A workflow diagram for the experiments is given in Figure 4.
Plants material and growth conditions
Upland cotton cultivars Coker 312 (R15) and TM-1 were kept in our laboratory, Lumian 21 and Zhongmian 49 were bought from Beijing Shunheyuan Seed Co. LTD. These four cotton cultivars were cultured in a growth chamber (16-h light/8-h dark; 28 ℃; 70% humidity). Plants were used for inducing hairy root when the two cotyledons had fully expanded (about 10 days after sowing).
Construction of CRISPR/Cas9 vector
The human codon-optimized Cas9 gene (35S-Cas9-SK), driven by the CaMV 35S promoter and the chimeric single-guide RNA (AtU6–26-SK), driven by the AtU6–26 promoter were gifts from Jian-Kang Zhu. In this study, one single-guide RNA (sgRNA) in the second exon of GhMYB25-like gene was selected. A pair of DNA oligonucleotides was synthesized and annealed to generate a dimer. The dimer was then cloned into the BbsI site of pAt-U6-26-SK to generate pSK-At-U6-sgRNA. pSK-At-U6-sgRNA was cut with restriction enzyme KpnI/SalI, and 35S-Cas9-SK was digested with restriction enzyme SalI/EcoRI. Two fragments were assembled into pCAMBIA2301 using KpnⅠ/EcoRⅠ restriction digestion followed by ligation to generate the p2301-GhMYB25-CRISPR construct. The kan gene, driven by the CaMV 35S promoter of pCAMBIA2301, was used as selection marker for cotton stable transformation. The positive plasmid was introduced into A. rhizogenes strain K599 to induce hairy roots.
Agrobacterium rhizogenes-mediated transient transformation of cotton
A. rhizogenes K599 harboring the p2301-GhMYB25-CRISPR construct was cultured at 28 ℃ with shaking (180 rpm) to different OD600s (0.4, 0.6, 0.8, 1.0). Then the apical meristem between the two cotyledons on 20 plants of cotton cv. R15 was injected with one of the concentrations. Plants were then grown in the growth chamber for 1 month, then the number of plants with hairy roots was counted. The other three cotton cultivars were then tested in the same way.
Genomic DNA extraction and mutations analyses
One month after injection, any hairy roots produced were collected, and DNA was extracted using the DNAsecure Plant Kit (TIANGEN, Beijing, China). Primers U6-F/R designed based on the AtU6-sgRNA sequence to detect exogenous T-DNA, and primers VirD-F/R were designed based on the VirD gene to detect A. rhizogenes contamination (Additional file1: Table S1). The region spanning the target gene GhMYB25-like in the A genome and D genome was amplified using 2´ Taq Plus Master Mix Ⅱ (Vazyme, Nanjing, China) with the specific primer pair (Additional file1: Table S1). The amplicons were sequenced using the platform HiTOM .
Samples were collected from more than three technical replicates for each injection. The number of plants with hairy roots data were analyzed using SPSS software (IBM, Armonk, NY, USA). Analysis of variance was used to compare the statistic difference based on Tukey’s HSD test at significance level of p < 0.05.