Enhancing the productivity of hybrid hardwood embryogenic cultures and genetic transformation of hybrid sweetgum for mercury phytoremediation
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Mercury pollution is a worldwide environmental problem with tens of thousands of contaminated sites. Phytoremediation is an environmentally friendly solution relying on long-lived plants like trees to clean contaminated soil, water and air. To explore the potential of transgenic hybrid hardwood trees to clean mercury pollutants, we first established efficient somatic embryogenesis systems for hybrid yellow-poplar (Liriodendron tulipifera × L. chinense) and hybrid sweetgum (Liquidambar styracifula × L. formosana). Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on induction-maintenance medium (IMM) supplemented with plant growth regulators (PGRs) and casein hydrolysate (CH). For hybrid yellow-poplar, a high number of germinable embryos were produced when PEMs were grown in liquid IMM lacking CH, followed by size fractionation and plating on semisolid embryo development medium without CH, but with abscisic acid. For hybrid sweetgum, a high number of germinable embryos were produced on development medium without any PGRs or other supplements after PEMs were grown in liquid IMM without CH, but with glutamine, asparagine and arginine. Using Agrobacterium-mediated gene transfer, transgenic hybrid sweetgum lines were generated either overexpressing a y -glutamylcysteine synthetase gene (y -ECS), or expressing modified mercuric ion reductase genes (merA). Hybrid sweetgum PEMs transformed with y -ECS driven by an actin promoter (ACT2) were able to grow in the presence of 50 µM HgCl2, which inhibited wild-type PEMs. However, abnormal plantlets were regenerated from the y -ECS PEMs and they did not survive for more than a few weeks following germination. Mature somatic embryos generated from 35S:merA9 and 35S:merA18 PEMs were capable of conversion to normal plantlets on germination medium containing 25 µM HgCl2, by converting ionic mercury into its volatile, elemental form, Hg(0). Somatic embryos generated from ACT2:merA77 PEMs converted to plantlets, but these remained unhealthy on Hg(II)-medium. Transgenic merA plantlets released Hg(0) 2-3 times more efficiently than the wild-type plantlets. These results indicate that the y -ECS gene may significantly affect the regeneration capacity of hybrid sweeetgum PEMs and the growth of the plants. Hybrid sweeetgum plants expressing modified merA genes driven by a 35S promoter may provide a means for phytoremediation of mercury pollution.