Marker free transgenic plants engineering the chloroplast genome without the use of antibiotic selection

Abstract

The present invention provides for a method to circumvent the problem of using antibiotic resistant selectable markers. In particular, target plants are transformed using a plastid vector which contains heterologous DNA sequences coding for a phytotoxin detoxifying enzyme or protein. The selection process involves converting an antibiotic-free phytotoxic agent by the expressed phytotoxin detoxifying enzyme or protein to yield a nontoxic compound. The invention provides for various methods to use antibiotic-free selection in chloroplast transformation.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001] This patent application claims the benefit of U.S. Provisional Application Nos. 60 / 208,763, filed Jun. 06, 2000, No. 60 / 257,406, filed Dec. 22, 2000 and No. 60 / 259,154, filed Dec. 28, 2000, Ser. No. 60 / 186,308, filed Mar. 02, 2000. All applications are hereby incorporated by reference.[0003] This application pertains to the field of genetic engineering of plant plastid genomes, particularly chloroplasts, and to methods of and engineered plants without the use of antibiotics.[0004] This application relates in particular to a method of selecting genetically engineered or transformed plants without the use of antibiotics as a selectable marker. The application also relates to a method of transforming plants to confer drought tolerance and to the transformed plants which are drought tolerant.DESCRIPTION OF THE RELATED ART[0005] Publications[0006] Various methods of selection of plants that employ antibiotic-free selectable marker, or non-an...

AI Technical Summary

Benefits of technology

0013] Disadvantages of the Antibiotic Selectable Marker System.

0014] Most transformation techniques co-introduce a gene that confers antibiotic resistance, along with the gene of interest to impart a desired trait. Regenerating transformed cells in antibiotic containing growth media permits selection of only those cells that have incorporated the foreign genes as the gene of interest. Once transgenic plants are regenerated, antibiotic resistance genes serve no useful purpose but they continue to produce their gene products. One of the primary concerns of genetically modified (GM) crops is the presence of clinically important antibiotic resistance gene products in transgecic plants that could inactivate oral doses of the antibiotic (reviewed by Puchta 2000; Daniell 1999A). Another concern is that the -antibiotic resistant genes could be transferred to pathogenic microbes in the gastrointestinal tract or soil rendering them resistant to treatment with such antibiotics. Antibiotic resistant bacteria are one of the major challenges of modem medicine. In Germany, GM crops containing antibiotic resistant genes have been banned from release (Peerenboom 2000).

0015] Plastid Genetic Engineering as an Alternative to Nuclear Genetic Engineering.

0016] Plastid genetic engineering, particularly chloroplast genetic engineering, is emerging as an alternative new technology to overcome some of the environmental concerns of nuclear genetic engineering (reviewed by Bogorad, 2000). One common environmental concern is the escape of foreign gene through pollen or seed dispersal from transgenic crop plants to their weedy relatives creating super weeds or causing genetic pollution among other crops (Daniell 1999B). Keeler et al. (1996) have summarized valuable data on the weedy wild relatives of sixty important crop plants and potential hybridization between crops and wild relatives. Among sixty crops, only eleven do not have congeners and the rest of the crops have wild relatives somewhere in the world. In addition, genetic pollution among crops has resulted in several lawsuits and shrunk the European market of Canadian organic farmers (Hoyle...

Problems solved by technology

Disadvantages of the Antibiotic Selectable Marker System.

Once transgenic plants are regenerated, antibiotic resistance genes serve no useful purpose but they continue to produce their gene products.

Antibiotic resistant bacteria are one of the major challenges of modem medicine.

In addition, genetic pollution among crops has resulted in several lawsuits and shrunk the European market of Canadian organic farmers (Hoyle 1999).

Yet another concern in the use of nuclear transgenic crops expressing the Bacillus thuringiensis (Bt) toxins is the sub-optimal production of toxins resulting in increased risk of pests developing Bt resistance.

Despite these advantages, one major disadvantage with chloroplast: genetic engineering in higher plants may be the utilization of the antibiotic resistance genes as the selectable marker to confer streptomycin / spectinomycin resistance.

Despite several advantages of plastid transformation, one major disadvantage with chloroplast genetic engineering in higher plants is the utilization of the antibiotic resistance genes as the selectable marker.

Unfortunately, bacterial infections in humans and animals are also controlled by using these antibiotics.

While it was known earlier that BADH was a plant enzyme, it could not be conclusively demonstrated that this was a chloroplast enzyme because it lacked the typical transit peptide found in all chloroplast proteins imported from the cytosol.

Accumulation of betaine aldehyde is toxic and lethal to plant cells.

While ancient plants (including pines) have the ability to synthesize chloroplhyll in the dark, flowering plants lost this capacity.

Additionally, the latter procedure results in rapid regeneration.

It is known that accumulation of betaine aldehyde is toxic and lethal to plant cells (Rathinasabapathi et. al.

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