Page 5 - mpbv3no5

Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.5, 50 -56
http://mpb.sophiapublisher.com
51
moderated levels of resistance are lost. Therefore
optimization of selective agent is very crucial for
selection of transgenic. Therefore, economical optimal
concentration of the selective agent is the most preferred
in the selection of stable transformed cells.
Selectable marker gene presence in transformed cells is
necessary for the survival of the transformed cells on the
selective agent. Until now almost 50 selectable marker
genes are used in transgenic development research (Miki
and Mchugh, 2004). Selective agent selection varies
from crop to crop. Hygromycin as selective agent did not
prove beneficial on the turf grass mature embryo
development (Cao et al., 2006) while in case of apricot,
kanamycin was proved to be excellent selective agent
because it also improved the proliferation rate of the
transformed tissues (Petri et al., 2005).
Basta is an efficient selective agent for monocot like
sugarcane (Chowdhury and Vasil, 1992), rice
(Christou et al., 1991), wheat (Vasil et al., 1992) and
maize (Fromm et al., 1990). Therefore for our study
basta was selected as selective agent for the selection
of transgenics in sugarcane. So, Basta dose was
optimized for sugarcane genotypes which were selected
for genetic transformation study.
1 Results
The concentration of selection agent in the tissue
culture media is very important. Less amount of
selective agent may give rise to many false positives.
Therefore an optimal amount of selective agent in the
media is too necessary. Selection pressure determines
the survival frequency of transformants and non
transformed cells. Optimization of basta dose for
genotypes viz., S-2003-us-359 and S-2003-us-127,
five different concentrations of Basta (1 mg/L, 3 mg/L,
5 mg/L, 7 mg/L, 10 mgl-) were studied for transgenic
selection on regeneration media. Basta concentrations
for both genotypes were found which becomes lethal
for non transformed cells. It was assumed that this
concentration will not be lethal for transformed cells.
1.1 Selection of optimal basta dose for genotype
S-2003-us-359 to be used in regeneration selection
media for transformation experiment
For the selection of optimal basta dose for regeneration
media, calli were induced on CIM1 (Table 1). Twenty
one (21) days old calli were transferred to a regime of
Regeneration Selection Media (RSM) containing 1, 3,
5,7,10 mg/L basta (Table 2) respectively to select the
optimal dose of basta, which was then used for the
selection of transgenic plants from bombarded tissues.
Calli on 10 mg/L basta showed death signs early. On
second day of shifting, approximately 15% embryos
died on this dose rate, and calli on 1 mg/L, 3 mg/L,
5 mg/L, 7 mg/L basta were unaffected. On the third
day of shifting, approximately 55% embryos died
on 10 mg/L and 10% embryos were died on 7 mg/L
basta, but other calli were still unaffected. On 4th day,
approximately 40%, 20%, and 10% death was observed
on 5 mg/L, 3 mg/L, 1 mg/L basta respectively. More
than 70 % death was observed on 10 mg/L basta and
50% embryos died on media having 7 mg/L basta. Fifth
day, all calli died on me1dia containing 10 mg/L basta
(Figure 2). But approximately 45%, 50%, 70% and
90% calli survived on 7 mg/L, 5 mg/L, 3 mg/L basta
respectively (Figure 1; Figure 2).
Complete calli death, in case of 7 mg/L, 5 mg/L, 3
mg/L basta was observed on 9th , 11th and 14th day
respectively. Up to 90% calli were survived on 1 mg/L
basta even on 14th day. Though death started earlier on
10 mg/L basta but it was not so sudden and it seemed,
on this basta dose if the calli are transgenic for bar
they will survive and if not then die in 5 days.
Figure 1 Optimization of basta dose for selection of transgenic
regenerants in genotype S-2003-us-359;
Note: 10 mg/L basta showed early death and after 5 days on
selection all calli were died. At 1 mg/L basta only 10%
embryos died even after 15 days, and survival rate was 90%