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In the June 2000 issue of Trends in Biotechnology (18:257-263), Professor Lawrence Bogorad email@example.com of Harvard University discusses the latest techniques of transforming plants via subcellular organelles such as chloroplasts. He initiated the discussion by saying that although we have a number of highly successful transgenic varieties of crops, the technique of transforming plants either by T-DNA or microprojectile has its limitations apart from the fact that it takes a very long time to market the product commercially. The limitations are 1) the expression of a transgene varies from plant to plant requiring it to be tested for further research or commercialization and 2) insertion of transgenes in host chromosomes occurs at a random location in the genome. This phenomenon creates “position effects” meaning that the specific position of a transgene in a particular chromosome determines the level of its phenotypic expression. The second limitation has been experienced by breeders when they have dealt with agronomically important quantitative traits because crops can rarely be transformed with more than one gene at a time.
The limitations of nuclear gene transformation methods necessitated devising a technique for introduction of foreign genes at specific locations on a particular chromosome; also needed was a technique to introduce block of genes conditioning a character such as nitrogen- or carbon- dioxide fixation
Compared to the nucelar-cytoplasmic compartment, plastids and mitochondria provide more favorable environments for certain biochemical reactions and for accumulating large amounts of some gene and enzyme products. In addition, these two organelles have the great advantage of having a smaller genomic size. Chloroplasts with 60 copies of a single circular chromosome have only 120 to150 genes.
In 1998, Boynton and his associates demonstrated that the defective trait of mutant atpB to carry out photosynthesis was corrected when the wild type allele ATPB replaced the mutated gene. The results showed that unlike nuclear transformation, DNA can be directed to a specific site. Not only that, McBride and his associates were able to show that the Bt gene could be directed for integration into the DNA of tobacco plants. Knoblauch and his associates confirmed the expression of DNA after it was injected directly into individual chloroplasts in the photosynthetic tobacco leaf cells.
Since many chloroplast genes occur in operons, it may be possible, to introduce blocks of foreign genes into a single operon. Furthermore, since the same kind of promoter is involved in transcription, location of a foreign gene in any position of the circular chloroplast chromosome will not make much difference in its expression. Transformation via chloroplasts has another great advantage. Since the chloroplasts are transmitted via ovules, there will be no pollen from transgenics to pass on the foreign gene(s) to nearby plants.
The author is of the opinion that with the techniques now available, it may be possible to bioengineer chloroplasts for nitrogen fixation. The present difficulty that prevents this is the sensitivity of nitrogenase to oxygen which is released as a result of photosynthesis. This problem may be solved by devising a method which Cyanobacteria have adopted by keeping the two processes, photosynthesis and nitrogen fixation separate temporally or by means of heterocysts. The latter is a kind of asexual spore with thickened walls which prevent the flow of oxygen to the cells where nitrogen fixation occurs. Watching the trend of current research in gene transfer via organelles, the author envisions that it may be possible to convert C3 plants to C4 plants by manipulating bundle sheath chloroplasts and their association with Rubisco