ABSTRACT
More than 20 years ago, the first genetically modified (GM) plants entered the seed market. The patents covering the first GM plants have begun to expire and these can now be considered as Off-Patent Events. Here we describe the challenges that will be faced by a Secondary Party by further use and development of these Off-Patent Events. Indeed, the conditions for Off-Patent Events are not available yet to form the basis for a new viable industry similar to the generic manufacturers of agrochemicals or pharmaceutical products, primarily because of (i) unharmonized global regulatory requirements for GM organisms, (ii) inaccessibility of regulatory submissions and data, and (iii) potential difficulties to obtain seeds and genetic material of the unique genotypes used to generate regulatory data. We propose certain adaptations by comparing what has been done in the agrochemical and pharmaceutical markets to facilitate the development of generics. Finally, we present opportunities that still exist for further development of Off-Patent Events in collaboration with Proprietary Regulatory Property Holders in emerging markets, provided (i) various countries approve these events without additional regulatory burdens (i.e., acceptance of the concept of data transportability), and (ii) local breeders agree to meet product stewardship requirements.
ABSTRACT
Aprotinin is a small serine protease inhibitor used in human health. Spirodela were transformed, via Agrobacterium, with a synthetic gene encoding the mature aprotinin sequence and a signal peptide for secretion which was driven by the CaMV 35S promoter. A total of 25 transgenic Spirodela lines were generated and aprotinin production was confirmed by northern and western blot analyses. Expression levels of up to 3.7% of water soluble proteins were detected in the plant and 0.65 mg/l in the growth medium. In addition, immunoaffinity purification of the protein followed by amino acid sequencing confirmed the correct splicing of the aprotinin produced in Spirodela and secreted into the growth medium.
Subject(s)
Aprotinin/metabolism , Araceae/metabolism , Plants, Genetically Modified/metabolism , Trypsin Inhibitors/pharmacology , Aprotinin/genetics , Araceae/genetics , Araceae/growth & development , Blotting, Northern , Blotting, Southern , Blotting, Western , Gene Expression Regulation, Plant , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plasmids , Promoter Regions, Genetic , Protein Sorting Signals/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transformation, Genetic , Transgenes/physiologyABSTRACT
The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2 days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18 months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA-DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant.
Subject(s)
Araceae/metabolism , Plant Proteins/metabolism , Transgenes , Blotting, Southern , Genome, Plant , Kanamycin Resistance , Microscopy, Fluorescence , Mutation/genetics , Plasmids , Subcellular Fractions/metabolism , Transformation, GeneticABSTRACT
Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.
