Transient expression of siRNA targeted against the TYLCV AV1, AC1 and AC3 genes for high resistance in tomato

Agrobacterium-mediated transformation of tomato (Lycopersicon esculentumL. cv. Hezuo 908) with improved efficiency

“…Tomato expression vectors pBIN438-AV1 (i/r), pBIN438-AC1(i/r), pBIN438-AC3(i/r) and pBIN438-AV1-AC1-AC3(i/r) were constructed previously in our laboratory. [9] The expression of the gene of interest was under the control of the double constitutive CaMV 35S promoter ( Figure 1). The plasmid was transferred into the competent cells of A. tumefaciens LBA4404 through the freezeÀthaw method.…”

“…[9] Untransformed tomato was selected as a negative control, whereas miRNA 166 was applied as a loading control. [9] The gels were blotted overnight on Hybond-N C membrane (Roche). The membrane was hybridized with PCR-labelled gene-specific Digoxin (DIG) probes (Roche).…”

“…Hezuo 908, which demonstrates inter determined growth type and high fruit-setting ability, is a commonly cultivated cultivar in several provinces in China. [9] However, efficient Agrobacterium-mediated genetic transformation methods are limited for this cultivar, when compared with other major commercial tomato cultivars. Several investigators have focused themselves extensively on the genetic transformation of this cultivar, but have achieved limited success.…”

“…[10,11] In our previous study, we developed an Agrobacteriummediated gene transient genetic transformation approach. [9] However, no gene was integrated into the genome and transgenes were unstable and could become lost through environmental factors. In this study, we established an efficient Agrobacterium-mediated gene transformation system for cv.…”

“…Genes AV1, AC1 and AC3 encode coat protein, replication-related factor and replication accessory factor, respectively. [9] Gene AV1-AC1-AC3 is a fusion gene that includes partial gene fragments of AV1 (151 bp), AC1 (150 bp) and AC3 (153 bp). Molecular characterization through the polymerase chain reaction (PCR) analysis, quantitative real-time PCR (qPCR) analysis, b-glucuronidase (GUS) activity staining and northern blot analysis revealed that 40% positive transgenic plants were successfully obtained.…”