Page 6 - Molecular Plant Breeding

Molecular Plant Breeding 2011, Vol.2, No.8, 48 -59
http://mpb.sophiapublisher.com
49
transformation. Although the generation of transgenic
plants is relatively easy for many rice varieties, the
transformation frequency is usually low and rather
genotype-dependent. Also, these gene delivery
techniques need undergo the obligatory processes of
tissue culture, which often results in phenotypic
abnormalities and reduced fertility of the transgenic
plants obtained (Zhang et al., 2005). Like mutants
providing useful traits, transgenic plants often have to
be used for relocating the gene in more suitable
genotypes (Horvath et al., 2001). Thus, the success of
plant genetic manipulation not only requires the stable
inheritance and expression of transgenes in the
transgenic plants across generations, but also depends
on whether the transgenic plant can be used as a
transgene donor in recombination crossbreeding.
Many studies have analysesed the progenies of the
primary rice transformants, revealing that transgene
stability was significantly related to differences in
transgene structure and expression levels between
transgenic lines, particularly in transgenic plants
derived from direct DNA transfer such as particle
bombardment (Vain et al., 2002; Altpeter et al., 2005).
In transgenic cereals, more than 50% of transgenes
can be inactivated over successive generations (Iyer et
al., 2000). These problems make molecular genetic
studies difficult, and frustrate attempts at crop
improvement through genetic engineering. Additionally,
they create difficulties in predicting transgene
behavior when transgene needs to be transferred by
conventional crossing (Vain et al., 2002). Altpeter et
al. (2005) speculated that particle bombardment might
be advantageous over Agrobacterium-mediated trans-
formation in respect of transferring the transgenes into
a new genetic background via traditional breeding,
because by particle bombardment multiple transgenes
are tend to be integrated into the same locus. But there
are few direct evidences for this question up to date.
We introduced the plasmid pCB1 carrying the selected
herbicide-resistant bar gene and the non-selected
cecropin B gene into four Japonica rice varieties via
particle bombardment between 1996 and 1998. Bar
gene was introduced into rice plant for resistance to
phosphinothricin (the active component of the
herbicide Basta) and the cecropin B gene was used to
resist a range of plant pathogenic bacteria including
Xanthmomonas compestris pv oryzae, which leads to
rice leaf bacterial blight disease. With obvious
phenotype and convenient detection, bar gene has
been proved to be a very useful marker to screen
transgenic hybrids. In the past ten years, the elite
transgenic rice plants harboring bar and cecropin B
gene were selected as transgene donors to cross to
different rice varieties. We constructed a population of
rice hybrids derived from multiple conventional
crosses. Here we report the inheritance and expression
behaviours of the foreign bar and cecropin B genes
during rice crossbreeding transfer.
1 Results and Analysis
1.1 Stability of transgene integration patterns in
mono-cross transmission
The stability of integration patterns for the selected
bar gene and non-selected cecropin B gene in the
transmission from transgenic donors to hybrid rice
plants was investigated using genomic DNA Southern
blotting analysis. Three transgene donors including
TR 5, TR 6, Ming B were used to produce hybrids, in
which several bar gene copies were inherited as a
single transgenic locus when tested by Basta
resistance (Hua et al., 2003). The transgene integration
patterns of transgene donor TR 5, TR 6, Ming B and
their corresponding hybrids were analysed and shown
in Figure 1A and 1B. Southern blotting results
revealed as follows: (i) The tightly linked bar and
cecropin B gene in the original plasmid exhibited
different integration patterns in the three transgene
donors, when hybridized with bar and cecropin B
probe respectively, after genomic DNA was digested
with Hind III, which cut once in plasmid pCB1. There
were two hybridization bands of bar gene and three
bands of cecropin B gene in transgene donor TR 5.
Transgene donor Ming B had four bar gene
hybridization bands and three cecropin B gene
hybridization bands. TR 6 donor plant possessed six
hybridization bands of bar gene and five bands of
cecropin B gene. These indicated that bar and
cecropin B might have different copies integrated in
the receptor genomes. (ii) In the self-pollinated
progenies of transgenic rice hybrids, the integration
patterns of non-selected cecropin B gene remained the