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Molecular Plant Breeding Provisional Publishing
Molecular Plant Breeding 2012, Vol.3, No.5, 50
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54
and 10 mg/L) were used. 10 mg/L basta was selected
for transformation study of genotype S-2003-us-359
and 5 mg/L basta was selected for the transformation
study of S-2003-us-127 because at these level early
calli death was observed but Ahmad (2009) used 3
mg/L basta dose for transgenic selection in wheat.
Manickavasagam et al (2004) suggested 5 mg/L basta
as most effective selective agent for transgenic
selection in sugarcane. These results showed, the dose
of selective agent (basta) varying in both genotypes
and these results reveal that dose rate of selective
agents is also genotype dependent. These results were
also agreed with the results of van Boxtel et al (1995),
who also reported that the sensitivity to selective
agents was genotype dependent. Therefore it
concluded that optimization of optimal dose of selective
agents should be done before the transformation of any
crop plant.
3 Materials and Methods
3.1 Germplasm/ plant materials
Two genotypes were selected viz., S-2003-us-359 and
S-2003-us-127, in which we have already established
an
in vitro
regeneration system (Ijaz et al., 2012;
Anjum et al., 2012).
3.2 Selection of basta dose rate for
in vitro
regeneration media to select transgenics
Young innermost leaves of sugarcane genotypes viz.,
S-2003-us-359 and S-2003-us-127 were cultured on
CIM 1 and CIM 2 respectively (Table 1). For determine
Table 1 Media composition for callus induction media (CIM)
Callus induction media (CIM)
CIM 1
CIM 2
MS Salt
4.33 g/L
4.33 g/L
Sucrose
40 g/L
30 g/L
2,4D
3 mg/L
3 mg/L
Kinetin
0
0.1 mg/L
Myoinositol
0.1 g/L
0.1 g/L
Nicotinic acid
1 g/L
1 g/L
Pyridoxine HCl
1 g/L
1 g/L
Thymine HCl
2 g/L
2 g/L
Glycine
4 g/L
4 g/L
Basta
0
0
Phyta gel
2.66 g/L
2.66 g/L
Note: CIM 1 is the callus induction medium for genotype
S-2003-us-359; CIM 2 is the callus induction medium for
genotype S-2003-us-127
the minimal inhibitory dose of basta on regeneration,
Calli of both genotypes were induced on their
respective callus induction media (CIM).
Twenty one (21) days old Calli of S-2003-us-359 and
28 days old calli of S-2003-us-127 were transferred to
regeneration selection medium 1 (RSM 1) and
regeneration selection medium 2 (RSM 2) respectively.
RSM 1 containing 2, 4
-
D (0.1 mg/L), BAP (0.25 mg/L),
sucrose (40 g/L) and basal MS Salt with different
levels (1 mg/L, 3 mg/L, 5 mg/L, 7 mg/L, and 10 mg/L)
of basta (selective agent) but RSM 2 containing 2, 4-D
(0.1 mg/L), BAP (1 mg/L), sucrose (30 g/L) and basal
MS Salt with different levels (1 mg/L, 3 mg/L, 5 mg/L,
7 mg/L, and 10 mg/L) of basta (Table 2). Experiment
was repeated three times and data were collected on
the basis of survival rate.
Table 2 Media composition for regeneration selection media
(RSM)
Regeneration selection media (RSM)
RSM 1
RSM 2
MS Salt
4.33 g/L
4.33 g/L
Sucrose
40 g/L
30 g/L
2,4D
0.1 mg/L
0.1 mg/L
BAP
0.25 mg/L
1 mg/L
Myoinositol
0.1 g/L
0.1 g/L
Nicotinic acid
1 g/L
1 g/L
Pyridoxine Hcl
1 g/L
1 g/L
Thymine Hcl
2 g/L
2 g/L
Glycine
4 g/L
4 g/L
Basta
1 mg/L, 3 mg/L, 5
mg/L, 7 mg/L, and 10
mg/L
1 mg/L, 3 mg/L, 5
mg/L, 7 mg/L, and
10 mg/L
Phyta gel
2.66 g/L
2.66 g/L
Note: RSM 1 is the medium for genotype S-2003-us-359; RSM
2 is the medium for genotype S-2003-us-127
3.3 Genetic transformation of both genotypes with
bar
gene to check the efficiency of selected basta
doses for transgenic selection
Genotype S-2003-us-359 and S-2003-us-127 were
genetically transformed with
bar
gene by using gene
gun delivery method to check the affectivity of
selected doses of basta for transgenic selection in both