HvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different phenotypes

Abstract

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 T1 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 T0 plants and T1 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 T1 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 T0 plants and T1 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.

Transgene integration complexity and expression stability following biolistic or Agrobacterium-mediated transformation of sugarcane

“…This is consistent with the recent findings of Jackson et al (2013) with cultivar Q117. However, earlier results in rice (Dai et al 2001;Breitler et al 2004), barley (Travella et al 2005;Zalewski et al 2012), maize (Shou et al 2004), and fescue (Gao et al 2008) showed that AMT transformants displayed higher expression than those of BLT. The main reason for the conflicting reports appears to be the use of small amounts of minimal expression cassettes by Jackson et al (2013), which dramatically increased the frequency of simple integration events.…”

“…Very few studies have directly compared integration complexity and expression levels following AMT vs. BLT in crops. Most of these studies have compared both methods side-by-side using genotypes that were amenable to both gene transfer systems but lacking commercial importance and/or based the comparison on a small number of transgenic lines (Snyder et al 1999;Dai et al 2001;Shou et al 2004;Travella et al 2005;Zalewski et al 2012;Jackson et al 2013;Joyce et al 2014). The transformation efficiency following AMT or BLT MC did not significantly differ in 12 independent experiments that generated 562 transgenic plants from the sugarcane cultivar CP88-1762.…”

Sina and Sinb genes in triticale do not determine grain hardness contrary to their orthologs Pina and Pinb in wheat

“…As was already documented in the case of gene silencing [28] as well as transgene expression [29,30], this method should be the method of choice. Segregation of silencing was observed among progeny of all tested T 1 lines documenting high efficiency of the RNAi silencing process.…”

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations

“…This method of genetic transformation is the method of choice since we documented that the biolistic approach might stimulate strong off-target effects in the case of gene silencing by siRNAs (Zalewski et al, 2012). The similar negative effect of biolistic transformation compared to the Agrobacterium -mediated method was documented in the case of transgene expression in cereals (Dai et al, 2001; Travella et al, 2006).…”