Effects of mir408 and bor2 overexpression in boron hyperaccumulator plants for boron phytoremediation

In this study, genetic transformation of miR408 gene to cotton and carnation plants was carried out via Agrobacterium. The regeneration of transgenic plants was performed successfully in both plants. However, F1 seed production could only be achieved in cotton plants. Boron phytoremediation potential of miR408 gene was investigated at the molecular and biochemical level by the use of F1 generation transgenic cotton plants. For this purpose, severe boron stress was applied to the transgenic and control cotton plants at two different durations, namely 1 day and 5 days at the rate of 7.5 mM B and the responses were examined comparatively. The mir408 gene was found to increase the expression level of PIP1;1 gene under long term boron stress in transgenic plants. Similarly, the PIP2;1 gene expression level increased with increasing boron stress in transgenic plants. Levels of MDA and ion leakage indicated that transgenic plants were more tolerant to boron stres when compared to control plants. Boron transporter 1 gene expression level decreased in transgenic plants when encountered with excesss boron on the 1st day of stress. However, at the end of the 5th stress day, increased expression of this gene was found to increase boron input to the plants. The miR408 gene was determined to regulate the expression level of boron transporter 1 gene in the provision of boron balance in plants. Similarly boron transporter 2 gene expression was increased with the exposure of longer stress duration in transgenic plants. The reason for the lower Boron transporter 2 gene expression in transgenic plants on the 1st day of stress might be resulted from the decrease of Boron transporter 1 gene expression and eventually decreased boron intake of plant tissues. Plant relative water content percentages (%) were higher and statistically significant in transgenic plants compared to control plants under boron stress on the 1st day of stress, while this percentage decreased and found to be statistically insignificant between transgenic and boron stressed control plants on the 5th day of stress. Plant proline contents did not exhibit statistically significant difference in transgenic plants when compared to boron stressed control plants on the first day of stress; however the difference increased at the duration of five days. These results also supported the relationship between Boron transporter 1 and Boron transporter 2 genes. The expression of plastocyanin gene was increased with the duration of stress. On the 1st day of stress, plastocyanin gene expression in transgenic plants was found to be lower than boron stressed control plants, whereas it was found to be higher on the 5th day of stress when compared to boron stressed control plants. Plastocyanin is a gene expressed in the lack of copper; however plants are unable to absorb essential copper under increased stress conditions. This might be the reason for the plastocyanin gene expression increase under long stress duration. miR408 gene seems to regulate plastocyanin gene expression under long stress duration in transgenic plants with 11% more expression compared to boron stressed control plants. Although the total chlorophyll content of transgenic plants was higher than boron stressed control plants at the 1st day of stress, the difference was not statistically significant. However, at the 5th day of boron stress, increase in the total chlorophyll content of transgenic plants compared to boron stressed control plants was statistically significant. Therefore, transgenic plants have been found to maintain their total chlorophyll contents under severe boron stress when compared to boron stressed control plants. The miR408 gene was found to play no effective role in the regulation of MAPK and CPR2 gene expressions under boron stress. EF1A8 was found to be a suitable referance gene which can be used under boron stres.