The transgene pyramiding tobacco with betaine synthesis and heterologous expression of AtNHX1 is more tolerant to salt stress than either of the tobacco lines with betaine synthesis or AtNHX1

Previous studies have shown that the overexpression of betA (encoding choline dehydrogenase from Escherichia coli) or AtNHX1 (a vacuolar Na+/H+ antiport from Arabidopsis thaliana) gene can improve the salt tolerance of transgenic plants. However, little is known about the effects of the transgene pyramiding of betA and AtNHX1. Here, betA + AtNHX1 transgene pyramiding tobacco was produced by sexual crossing, and the salt tolerance was evaluated at the cellular and plant levels. In NaCl stress, the Na+ concentration in vacuoles and vacuolar membrane potential of transgene pyramiding cells were similar to those of AtNHX1-transgenics, and much higher than those of betA-transgenics when detected using fluorescent dye staining; transgene pyramiding cells showed a higher protoplast viability and comparable mitochondrial activity as compared with single transgenics; and transgene pyramiding plants showed comparable Na+ content in leaves as compared with AtNHX1-transgenics and remarkably higher than betA-transgenics; and transgene pyramiding lines exhibited higher percentage of seed germination, better seedling growth and higher fresh weight than lines that had betA or AtNHX1 alone. Based on the integrative analysis of salt tolerance, the consistency between the cellular level and the whole plant level was confirmed and the transgene pyramiding plants exhibited improved salt tolerance, but compared with the plants with betA or AtNHX1 alone, the differences were relatively small. Other mechanisms involved in salt tolerance should be considered to further enhance transgene pyramiding plants salt tolerance.