Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-06-10
2002-04-09
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C047S05810R
Reexamination Certificate
active
06369299
ABSTRACT:
BACKGROUND
More than 8 million organic compounds are known and many are thought to be biodegradable by microorganisms, the principle agents for recycling organic matter on Earth. In this context, microbial enzymes represent the greatest diversity of novel catalysts. This is why microbial enzymes are predominant in industrial enzyme technology and in bioremediation, whether used as purified enzymes or in whole cell systems.
Effects of s-triazine Herbicide Use
Modern agricultural practices rely heavily on the use of herbicides to control weed populations. S-triazine (i.e., symetric triazine) herbicides, primarily atrazine and simazine, are widely used herbicides for selective control of broadleaf weeds and some grasses in a variety of crops. Since atrazine and other s-triazine herbicides biodegrade relatively slowly in soils, label directions for the use of atrazine restrict the types of crops that can be planted to prevent carryover problems in the next growing season. For example, alfalfa and soybeans are susceptible to atrazine concentrations in soil ranging from 0.09 mg/Kg to 0.53 mg/Kg, depending on the concentration of soil organic matter.
Numerous studies on the environmental fate of atrazine have shown that atrazine is a moderately persistant compound that is transformed to CO
2
very slowly, if at all, under aerobic or anaerobic conditions. It has a water solubility of 33 mg/l at 27° C. Its half-life (i.e., time required for half of the original concentration to dissipate) can vary from about 4 weeks to about 57 weeks when present at a low concentration (i.e., less than about 2 parts per million (ppm)) in soil. High concentrations of atrazine, such as those occurring in spill sites have been reported to dissipate even more slowly.
As a result of its widespread use, atrazine is sometimes detected in water in concentrations exceeding the maximum contaminant level (MCL) of 3 &mgr;g/l (i.e., 3 parts per billion (ppb)), a regulatory level that took effect in 1992. Point source spills of atrazine have resulted in levels as high as 25 ppb in some wells. Levels of up to 40,000 mg/l (i.e., 40,000 ppm) atrazine have been found in the soil at spill sites more than ten years after the spill incident. Point source spills and subsequent runoff can result in the presence of atrazine in surface, subsurface, and ground water.
While earlier studies have reported atrazine degradation only by mixed microbial consortia, more recent reports have indicated that several isolated bacterial strains can degrade atrazine. In fact, research groups have identified atrazine-degrading bacteria classified in different genera, including Rhodococcus sp. and Pseudomonas sp., from several different locations in the U.S. (e.g., Minnesota, Iowa, Louisiana, and Ohio) and Switzerland (Basel).
An atrazine-degrading bacterial culture, identified as Pseudomonas sp. strain ADP, ATCC No. 55464, was isolated and was found to degrade atrazine at concentrations greater than about 1,000 &mgr;g/ml under growth and non-growth conditions. See Mandelbaum, et al. (U.S. Pat. No. 5,508,193). Pseudomonas sp. strain ADP (Atrazine Degrading Pseudomonas) uses atrazine as a sole source of nitrogen for growth. The organism completely mineralizes the s-triazine ring of atrazine under aerobic growth conditions. That is, this bacteria is capable of degrading the s-triazine ring and mineralizing organic intermediates to inorganic compounds and/or ions (e.g., CO
2
and NH
4
).
Herbicide Resistant Plants
More than 35 species of plants have been reported to be naturally resistant to s-triazine herbicides. Typically, these plants degrade atrazine via glutathione s-transferase reactions (i.e., the atrazine is conjugated to glutathione and subsequently degraded). Alternatively, these plants alter the protein atrazine binds, quinone-binding (Q
B
) protein, which is a component of photosystem II in the chloroplast. Atrazine resistant weeds have been reported to have an altered Q
B
protein which has a 1000-fold reduced affinity for atrazine; however, these plants typically do not compete well in natural systems due to decreased photosynthesis efficiency. Furthermore, these plants do not degrade s-triazines. In the case of atrazine resistant plants, cross resistance to other s-triazine herbicides appears to be relatively common (Ottmeier, W. et al.,
Pesticide Biochem. Physiol.,
18, 357-367. (1982)). Thus, there is a need for methods to remove s-triazines from the environment.
SUMMARY OF THE INVENTION
In view of the occasional prevalence of s-triazines in the environment at levels above regulatory standards, there is a need in the art for methods to remediate, i.e., remove, s-triazines present in the environment, including soil and water. Thus, preferred aspects of the present invention provide transgenic plants that are resistant to s-triazine compounds, and methods of making and using such plants. Preferably these plants will degrade s-triazines, more preferably detoxify s-triazines, to more quickly reduce the occurrence of s-triazines in soil and water.
In a preferred embodiment, the present invention provides transgenic alfalfa plants that express a bacterial atrazine chlorohydrolase enzyme, AtzA. AtzA converts atrazine to hydroxyatrazine, which appears to have no herbicidal activity and which is relatively immobile in soil. Alfalfa has rooting characteristics which allow it to explore shallow and deep soils, thus it provides for the remediation of contaminated soils and possibility of remediating contaminated surface and subsurface water.
The present invention provides a transgenic plant including an exogenous coding region encoding an enzyme that imparts resistance to, and optionally degrades, at least one s-triazine. The s-triazine can be atrazine. The enzyme can dehalogenate at least one s-triazine, and if the s-triazine is atrazine, the atrazine can be converted to hydroxyatrazine.
The nucleotide sequence of the exogenous coding region can be nucleotides 58-1480 of SEQ ID NO:1. Alternatively, the complement of the nucleotide sequence of the exogenous coding region hybridizes to the nucleotide sequence set forth at nucleotides 58-1480 of SEQ ID NO:1 in a solution containing 250 mM Na
2
HPO
4
, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65° C. for at least 4 hours, followed by three washes for twenty minutes each at 65° C. in a solution containing 2×SSC and 0.1% SDS.
The invention includes seeds of the transgenic plant, the progeny of a first or subsequent generation of the transgenic plant and the seeds thereof, and the seeds of the progeny of the first or subsequent generation of the transgenic plant. The plant can be a dicot, including an alfalfa plant, or a monocot, including a grass. A hybrid plant resistant to at least one s-triazine and an inbred plant resistant to at least one s-triazine, prepared from the transgenic plant, is also included in the present invention. In the transgenic plants, the exogenous coding region can impart resistance to levels of at least one triazine that inhibit the growth of a nontransgenic plant.
The invention is also directed at a method for degrading at least one s-triazine, including planting a plant in a composition containing an s-triazine wherein the plant degrades, and optionally detoxifies, at least one s-triazine in the composition, and growing the plant in the composition so that the plant degrades, and optionally detoxifies, at least one s-triazine. The plant can include an exogenous coding region that produces an enzyme capable of degrading, and optionally detoxifying, at least one s-triazine. The composition can include soil and/or water, and the plant can decrease the concentration of at least one s-triazine in the soil and/or the water. The at least one s-triazine can be selected from the group of atrazine, desethylatrazine, deisopropylatrazine, desethylhydroxyatrazine, desisopropylhydroxyatrazine, desethyldesisopropylatrazine, simazine, terbuthylazine, melamine, ammelide, ammeline, prometryn, ametryn, and propazine. The plant can be a dicot
Sadowsky Michael J.
Samac Deborah A.
Vance Carroll P.
Wackett Lawrence P.
Kruse David H
Mueting Raasch & Gebhardt, P.A.
Nelson Amy J.
Regents of the University of Minnesota
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