Yuri T. Yamamoto

Genetic transformation of duckweed Lemna Gibba and Lemna Minor

SummaryWe developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer. Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing β-glucuronidase and nptII expression cassettes. Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin. Transgenic duckweed fronds were regenerated from selected nodules. We demonstrated that transgenic duckweed could be regenerated within 3 mo. after Agrobacterium-mediated transformation of nodules. Furthermore, we developed a method for transforming L. minor 8627 in 6 wk. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins.

NUTRIENT REMOVAL FROM SWINE LAGOON LIQUID BY LEMNA MINOR 8627

Nitrogen and phosphorus removal from swine lagoon liquid by growing Lemna minor 8627, a promising duckweed identified in previous studies, was investigated under in vitro and field conditions. The rates of nitrogen and phosphorus uptake by the duckweed growing in the in vitro system were as high as 3.36 g m–2 day–1 and 0.20 g m–2 day–1, respectively. The highest nitrogen and phosphorus removal rates in the field duckweed system were 2.11 g m–2 day–1 and 0.59 g m–2 day–1, respectively. The highest observed duckweed growth rate was close to 29 g m–2 day–1 in both conditions. Wastewater concentrations and seasonal climate conditions had direct impacts on the duckweed growth and nutrient removal in outdoor tanks. The rate of duckweed production in diluted swine lagoon liquid increased as the dilution rate increased. Duckweed assimilation was the dominant mechanism for nitrogen and phosphorus removal from the swine lagoon liquid when the nutrient concentration in the wastewater was low, but became less important as nutrient concentration increased. Reasonably high light intensity and a longer period of warm temperature could result in a higher growth rate for the duckweed. Pre–acclimation of the duckweed with swine lagoon liquid could accelerate the start–up of a duckweed system to remove nutrients from the wastewater by preventing the lag phase of duckweed growth.

Immunolocalization of Mannitol Dehydrogenase in Celery Plants and Cells

Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme. MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles. Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction. Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts. The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent w ith the hypothesis that the Mtd gene may be regulated by sugar repression. Additionally, a developmental component may regulate the distribution of MTD within celery plants.

Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. Cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown.