Brassica campestris L. ssp. chinensis M

450
the BcMF5 transgenic plants of flowering Chinese
cabbage.
Integration and transcription confirmation of
the hpRNA of the BcMF5 gene in transgenic
plants of pBIA9-RMF5
To confirm the hpRNA of the BcMF5 gene in the
regenerated plants of flowering Chinese cabbage,
19 T0 plants of pBIA9-RMF5 were subjected to PCR
analysis with the primers specific for the uidA gene.
Both PCR (Figure 5A) and the two transgenic lines
TB6 and TB13 for DNA gel blot analysis (Figure 5B)
confirmed that the hpRNA of the BcMF5 gene had
integrated into the genome of the flowering
Chinese cabbage transgenic seedling of pBIA9-
RMF5. Data from the DNA hybridization showed
that it was a single copy in the assayed transgenic
lines TB6 and TB13 (Figure 5B). In addition, the RNA
hybridization results for both TB6 and TB13
indicated that the expression of the BcMF5 gene
decreased seriously in the transgenic line of pBIA9-
RMF5 (Figure 5C), thus confirming that the normal
expression of BcMF5 in the flowering Chinese
cabbage was inhibited by hpRNA-mediated RNA
interference in the transgenic plant.
ARTICLE IN PRESS
Q. Zhang et al.
Figure 4. The BcMF5 RNAi transgenic plantlets of flowering Chinese cabbage were obtained by the Agrobacterium
tumefaciens-mediated genetic transformation method. (A) Seedling for 4–5 d; (B) cotyledon-hypocotyl explants during
co-culture; (C) after Kan R buds were induced. (D) Roots induced from the Kan R seedling; (E) transgenic plantlets of
flower Chinese cabbage flowering in the test tube; and (F) regenerated plants transferred to the plot.
Pollen germination test and scanning
electron microspore analysis of transgenic
plants
To further understand the effect of RNA interference
on pollen development of the transgenic
plant, the germination ability of the pollen was
tested (Figure 6). The results showed that the
pollen germination percentage of the transgenic
plant of pBIA9-RMF5 was 38.74%. In contrast, pollen
from the non-transgenic plant could germinate with
a frequency of approximately 80.24% (Table 1).
Further, pollen of the transgenic plant of pBIA9-
RMF5 and the non-transgenic plant was detected by
use of a scanning electron microscope, and the
results indicated that there was a significant
difference between transgenic plants and their
non-transgenic counterparts (Figure 7). Compared
with the pollen of the non-transgenic plant, most of
the pollen of the transgenic plant of pBIA9-RMF5
was abnormal and the pollen germination furrow
collapsed. The results showed that the percentage
of abnormal pollen of pBIA9-RMF5 in the 402 pollen
grains assayed was 88.37%, while that of the nontransgenic
plant in the 282 pollen grains assayed
was 7.9% (Table 2). This illustrated that RNA