This junction was found in 77 of 81 candidates. The four plants that failed to yield a PCR product may possibly be because of hpt fusion to an endogenous professional moter while in the genome. The 77 plants have been analyzed by Southern hybridization. DNA handled with EcoRI was probed with a 35S cre fragment. An intact left and appropriate junction was confirmed in 73 from the 77 plants as a result of detection of your one. seven kb 35S lox hpt plus the one. four kb gus lox cre band, and the reduction with the 2. 4 kb parental 35 lox cre fragment. With EcoRV taken care of DNA, a gus particular probe hybridized to only one two. 0 kb gus lox cre fragment in 35 in the 73 plants. The presence of a single or a lot more further bands during the other plants indi cated a lot more than just one copy of Cp gus DNA, no matter whether located in tandem with the exact same web page, or elsewhere within the genome. These 35 plants were examined by hpt particular hybrid ization to EcoRV handled DNA that need to detect a four.
6 kb 35S lox hpt pUC backbone fragment. They were also examined this article by gus certain hybridization to EcoRI cleaved DNA that really should reveal a five. 6 kb pUC Cp gus fragment. Fragments in the predicted sizes have been found in 22 on the 35 single copy integrant plants. With each other, the detection of all five overlapping fragments demonstrates a contiguous ar rangement within the pEL1 insertion as illustrated in Figure 1A. The failure to detect fragments within the anticipated sizes could be attributable to both of two occasions, random integration of pEL1 followed by recombination involving the two lox web-sites,in these occurrences, the 35S lox hpt and gus lox cre junctions are observed, nevertheless they are usually not con tiguous,and site unique integration of pEL1, fol lowed by DNA rearrangement, such as insertion or de letion amongst the two lox junctions, such the ex pected dimension bands aren’t uncovered.
Reporter gene expression pattern Table one lists the 22 plants discovered using a exact single copy integration of pEL1. For the plants Trichostatin A structure through the identical transformation experiment, each was regenerated from a diverse cluster of immobilized cells. The probabilities are remote for

any two plants for being derived through the very same protoplast through the brief incubation time period in advance of im bedding the protoplasts in agarose. Nevertheless, this possibility could not be totally ruled out. Therefore, despite the fact that we usually do not believe this might be the situation, we yield to the likelihood that eight lines may well be sibling clones on the other 14 representative lines. The main transformants of your lines listed in Table 1 had been crossed to wild type tobacco to produce F1 progeny hemizygous for Cp gus. Seedlings had been germi nated from the presence of hygromycin, and GUS enzyme action was examined at the two leaf stage by histo chemical staining. Amongst the progeny of any given plant line, the staining pattern is highly constant,consequently, sib ling seedlings are alike.