Advances in Agrobacterium-mediated Maize Transformation

Abstract

One of the major limitations of maize transformation is the isolation of a large number of immature embryos using the time-consuming manual extraction method. In this article, we describe a novel bulk embryo extraction method for fast isolation of a large number of embryos suitable for both biolistic- and Agrobacterium-mediated transformation. Optimal gene delivery and tissue culture conditions are also described for achieving high efficiency in Agrobacterium-mediated maize transformation using phosphomannose isomerase (PMI) as a selectable marker.

Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize

“…Since reasonable editing efficiencies were achieved using AsCas12a RNP in direct immature embryo bombardment, we also tested whether stably edited lines can be recovered from immature embryos bombarded with RNPs targeting Bx9 gene. We co-delivered RNP with a plasmid named pBSC12672 which contains the PMI selectable marker gene (Zhong H. et al, 2018 ). We tested three different versions of AsCas12a nuclease: AsCas12a-WT, AsCas12a-V3 and AsCas12a-Ultra; the AsCas12a-WT and AsCas12a-V3 proteins contain the wild-type AsCas12a sequence with AsCas12a-V3 also containing an optimized NLS sequence, whereas the AsCas12a-Ultra is an engineered-version of AsCas12a-V3 with mutations that were isolated using directed evolution in bacteria to achieve higher editing efficiencies in living cells (Zhang L. et al, 2020 ) ( Supplementary Table 1 ).…”

“…Syngenta's proprietary elite maize inbred variety NP2222 was used in all experiments. Stock plant care, maize ear production, immature embryo extraction and transformation with PMI as selectable marker gene was carried out as described before (Zhong H. et al, 2018 ).…”

“…Binary transformation vector 12672 contains 2 gene expression cassettes, one for the PMI selectable marker gene driven by the maize Ubiquitin-1 promoter and another for the AmCyan fluorescent protein gene driven by the maize Ubiquitin-1 promoter (Zhong H. et al, 2018 ). For expression of LbCas12a, the rice codon-optimized coding sequence (Tang et al, 2017 ) was used with 3 bp changes to remove 2 Bsp119I and one RsrII recognition sites.…”

“…The Bx9 gene target sequences for the crRNAs of these Cas12a vectors are listed in Supplementary Table 3 . Transgenic plants were generated through particle bombardment using isolated immature embryos as targets and PMI as a selectable marker (Wright et al, 2001 ; Chen et al, 2018 ; Zhong H. et al, 2018 ). Briefly, immature embryos were isolated from harvested ears at about 9–11 days after pollination and pre-cultured for 1–3 days on osmoticum media.…”

Complete Genome Sequence of a Novel Latent Tobamovirus Infecting Hevea Brasiliensis in China

“…Agrobacterium tumefaciens-mediated transformation has been widely used in plant genetics research [10,11] and has been successfully applied in rubber tree [5], but Agrobacterium-mediated transformation has rarely been used in the research of rubber tree due to the low transformation e ciency and time cost.…”