overexpression vectors pcbl101  (Addgene inc)

Journal: Plant physiology

Article Title: New T-DNA binary vectors with NptII selection and RUBY reporter for efficient maize transformation and targeted mutagenesis.

doi: 10.1093/plphys/kiad231

Figure Lengend Snippet: Figure 1. A ternary vector system for Agrobacterium-mediated plant transformation. A) Schematic illustration of a ternary vector system consisting of 3 compatible plasmid vectors: a T-DNA binary vector, a helper plasmid, and a disarmed tumor-inducing (Ti) plasmid. The T-DNA binary vector carries GOI that will be transferred to the plant cells. The helper plasmid carries extra copies of essential virulence genes (VIR), whereas the disarmed Ti plasmid encodes all the necessary virulence (VIR) genes for the T-DNA transfer. All 3 plasmids must have compatible origins of replication to stably coexist in the same cells. C1, C2, pAT, Agrobacterium chromosomes C1 and C2, and the pAT plasmid. Empty T-DNA binary vectors with pVS1 ORI for transgene overexpression B) or CRISPR–Cas-mediated gene mutagenesis C). These vectors are compatible with ternary helper plas mids such as pKL2299 (Kang et al. 2022; Addgene #186332) with an RK2 ORI. pCBL101 (Addgene #199722), pCBL101.1 (Addgene #199724), and pCBL101gw (Addgene #199725) are identical, except that pCBL101 and pCBL101.1 have MCS, whereas pCBL101gw has Gateway attR1/R2 cassettes for LR clonase reaction for cloning a GOI into the vector. pCBL101.1 was generated by removing a BsaI restriction site within the pVS1 ORI and inserting new MCS with dual BsaI restriction sites for Golden Gate assembly. pKL2351 (Addgene #199721) is a Gateway destination vector (attR1/R2) and has a red fluorescent protein (mCherry) gene driven by the 35S promoter as a visible marker and a maize ubiquitin gene promoter (PZmUbi; Christensen et al. 1992) for transgene expression. All 3 vectors contain neomycin phosphotransferase II (NptII; Fraley et al. 1983) gene driven by PZmUbi as a plant selectable marker. D) T-DNA binary vectors used for maize B104 transformation. pCBL101-RUBY (left; Addgene #199723) carries the RUBY reporter (He et al. 2020) that contains betalain biosynthesis genes (CYP76AD1, DODA, and Glucosyl transferase). pKL2359 (right) carries a maize codon–optimized Cas9 from Streptococcus pyogenes (SpCas9) and a sgRNA1 targeting maize glossy2 gene. RB, T-DNA right border; LB, T-DNA left border; P35S, CaMV 35S promoter; CmR, chloramphenicol resistance gene; ccdB gene codes for toxin protein CcdB; pVS1, a broad host range ORI from Pseudomonas pVS1 plasmid for Agrobacterium; pBR322, a high copy number ORI for Escherichia coli; KmR, Kanamycin resistance gene; SpR, spectinomycin resistance gene. E) Summary of maize B104 transformation. F) Summary of CRISPR–Cas9-mediated Glossy2 mutagenesis. HO, homo zygous; BI, biallelic; HT, heterozygous; MO, mosaic mutations. G) Scutella tissue of immature embryo, 3 d postinfection. H) Embryogenic callus tissue on maturation medium, 3 wk postinfection. I) Immature shoots on maturation medium, 5 wk postinfection. J) Transgenic plantlets, 7 wk postinfection. The immature embryo and embryogenic callus tissues G and H) were infected with Agrobacterium LBA4404Thy- strain carrying a binary vector pCBL102-RUBY (Supplemental Fig. S1) and other images were taken from transgenic plants produced by pCBL101-RUBY D).

Article Snippet: The overexpression vectors pCBL101 (Addgene #199722), pCBL101.1 (Addgene #199724), and pCB101gw (Addgene #199725; Fig. 1B) have spectinomycin-resistant gene, while https://doi.org/10.1093/plphys/kiad231 PLANT PHYSIOLOGY 2023: 192: 2598–2603

Techniques: Plasmid Preparation, Transformation Assay, Stable Transfection, Over Expression, CRISPR, Mutagenesis, Cloning, Generated, Marker, Ubiquitin Proteomics, Expressing, Transgenic Assay, Infection, Produced

Journal: Plant physiology

Article Title: New T-DNA binary vectors with NptII selection and RUBY reporter for efficient maize transformation and targeted mutagenesis.

doi: 10.1093/plphys/kiad231

Figure Lengend Snippet: Figure 2. Betalain accumulation pattern in various transgenic maize tissues. A) Transgenic plants with varying levels of betalain accumulation. Three plants of the same age but different betalain accumulation in tissues. Heavily pigmented plants (middle and right) exhibited stunted growth. B) Transgenic plant with betalain strips in the leaves. C) Variation of betalain accumulation in T0 leaf tissues. Wild-type B104 (W); transgenic T0 plant (T). D) Transgenic root tissue. E) Cross section of the T0 root tissue. F) Cross section of the T0 stem. G) Close-up of a stem cross section with vascular bundles showing betalain accumulation. H) Tassel and pollen of the T0 plant. No betalain accumulation was observed in pollen. I) Anthers of the T0 plant. J) Young ear tip of the T0 plant. K) Cross section of a young ear (bottom part of J). L) Cross section of a kernel (23 d after pollination, T0 × WT B104 pollen). Betalain is mainly accumulated in the endosperm and at the tip of the scutellum. M to P) Ears from different transgenic events. All images were taken from transgenic plants produced by pCBL101-RUBY (Fig. 1D) except for O [T0 produced by pCBL102-RUBY (Supplemental Fig. S1) × WT B104 pollen] and P (WT B104 × pCBL102-RUBY transgenic pollen).

Article Snippet: The overexpression vectors pCBL101 (Addgene #199722), pCBL101.1 (Addgene #199724), and pCB101gw (Addgene #199725; Fig. 1B) have spectinomycin-resistant gene, while https://doi.org/10.1093/plphys/kiad231 PLANT PHYSIOLOGY 2023: 192: 2598–2603

Techniques: Transgenic Assay, Produced