In planta particle bombardment (iPB): A new method for plant transformation and genome editing

Abstract

Transformation is a key step in modern breeding technology that involves genome editing. The requirement for in vitro tissue culture and regeneration hampers application of this technology to commercially important varieties of many crop species. To overcome this problem, we developed a simple and reproducible in planta transformation method in wheat (Tritticum aestivum L.). Our in planta particle bombardment (iPB) method utilizes the shoot apical meristem (SAM) as a target tissue. The SAM contains a subepidermal cell layer termed L2, from which germ cells later develop during floral organogenesis. The iPB method can also be used for genome editing through transient CRISPR/Cas9 expression or direct delivery of the CRISPR/Cas9 ribonucleoprotein. In this review, we describe the iPB technology and provide an overview of its current and future applications in plant transformation and genome editing.

Keywords: SAM; biolistic; genome editing; wheat.

Figure 1. Outline of the iPB method for transformation and genome editing. See text for description of the procedure.

Figure 2. Analysis of transgenic plants. A. Genomic PCR analysis of a putative transgenic ‘Fielder’ line and wild-type (Wt) line. Genomic DNA was extracted from the indicated leaves of a putative T₀ wheat plant. B. PCR analysis of five independent transgenic T₁ (L1–L5) and wild-type (Wt) ‘Fielder’ lines. Genomic DNA was extracted from the first leaf of each T₁ progeny. C. GFP accumulation in T₁ seeds of the L1 transgenic plant. Left: fluorescence image of whole Wt (upper) and L1 (lower) seeds. Right: fluorescence images of sections of Wt (upper) and L1 (lower) seeds. D. GFP image of T₁ leaf. Young leaves of L1 (T₁ progeny) and wild-type (Wt) plants. E. Total protein from transgenic (L1) and Wt plants. Fluorescent images captured by FUJIFILM LAS3000 equipped with a GFP filter (Ex: 460, Em: 510).

Figure 3. Principle of the iPB method. Gold nanoparticles can bind DNA, RNA, and protein and deliver these materials into L2 layer cells, which develop into the pollen and embryo sac.

Figure 4. Genome editing using the iPB-DNA method. A. CAPS assay of TaLOX2 candidate edited plants. The TaLOX2 target region was amplified by PCR from total DNA isolated from the fifth leaf of T₀ plants, digested with SacI and separated on an agarose gel. B. Genome editing efficiency of TaLOX2 and TaGASR7.