Light-Dependent Seedling Development
Objective: Assessment of seedling development and hypocotyl length in darkness or continuous red light under various chemical treatments
Protocol Steps
Generate transgenic 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
Note: Fragment cloned into pENTRY S/D-TOPO vector
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”
Create PIF41A construct
Substitute NcoI–AgeI fragment of pPIF4::PIF4 TOPO plasmid with corresponding fragment including PIF41A mutation
Note: Generates 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 into destination vector
Use LR clonase to mobilize genomic regions into pGWB13-pPZP destination vector
Note: Prepares construct for Agrobacterium transformation
View evidence from paper
“These genomic regions were mobilized by LR clonase into the pGWB13-pPZP destination vector”
Transform into pif4pif5 mutant
Transform destination vector via Agrobacterium into pif4pif5 mutant plants
Note: Generates pPIF4::PIF4-HA pif4pif5 transgenic lines
View evidence from paper
“transformed via Agrobacterium into the pif4pif5 mutant”
Generate bes1-D PIF41A plants
Cross pPIF4::PIF41A-HA plants into bes1-D pif4pif5 mutants
Note: Creates double mutant transgenic 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 seedling growth plates
Transfer seeds to vertical MS plates supplemented with different chemical treatments as specified
Note: Plates oriented vertically for growth measurement
View evidence from paper
“Seeds were transferred to vertical MS plates supplemented with the different chemical treatments”
Grow seedlings in light conditions
Grow seedlings in darkness or continuous red light for 5–7 days as specified
Note: Light condition depends on experimental group
View evidence from paper
“grown in darkness or continuous red light for 5–7 d, as specified”
Photograph plates
Photograph seedling plates 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”
Measure hypocotyl length
Use ImageJ software to measure seedlings' hypocotyl length from plate photographs
Note: Primary outcome measure for seedling development
View evidence from paper
“the ImageJ software was used to measure the seedlings' hypocotyl length”
Perform diurnal growth studies
Conduct diurnal growth and protein accumulation studies in plants grown in short days (8 h light/16 h dark)
Note: Separate experimental condition for temporal analysis
View evidence from paper
“Diurnal growth and protein accumulation studies were performed in plants grown in short days (8 h light/16 h dark)”