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Final Report: Genetic Improvement of Duckweed (Lemma Gibba) Wastewater TreatmentEPA Grant Number: R823570
Title: Genetic Improvement of Duckweed (Lemma Gibba) Wastewater Treatment
Investigators: Stomp, Ann-Marie
Institution: North Carolina State University
EPA Project Officer: Manty, Dale
Project Period: June 26, 1995 through June 25, 1998
Project Amount: $143,962
RFA: Exploratory Research - Environmental Biology (1995) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Health , Ecosystems
Objective:To develop method for genetic engineering of the aquatic plant, duckweed (Lemnaceae) as a tool to improve the plant for bioremediation of wastewater.
Summary/Accomplishments (Outputs/Outcomes):Constructed wetlands are an effective wastewater remediation strategy that satisfy the conditions of low cost, simple technology and large water volume (Hammer 1989). The aquatic plants used for constructed wetlands are wild, random selections and have not been genetically selected for superior ability to sequester or degrade contaminating substances. Following the agricultural, crop plant model of genetic improvement for improved characteristics, it is reasonable to hypothesize that genetic methods, including biotechnology and genetic engineering, could be used to improve aquatic plants for their remediation ability.
A genetic improvement program is built upon the foundation of basic understanding of the biology of the organism. Of all the aquatic plants used for wastewater remediation, more basic biology and remediation application work has been described for species of the family Lemnaceae, commonly called duckweed (Landolt and Kandeler 1987). The plant is easy to grow under simple laboratory conditions (Hillman 1961), flower induction and controlled crossing methods are known (Yukawa and Takimoto 1976), tissue culture methods have been explored (Chang and Chui 1976; Frick 1991), and basic molecular biology approaches to gene isolation and characterization have been successful (Rolfe and Tobin 1991; Smart and Fleming 1996). Growth, harvesting and dewatering systems have been engineered for duckweed grown on wastewater laggons (Skillicorn P, et al. 1994). This research lays a sufficient foundation for exploring genetic improvement in duckweed.
Our previous work with genetic transformation of forest tree species (Sederoff and Stomp 1992; Chen and Stomp 1992) and our work on remediation (Stomp et al., 1993) gave us sufficient expertise for initiating the development of gene transfer methods in duckweed. Preliminary work had established duckweed frond culture in our laboratory and the extensive 1000 strain collection of Lemnaceae species and strains amassed over the last 40 years by Dr. E. Landolt of ETH, Zurich. These duckweed strains proved invaluable as there are significant differences in from strain to strain in their ability to be tissue cultured or transformed.
In the current project we were successful in developing Agrobacterium-mediated transformation in two species of duckweed, Lemna gibba and Lemna minor. The first round of frond co-cultivation experiments, using b-glucuronidase (GUS; with an intron in the coding sequence) as the visual marker and neomycin phosphotransferase as the selectable marker, showed that transient expression was readily achieved at high expression levels. Further experiments were done to optimize bacterial concentration, co-cultivation time, medium components, frond pre-treatment, and co-cultivation physical environment. These experiments improved the frequency of transient expression (the number of fronds exhibiting GUS transient expression/total number of fronds co-cultivated) to 90-95%. GUS expression was associated with new budding fronds, meristematic tissue, and roots. Although encouraging, production of stablely transformed fronds was not obtained from direct transformation of fronds.
Duckweed callus culture was developed simultaneously to our frond experiments to provide an alternative to frond co-cultivation. Extensive callus induction and callus culture experiments were conducted (Moon and Stomp 1997; Moon, Rajbhandari and Stomp 1998) to establish robust callus cultures in a number of duckweed species. These cultures were used in callus co-cultivation experiments with Agrobacterium configured as for frond co-cultivation. Fully transformed fronds were regenerated from callus cultures first in L. gibba and then in one strain of L. minor. Stable transformation was confirmed using PCR, Southern hybridization and ELISA for nptII and GUS. A patent was filed (Stomp and Rajbhandari 1997) and the first divisional allowance has recently been granted (in August 1999).
Success achieved with this support has allowed us to secure a follow on grant from USDA (Genetic and engineering improvement of a swine wastewater plant treatment system, USDA CSRS-NRIGP Proposal #9800906).
Journal Articles on this Report : 2 Displayed | Download in RIS Format
|Other project views:||All 3 publications||2 publications in selected types||All 2 journal articles|
||Moon HK, Stomp AM. Effects of medium components and light on callus induction, growth, and frond regeneration in Lemna gibba (Duckweed). In Vitro Cellular & Developmental Biology-Plant 1997;33(1):20-25.||
||Moon HK, Stomp AM. Effects of media components and phytohormones on in vitro frond proliferation of Lemna gibba G3 and 24 additional Lemna gibba strains. Plant Research 1998;1(2):98-104.||