0.2 glufosinate (BASTA), and transgenic lines have been obtained by continuous self-crossing of single-locus transgenic plants and screening by Basta.Subcellular localization of TaCYP78A5 in wheatThe coding region of TaCYP78A5-2A from +1 to +1017 bp that consists of the hydrophobic domain and oxygen-binding motif was inserted into 35S::GFP of expression vector p16318 by utilizing restriction endonuclease HindIII and SalI web site to produce the construct 35S::TaCYP78A5-GFP. The resulted construct along with the vector handle (35S::GFP) were transferred into wheat protoplast ready based on previous study (Yoo et al., 2007). GFP was observed by inverted fluorescence microscope (DMi8, Leica, Germany). The primers utilised for developing the constructs are listed in Table S8.RNA sequencing and information analysisThe RNA samples isolated in the 1-mm size ovaries of transgenic wheat line pINO-24 and WT were utilised for RNA sequencing. The differentially expressed genes were identified as described in Appendix S1. All the genes detected have been subjected to GO (http://gene ontology.org/) to acquire GO annotations, GO and KEGG enrichment had been performed as previously described (Chi et al., 2019).Genetic mapping of TaCYP78A5 in wheatThe physical areas of 3 homoeologs of TaCYP78A5 were localized depending on physical maps and wheat genome reference sequence IWGSC Ref v1.0 (IWGSC, 2018). The known genetic2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology plus the Association of Applied Biologists and John Wiley Sons Ltd., 20, 168180 Lijian Guo et al.Determination of auxin and cytokinin metabolite levels in wheatThe 1-mm size ovaries with the pINO lines and WT were employed to detect auxin levels, and auxin was determined by electrospray ionization igh-performance liquid chromatography andem mass spectrometry (PI4KIIIβ Storage & Stability ESI-HPLC-MS/MS) strategy. The spikes of the pINO lines and WT at heading stage have been used for testing relative quantification of auxin precursors, auxin conjugates and cytokinin. The protocols of auxin and cytokinin metabolite determination are shown in Appendix S1.Statistical analysisAll data obtained had been processed in SPSS, and statistical evaluation was carried out by one-way ANOVA or unpaired Student’s t-test. Considerable variations were regarded as if P-values 0.05.AcknowledgementsWe thank Jinan Bondi Biotechnology Co., Ltd and Wheat Transformation Platform, State Key Laboratory of Crop Pressure Biology for Arid PAK3 custom synthesis Regions, Northwest A F University, for help in generation of transgenic wheat plants. We thank Dr. Li Huang, Division of Plant Sciences Plant Pathology, Montana State University, for kindly giving us with BSMV vector employed in this study. We also thank Prof. Wanquan Ji, College of Agronomy, Northwest A F University, for kindly delivering with the Experimental Base of Transgenic Plants for us to grow transgenic wheat employed within this study. Ultimately, we thank Prof. Rudi Appels, Honorary Professor, University of Melbourne, for enhancing the draft of this manuscript. This study was financially supported by the Organic Science Foundation of China (32072003 and 32072059), and Key Study and Development Program of Shaanxi Province (2021NY-079) along with the Organic Science Foundation of Shaanxi province (2017JQ3032).Quantitative real-time reverse transcriptase olymerase chain reaction (qRT-PCR)The qRT-PCR was performed using the SYBR Green premix Pro Taq HS qPCR Kit (Precise Biotechnology) on a CFX96TM Realtime PCR Detection Sys
