Efficient Gene Stacking in Rice Using the GAANTRY System

Abstract

Genetic engineering of rice provides a means for improving rice grain quality and yield, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was previously shown to be a precise and robust system to stably stack ten genes (28 kilobases (kb)) within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis and potato transgenic events. To determine whether the GAANTRY system can be used to engineer a monocotyledonous crop, two new T-DNA constructs, carrying five (16.9 kb) or eleven (37.4 kb) cargo sequences were assembled and transformed into rice. Characterization of 53 independent transgenic events demonstrated that more than 50% of the plants carried all of the desired cargo sequences and exhibited the introduced traits. Additionally, more than 18% of the lines were high-quality events containing a single copy of the introduced transgenes and were free of sequences from outside of the T-DNA. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking in rice and potentially other cereal grain crops.

Molecular Mechanisms of Diverse Auxin Responses during Plant Growth and Development

“…We can adapt the CRISPR/dCas9-act gene activation system [ 135 ] to enhance some gene expression and CRISPR/Cas13 gene silencing system that uses sgRNAs target message RNAs to reduce gene expression [ 136 ].Third, we can use recombinase technology, which is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid transfer DNA. With this method, it is possible to assemble and deliver a 28 kilobase (kb) T-DNA with 10 cargo sequences into Arabidopsis [ 137 ] and Solanum tuberosum [ 138 ] and a 37 kb, 11-stack T-DNA into Oryza sativa [ 139 ], which allows us to modify multiple genes related to diverse auxin responses to manipulate multiple traits with higher efficacy in one effort. Finally, some regulatory mechanisms in diverse auxin responses can be used to construct the synthetic promoters or regulatory systems.…”

The Future of Essentially Derived Variety (EDV) Status: Predominantly More Explanations or Essential Change

“…However, as technological progress continues, variety development through the simultaneous addition and/or changes in expression of more than "a few" genes will occur [87][88][89][90][91][92]. Also, it may be that judicial precedent on the definitions of: "a few", "predominant", and "essential" becomes problematic or contradictory in terms of supporting the advancement of genetic gain through plant breeding.…”

All‐in‐one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants

“…The improved TransGene Stacking II (TGSII) system utilizes modified Cre/ loxP recombination based on a transformation‐competent artificial chromosome and rare‐cutting homing endonuclease sites to stack multiple genes (Lin et al ., 2003 ; Zhu et al ., 2017 , 2018 ). The Agrobacterium‐ based GA A NTRY (gene assembly in Agrobacterium by nucleic acid transfer using recombinase technology) system uses unidirectional site‐specific recombinases to assemble transgene cassettes (Collier et al ., 2018 ; Hathwaik et al ., 2021 ). The advanced multisite Gateway cloning system allows two or three DNA fragments to be simultaneously transferred into destination vectors in a single recombination step, for use in BiFC and Forster resonance energy transfer (FRET) analyses (Grefen and Blatt, 2012 ; Hecker et al ., 2015 ; Karimi et al ., 2005 ; Martin et al ., 2009 ).…”