Page 13 - BM 2012 vol.3 No.2

Bioscience Methods 2012, Vol.3, No.2, 7-20
http://bm.sophiapublisher.com
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combination with kinetin because at high 2,4- D level
might disrupt genetic stability of in vitro regenerants
which is not desired for genetic transformation
approaches. Same criteria of CFM selection were kept
for genotype S-2003-us -371. High dose rate of this
auxin also decrease regeneration potential (Ather et al.,
2009). Due to an excellent response of both genotypes
on CFM11 and CFM3 are selected as the best media
among others for good callus formation. Low dose
rate of auxin, cytokinin and amino acid are found to
be good for getting better regeneration. BAP at 1 mg/L
dose rate has significant impact for getting good
regeneration. Genotype S-2003-us-127 showed excellent
shoot formation on RM2 in which 1 mg/L dose rate
of BAP was used for the 28 days old calli from
CFM11. Sadat et al (2011) also obtained an excellent
regeneration at 1 mg/L BAP level. Similarly Gopitha
et al (2010) also observed excellent shoot formation at
1 mg/L BAP level. Similar types of results were also
described found by Ather et al (2009). Our results are
also matched with the findings of Khan et al (2009)
and Khan et al (2006) in which they got significant
shoot formation response with the inclusion of BAP at
1 mg/L dose rate.
Response of genotype S-2003-us -127 at 0.5 mg/L is
less than the response of this genotype at 1 mg/L BAP
but excellent regeneration response at 0.5 mg/L BAP
was observed by Pathak (2009); Baksha et al (2002)
and Goel et al (2011). BAP is a synthetic cytokinin
which is used by many researchers for regeneration
experiment. Dibax et al (2011) found 0.25 mg/L BAP
dose rate to be good for regeneration. Khan et al (2009)
used 1.5 BAP level while Behera and Sahoo (2009)
studied 2 mg/L BAP level and found good shoot
formation.
Contrary to this, calli CFM11 showed poor response
on RM1 and RM2 for the genotype S-2003-us- 371.
35 days old calli of this genotype from CFM11
showed good response at 1 mg/L BAP but in
combination with 0.25 mg/L kinetin and 250 mg/L
proline. Here our results were also similar with the
study of Ali et al (2008) and Baksha et al (2002) in
which he found excellent regeneration response at
0.25 mg/L kinetin level and observed that proline has
significant impact on shoot formation.
Molecular approaches are more convincing and more
reliable as compared with phenotypic observations for
determining variations (Leroy et al., 2000). Among
various molecular marker techniques, RAPD technique
was found to be more powerful tool to determine
variation in vitro-regenerated plants (Isabel et al.,
1993; Rani et al., 1995; Shoyama et al., 1997; Goto et
al., 1998). Jain et al., 2005 used the RAPD marker and
Isozyme to find out the genetic purity of in vitro
regenerants of sugarcane. 28 days calli from CFM 11
was selected in which 3 mg/L 2,4 -D was used in
combination with 0.1 mg/L kinetin and these calli
were shifted to RM 2 (0.1 mg/L 2,4 -D and 1 mg/L
BAP) gave maximum number of plants and became
the media combination of choice. The regenerants of
this media combination of choice were characterized
at molecular level by using RAPD analysis to verify
their genetic stability. Because in this study our
objective is to generate variability, due to our transgene
and not due to tissue culture regime. These regenerants
of selected media combination showed genetic
stability by giving same banding pattern as found in
wild type pant. This showed that for callus induction
exposure of ex-pant to 3 mg/L 2,4-D brings no genetic
change in plant genome. Jayanthi and Mandal (2001)
also observed no genetic variation at 3 mg/L 2,4- D.
Mohanty et al (2011) also confirmed genetic uniformity
in in vitro regenerants in which they used Kinetin
(1.0~3.0 mg/dm 3) along with different levels of NAA,
IAA and adenine sulphate. Similar finding was in the
study of Ijaz et al (2012) in which they proved that
3 mg/L 2,4- D level did not bring any genetic change
in the regenerants of selected combination.
3 Material and Methods
3.1 Germplasm collection
Two sugarcane genotypes viz., S- 2003-us- 127 and
S-2003 -us-371 with good agronomic traits but
susceptible to U. scitaminea were selected.
3.2 Callus mass formation
Young immature leaf was used as explants which was
collected from six month old healthy plants. The
field-collected material was washed two to three times