Pollen Tube Pathway
In all fields of the sciences, premature claims are made which are often inadequately substantiated. The plant transformation sciences is certainly not exempt from this type of activity as new or more efficient methods for transformation are valuable and any success can accelerate career development. This "rush to publish" mentality has yielded numerous reports of new and exciting transformation methods which have not stood the test of time. The mixing of pollen with DNA and injecting DNA into the meristem and ovules have yielded some very provocative results which have not been repeated.
One method of transformation which enjoyed some major attention during the early days of plant transformation, and has seen resurgence, is transformation via the pollen tube pathway (Luo and Wu 1988). Soon after this early report with rice, the method was informally confirmed by others working with different crops. These follow-up early reports were never published. Over the years, transformation via the pollen tube pathway has been both ridiculed and praised but it has neither seen wide adoption nor been swept under the scientific carpet. This method is currently being actively used by one laboratory in China, which is quite aggressive with publication efforts (Yang 2009a, b).
For transformation via the pollen tube pathway, pollen is placed on the stigma and allowed to germinate and grow down the style to the ovary. The growing pollen tube contains the pollen nuclei and once the pollen tube grows down to the egg, one pollen nucleus fuses with the egg to form the zygote. When the pollen tube reaches the egg, the style is severed using a scalpel, supposedly leaving an open pollen tube. The success of this procedure is grounded in the concept of using a hollow pollen tube as a transport vehicle for direct DNA introduction into the freshly-fertilized egg. It is not clear whether the pollen tube is actually hollow. It is also unclear whether any DNA is able to enter the ovule. The timing of the cutting of the pollen tube and subsequent DNA introduction must be very precise, to have the DNA enter the cell with the pollen nucleus. The reported efficiency of the process is inexplicably high, considering that <1% of cells that contain DNA introduced via particle bombardment are able to integrate that DNA into their genome. Extensive analysis of soybean plants obtained through the pollen tube pathway suggests that this method is not reproducible (Shou et al. 2002).
In spite of these problems, the pollen tube pathway continues to receive positive validation in peer-reviewed literature, often in respected journals. Two recent reports (Yang 2009a, b) deserve special attention here, as the results should raise major concerns of scientific rigor. One of these papers reports a comparison of transformation via the "ovary drip" method (variant of the pollen tube pathway) and the pollen tube pathway (Yang et al. 2009a) while the other paper reports the results of a pollen tube pathway study (Yang et al. 2009b). In spite of reportedly using different methods and different maize lines in the two papers, the authors show the exact same image of a GFP-expressing root in both papers, as one piece of evidence for transformation. Further scrutiny of these papers reveals additional problems but this one duplicated image is indicative of a basic problem with scientific accuracy. It appears that the pollen tube pathway method for DNA introduction has not yet been convincingly validated.
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