Dataset Information

TRA1: A Locus Responsible for Controlling Agrobacterium-Mediated Transformability in Barley.

ABSTRACT: In barley (Hordeum vulgare L.), Agrobacterium-mediated transformation efficiency is highly dependent on genotype with very few cultivars being amenable to transformation. Golden Promise is the cultivar most widely used for barley transformation and developing embryos are the most common donor tissue. We tested whether barley mutants with abnormally large embryos were more or less amenable to transformation and discovered that mutant M1460 had a transformation efficiency similar to that of Golden Promise. The large-embryo phenotype of M1460 is due to mutation at the LYS3 locus. There are three other barley lines with independent mutations at the same LYS3 locus, and one of these, Risø1508 has an identical missense mutation to that in M1460. However, none of the lys3 mutants except M1460 were transformable showing that the locus responsible for transformation efficiency, TRA1, was not LYS3 but another locus unique to M1460. To identify TRA1, we generated a segregating population by crossing M1460 to the cultivar Optic, which is recalcitrant to transformation. After four rounds of backcrossing to Optic, plants were genotyped and their progeny were tested for transformability. Some of the progeny lines were transformable at high efficiencies similar to those seen for the parent M1460 and some were not transformable, like Optic. A region on chromosome 2H inherited from M1460 is present in transformable lines only. We propose that one of the 225 genes in this region is TRA1.

SUBMITTER: Orman-Ligeza B

PROVIDER: S-EPMC7176908 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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TRA1: A Locus Responsible for Controlling Agrobacterium-Mediated Transformability in Barley.

Orman-Ligeza Beata B Harwood Wendy W Hedley Pete E PE Hinchcliffe Alison A Macaulay Malcolm M Uauy Cristobal C Trafford Kay K

Frontiers in plant science 20200416

In barley (Hordeum vulgare L.), Agrobacterium-mediated transformation efficiency is highly dependent on genotype with very few cultivars being amenable to transformation. Golden Promise is the cultivar most widely used for barley transformation and developing embryos are the most common donor tissue. We tested whether barley mutants with abnormally large embryos were more or less amenable to transformation and discovered that mutant M1460 had a transformation efficiency similar to ...[more]

PMID: 32373138

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Project description:BackgroundCKX genes encode cytokinin dehydrogenase enzymes (CKX), which metabolize cytokinins in plants and influence developmental processes. The genes are expressed in different tissues and organs during development; however, their exact role in barley is poorly understood. It has already been proven that RNA interference (RNAi)-based silencing of HvCKX1 decreased the CKX level, especially in those organs which showed the highest expression, i.e. developing kernels and roots, leading to higher plant productivity and higher mass of the roots [1]. The same type of RNAi construct was applied to silence HvCKX2 and analyze the function of the gene. Two cultivars of barley were transformed with the same silencing and selection cassettes by two different methods: biolistic and via Agrobacterium.ResultsThe mean Agrobacterium-mediated transformation efficiency of Golden Promise was 3.47% (±2.82). The transcript level of HvCKX2 in segregating progeny of T(1) lines was decreased to 34%. The reduction of the transcript in Agrobacterium-derived plants resulted in decreased CKX activity in the developing and developed leaves as well as in 7 DAP (days after pollination) spikes. The final phenotypic effect was increased productivity of T(0) plants and T(1) lines. Higher productivity was the result of the higher number of seeds and higher grain yield. It was also correlated with the higher 1000 grain weight, increased (by 7.5%) height of the plants and higher (from 0.5 to 2) numbers of spikes. The transformation efficiency of Golden Promise after biolistic transformation was more than twice as low compared to Agrobacterium. The transcript level in segregating progeny of T(1) lines was decreased to 24%. Otherwise, the enzyme activity found in the leaves of the lines after biolistic transformation, especially in cv. Golden Promise, was very high, exceeding the relative level of the control lines. These unbalanced ratios of the transcript level and the activity of the CKX enzyme negatively affected kernel germination or anther development and as a consequence setting the seeds. The final phenotypic effect was the decreased productivity of T(0) plants and T(1) lines obtained via the biolistic silencing of HvCKX2.ConclusionThe phenotypic result, which was higher productivity of silenced lines obtained via Agrobacterium, confirms the hypothesis that spatial and temporal differences in expression contributed to functional differentiation. The applicability of Agrobacterium-mediated transformation for gene silencing of developmentally regulated genes, like HvCKX2, was proven. Otherwise low productivity and disturbances in plant development of biolistic-silenced lines documented the unsuitability of the method. The possible reasons are discussed.

Dataset Information

TRA1: A Locus Responsible for Controlling Agrobacterium-Mediated Transformability in Barley.

Publications

<i>TRA1</i>: A Locus Responsible for Controlling <i>Agrobacterium</i>-Mediated Transformability in Barley.

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