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Bioscience Methods 2012, Vol.3, No.2, 7
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17
with running tap water and then sterilized with 70%
ethanol. Leaf rolls were peeled off under sterile
conditions inside horizontal laminar air flow hood
and cut into cylindrical pieces approximately 2~3
mm in diameter. Five slices were cultured per plate
on twelve callus formation media (CFM), which were
supplemented with different plant growth regulators
and basal MS salts in common. In callus formation
media, 2,4
-
D, BAP and Kinetin were used in different
concentrations (Table 5). The culture plates were placed
in a controlled temperature room at (26
±
2)
℃
under
dark conditions. The cultures were transferred on to
fresh callus formation media (CFM) after twenty days.
Data of callus formation were recorded in the form of
callus score.
3.3 Shoot induction
Proliferated calli of both genotypes with different
ages viz., 21 days old, 28 days old and 35 days old,
were shifted to four different regeneration media.
Regeneration media were composed of basal MS salt
in common with different levels of 2,4
-
D, BAP,
Kinetin and Proline (Table 6).
3.4 Root induction
For the development of roots, 1/2 MS medium was
used (Table 7). Plantlets were shifted to 1/2 MS medium,
for root induction. After the formation of profuse root
system, plants of all regeneration media were counted
and shifted to peat and moss in poly ethylene bags.
Acclimatization After shifting to peat moss, these
plants were acclimatized in green house (Figure 12).
3.5 Genome stability analysis
DNA was isolated by the method described by Pallotta
et al (2000). Young leaf tissues (200 mg) were ground
Table 6 Regeneration media (RM) with different plant growth
regulators (2,4
-
D, BAP, Kinetin and Proline
Ingredients
Regeneration media
RM1
RM2
RM3
RM4
Ms Salts (g/L)
4.33
4.33
4.33
4.33
Sucrose (g/L)
30
30
30
30
Myoinositol (mg/L)
100
100
100
100
Thiamine HCL (g/L)
2
2
2
2
Nicotinic acid (g/L)
1
1
1
1
Glycine (g/L)
4
4
4
4
Pyridoxine HCl (g/L)
1
1
1
1
2,4
-
D (mg/L)
0.1
0.1
0.1
0.1
BAP (mg/L)
0.5
1
0.5
1
Kinetin (mg/L)
0
0
0.30
0.30
Proline (mg/L)
0
0
250
250
Phytagel (g/L)
2.6
2.6
2.6
2.6
Table 7 Root induction medium for root development
½ MS medium
Content
Ms Salts (g/L)
2.165
Sucrose (g/L)
30
Thiamine HCL (g/L)
2
Nicotinic acid (g/L)
1
Glycine (g/L)
4
Pyridoxine HCL (g/L)
1
Phytagel (g/L)
2.6
Table 5 Callus Formation Media (CFM) with different plant growth regulators (2,4
-
D, BAP and Kinetin)
Ingredients
Callus formation media
CFM1 CFM2 CFM3 CFM4 CFM5 CFM6 CFM7 CFM8 CFM9 CFM10 CFM11 CFM12
Ms Salts (g/L)
4.33
4.33
4.33
4.33
4.33
4.33
4.33
4.33
4.33
4.33
4.33
4.33
Sucrose (g/L)
30
30
30
30
30
30
30
30
30
30
30
30
Myoinositol (mg/L)
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Thiamine HCL (g/L)
2
2
2
2
2
2
2
2
2
2
2
2
Nicotinic acid (g/L)
1
1
1
1
1
1
1
1
1
1
1
1
Glycine (g/L)
4
4
4
4
4
4
4
4
4
4
4
4
Pyridoxine HCl (g/L)
1
1
1
1
1
1
1
1
1
1
1
1
2,4
-
D (mg/L)
1
2
3
4
1
2
3
4
1
2
3
4
BAP (mg/L)
0
0
0
0
0.1
0.1
0.1
0.1
0
0
0
0
Kinetin (mg/L)
0
0
0
0
0
0
0
0
0.1
0.1
0.1
0.1
Phytagel (g/L)
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6