Agrobacterium. Although transgenic plants were not recovered, the authors reported that the
seedlings tested positive for the presence of opine synthase enzymes. These specific
enzymes can be produced only after successful T-DNA transfer, and opine synthase analy-
sis was one of the only tests for successful transformation at the time. This work was done
before the optimization treatments, which were described earlier in this chapter, were even
known. Moreover, the transformation efficiency was 60–80%, which is high by today’s
standards. If photocopies of this paper are inspected, inconsistencies cannot be detected.
However, if the original paper is carefully examined, one can see that the differences
between the control and experimental treatments disappear when the images showing
opine production are digitally lightened or darkened to provide similar background
levels. This paper is continuously referenced in the transformation literature, but it probably
should not be.
10.6.1.3. Pollen Tube Pathway. The pollen tube pathway method (Luo and Wu 1988)
for transformation is different from pollen transformation method (discussed above) as the
pollen is not transformed, but the pollen tube is used as a vehicle for the delivery of DNA.
The basis of this method is the inoculation of DNA into the hollow pollen tube, where it
finds its way to the freshly fertilized egg for incorporation into the DNA of the young
zygote. Timing was reported to be critical as the pollen is first placed on the stigma for ger-
mination. After the pollen tube grows down the style to the ovary, the stigma is severed,
leaving a narrow hollow pollen tube as an open pathway to the fertilized egg. DNA is
then inoculated onto the open pollen tube, where it was believed that capillary action
drew the DNA in solution to the zygote. On the surface, this method appears to have
some merit, but the pollen nuclei are the only things to enter the egg, and the fertilized
egg or zygote has the same barriers as any other young plant cell, notably the cell wall.
From the 1980s through the 1990s, there were many additional reports in the literature of
the successful use of the pollen tube pathway for many different crops; almost all of
these reports originate from China. Although it is very difficult to publish negative
results, Shou et al. (2002) performed a very extensive study of the pollen tube pathway
method in soybean and concluded that it was not reproducible. It appears that the pollen
tube pathway method for DNA introduction is invalid. In the first published report (Luo
and Wu 1988), transformation was confirmed using reliable molecular techniques but the
patterns of DNA hybridization (see Section 10.6.2, later in this chapter) were a little
unusual and may have been misinterpreted.
10.6.1.4. Rye Floral Tiller Injection. In this early report of plant transformation,
young floral tillers of rye were injected with DNA carrying a kanamycin resistance gene
(de la Pen ̃a et al. 1987). The authors speculated that the DNA was transported through
the plant’s vascular system to the germ cells, where it was taken up and incorporated.
They suggested that the cells that ended up forming pollen were probably transformed
with this injected DNA. The end result from floral tiller injection was the production of
seeds carrying a kanamycin resistance gene. Molecular analysis seemed to show the pre-
sence of an intact gene in the rye DNA, but the most important results were only briefly
described in the paper and presented as “data not shown.” The “data not shown” term is
used in situations where it may not be necessary to present data or images, but these
data should have been presented for this work. In this paper, the authors also claim that
the experiment was repeated (again, repeatability is expected for scientific reports) with
similar results of recovery of transgenic rye plants. The authors write, “We are confident
266 TRANSGENIC PLANT PRODUCTION