Glufosinate

The gene-specific primer pair 5′-CATGCCATGGATGGCTTTGTCTCTTATCTTTC-3′ and 5′-CATGCCATGGCTAACATTCTGGTGTTTGGG-3′ was designed to isolate the full-length CDS of AtXTH31 from Arabidopsis. The PCR product (902 bp) was cloned into the pCR4-TOPO vector, and the positive plasmid was fully sequenced with M13 sequencing primers. The AtXTH31 gene sequence was inserted into the pCNSH.131. AtMyb2p-Gus vector, which contained the Myb2 promoter. Finally, the whole cassette contained a promoter, and the gene sequence was moved into the pZY101-Asc binary vector. An improved Agrobacterium-mediated transformation of the soybean cotyledonary node system [65 (link)] was performed using the elite genotype “Maverick”. To determine the segregation of gene of interest and selectable marker gene, at least 30 plants from each T0 event were screened using leaf paint (100 mg/L glufosinate, Sigma, St. Louis, MO, USA) analysis carried out for the T0 generation. T2 progeny from the T1 generation was similarly analyzed to identify homozygous T1 lines for subsequent study.

For constitutive overexpression of ZmVQ52, the ZmVQ52 ORF was PCR amplified with cDNA of B73 and subsequently cloned into the binary vector pCAMBIA3301. The primers VQ52-F and VQ52-R are listed in the S1 Table. For the construction of transgenic plants, the vector CaMV35S::ZmVQ52 was transformed into Arabidopsis ecotype Col-0 using the floral dip method mediated by the Agrobacterium tumefaciens strain EHA105. Transgenic plants were screened with Glufosinate (5 mg/L, Sigma) and confirmed by PCR analysis.

The gene-specific primer pair 5′-GGAGCTCATGGCATCCCATAGGCAAAGC-3′ and 5′-TCCCCGGGGTAATTTCTGCGGTTGTCTTG-3′ was designed to isolate the full-length CDS of GmLEA4_19 from soybean. The PCR product (423bp) was cloned into the Topo vector for sequencing. The positive clone was cut with SacI and SmaI to make the pRTL2-ABRC3 subcloning vector before fusion with the ABRC3 promoter. Finally, the whole cassata was cloned into the pPTN200 binary vector. For the soybean transformation, an improved Agrobacterium-mediated transformation of the soybean cotyledonary node system was performed using the elite genotype “Thorne” [63 (link)]. The transgenic soybean plants were screened using the leaf paint (100 mg/L glufosinate, Sigma, St. Louis, MO, USA) analysis and the transgenic Arabidopsis were screened using 10 μg/mL of basta. The abiotic-resistance seedlings were verified via PCR analysis using specific primers. The homozygous lines used for subsequent phenotype studies.

For gene expression construct, the coding sequence of Glyma02g07650 (GmFT7) was amplified (with XhoI and BamHI restriction enzyme sites on 5′ and 3′ end, respectively) and ligated to pGEM-T Easy vector (Promega). The vector was digested, and gene fragment was ligated to pRT101 at Xho I and Bam HI sites, to obtain 35S promoter and polyA terminator. Then the 35S:GmFT7:polyA cassette was excised using Hind III and ligated to pUQC10255 expression vector (Supplementary Fig. S4). Transformation of Arabidopsis Col (WT) and ft-10 mutant was performed using the floral dip method^{53 (link)}.
Bragg cultivar was used for soybean transformation following the protocol by Li et al.^{40 (link)} with modifications; selection on SIM medium containing 4 mg/L glufosinate (Sigma, USA) was started during the second subculture step SIM and continued till SEM medium. 100 mg/L Cefotaxime (GoldBio, USA), 50 mg/L Vancomycin (GoldBio, USA) and 50 mg/L Ticarcillin (GoldBio, USA) were used instead of carbenicillin on SIM, SEM and RM medium. Five independent experiments were conducted using an average of 110 seed explants per experiment and recovered five transgenic shoots. However, one-line produced seeds. The transgenic seeds were multiplied and selected for the Bar gene. Three T2 lines were used for analysis.

Tetraethyl orthosilicate (TEOS, 98%, analytical reagent) was purchased from Acros Organics. Absolute ethanol (99.9%, analytical reagent) and ammonia water (NH₄OH, analytical reagent) were obtained from Sigma Aldrich Co., St. Louis, MO, USA. To prepare patterned MIP films, MAA (Daejung Chemicals & Metals Co., Siheung, Korea) was used as a functional monomer. EGDMA, 2,4-dichlorophenoxyacetic acid (2,4-D; M_w 221.04 g/mol), and dimethylformamide (DMF) were purchased from Tokyo Chemical Industry Co (Tokyo, Japan). use as cross-linker, template molecule, and solvent, respectively. 2,2′-azobisisobutyronitrile (AIBN), used as an initiator, was purchased from Daejung Chemicals & Metals Co. Atrazine, ametryn, glufosinate (Sigma-Aldrich Co.), and (4-chloro-2-methylphenoxy)acetic acid (MCPA, Tokyo Chemical Industry Co.) were used as 2,4-D-analogous chemicals for the MIPs’ selectivity test. Deionized (DI) water (Pure & Ultra pure water system, pure ROUP30, Pure Water Co., Namyangju, Korea) and all other chemicals were used as received.

Genetic transformation of Nicotiana benthamiana followed the method of Regner et al.^{109 (link)} and Bardonn et al.^{110 (link)}. Leaf discs of Nicotiana benthamiana were transformed with GV3101 cells bearing various vectors (35Spro:CaKAN3-GFP or 35Spro:CaHSF8-GFP). Independent T₀ transgenic Nicotiana benthamiana plants were selected on 10% PPT (glufosinate, Sigma, 45520) and later confirmed by PCR with specific primers (Supplementary Table 1). T₀ plants were self-pollinated, and seeds of each plant were separately harvested. The T₁ plants were selected by germination of seeds harvested from T₀ plants on MS medium (PhytoTech, M519) supplemented with PPT (1:250), which was further confirmed by PCR using specific primers (Supplementary Table 1). Similarly, seeds of the T₂ and T₃ lines were acquired, and the homozygous T₃ lines were used for functional assays of the tested genes.

The genetic transformation of Nicotiana benthamiana followed the method used by Regner et al. [64 (link)] and Bardonn et al. [65 (link)]. Leaf discs were transformed with GV3101 cells containing objective vector and the acquired T₀ plants were selected with 10% PPT (glufosinate, Sigma-Aldrich, Shanghai, China, 45520) and later confirmed by using PCR with specific primers (Table S1). The confirmed T₀ plants were self-pollinated to produce the seeds of T1 lines, which were separately harvested; the acquired seeds were selected with 10% PPT during germination. Similarly, the seeds of T₂ and T₃ lines were acquired, and the homozygous T3 line plants were used for functional assays of the tested genes.