Noguera, L. M. | Search bioRxiv

bioRxiv2023

Factors influencing Agrobacterium-mediated genetic transformation efficiency in wheat.

Assessment of the efficiency of Agrobacterium tumefaciens mediated transformation in four bread wheat varieties was focused and upon optimization of experiments done to obtain tangible results. The four varieties namely Fielder, Navojoa, Baj and Kachu/saul were transformed with optimized protocols, subsequently randomly selected from selection medium amalgamated with phosphinothricin (Glufosinate). Initially the variety Fielder was proceeded with 36 different concentrations of synthetic auxins 2,4-D and Picloram as 36 treatments. The treatment 30 (48 g of 2,4-D and 64 g of Picloram) has shown higher efficiency and is comparable in terms of the three traits studied. Additionally, antioxidants and growth regulators were adjusted to obtain better result towards reasonable transformation frequency. Based on the optimization for the variety Fielder, additional varieties were also tested and selected against the selectable marker PPT. Selected genotypes were subjected for gusA or Bar gene PCR amplification. The result revealed the frequency of transgenesis with the range of 60-70% with the varieties studied, however the whole experiment needs further repetitions for confirmatory result. Transgenic genotypes of the Fielder variety showed moderate to strong GUS expression in leaves, anthers, seeds, seed coats, roots compare to non-transgenic control plant tissues. PPT leaf painting assay showed the lack of necrosis on painted area of leaves in transgenic genotypes compared to the non-transgenic ones.

plant biology↗

bioRxiv2022

Gene editing for accelerated breeding in wheat

Diseases adversely affect grain yield of crop plants. Leaf rust is a major disease of wheat. As race-specific resistance breaks down, introduction of newer sources of resistance often from older accessions is necessary. Linkage drag from the donor accessions adversely impacts grain yield. As a result, CIMMYT breeding effort has shifted to using durable resistance, also referred to as race non-specific host resistance, which allows slow rusting but maintains grain yield. One of the key genes for durable resistance is Lr67 and the resistant form of this gene is absent in CIMMYT elite lines. Hence, we have initiated efforts to convert the susceptible copy of Lr67 into its resistant form directly in these elite lines using gene editing. This would eliminate backcrossing and thus save time as well as eliminate linkage drag that would accompany the resistant copy of the gene if it were to be introgressed from an older accession. As first steps, we have isolated and sequenced the genomic copies from each of the A, B, and D genome of the Lr67 gene from three elite lines and an experimental line. Identification of more than 50 single nucleotide polymorphisms (SNPs) in the open reading frames (ORF) among these lines would be useful in designing the guide RNA molecules with precision. Further, we have streamlined genetic transformation of these elite lines, a prerequisite step for gene editing.

molecular biology↗