Generating Arabidopsis double mutants

Arabidopsis thaliana wild-type (ecotype Columbia) and mutant plants were grown in soil supplemented with Hoagland medium in growth chambers under long-day conditions (16 h light/8 h darkness) at 22 °C during the day and 20 °C during the night. The light intensity was set at 140 μE m⁻² s⁻¹. The 2-Cys Prx A–2-Cys Prx B double mutant, denoted Δ2cp, was obtained by manual crossing of the single mutants 2cpA, line SALK_065264, and 2cpB, line SALK_017213, previously characterized (Kirchsteiger et al., 2009 (link)). Seeds resulting from this cross were checked for heterozygosity of T-DNA insertions in the 2cpA and 2cpB genes. The plants were then selfed and double homozygous plants were detected in the progeny by PCR analysis of genomic DNA using oligonucleotides a (5′-GAGAAGTTGAACACCGA-3′) and b (5′-GGGGACAAAGTGAGAATC-3′) for the 2cpA gene, and a′ (5′-CCACCTGAACCAAGAAAG-3′) and b′ (5′-CCTGCAAGACAACATCAC-3′) for the 2cpB gene in conjunction with the oligonucleotide T (5′-TGGTTCACGTAGTGGGCCATCG-3′) located in the T-DNA. A T-DNA homozygous line SALK_128914 (Alonso et al., 2003 (link)) in the single gene encoding Trx x of Arabidopsis (At1g50320 locus) was selected by PCR analysis of genomic DNA with oligonucleotides c (5′-GCCATGGACTCTATCGTCTC-3′) and d (5′-CCTTCCCTTCTGCTCCCT-3′) in conjunction with the T oligonucleotide. The NTRC knock-out mutant was described previously (Serrato et al., 2004 (link)).