|Genome Project and Cloning Duckweed Genes
|Duckweed Chromosomes: Under the Microscope | Counting Chromosomes | DNA Content|
|Duckweed Genetic Engineering: GM Duckweed|
|Pharmaceutical Proteins from Duckweed|
(1) Mardanov, A.V. et al. (2008) Complete Sequence of the Duckweed ( Lemna minor ) Chloroplast Genome: Structural Organization and Phylogenetic Relationships to Other Angiosperms. J. Molec. Evol. 66(6):555-564. http://www.springerlink.com/content/8042060xp665646x/
(2) Soltis, D.E. et al. (2003) American Journal of Botany. 90:1596-1603. http://www.amjbot.org/cgi/content/full/90/11/1596#F1
(3) Duckweed genome sequencing has global implications. E! Science News. http://esciencenews.com/articles/2008/07/08/duckweed.genome.sequencing.has.global.implications
Cloning Duckweed Genes
Duckweed genes are similar to those of other plants. Many laboratories have cloned (isolated) genes from duckweeds for research in several areas, particularly photosynthesis and phytochrome regulation of plant development. Elaine Tobin's lab (UCLA) has studied the regulation of chloroplast development by phytochrome. Cheryl Smart (ETH, Zurich) has cloned a plant ABC transporter regulated by Abscisic Acid (ABA) from Spirodela . ABC transporters protect against toxic substances by pumping them out of the cell at the expense of ATP.
Bibliography of duckweed population genetics, molecular biology and tissue culture.
Duckweed Genetic Engineering
The desirable characteristics of duckweeds in agriculture and the environment spurred scientists to think of more and more novel uses for these plants. Many of these new applications may require new capabilities for duckweed metabolism, growth or nutritional composition. Genetic modifications will be required to give duckweeds these new characteristics, and the new technology of genetic engineering is a powerful technique that can help bring many of these dreams to reality. This technology is being used to modify the activity of existing duckweed genes, or to introduce new genes into duckweeds.
Some of the most exciting prospects in duckweed technology are aimed at using this plant as a factory for biopharmaceuticals. The advantages of this technology are many:
inexpensive culture techniques,
complete containment of the culture system,
easy scale-up from laboratory to factory, and
freedom from animal materials in the production process.
glycosylated proteins can be engineered.
The total absence of potentially hazardous animal proteins, cells and viruses means that many of the challenges of producing biopharmaceuticals are vastly simplified and the cost of production reduced accordingly. At first these advantages were merely theoretical, but now this technology is making rapid strides towards practical commercialization.
A North Carolina State University scientist, Anne-Marie Stomp, developed the first procedure to genetically engineer duckweed, a common aquatic weed, to produce therapeutic proteins. Stomp's spin-off company, Biolex, has achieved promising levels of recombinant protein expression in duckweed.
Stomp estimates the market for pharmaceutical proteins
like insulin is worth about $10 billion
Dr. Stomp made this advance with the aid of an EPA research grant, Genetic Improvement of Duckweed (Lemna gibba) for Wastewater Treatment. The final report of this grant is still available on line.
Yamamoto Y.T.*; Rajbhandari N.; Lin X.; Bergmann B.A.; Nishimura Y.; Stomp A.M. (2001) "Genetic transformation of duckweed Lemna gibba and Lemna minor." In Vitro Cellular and Development Biology - Plant, 37(3):349-353.
"We 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 -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
months after Agrobacterium-mediated transformation of
nodules. Furthermore, we developed a method for
transforming L. minor 8627 in 6 weeks. These
transformation protocols will facilitate genetic
engineering of duckweed, ideal plants for bioremediation
and large-scale industrial production of biomass and
Duckweed tissue culture research from this lab.
H. K. Moon and A. M. Stomp (1996) Effects of Medium Components and Light on Callus Induction, Growth, and Frond Regeneration in Lemna gibba (Duckweed). In Vitro Plant Cellular & Developmental Biology 33(1):20-25.Tamara Lynn Tatroe. (1998)
Investigation of growth parameters for a plant-based gene expression system using duckweed. M.S. Thesis, Biological and Agricultural Engineering, N.C. State University.
Identification of superior clones for bioremedation:
Bergmann, B.A., Cheng, J., Classen, J., Stomp, A.M. (2000) In vitro selection of duckweed geographical isolates for potential use in swine lagoon effluent renovation. Bioresource technology. 73 (1):13-20.
"The objective of this study was to select superior duckweed (Lemnaceae) genotypes for the utilization of nutrients in animal wastes. A two-step protocol was used to select promising duckweed geographic isolates to be grown on swine lagoon effluent. Forty-one geographic isolates from the worldwide germplasm collection were used in an in vitro screening test, because they were noted to be fast-growing genotypes during routine collection maintenance. In vitro screening was accomplished by growing geographic isolates on a synthetic medium that approximated swine lagoon effluent in terms of nutrient profile, total ionic strength, pH, and buffering capacity.
"Large differences among geographic isolates were observed for wet weight gain during the 11-day growing period, percent dry weight, and percent protein in dry biomass. Total protein production per culture jar differed 28-fold between the most disparate of the 41 geographic isolates and was the variable used for selection of superior geographic isolates. The challenge of eight of the 41 geographic isolates with full-strength swine lagoon effluent in the greenhouse led to the selection of three that are promising as genotypes to be grown on lagoon effluent."
Cox KM et al. Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol. 2006 Dec;24(12):1591-7.
“N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.”
Biolex developed processes for producing pharmaceutical proteins in duckweed. Although the technology was successful, its pharmaceutical business ventures were not, and the company declared bankruptcy in July 2012. Biolex's Lemna-based technology was sold to Synthon, a Netherlands-based company with a presence in Research Triangle Park, North Carolina.
Much of the information below comes from the former Biolex corporate website, which is no longer on-line:
"Biolex, Inc. is a biotechnology company based in Pittsboro, North Carolina, just outside of Research Triangle Park. Biolex is focused on developing the next generation of patented, recombinant protein production technologies in a plant based system. The foundation of the Biolex technology is its patented Lemna System™. This system couples the natural characteristics of the green plant, Lemna, with advanced genetic engineering and protein recovery methods to create a recombinant protein production platform."
"The Lemna System™ has unique, innate characteristics that provide enormous value for recombinant protein production" The stated advantages include: versatility, efficient genetic engineering, fast and flexible operation, low capital costs for facilities, low operating costs, high protein recovery and simple purification, product safety, and environmental safety."
Biolex was working with several pharmaceutical companies on production of several important therapeutic proteins, including plasmin, alpha-interferon-2b and human growth hormone. Biolex claimed to have successfully expressed twelve recombinant proteins in duckweeds, including small peptides, Fab fragments (Fabs), monoclonal antibodies (mAbs), and large multimeric enzymes. These include interferon alpha 2b (IFN), human growth hormone (hGH), a Fab, and a mAb.
A Biolex publication stated that IFN and hGH was successfully isolated from Lemna growth medium,
"At least 50% of IFN and hGH was secreted into asceptic growth media with pre-purification titers as high as 609 mg/L. Both proteins were found to be biologically active with IFN shown to be at least equal to the commercial source, Intron A. Efficient secretion of these recombinant proteins into an inorganic media with no need for viral inactivation offers substantial cost advantages in downstream purification."
It should be noted that Biolex defines "pre-purification titer" to include a 50x concentration step, so that their actual titer in Lemna medium would be 609/50 = 12 mg/L. Biolex indicates that it is working on optimization of protein secretion from duckweed cultures.
Another key advantage of recombinant Lemna cultures is that, unlike mammalian tissue culture systems, animal viruses cannot multiply. Therefore, this system should provide an increased level of pathogen safety in the final product. Currently many recombinant proteins are produced in mammalian cell cultures, where processes and tests to assure pathogen safety are major costs of production.
According to an article in Fierce
Biotech, "Biolex's plan was to wrap mid-stage studies and
sign on a partner for pivotal trials, But by early this year,
Biolex's once bustling operation was reduced to a much smaller
group of 13 staffers who were scrambling to find some way to raise
new money. Hepatitis C is one of the hottest fields in the
business, but the spotlight now is on a new generation of drugs
which will do away with the need for interferon. And some of the
biggest, most ambitious players in the business have jumped in,
eclipsing drugs like Locteron."
John R. Gasdaska, David Spencer and Lynn Dickey "Advantages of Therapeutic Protein Production in the Aquatic Plant Lemna" BioProcessing Journal, Mar/Apr 2003.
Katharina Schoebi, "One weed you do not want to get rid of" Checkbiotech April 15, 2005.
Duckweeds Engineered for Bioremediation.
USDA scientists in Peoria, Illinois are developing ways to convert renewable agricultural resources into value-added products. Their research may also show how genetically modified duckweeds can improve waste-water processing systems. One object of this research is to develop a duckweed which produces cellulase, the enzyme that converts cellulose into simple sugars. By converting cellulose into sugars, agricultural wastes containing cellulose could be used to make many valuable products, including lactic acid, ethanol, and glycerol.
Patents for GM Duckweed Applications.
Much of the technology for genetic modification of duckweeds can be found in patent literature. These patents can be read on-line and have considerable information about this technology.
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Arabidopsis is a small member of the Brassica family. It is the subject of a concentrated genome sequencing project, the Arabidopsis Genome Initiative (AGI). For more information, visit the Arabidopsis Information Resource (TAIR) and read "All about Arabidopsis" by Amy Fluet.
Cytogenetics (cyto = cell + genetics) is the study of chromosomes using the microscope.
Chromosomes are the small bodies in the cell that contain DNA. During cell division (mitosis), chromosomes condense from the nucleus and become discrete and compact. They can be identified and counted under the microscope only during mitosis and meiosis.
GM (genetically-modified). Human beings have been modifying plant genes in many ways for centuries. For example, corn and wheat do not even exist in the wild. These domesticated plants and others are the product of thousands of years of human intervention. However, today, GM refers specifically to the precise modifications that are possible by the use of DNA technology.
Genetic engineering is the use of DNA technology to genetically-modify organisms. This technology includes cloning (isolating) specific genes, copying, sequencing, editing, and splicing the isolated genes, and re-introducing the genes into other organisms to produce genetically-modified organisms. This technology is precise and controllable so that the results of each step are known. This is in contrast to conventional breeding, which results in a random assortment of the genes from the parents.
Background Information on Plant Genetic Engineering
Techniques of Plant Molecular Biology
Research Tools for Plant Nucleic Acids: Isolating and manipulating the plant genome by Christopher M. Smith, San Diego Supercomputer Center (SDSC), published in The Scientist 14:26, Nov. 13, 2000.
Extraction, Purification, and Cloning of Plant Genomic DNA/RNA,
Reporter Genes and Proteins, Gene Transfer,
A Shot in the Dark: Invention of the Gene Gun. How a squirrel problem lead to a breakthrough in biotechnology.
Public Issues: GMO - Genetically Modified Organisms and Plant Agriculture
Information Systems for Biotechnology (ISB) A national resource in agbiotech information from Virginia Tech.
"...provides information resources to support the environmentally responsible use of agricultural biotechnology products. Here you will find documents and searchable databases pertaining to the development, testing and regulatory review of genetically modified plants, animals and microorganisms within the U.S. and abroad. "
Transgenic Crops, An Introduction and Resource Guide, from the Department of Crop and Soil Sciences, Colorado State University.
History of Plant Breeding,
What are Transgenic Plants?
How do you make Transgenic Plants?
How to make Transgenic Plants: Animation Demo
Evaluation & Regulation,
Current Transgenic Products,
Future Transgenic Products,
Risks & Concerns,
Agricultural Biotechnology Links from the American Society of Plant Biologists (ASPB), including the ASPB "Statement on Genetic Modification of Plants Using Biotechnology".
Public Outreach information from the University of California slides and text from talks to farmers, scientists and the general public from UC Extension Specialists.
Crop Specific Issues
What's in the Pipeline: Looking in the Crystal Ball at Agricultural Biotechnology
AgBioWorld Foundation is devoted to bringing information about technological advances in agriculture to the developing world.
"AgBioWorld seeks to provide information
to scientists, policymakers, journalists and the general public on
the relevance of agricultural biotechnology to sustainable
AgBioWorld's President, Dr. C.S. Prakash, is also the Director of the Center for Plant Biotechnology Research at Tuskegee University and a member of the USDA Advisory Committee on Agricultural Biotechnology.
AgBioView - a discussion group on agricultural biotechnology, features discussion of hot issues in GMO and agriculture.
Benefits of Biotechnology for Developing Countries
About Food Security
2. Questions About Environmental Protection
3. Questions About Human Health
4. Questions About Socio-economic Issues
Urban Myths about Organic Agriculture. by Prof. Tony Trewavas, University of Edinburgh, Scotland.
"There is a widespread belief that farming systems with lower yields and lower use of inputs are more friendly to the environment, and more sustainable than higher producing systems. Organic food is often viewed as healthier and benign. However the information below rarely finds its way into discussion on organic food but is essential for a critical assessment."
Agricultural Biotechnology and Genetically Engineered Foods - documents from various perspectives from the Food Safety Network.
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Revised: October 6, 2012