Abstract
Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed (control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving the gus gene in different transgenic lines.
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Abbreviations
- bspA:
-
boiling stable protein gene of aspen
- DIG:
-
digoxigenin
- EDTA:
-
ethylenediamine tetra acetic acid
- EtBr:
-
ethidium bromide
- GUS:
-
β-glucuronidase enzyme
- hpt:
-
hygromycin phosphotransferase gene
- IAA:
-
indole-3-acetic acid
- LB:
-
Luria-Bertani
- MS:
-
Murashige and Skoog
- PCR:
-
polymerase chain reaction
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Roy, R., Purty, R.S., Agrawal, V. et al. Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance. Biol Plant 50, 352–358 (2006). https://doi.org/10.1007/s10535-006-0049-6
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DOI: https://doi.org/10.1007/s10535-006-0049-6