Based on RNAi processes, gene silencing has already been proven to be an efficient approach for functional genomics in cereals [1, 4, 24, 25]. The stability of silencing cassette expression as well as directed gene silencing is one of the key requirements for the successful application of transgenic lines in basic research of gene function and agricultural genetic improvement. In this study we applied the posttranscriptional gene silencing process (PTGS) to silenced HvCKX2 in two barley cultivars by two different transformation methods: biolistic and Agrobacterium.
Golden Promise cultivar is widely used in barley biotechnology/functional genomics, because of its susceptibility to Agrobacterium-mediated transformation, which was also confirmed in this paper. The second one, cv. Scarlett, showed very low transformation ability. The data obtained for Golden Promise with the bar selection system were 3.5% for Agrobacterium-mediated transformation and less than half this value, 1.6%, after biolistic transformation. A comparable result for the same cultivar of barley, doubled transformation efficiency with Agrobacterium compared to particle bombardment, was obtained by Travella et al. . These results are also in agreement with those obtained for rice [21, 26]. The second cultivar tested, expressing very low efficiency after Agrobacterium-mediated transformation, also showed very low efficiency after the biolistic method. We might assume that the susceptibility to bacteria was not the only limiting factor for cv. Scarlett transformation. This result is against the hypothesis that genotype dependence of susceptibility to Agrobacterium might be the limiting factor in applying the Agrobacterium-mediated method, which should not appear in the biolistic method .
The silencing of HvCKX2 by the biolistic method determined low productivity and by Agrobacterium high productivity of T0 plants. This general tendency of plant productivity was also transmitted to the next generation. Higher productivity was the result of a higher number of seeds and grain yield, higher 1000 grain weight as well as increased (by 7.5%) height of plants and higher (from 0.5 to 2.3) numbers of spikes. We also documented that this higher productivity was correlated with lower levels of HvCKX2 transcript in 7 DAP spikes and decreased CKX activity in leaves and 7 DAP spikes in the progeny of lines silenced via Agrobacterium. The tissues/organs appropriate for analysis were chosen based on temporal and spatial expression of HvCKX2 measured in developing wild barley cultivars of Golden Promise and Scarlett. These data were highest in 14 DAP spikes, followed by 7 DAP and 0 DAP, as well as in the leaves (not yet published). The phenotypic result of silencing of HvCKX2 in these tissues via Agrobacterium was higher productivity and increased height of silenced lines. These results confirm once again the hypothesis that spatial and temporal differences in expression contributed to functional differentiation [1, 4]. The higher productivity might be the result of lower HvCKX2 transcript in developing spikes and decreased CKX activity. Increased height of silenced plants may be dependent on decreased CKX activity in the leaves, estimated at a high level in the wild plants. Similar results of HvCKX1 silencing via Agrobacterium in Golden Promise, which correlated with the specific organs, were observed in our previous research . The highest expression of the gene in wild-type plants was in 7 DAP spikes, followed by 14 DAP and 0 DAP, as well as in the roots. In that experiment, the silencing of HvCKX1 led to higher plant productivity as well as higher mass of the roots, although the height of the plants was reduced. The reduced expression of another CKX gene in rice, OsCKX2, caused cytokinin accumulation in the inflorescence meristems, increased the number of reproductive organs, and increased grain number and yield . It was also documented that halophyte variants of TaCKX6
D1, a wheat ortholog of rice OsCKX2, was associated with grain weight in hexaploid wheat . Newly published research on phylogenic and sequence analysis showed that CKX1 and CKX2 are closely related in clade Ia of Poaceae and are physically linked . The authors hypothesized that both genes might have similar functions, which is supported by our earlier research on HvCKX1 and HvCKX2 in this paper.
The opposite result of the plant productivity obtained in T0 and T1 lines, when HvCKX2 was silenced with the biolistic method, might be explained by somaclonal variation . This term, describing the phenotypic variability among plants of in vitro origin, includes genetic and epigenetic modifications . Both types of modifications in biolistic-derived plants are caused by a physically destructive method and integration of many, mostly rearranged copies of a transgene, the result of which is frequently determined transgene silencing . Due to resource limitation, the copy number was not examined in this study. However based on the knowledge from previously published papers [16, 21, 22, 30] the phenotypic differences in lines generated by two transformation methods might be attributed to different copy numbers of transgene integrated to the genome as well as the DNA/transgene rearrangements. In such situations our silencing cassette introduced by the biolistic method might disturb the effect of silencing and the whole phenotype, otherwise visible in the group of Agrobacterium-silenced plants. This effect, depending on the method of transformation, might be especially distinct in the case of the silencing of developmentally regulated genes, like CKXs. Besides lower plant productivity, it also caused a lack of germination in one line and inability of seed setting in half of the progeny of another line (out of five tested). The primary reason was a lack of functional anthers. This was correlated with the lowered to 50% - 60% CKX activity in 7 DAP spikes and it was not observed in seed-setting plants of biolistic origin. However, lowered CKX activity has also been proven in Agrobacterium-silenced lines, where it resulted in higher productivity. The explanation of these differences might be observed, unbalanced CKX activity in the whole biolistic-derived plants – very high in the leaves and very low in the 7 DAP spikes. This result proved the earlier reported observations in other plant species that CKXs respond differently to various stresses [31, 32]. The effect of SBEIIa silencing on starch metabolism in durum wheat lines obtained with the two methods of transformation, biolistic and Agrobacterium, was genotype and protocol independent . However, these results are not directly comparable with ours, because in that report two different cultivars were transformed with one of two methods, the silenced genes influenced only starch metabolism (causing alterations in granule morphology and starch composition, leading to high amylose wheat), and there was a lack of detailed data on productivity, possibly because of the character of the genes tested.
The final effect of the silencing observed in biolistic-derived and Agrobacterium-derived plants was also different. The transcript level in segregating progeny of lines transformed by both methods was similar, decreased to 24% (biolistic) and to 34% (Agrobacterium), and it was reduced in about 1/3 of plants by more than 50%. The consequence of this reduction of transcript in Agrobacterium-derived plants was decreased CKX activity in developing and developed leaves as well as in 7 DAP spikes. Otherwise the enzyme activity for developing and developed leaves of the lines of biolistic origin, especially in cv. Golden Promise, was very high, exceeding the relative level for control lines. This imbalanced effect of the low level of the transcript and very high CKX activity suggest disturbances of developmental processes, which are naturally guided by small RNA at the transcriptional (DNA) and posttranscriptional (RNA) levels [34, 35]. Both PTGS and TGS may be influenced by environmental and developmental factors . Such disturbances in HvCKX2 experimentally silenced by Agrobacterium have not occurred, proving the applicability of the method for gene silencing of developmentally regulated genes.