Page 13 - Molecular Plant Breeding

Molecular Plant Breeding 2011, Vol.2, No.8, 48 -59
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56
expression and methylation patterns established in the
hybrid plants. Thus we speculated the inactivation of
cecropin B gene occurred in F1 hybrids and the
silencing status was passed on through sexual
reproduction.
In conclusion, transgenes in rice genome introduced
by particle bombardment could be easily transferred
by conventional crossbreeding. The stability of
transgene integration patterns and expression status in
crossbreeding transmission was mainly determined by
the primary transgenic donor plant. In the course of
cross transmission, the variance in integration patterns
of selected marker bar gene did not influence its stable
expression under selection, whereas the expression
status of non-selected cecropin B gene is affected by
cross recombination. And more importantly, transgenic
plant can be produced by particle bombardment in
which transgenes sustains inheritance and expression
activity over long-term generations in both sexual
reproduction and conventional crossbreeding.
3 Materials and methods
3.1 Generation of transgenic plants
Four transgenic rice lines TR 5, TR 6, Ming B and
Jingyin 119 transformed via particle bombardment
were used as transgene donors to create transgenic
hybrids. All the above transgenic donors were
generated as described in previous reports, harboring
plasmid pCB1, which contains the selected
marker-gene bar and non-selected cecropin B gene.
The bar gene is controlled by cauliflower mosaic virus
35S (CaMV 35S) promoter, cecropin B gene is controlled
by the rice actin-1 promoter (Huang et al., 1996).
3.2 Crosses
The transgenic plants of each rice variety were
identified by both Basta-resistant phenotype analyses
and Southern-blotting hybridization. Bagged seeds of
transgenic T0 plants of Jingyin 119, TR 5, TR 6 and
Ming B variety were planted in the experimental farm
of China National Rice Research Institute between
1996 and 1998. Seeds of each plant were harvested
individually and the elite homozygous transgenic
lines were selected as transgene donors for cross
experiments.
All cross experiments were conducted in the field in
the rice growing seasons between 1996 and 2009 both
in Zhejiang Province and Hainan Province. A cross
between transgene donor TR 5 (T5) and TR 6 (T6)
was made to investigate the relationship between
integration and expression of transgenes in different
transgene donors. The homozygous transgenic line
Jingyin 119 (T3 generation) was used as transgene
donor to cross with different Japonica rice varieties.
Some of the resulting F1 hybrids were used as new
transgene donors for next crosses to produce hybrids
of multiple crosses. All the selected transgene donors
and hybrids were subjected to Basta resistant assay in
every generation. We sprayed 0.32% Basta to whole
plant at their seedling stage. After one week, the
susceptible plants were dead. The selected transgenic
plants were grown in the field for seeding or cross
experiment. The transgenic hybrids were self-crossed
to produce homozygotes for the bar gene, and
progenies were selected based on Basta resistance and
Southern hybridization. All homozygous transgenic
hybrids and their transgenic donors were planted in
the experimental farm of China National Rice
Research Institute. The information of rice materials
for experiments was detailed in Table 1.
3.3 Southern blot hybridization
Genomic DNA was isolated from rice leaves using the
SDS DNA extraction method as described by Lu and
Zheng (1992) Aliquots (5 μg) were digested overnight
with appropriate restriction enzymes, fractionated by
0.8% agarose gel electrophoresis and blotted onto
Amersham N + Hybond membranes according to the
Southern blotting method (Sambrook et al., 1989).
The linear fragment (0.9 kb) comprising the open
reading frame of bar gene and nos terminator from
pCB 1 digested with Eco RⅤ was used as probe for bar
gene. The linear fragment (1.12 kb) from pCB1 after
digestion with HindⅢ and Pst Ⅰ, which comprises
cecropin B coding region and Pin terminator was used
as probe for cecropin B gene. The probe structure and
sites on pCB1 are illustrated in Figure 3.
The probe labeled by random priming was conducted
using DIG DNA Labeling Kit (Roche Company)
according to the manufacture’s instructions. Southern