Computational protocol: Low Agrobacterium tumefaciens inoculum levels and a long co-culture period lead to reduced plant defense responses and increase transgenic shoot production of sunflower (Helianthus annuus L.)

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Protocol publication

[…] Upregulated genes, associated with plant defense response to A. tumefaciens infection, were selected from studies of Agrobacterium inoculation of A. thaliana cell cultures (Ditt et al.), inflorescence stalks (Lee et al.), tobacco cell cultures (Veena et al.), and wheat embryogenic calluses (Zhou et al.). Orthologs of the selected genes were identified in the sunflower genome by running basic local alignment search tool (BLAST) using the HeliaGene database (www.heliagene.org/HA412.v1.1.bronze.20141015) with the amino acid sequences of the selected A. thaliana genes (Table ), and the best hit with the highest percentage of identity and the lowest expectation value was chosen for each gene. Afterwards, the amino acid sequence of the best hit for each sunflower gene was used to run BLAST using The Arabidopsis Information Resource (TAIR; www.arabidopsis.org) database to confirm that the selected sunflower gene and the best hit of A. thaliana gene belonged to the same gene family. The selected genes were HaPR1, HaPR2, HaMBL, HaWRKY53, and HaOxo (Table ). In addition, a sunflower ortholog of the Arabidopsis shoot meristemless (STM) gene (HaSTM) was identified and included in this study to monitor how shoot induction was influenced by different inoculation methods (Table ). Specific primers (Table ) for qRT-PCR were then designed using the real-time qPCR Assay Tool (Integrated DNA Technologies, Coralville, IA) or the PrimerQuest® Tool (Integrated DNA Technologies).Cotyledons were inoculated with either low or high inoculum, and explants were prepared and plated on SIM for co-culture as previously described. Explants derived from cotyledons, immersed in inoculation medium without A. tumefaciens for 10 min, were used as a non-inoculated control. Seven cotyledon explants were removed from culture at 3 h, or 3, 6, 9, 12, and 15 d after inoculation, frozen in liquid nitrogen, and stored at −80°C. RNA extraction was performed within 1 mo. Three independent experiments were conducted.Total RNA was isolated by using the RNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany), and genomic DNA was removed using the on-column RNase-Free DNase Set (QIAGEN) according to the manufacturer’s instructions. RNA samples were screened by PCR with HaOxo primers (Table ), which spanned an 887 bp intron, and the detection of a 1011 bp amplicon in PCR products after electrophoresis indicated the presence of genomic DNA contaminant. The samples with detectable genomic DNA contamination were further treated with the Ambion® DNA-Free Removal Kit (Thermo-Fisher Scientific) according to the manufacturer’s instructions until genomic DNA was undetectable. RNA concentration was quantified using a Nanodrop® ND-1000 spectrophotometer (Thermo-Fisher Scientific), and RNA integrity was determined by gel electrophoresis. Single-strand cDNA was synthesized with a RETROscript® Reverse Transcription Kit (Thermo-Fisher Scientific) from 1–2 μg of total RNA according to the manufacturer’s instructions. The products were diluted, and 5 ng cDNA was used as template for qRT-PCR.Quantitative RT-PCRs were conducted in 20 μL reactions using iQ™ SYBR® Green Supermix (Bio-Rad, Hercules, CA) following the manufacturer’s instructions, and the iQ™5 optical system (Bio-Rad) was used to measure target cDNA levels. The PCR cycling conditions were 3 min at 95°C; 40 cycles of 10 s at 95°C and 30 s at 60°C; and a melt curve profiling program with a constant increase by 0.5°C every 30 s from 55 to 95°C. Each gene assay was conducted three times for each sample. The data (quantification cycle, Cq) were obtained from the iQ™5 optical system software (Bio-Rad), and analyzed according to the qBase relative quantification framework (Hellemans et al.). Amplification efficiency of each assay was estimated based on the qRT-PCR data of a 5-log serial dilution (0.005, 0.05, 0.5, 5, 50 ng μL−1) of the pooled cDNA from all samples in each independent experiment. The Cq values were converted into relative quantity and then normalized by three reference genes (Table ) using the geNorm method (Vandesompele et al.). The reference genes were HaActin, HaRPS2, and HaEFh (Table ), and the stability of the reference genes was determined by the gene-stability measure (Vandesompele et al.; Hellemans et al.) (M = 0.72). The means of normalized relative quantity were calculated from three independent experiments for each gene/treatment/time point. […]

Pipeline specifications

Software tools BLASTN, PrimerQuest
Databases TAIR Heliagene
Application qPCR
Diseases Blind Loop Syndrome