--Yong-Bao Pan, Hua Zhou, Peter A. Peterson and Alan G. Atherly
In our initial attempt with maize pollen transformation, there were five treatments (see footnote in Table) that were conducted under two different conditions. Using the original method at 29 C, maize pollen paste of one genotype from the 5 treatments was directly applied to freshly nicked silks of another genotype in a "pollen-free" greenhouse room for 3 consecutive days. Under a revised procedure in a 20 C "pollen-free" room, pollen samples of the same genotype were put through the 5 treatments and were applied to the freshly nicked silks pretapped at their base (i.e., ear tip) in order to hold the applied pollen paste. On day 2 after the treatments, the same procedure was repeated an additional time. On day 4, these plants were moved into the 29 C greenhouse room and grown to maturity.
Table. Effect of temperature (T C), pollination method and treatment on seed setting in pollen transformation.
Upon harvest, we observed a very strong effect of temperature, pollination method, and treatment on the average seed set per ear (Table). First, a lower temperature at around 20 C in combination with two-day cycles of nick-tape-pollen application method indeed improved seed setting (Table and Figure). With this procedure, we were able to increase seed setting in treatment 2 from 1.2 seeds per ear to 26.5 seeds per ear. Second, even though we failed to obtain any seed from electroporated pollen samples in the original experiment, we were able to get 3.4 to 4.1 seeds per ear from two electroporated pollen samples under the revised procedure. Currently we are analyzing these maize kernels in order to determine if the DNA markers are present that will be a confirmation of successful transformation.
Figure. Sample ears from pollen transformation experiment.
Top row: ears from treatment 2 by the original method; Bottom row: ears
from treatment 2 by the revised method.
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