organism: Hypericum perforatum
description: Protocols: About 100 mg of cells or plantlets were collected and powdered using liquid nitrogen. Hypericum perforatum cell suspension cultures established from root tissues by germinating seeds of the variety 'Helos' (Richters seeds, Ontario, Canada) on Murashige and Skoog's (MS - Duchefa Biochemie, Haarlem, Netherlands) medium containing 2.0 mg/l 2,4-dichlorophenoxy-acetic acid (2,4 D– Sigma-Aldrich, USA) . The established cell suspension cultures were maintained in Erlenmeyer flasks (250 ml) containing 80 ml Murashige and Skoog's (MS - Duchefa Biochemie, Haarlem, Netherlands) medium augmented with 0.5 mg/L α-naphthaleneacetic acid (NAA) on an Orbi-Shaker (Benchmark Scientific, USA) at 110 rpm kept in a growth chamber programmed to provide 16/8 (day/night) photoperiod, 80 µmol m−2 s−1 light intensity, 70 % relative humidity and 25 °C temperature. To maintain the cells in the growth phase, 10 ml of grown culture was subcultured to 70 ml of fresh medium once in 7 days. RNA extracted from cultures 5 days after subculture for sequencing. Hypericum perforatum plantlets were established from root tissues obtained from aseptic seedlings of the variety 'Helos' (Richters seeds, Ontario, Canada) as follows. Roots of 25 days old in vitro-grown seedlings were excised and cultured in half strength liquid Murashige and Skoog medium (MS, Duchefa, Netherlands) supplemented with 0.5 mg/l indole-3-butyric acid (IBA, Sigma-Aldrich, USA) and incubated in dark conditions at 25 °C to establish root cultures. Root explants were cultured on semisolid MS basal medium (Duchefa Biochemie, Netherlands) supplemented with 0.05 mg/l α-naphthaleneacetic acid (NAA, Sigma-Aldrich, USA) and 0.5 mg/l 6-benzyladenine (BA, Sigma-Aldrich, USA) for shoot regeneration. Then, the shoot cultures were maintained in liquid MS medium augmented with 0.05 mg/L NAA and 0.5 mg/L BA by regular subculture. For rooting, shoots were cut and transferred to ½ strength MS liquid medium containing 0.5 mg/l IBA. After 15 days, rooted plantlets were used for RNA extraction. Plantlets were maintained in MS liquid medium augmented with 0.1 mg/L NAA and 1 mg/L 6-benzyladenine. Cell suspension cultures were maintained in Erlenmeyer flasks (250 ml) containing Murashige and Skoog's (MS) medium (Duchefa Biochemie, Haarlem, Netherlands) augmented with 0.5 mg/L α-naphthaleneacetic acid (NAA) on an Orbi-Shaker (Benchmark Scientific, USA) at 110 rpm kept in a growth chamber programmed to provide 16/8 (day/night) photoperiod, 80 µmol m−2 s−1 light intensity, 70 % relative humidity and 25 °C temperature. To keep the cells in growth phase, 10 ml of grown culture was subcultured to 70 ml of fresh medium once in 7 days. Total RNA was isolated using Sigma spectrum RNA extraction kit (Sigma-Aldrich, USA). On column DNase treatment (Sigma-Aldrich, USA) was given to clean the DNA contamination. Suppliers' information was followed for extraction and purification, and the RNA was eluted in 100 µl elution buffer provided in the kit. Quantity of the RNA was measured using NanoDrop™ OneC and the RNA integrity was measured using Agilent Bioanalyzer 2100. mRNA was fragmented randomly and cDNA was synthesized using random hexamers primer. Second strand was synthesized using custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA12548280
attributes.tag: INSDC center alias
attributes.value: Department of Biometry and Bioinformatics, Institute of Plant Genetics, Polish Academy of Sciences
attributes.tag: INSDC center name
attributes.value: Department of Biometry and Bioinformatics, Institute of Plant Genetics, Polish Academy of Sciences
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:57:45Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:57:45Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11325:Sample 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cultivar
attributes.value: Helos
attributes.tag: developmental stage
attributes.value: sporophyte vegetative stage
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: cell suspension culture
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: root
attributes.tag: sample name
attributes.value: E-MTAB-11325:Sample 1