Brassica rapa var. rapa Landrace KTRG-B-103 (Xichang, Sichuan, China) was selected owing to its high differentiation rate determined in a preliminary experiment. KTRG-B-103 homozygous F1 seeds were harvested after self-pollination. The seeds were sterilized in petri dishes containing 1% sodium hypochlorite solution for 10 min and rinsed five times with sterilized deionized water. The sterilized seeds were sown on half-strength MS medium (pH 5.8) supplemented with 3% (w/v) sucrose and 0.5% (w/v) Phytagel. The seeds germinated for 2 days in the darkness, and then transferred to long-day condition (16-h light/8-h darkness, 22 ℃) and geminated for 2 days. Subsequently, hypocotyl explants (about 3–5 mm in length) were prepared with a scalpel and infected by Agrobacterium.
Plasmid constructions
The full-length coding DNA sequence of BrrWUSa was cloned in accordance with the method of Li et al. [18] and subcloned into the pRI101-GFP vector using the ClonExpress II One Step Cloning Kit (Vazyme Biotech) (Additional file 1: Fig. S3a). For cloning, the primers F (5ʹ-GCAGCGGCCGTCGACATGGAGCAACCGCAACATCA-3ʹ) and R (5ʹ-GTTGATTCAGAATTC TTAATCCGGTGAGACGCCTG-3ʹ) were used.
The pER8-BrrWUS plasmid was reconstructed in accordance with the method of Guo et al., [19]. BrrWUSa was subcloned into pENTR™/D-TOPO (Invitrogen) using the primers F (5ʹ-CACCatggagcaaccgcaacatca-3ʹ) and R (5ʹ-atccggtgagacgcctg-3ʹ) for the entry clone. The pENTR-BrrWUSa plasmid was chosen to proceed to the LR reaction with pER8-GATEWAY-3Flag and generated the pER8-BrrWUSa plasmid (Additional file 1: Fig. S3b). Then BrrTCP4b target sequence was fused into CRISPR/Cas9 vector to generate the genome-editing construct (Additional file 1: Fig. S3c).
Agrobacterium-mediated transformation
The reconstructed plasmids pRI101-BrrWUSa-GFP and pER8-BrrWUSa were transferred into A. tumefactions strain EHA105 by electroporation, respectively. Positive transformants were identified by PCR, then incubated for two days in 50 mL liquid YEB medium at the ratio of 1:50. The culture was centrifuged at 5000×g for 10 min and adjusted to OD600 = 0.3 for the inoculation buffer (half-strength MS supplemented with 100 mM acetosyringone). The explants were infected for 15 min and then transferred to the MS medium. After co-cultivation for 3 days in the dark, the explants were rinsed five times with sterile water supplemented with 500 mg/L cefalexin and transferred to MS medium supplemented with 6-benzylaminopurine (4.0 mg/L), naphthaleneacetic acid (1.5 mg/L), and Timentin (100 mg/L). The medium was renewed every 2 weeks. The regenerated shoots were excised and transferred to root induction medium (MS medium).
Gene expression analysis
Total RNA was extracted using the Eastep® Super Total RNA Extraction Kit (Promega, Beijing, China) according to the manufacturer's instructions. One microgram total RNA was used for the first-strand cDNA synthesis in 20µL reaction volumes containing GoScript™ Reverse Transcriptase (Promega). 2 µL cDNA, 6 µL distilled water, 10 µL FastStart Universal SYBR Green Master Mix (ROX), and gene-specific primers was mixed to generate 20 μL qPCR reaction mixtures. The Step One Plus Real-Time PCR System (Applied Biosystems) was used for the PCR program, which comprised one cycle (50 °C, 2 min), one cycle (95 °C, 10 min), 40 cycles (95 ℃ for 5 s, 60 ℃ for 15 s, 72 ℃ for 5 s), and one cycle (72 ℃, 10 min). Three independent biological replicates were performed. Tubulin was amplified as a constitutive control. Relative expression levels were calculated using 2−ΔΔCt method. The primers used for quantitative RT-PCR are listed in Additional file 1: Table S1.
Fluorescence microscopy
Root tips from transgenic plants were excised and GFP fluorescence was observed with an OLYMPUS confocal microscope equipped with a 480/530 nm excitation filter.
Cryo-scanning electron microscopy
Cryo-scanning electron microscopy was used to observe the trichomes on the leaf. A section of leaf (approximately 3- × 3-mm) along the length of the lamina and midway between the margin and the mid-vein was fixed in a custom sample holder and submerged in liquid nitrogen for 2 min. A cryo-transfer shuttle Quorum pp3010t was used to transfer the sample to a precooled chamber under vacuum for coating. The samples were then observed at an accelerating voltage of 7.0 kV with a scanning electron microscopy (ZEISS Sigma 300, Zeiss, Oberkochen, Germany) with a cryogenic stage maintained at – 140 ℃.
Molecular analysis of CRISPR/Cas9 target sites
Genomic DNA was extracted from seedlings according to CTAB method. The primers BrrTCP4bF and BrrTCP4bR were used to amplify BrrTCP4b. The PCR products were sequenced and aligned with the MEGA11 [20].
Statistical analysis
Statistical analyses of the experimental data were performed using single-factor analysis of variance or Student’s t-test implemented in SPSS (Version 17.0).
Accession numbers
Sequence data for most genes studied in this article can be found in the National Center for Biotechnology Information under the following accession numbers: BrrTCP4b (KY608005); BrrWUSa (MN481054); BrrGIS (ON887158) and BrrLOX2 (ON887159).