Page 12 - BM 2012 vol.3 No.2

Bioscience Methods 2012, Vol.3, No.2, 7-20
http://bm.sophiapublisher.com
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introduction of desired gene into this grass Synman et
al (1996). Immature young leaves was used as an
explant because of high differentiation potential for
regeneration system and reported as an ideal source
for rapid callus formation Niaz and Quraishi, (2002).
For callus induction, four levels of 2,4-D (1 mg/L,
2 mg/L, 3 mg/L and 4 mg/L), two levels of BAP
(0 mg/L and 0.1 mg/L) and two levels of Kinetin
(0 and 0.1 mg/L) were studied for both genotypes viz.,
S-2003-us- 127 and S-2003-us- 371. Both genotypes
showed different response on all CFM which depicts
genotype based response. Similar results were observed
by Gandonou et al (2005). In their experiment callus
formation differ in genotypes studied which depicts
that callus formation ability is highly genotype
dependent. Genotype response is more influential for
callus induction in sugarcane (Badawy et al., 2008;
Burner, 1992). Auxin and cytokinin play key role in
callus induction (Bhansali and Singh, 1984). For
callus induction both genotypes S-2003-us -127 and
S-2003-us- 371 responded well when 2,4-D was used
alone at the level of 3 mg/L. These results are agreed
with the results of Sadat et al (2011) and Goel et al
(2010) in sugarcane in which he observed good callus
induction at 3 mg/L 2,4-D. Ather et al (2009) also
observed that 3 mg/L dose rate of 2,4-D gave 100
percent callus induction.
Genotype S- 2003-us-127 also showed better callus
mass proliferation on CFM4 when 2,4-D was used
alone with dose rate of 4 mg/L. Similar results were
observed by Ramanand et al (2006) when he used
2,4-D at 4 mg/L. While the genotype S-2003 -us-371
showed less response at the same level of 2,4-D. This
type of response indicates that genotype play key role
in callus formation. Contrary to this, both genotypes
showed less response at 1 mg/L and 2 mg/L 2,4- D
levels. Our results are matched with Goel et al (2010)
finding in which he observed low callus induction
frequency at 1 mg/L and 2 mg/L 2,4-D. But our
results deviate from the results of Behera and Sahoo,
(2009) in which they observed maximum callus
formation at 2.5 mg/L 2,4-D.
Kinetin also plays critical role for callus formation. In
our study, the addition of kinetin in CFM at 0.1 mg/L
gave an excellent response in both genotypes. Both
genotypes showed an excellent response at CFM11
and CFM12 with mean score of 5 and 4 respectively.
Similar results were produced by Khan et al (2006).
Excellent shoots were developed when he used kinetin
at 0.1 mg/L. While Ali et al (2010) used kinetin at
1 mg/L and observed excellent callus proliferation in
all genotypes.
When 0.1 mg/L BAP was used in combination with
2,4 -D (1 mg/L, 2 mg/L, 3 mg/L and 4 mg/L) in CFM,
we obtained different results. Genotype S-200-us-371
gave very little callus proliferation with mean of 1.00,
but genotype S-2003-us-127 showed no callus induction
and just swallowing of explants was observed. Ali et
al (2008) also observed less response when he used
BAP for callus induction. But our results have no
similarity with the results of Sadat et al., 2011 in
which he used BAP at 0.1 mg/L and 0.3 mg/L and
found an excellent callus growth at both levels. Naz
and Jahangir, 2008 obtained callogenesis at 2 mg/L
and 3 mg/L BAP level. Behera and Sahoo (2009)
and Goel et al (2010) used NAA and IBA for callus
induction.
Callus age is also an important factor in in vitro
regeneration and has significant impact in regeneration.
Effect of callus age on in vitro regeneration was first
reported by Ijaz et al (2012). They used 15 days, 21
days, 28 days and 35 days old calli in regeneration.
Here in this study, 21 days, 28 days and 35 days old
calli were used for in vitro regeneration and 28 days
old calli gave maximum number of shoots per explant
in genotype S-2003- us-127 and 35 days old calli
gave maximum number of shoots in case of genotypes
S-2003 -us-371. But this results deviate with the
study of Ijaz et al (2012), in which they obtained
maximum numbers of shoots from 21 days old calli.
These results depicts that this factor is also genotype
dependent.
Same response of genotype S-2003- us-127 at 3 mg/L
and 4 mg/L 2,4 -D alone and even in combination
with kinetin was observed but 3 mg/L 2,4 -D level
alone or in combination with kinetin was selected for
further studies instead of 4 mg/L 2,4 -D alone or in