The basal MS29 (link) medium supplemented with 20 mg/L casein hydrolysate30 (link) (Duchefa, Germany), 100 mg/L phloroglucinol31 (link), 0.2% activated charcoal23 (link),32 (link) (used to reduce tissue oxidation due to phenolic compounds during explant establishment), 3% (w/v) sucrose and 7 g/L agar was used in this study. Different PGRs including 6-benzylamino purine (BAP) as cytokinin and indol-3-butyric acid (IBA) and naphthalene acetic acid (NAA) as auxin (Duchefa, Germany) are used at different concentrations (mg/L). The medium was adjusted to 5.8 ± 0.05 prior to autoclaving at 121 °C for 15 min. The cultures were incubated in phytotron chambers at 24 ± 2 °C under a 16/8-h (light/dark cycle) photoperiod provided by cool white fluorescent lights (1500–3000 Lx)33 (link).
6 benzylaminopurine
Manufactured by Duchefa Biochemie
Sourced in Germany, United States
6-benzylaminopurine is a plant growth regulator that belongs to the class of cytokinins. It is a synthetic compound used to promote cell division and differentiation in plant tissues. The primary function of 6-benzylaminopurine is to stimulate the growth and development of various plant parts, including shoots, roots, and leaves.
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Lab products found in correlation
5 protocols using 6 benzylaminopurine
1
Plant Tissue Culture Protocol for Regeneration
2
Cytokinin Induction of Ectopic Pistil Outgrowth
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Inflorescences were treated with cytokinin 6-benzylaminopurine (BAP) as previously described [32 (link)]. In summary, one week after bolting, BAP solution drops were placed on the inflorescences once a day for 2 (48-hour period) or 5 to 10 (repeated applications) consecutive days. To observe ectopic outgrowths from the medial domain of the pistil, a BAP treatment for five days is given and after three to four weeks observations are made. The BAP solution contains 100 μM 6-benzylaminopurine (BAP; Duchefa Biochemie) and 0.01% Silwet L-77 (Lehle Seeds) in distilled water. Mock treatments contained only 0.01% Silwet L-77 in distilled water. All treated plants with their respective controls were cultivated simultaneously under the same growth conditions.
3
Cytokinin Treatments for Inflorescence Development
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Cytokinin treatments were performed in a similar way as described by Zuñiga-Mayo et al. (2014) (link). The experiment was carried out in greenhouse conditions with natural light in autumn, and all plants (Ler and Col as WT, drnl-2 and ahp6) were grown simultaneously under the same conditions. When the first fruits were observed in the inflorescence stem, those fruits were removed, leaving only closed buds in the inflorescences. Once this was done, BAP solution drops (100 μM 6-benzylaminopurine; Duchefa Biochemie, Haarlem, Netherlands) and 0.01% Silwet L-77 (in distilled water) were applied on the inflorescences for five consecutive days. Sixteen days after treatment the gynoecia were collected and analyzed in chronological order of development. The mock solution contained Silwet L-77 and the same concentration of NaOH (0.2 mN) used to prepare the hormone solution.
4
Inflorescence Hormone Treatments in B. napus and A. thaliana
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Treatments to B. napus plants were started one week after bolting, employing two different methods. In the first, the inflorescences were sprayed five days a week for three weeks, with a solution containing 200 µM BAP (6-Benzyl aminopurine; Duchefa Biochemie) and 0.02% Silwet L-77 (Lehle Seeds, Round Rock, TX, USA) or a mock solution containing only 0.02% Silwet L-77. In the second treatment, inflorescences were treated with a single application of lanolin containing 500 µM BAP or a mock treatment with lanolin alone. All treated plants with their respective controls were grown simultaneously under the same conditions. The flowers were analyzed after anthesis.
Hormone treatments to A. thaliana plants were performed one week after bolting. The inflorescences were dipped twice the same day in an eight-hour interval with a solution containing 100 µM BAP and 0.01% Silwet L-77 or a mock solution containing only 0.01% Silwet L-77. Three weeks after the treatment, the first three fruits produced by the main or secondary inflorescences were analyzed.
Hormone treatments to A. thaliana plants were performed one week after bolting. The inflorescences were dipped twice the same day in an eight-hour interval with a solution containing 100 µM BAP and 0.01% Silwet L-77 or a mock solution containing only 0.01% Silwet L-77. Three weeks after the treatment, the first three fruits produced by the main or secondary inflorescences were analyzed.
5
Transcriptional Profiling of Hormone-Treated Plants
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For dGR induction, plants were grown on ½ MS (1% agar) for 5 days before transferring to either mock or 10 µM DEX for 2h. For CK treatment, 5-day-old seedlings were transferred to medium containing 10 µM 6-benzylaminopurine (6-BAP; Duchefa) from a 10 mM DMSO stock solution. All samples were ground in liquid nitrogen and RNA was extracted using RNA isolation protocol for non-fiberous tissue by the RNA Tissue Miniprep System (Promega).
cDNA synthesis was done using 1µg of total RNA with the qScriptTM cDNA Supermix kit (Quanta BioSciences). The qRT-PCR primers were designed by Universal Probe Library Design Center (Roche) (Table S3). The qRT-PCR was performed using UBC and EEF as reference genes on a Roche Lightcycler 480 device (Roche Molecular Systems Inc.) with SYBR Green I Master kit (Roche). The gene expression analysis was done using qBase v3.2 software (Biogazelle, Zwijnaarde, Belgium -www.qbaseplus.com) .
cDNA synthesis was done using 1µg of total RNA with the qScriptTM cDNA Supermix kit (Quanta BioSciences). The qRT-PCR primers were designed by Universal Probe Library Design Center (Roche) (Table S3). The qRT-PCR was performed using UBC and EEF as reference genes on a Roche Lightcycler 480 device (Roche Molecular Systems Inc.) with SYBR Green I Master kit (Roche). The gene expression analysis was done using qBase v3.2 software (Biogazelle, Zwijnaarde, Belgium -www.qbaseplus.com) .
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