Plasmid dna extraction kit

Plant DNA and plasmid DNA isolation was performed using a plant DNA isolation kit (Tiangen Biotech) and a plasmid DNA extraction kit (Tiangen Biotech), respectively, following the manufacturer’s instructions. For RNA isolation, all tissues were collected fresh, flash-frozen in liquid nitrogen, and total RNA was extracted using a plant total RNA isolation kit (Tiangen Biotech) following the manufacturer’s instructions. First-strand cDNA was synthesized using a reverse transcription kit (TAKARA) as directed by the manufacturer. For qPCR, 2 μl of cDNA was used as template in 20 μl reaction volumes, and the qPCR amplifications were performed using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus; TAKARA) on a LightCycler® 480 instrument (Bio-Rad). The rice UBIQUITIN10 (OsUBQ10) or ACTIN1 genes were used as the internal controls for normalization of gene expression. All oligonucleotide primers used in this study are given in Supplementary Table 1.

The primers M13-47 and RV-M were used for PCR amplification of cDNA inserts from white colonies. The PCR reactions were performed in a total volume of 20 μL and included 15.2 μL sterile water, 2 μL 10× buffer, 0.5 μL of each primer (20 μM), 0.5 μL dNTPs (10 mM), 0.3 μL Taq DNA polymerase (5 U μL⁻¹) (Invitrogen) and 1 μL bacterial culture. Each PCR was performed as follows: 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 3 min, and a final extension at 72°C for 10 min. The PCR products were electrophoresed in 1% agarose gel to confirm the amplification. A subset of positive clones for which PCR products were longer than 100 bp was selected for preparation of plasmid DNA using the Plasmid DNA Extraction Kit (TIANGEN).

A plasmid containing RNAi and PvPIN1 gene expression vectors was extracted according to the manufacturer's protocol (Plasmid DNA extraction kit, Tiangen Biotech). Before particle bombardment, the embryogenic calli were placed in a 4-cm² circle monolayer on MSH (Hypertonic MS) medium in a 9-cm dish containing MS medium basal medium with 22.6 μM 2,4-D and 0.4 M mannitol (Sigma, St. Louis, MO) followed by a 4–6 h osmotic treatment in the dark at 25 °C. Gold particles (1 μm, Bio-Rad, Hercules, CA) were soaked with the above-mentioned plasmid DNA (Vain et al,. 1993 (link)). For each bombardment shot, 1 μg of plasmid DNA and 250 μg of Gold particles were used. The bombardment was done using a 1100 psi He inflow with a target distance of 9 cm. Thereafter, the bombarded calli were incubated on MSH medium overnight.