The pBY520 containing the Hordeum vulgare？？HVA1 regulated by the rice actin promoter (Act1 5′) or the JS101 containing the bacterial mannitol-1-phosphate dehydrogenase (mtlD) also regulated by rice Act1 5′ and a combination of these two plasmids were transferred into the maize genome, and their stable expressions were confirmed through fourth generations. Plants transcribing a combination of the HVA1+mtlD showed higher leaf relative water content (RWC) and greater plant survival as compared with their single transgene transgenic plants and with their control plants under drought stress. When exposed to various salt concentrations, plants transcribing the HVA1+mtlD showed higher fresh and dry shoot and dry root matter as compared with single transgene transgenic plants and with their control plants. Furthermore, the leaves of plants expressing the mtlD accumulated higher levels of mannitol. Plants expressing the HVA1+mtlD improved plant survival rate under drought stress and enhanced shoot and root biomass under salt stress when compared with single transgene transgenic plants and with their wild-type control plants. The research presented here shows the effectiveness of coexpressing of two heterologous abiotic stress tolerance genes in the maize genome. Future field tests are needed to assure the application of this research. 1. Introductions In 2007, 60% of the total biotech maize in the United States carried transgenes stacked for both herbicide and insect resistance [1]. Another transgene pyramiding approach has been to express multiple insect and/or disease resistance genes in plants to avoid the possibility that the insect or pathogen develops resistance against the gene products under extreme pressures. For example, transgene stacking delayed the emergence of Bacillus thuringiensis (Bt) resistance in insects (biotype) of broccoli [2] and a combination of transgenes improved resistance to a pest and a disease in rice [3]. There are three strategies on how to stack transgenes in plants. The first strategy is cross-breeding of single gene transgenic plants for gene stacking. For example, cross-breeding of different Bacillus thuringiensis (Bt) and the phosphinothricin acetyltransferase (PAT) herbicide resistant transgenic plants have enhanced corn borer and rootworm resistance along with herbicide tolerance in maize [4]. Wei et al. [5] crossed two transgenic parents, one expressing the BetA gene (encoding for choline sulfatase) and the other expressing the H+-PPase (TsVP) gene(encoding the vacuolar H+ pyrophosphatase of Thellungiella halophila)
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