Diurnal Growth Analysis
Objective: Analysis of plant growth patterns and protein accumulation under short day photoperiod conditions (8h light/16h dark)
Gather these items before starting the experiment. Check off items as you prepare.
Equipment2
Materials3
Software1
Not specified • Not mentioned
As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
Protocol Steps
Generate transgenic plant lines
Amplify genomic fragment including 2.4-kb promoter region and full-length PIF4 coding sequence without stop codon using PIF4promoter-f and PIF4_YFPr primers. Clone into pENTRY S/D-TOPO vector.
Note: For pPIF4::PIF4-HA pif4pif5 transgenic lines
View evidence from paper
“a genomic fragment, including a 2.4-kb promoter region and the full-length PIF4 coding sequence without stop codon, was amplified with the PIF4promoter-f and PIF4_YFPr primers and cloned into the pENTRY S/D-TOPO (Invitrogen) vector”
Generate PIF41A construct
Substitute the NcoI–AgeI fragment of the pPIF4::PIF4 TOPO plasmid with the corresponding fragment including the PIF41A mutation.
Note: Creates pPIF4::PIF41A construct
View evidence from paper
“The pPIF4 ⊳PIF41A construct was obtained by substituting the NcoI–AgeI fragment of the pPIF4 ⊳PIF4 TOPO plasmid with the corresponding fragment, including the PIF41A mutation”
Mobilize genomic regions and transform
Mobilize genomic regions by LR clonase into the pGWB13-pPZP destination vector and transform via Agrobacterium into the pif4pif5 mutant.
Note: Generates transgenic lines
View evidence from paper
“These genomic regions were mobilized by LR clonase into the pGWB13-pPZP destination vector and transformed via Agrobacterium into the pif4pif5 mutant”
Generate bes1-D PIF41A plants
Cross pPIF4::PIF41A-HA plants into the bes1-D pif4pif5 mutants.
Note: Creates double mutant line
View evidence from paper
“bes1-D PIF41A plants were generated by crossing the pPIF4 ⊳PIF41A-HA plants into the bes1-D pif4pif5 mutants”
Prepare seeds on growth medium
Transfer seeds to vertical MS plates supplemented with different chemical treatments as specified.
Note: Plates are vertical orientation
View evidence from paper
“Seeds were transferred to vertical MS plates supplemented with the different chemical treatments”
Grow seedlings under controlled conditions
Grow plants in darkness or continuous red light for 5–7 days as specified.
Note: Light conditions vary by experimental condition
View evidence from paper
“grown in darkness or continuous red light for 5–7 d, as specified”
Photograph plates
Photograph plates containing seedlings for subsequent analysis.
Note: Images used for hypocotyl length measurement
View evidence from paper
“Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length”
Measure hypocotyl length
Use ImageJ software to measure seedlings' hypocotyl length from photographs.
Note: Quantitative measurement of growth
View evidence from paper
“the ImageJ software was used to measure the seedlings' hypocotyl length”
Perform diurnal growth and protein accumulation studies
Conduct diurnal growth and protein accumulation studies in plants grown under short day photoperiod conditions.
Note: Short day conditions are 8 hours light/16 hours dark
View evidence from paper
“Diurnal growth and protein accumulation studies were performed in plants grown in short days (8 h light/16 h dark)”