Page 6 - Maize Genomics and Genetics

Maize Genomics and Genetics 2012, Vol.3, No.1, 1- 5
ISSN 1925-1971 http://mgg.sophiapublisher.com
3
Figure 4 Regeneration of transgenic maize plants
Note: A: Callus was obtained after subculture for 20 days; B:
Co-cultured for 3 days after infection; C: Differentiation culture
for 15 days; D: Seedling after differentiation; E: Transplanting
seedlings; F: Transgenic plants in greenhouse
amplify the same size electrophoretic bands with
the target fragment, while the blank control and
untransformed plants had no corresponding band. All
the detected positive plants were handled with specific
primers, which were designed with part of the promoter
and gene fragment, for PCR detection, which also
amplified the same bands as target fragment (Figure 6).
These preliminary demonstrated that the target gene
had been integrated into the regeneration maize plant,
and the positive rate of which was about 15%.
2 Discussion
In this research, we constructed a plant expression
vector pHorD-GBSS Ⅰ. We used barley endosperm
specific expression promoter during this process,
which has experienced GUS detection in callus and
expressed successfully. There are some studies have
Figure 5 Part of transgenic events detected by PCR for bar gene
identification
Note: M: DL2000 marker; 22: Blank (H2O) control; 23:
Positive control (non-transgenic plant); 24: Negative control
(pHorD-GBSS Ⅰ plasmid); 4,5,10,12,15: Transgenic plants;
1~3,6~9,11,13,14,16~21: Non-transgenic plants
Figure 6 Part of the transgenic plants detected by PCR for
targeted gene-specific fragments
Note: M: DL2000 marker; 23: Blank (H2O); 24: Negative
(non-transgenic plants); 22: Control Positive control (pHorD-
GBSS Ⅰ plasmid); 6,12,14,21: The transgenic plants; 1~5,
7~11,13,15~20: Non-transgenic plants
shown that Hord gene has endosperm specific
expression in transgenic plants (Cho et al., 1999). The
purpose of this research is to make GBSS Ⅰgene over
expressed in a specific period to improve the amylose
content in maize endosperm. So far, the regeneration
plant contain GBSS Ⅰgene regeneration plant has
experienced PCR initial detection. However, among
the positive plants, whether expression of GBSS Ⅰ
gene does improve, and whether amylose content is
increased, and how much the amylase content has
increased, need further studies, there are some studies
have shown that wheat GBSS Ⅰgene can be expressed
in transgenic plants (Kuipers et al., 1992).
At present, using Agrobacterium mediated method,
has successfully obtained transgenic plants regeneration
from corn sprouts and young embryo explants (Liu,
2011). According to some statistics, infected with
maize immature embryo by Agrobacterium mediated
transformation method, the rate of positive plant
conversion was reached to 5%~10%. By infected on
the shoot tip of maize, the rate of positive plant
conversion was 0.5%~6% (Oltmanns et al., 2010).
Therefore, we found that a good tissue culture
regeneration system was one of the key factors affected
maize genetic transformation efficiency. In this
investigation, we used somatic cell embryos for
genetic transformation, and adopted the optimum
condition of various stages obtained by other
researchers in the transformation process, and we
improved the quantity and positive rate of regeneration
plant successfully (Li, 2011). Based on the obtained
regenerated plants, we found some changes on the
phenotypes of positive plants. Firstly, the positive
plants which were obtained by transformation were
smaller than normal corn plants, and the number of
leaves was also less than the normal corn plants