Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application

Abstract

Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free 'quality events' ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84-7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9-mediated gene knockouts in Tx430.

Keywords: Agrobacterium; Africa sorghum varieties; CRISPR/Cas9; genome modification; sorghum transformation; ternary vector.

Figure 1

Schematic representation of the molecular components of (a) CIV and (b) ternary vector systems. pPHP71539 works as the accessory plasmid for the ternary vector system.

Figure 2

Effect of surfactant Silwet‐70 on transformation frequency in Tx430. Error bars indicate the ±SD from three treatments with more than 100 embryos per treatment.

Figure 3

T‐DNA delivery determined by YFP transient assay. Sorghum Tx430 immature embryos were infected with Agrobacterium carrying either pPHP38331(CIV) or pPHP45981(ternary) with pPHP71539 as accessory plasmid. T‐DNA delivery was represented by the number of transgenic cells exhibiting YFP fluorescence and the fluorescence intensity on the surface of sorghum embryos.

Figure 4

Stringent PTXD /Phi selectable system. Picture represents the development of 6 weeks old transgenic and nontransgenic callus on callus induction medium containing 300 mg/L Phi_. The transgenic callus is indicated by the arrow.

Figure 5

Diagram of the DNA sequence of the target region of Sb‐CENH3 gene. The three‐nucleotide sequences highlighted in grey indicate the corresponding PAM motifs recognized by the Cas9 protein. crRNA hybridization targets for three different gRNAs are underlined. Exons and introns are represented by the uppercase and lowercase letters, respectively.