Thus we could generate bentazon sensitive rice plants by suppressing the expression of this detoxification gene thorough antisense RNA or RNA interference.
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Here we report the creation of the bentazon sensitive transgenic rice with high glyphosate tolerance. We demonstrated that such transgenic rice could be selectively eliminated by bentazon. The polyubiqitin-1 promoter of Zea mays  is used to drive the expression of this gene.
G6 serves as the transformation selection marker as well as the gene of interest for conferring glyphosate tolerance to facilitate weed control of the transgenic rice. Total of over 40 independent transgenic events were obtained in our transformation. We noticed no significant difference in transformation efficiency compared to other transformation experiments using the same G6 gene as the selection maker but without the RNA interference cassette, suggesting that this RNA interference cassette likely does not affect the transformation.
The insertion of the transgene in the selected transgenic events, R, R, R and R, was further confirmed by PCR and immunoblot analysis Fig. The non-transgenic control plants were negative in both PCR and immunoblot analysis.
The G6 expressed in transgenic rice plants were detected by its antiserum from rabbits B. Lane 1 to 4, transgenic event R, R, R and R, respectively; CK, non-transformed rice as the negative control; M, bp ladder A or 48 kDa pre-stained protein size maker B. The T 0 plants from eight transgenic events were selected for determining their sensitivity to bentazon along with the non-transgenic plants.
In contrast, the 20 mM glyphosate spray killed all the conventional rice plants but not any of the transgenic rice plants in 7 days Fig. The transgenic plants that were not sprayed with either bentazon or glyphosate appeared to be healthy and grew normally Fig.
The plants in panel C were not sprayed. The pictures were taken 7 days after the spray. The plants in each panel were in the same order. Therefore, the transgenic rice plants we generated were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice plants. This striking difference in response to the two herbicides between the transgenic and the non-transgenic rice makes the selection or termination of the transgenic rice plants to be extremely convenient and effective.
The total RNAs were isolated from the non-transgenic control plants and the transgenic plants of each event of R, R, R, and R, respectively. We found that the amount of the bp CYP81A6 RT-PCR product was greatly reduced from the transgenic plants compared to that from the non-transgenic control plants, while the amount of the bp Actin RT-PCR product was roughly equal among the transgenic and non-transgenic plants Fig. This is in agreement with our assumption that the RNA interference cassette introduced in tandem with the gene of interest in these transgenic rice plants is responsible for their sensitivity to bentazon.
The rice Actin gene was used as the control. The T 1 plants of events R and R were further tested for their sensitivity to bentazon in a field trial. The T 1 seedlings of the events R and R along with the non-transgenic conventional rice seedlings of the same cultivar were replanted individually in the trial field. All of the individual F1 plants were analyzed by PCR to determine if they were transgenic plants or segregates without the transgene.
Thirteen of the total 23 individually replanted plants of the event R are transgenic, while all of the plants of the event R are transgenic, as determined by PCR analysis. All of the transgenic plants of both events R and R died within 6 days after spray, while all the conventional rice plants and the negative segregates without the transgene survived as expected Fig. The event R may have multiple copies of insert in its genome, which may explain the lack of negative segregates in its T1 population.
The picture was taken 7 days after the spray. The surviving plants in the R rows were segregates not carrying the transgene. Furthermore, we did not observed any yield penalty or other abberations of phenotype over the transgenic plants. We measured the plant height, number of panicles per plant, average panicle length, number of grains per panicles and weight of per grains of the F1 transgenic rice plants of both event R and R Table 1.
Once an event of transgenic rice is released for commercial planting, it is hard to ensure the total containment. Thus, it is important to develop technology to selectively terminate the escapes to ensure the decontamination of the concerned fields. Normally it is difficult even to detect let alone to selectively terminate the transgenic plants from the non-transgenic ones in large area of crops. However, the strategy we report here will make the detection and selective termination of the transgenic rice plants quite convenient. In this strategy, the transgenic rice could be detected and terminated selectively by a registered agrichemical.
This unique technology is especially useful for creating transgenic rice plants as bioreactors for molecular farming, an emerging technology for production of pharmaceutical and industrial proteins. Currently many pharmaceutical proteins produced from plants are under clinical trials . These otherwise high value proteins could be harmful if consumed unintentionally though the contamination from food or feed supply.
This novel containment technology could in essence serves as an insurance policy of no contamination for food or feed supply. Therefore, we suggest that all transgenic rice for molecular farming should be generated with a controllable maker such as the one described in this report. Moreover, this technology may also be used for creating transgenic rice with genes that are currently regarded as safe.
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The safety regarding a gene used in a transgenic crop may be conditional and subject to change with our better understanding over time. A safe gene now could be considered as undesirable in the future. Transgenic plants created by this strategy could make any recall of a released transgene much easier if ever need.
This novel strategy appears simple, reliable and inexpensive for implementation. To create terminable transgenic rice, the T-DNA binary plasmid pGi reported in this paper could be used as a basic backbone for inserting any genes of interest. This strategy is also highly reliable. The gap of the killing dose of bentazon between the transgenic and non-transgenic plants is quite wide.
This wide gap will make the termination of transgenic rice plants quite feasible and flexible. Our field trials demonstrated that all the transgenic rice plants were killed efficiently by one spray of bentazon at regular weed control dose. Thus, the reliability of this strategy is as high as that of bentazon for weed control. Finally, because bentazon is an herbicide that has been registered for weed control in rice, it is ready for use. There will be almost no extra cost incurred for decontaminating transgenic rice if bentazon is incorporated into the practice of weed control for conventional rice production.
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