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

• Main Authors: Zalewski Wojciech, Orczyk Wacław, Gasparis Sebastian, Nadolska-Orczyk Anna
• Format: Article
• Language: English
• Published: BMC 2012-11-01
• Series: BMC Plant Biology
• Subjects: RNAi silencing; <it>HvCKX2</it>; Barley; Genetic transformation; <it>Agrobacterium</it>; Microprojectile bombardment
• Online Access: http://www.biomedcentral.com/1471-2229/12/206

<p>Abstract</p> <p>Background</p> <p><it>CKX</it> 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 <it>HvCKX1</it> 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 <it>HvCKX2</it> 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 <it>Agrobacterium</it>.</p> <p>Results</p> <p>The mean <it>Agrobacterium</it>-mediated transformation efficiency of Golden Promise was 3.47% (±2.82). The transcript level of <it>HvCKX2</it> in segregating progeny of T₁ lines was decreased to 34%. The reduction of the transcript in <it>Agrobacterium</it>-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.</p> <p>The transformation efficiency of Golden Promise after biolistic transformation was more than twice as low compared to <it>Agrobacterium</it>. 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 <it>HvCKX2</it>.</p> <p>Conclusion</p> <p>The phenotypic result, which was higher productivity of silenced lines obtained via <it>Agrobacterium</it>, confirms the hypothesis that spatial and temporal differences in expression contributed to functional differentiation. The applicability of <it>Agrobacterium</it>-mediated transformation for gene silencing of developmentally regulated genes, like <it>HvCKX2</it>, 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.</p>