A competence of embryo-derived tissues of tetraploid cultivated wheat species Triticum dicoccum and Triticum timopheevii for efficient and stable transgenesis mediated by particle inflow gun

Abstract

Background The ability to engineer cereal crops by gene transfer technology is a powerful and informative tool for discovering and studying functions of genes controlling environmental adaptability and nutritional value. Tetraploid wheat species such as emmer wheat and Timopheevi wheat are the oldest cereal crops cultivated in various world areas long before the Christian era. Nowadays, these hulled wheat species are gaining new interest as donors for gene pools responsible for the improved grain yield and quality, tolerance for abiotic and biotic stress, resistance to pests and disease. The establishing of efficient gene transfer techniques for emmer and Timopheevi wheat may help in creation of modern polyploid wheat varieties. Results In the present study, we describe a robust protocol for the production of fertile transgenic plants of cultivated emmer wheat (Russian cv. ‘Runo’) using a biolistic delivery of a plasmid encoding the gene of green fluorescent protein (GFP) and an herbicide resistance gene (BAR). Both the origin of target tissues (mature or immature embryos) and the type of morphogenic calli (white or translucent) influenced the efficiency of stable transgenic plant production in emmer wheat. The bombardment of nodular white compact calluses is a major factor allowed to achieve the highest transformation efficiency of emmer wheat (on average, 12.9%) confirmed by fluorescence, PCR, and Southern blot. In the absence of donor plants for isolation of immature embryos, mature embryo-derived calluses could be used as alternative tissues for recovering transgenic emmer plants with a frequency of 2.1%. The biolistic procedure based on the bombardment of immature embryo-derived calluses was also successful for the generation of transgenic Triticum timopheevii wheat plants (transformation efficiency of 0.5%). Most of the primary events transmitted the transgene expression to the sexual progeny. Conclusion The procedures described here can be further used to study the functional biology and contribute to the agronomic improvement of wheat. We also recommend involving in such research the Russian emmer wheat cv. ‘Runo’, which demonstrates a high capacity for biolistic-mediated transformation, exceeding the previously reported values for different genotypes of polyploid wheat.

Agrobacterium-mediated direct transformation of wheat mature embryos through organogenesis

“…Mature embryos (MEs) from wheat seeds have also been used for transformation through somatic embryogenesis. Transgenic wheat plants were obtained by bombardment of ME-derived embryogenic calli ( Patnaik and Khurana, 2003 ; Miroshnichenko et al., 2020 ) or by Agrobacterium -mediated transformation of precultured MEs on auxin media followed by callus induction and shoot regeneration ( Ding et al., 2009 ; Moghaieb et al., 2010 ; Aadel et al., 2018 ; Kumar et al., 2019 ). In planta wheat transformation by direct bombardment of the meristematic tissues of MEs has also been reported.…”

CRISPR/Cas9-induced modification of the conservative promoter region of VRN-A1 alters the heading time of hexaploid bread wheat

“…Three uniform plasmids carrying one of the sgRNAs were independently co-transformed with Cas9-expression cassette pGCB ( Figure 1B ) into ‘Chinese Spring’ embryogenic calli using a particle inflow gun. Following the dual selection protocol ( Miroshnichenko et al., 2020 ) we obtained 23 independent T0 transgenic plants carrying both the Cas9 gene and the sgRNA sequence ( Supplementary Table S3 and Supplementary Figure S4 ). All Cas9+sgRNA positive plants, including nine T0 plants carrying vr-30, eight T0 plants carrying vr-31, and six T0 plants carrying vr-34, were screened for indels.…”

“…The biolistic-mediated transformation was performed with the help of a particle inflow gun using 7–15-day-old embryogenic calli induced from isolated immature embryos as earlier described ( Miroshnichenko et al., 2020 ). To avoid the formation of escapes, a dual selection strategy was used that is based on the continuous visual selection of transgenic tissues/plants displaying GFP expression on a phosphinotricin-enriched medium.…”

An established protocol for generating transgenic wheat for wheat functional genomics via particle bombardment

“…Recently, using dephosphorylation cassettes and decreasing the amount of DNA during particle bombardment were found to be an effective method to produce transgenic plants with simple integration patterns and a high transmission rate of the expressed transgene to the progeny ( Tassy et al., 2014 ). In addition, a new bombardment-based transgenic protocol has been developed, allowing efficient transformation in cultivated emmer and Triticum timopheevii accessions ( Miroshnichenko et al., 2020 ).…”