATH1-121501] to document changes in response to the MADS-domain transcription factor AGAMOUS-Like 15 during somatic embryogenesis. A somatic embryo system was used where mature seed is allowed to complete germination in liquid MS media containing 2,4-D and seedlings produce somatic embryos from the shoot apical meristem (SAM) region. The frequency with which these embryos are produced directly correlates with AGL15 accumulation.
Project description:A soybean ortholog of the Arabidopsis MADS-domain transcription factor (called GmAGL15) enhanced somatic embryogenesis from immature cotyledon explants of soybean when expressed via the 35S promoter compared to non transgenic tissue (cultivar Jack). To better understand how this occurs an expression microarray experiment was performed. publication: Q. Zheng and S.E. Perry. (2014). Alterations in the transcriptome of soybean in response to enhance somatic embryogenesis promoted by orthologs of AGAMOUS-Like 15 and AGAMOUS-Like 18. Plant Physiology, in press. Comparison of transcriptomes of non-transgenic tissue to 35S:GmAGL15 tissue at the time of explant preparation (0 days) and 3 and 7 days after induction of somatic embryogenesis by placement on medium containing 2,4-D.
Project description:Several factors influence the culture conditions and somatic embryogenesis responses, such as the role of plant growth regulators (PGRs) during the establishment of in vitro cultures. For instance, auxin is required for callus induction and the acquisition of embryogenic capacity in different species, but the removal of auxin is needed for the further differentiation of somatic embryos. Thus, we aimed to evaluate the effects of residual 2,4-dichlorophenoxyacetic acid (2,4-D) on the maturation of sugarcane somatic embryos. First, embryogenic calli were separated into two groups: the PGR-free group was cultured without 2,4-D and b) the control group was maintained on proliferation culture medium, which contains 2,4-D. After 21 days of culture in the dark, both groups were transferred to maturation culture medium under light. Our results showed that calli grown in PGR-free culture medium yielded more somatic embryos and exhibited a higher accumulation of protein and starch reserves. A proteomic analysis showed a decrease in the abundance of abscisic acid (ABA)-induced proteins in the control group. The levels of residual 2,4-D and endogenous 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, were higher in the control group than in the PGR-free group. Conversely, the level of ABA was higher in the PGR-free group than in the control group. A disruption in the ABA and ethylene levels might be responsible for the observed delay in the accumulation of storage reserves in the embryogenic calli of the control group, which might have affected the development of somatic embryos. Our results also showed that the efficient development of sugarcane somatic embryos appears to be preceded by an efficient accumulation of storage reserves in calli, which is related to hormone homeostasis.
Project description:Transcript accumulation was measured using the Affymetrix Arabidopsis ATH1 Genome Array [ATH1-121501] to document changes in response to the MADS-domain transcription factor AGAMOUS-Like 15 during somatic embryogenesis. A somatic embryo system was used where mature seed is allowed to complete germination in liquid MS media containing 2,4-D and seedlings produce somatic embryos from the shoot apical meristem (SAM) region. The frequency with which these embryos are produced directly correlates with AGL15 accumulation. Experiment Overall Design: Wild type Arabidopsis (Columbia ecotype) with normal amounts of AGL15 were compared to (1) 35S:AGL15 that constitutively expresses AGL15 and produces more somatic embryos from the shoot apical region than wild type and (2) agl15 agl18 double loss-of-function mutants that produce less somatic embryos from the shoot apical region than wild type.
Project description:This investigation was aimed at studying global gene expression during the process of somatic embryogenesis in potato using internodal segments (INS) as explants. The INS explants from 4 to 6 week old potato cv. Desiree in vitro cultures were cultured on somatic embryogenesis induction for 2 weeks (stage-I) and expression/regeneration modified MS media for 3 weeks, respectively with (stage-II) and without (stage-III) 2, 4-dichlorophenoxy acetic acid (5 µM). The culturing frequency was 10 INS/petridish with 25 ml medium. The in vitro cultures were incubated at 19±1°C, 16/8 hrs light/dark cycles and 90 µmol/m2/s light intensity. To study the molecular events of SE as affected by the absence or presence of auxin, the INS explants were divided into 2 groups after the induction phase. One-half of the induced INS explants were transferred to auxin free medium to evoke SE, the other half were subjected to a prolonged induction phase. The total RNA from INS explants at these critical stages of SE progression was isolated using TRIzol (Invitrogen) as per the manufacturer’s instructions. The on-column DNase treatment and clean-up steps were performed using Qiagen RNase free DNase and RNeasy midi kits. The selected time points used for the RNA isolation were day 0 (freshly cut INS), day 14 (INS on induction medium) and day 21, 28 & 35 (INS on expression medium as well as from prolonged induced phase). Except day 0, where the whole INS were used for RNA isolation, all other time point RNA was isolated from the callused part of the INS distal end. Each time point was replicated 3 times. The number of explants for each biological replicate varied from 30 to 300 owing to variability in tissue availability and corresponding RNA yield from explants of each time-point. The entire process from tissue excision to RNA isolation was completed on the same day to avoid any unintended change in tissue condition by storage. Keywords: Direct comparison
Project description:We show that isolated zygotic embryos of Ulmus minor and U. glabra can produce embryogenic cultures provided they are isolated from immature seeds before storage proteins begin to accumulate. Rates of somatic embryogenesis were highest among zygotic embryos collected 6 weeks post-anthesis when they were at the midcotyledonary stage, were about 5 mm long and had a fresh weight of approx. 10 mg. At this time, induction was even possible in Murashige and Skoog basal medium with no plant growth regulators, but addition of 2,4-dichlorophenoxyacetic acid was necessary at earlier stages of zygotic development. In medium supplemented with benzyladenine (BA) only, no embryogenic induction was observed. The formation of callus was an essential step not only for the induction of embryogenic masses, but also for the maintenance of embryogenic competence through successive subculture of callus on induction media supplemented with 0.1 mg l(-1) BA. Nine embryogenic U. minor lines and 24 U. glabra lines have been maintained in this way for 3 years. However, conversion into plantlets has occurred only rarely.
Project description:This study is to develop a short term and highly accurate prediction method of renal carcinogenicity based on gene expression profile of rats administrated by carcinogens. We conducted 28 days-repeated dose experiments in male SD rats with 1-amino-2,4-dibromoanthraquinone, and the gene expression profiles of renal cortex were analyzed using custom microarrays.