Page 7 - BM 2012 vol.3 No.2

Bioscience Methods 2012, Vol.3, No.2, 7-20
http://bm.sophiapublisher.com
10
conducted to develop stable in vitro regeneration
system for genetic transformation experiment in selected
genotypes.
1.2 Callus formation
Two whip smut susceptible genotypes S-2003 -us-127
and S-2003-us -371 with good agronomic features
and an excellent sugar recovery were selected for
this study. Good callus mass is required for efficient
regeneration. Embryogenic callus also have significant
role in effective regeneration. In the present study,
immature young leaves of selected sugarcane geno-
types were surface sterilized and cultured on callus
formation media (CFM). Combinations of different
plant growth regulators (2,4-D, BAP and Kinetin)
were used in CFM. For callus induction study, 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 mg/L and 0.1 mg/L) were used for
both genotypes. Data for callus formation response of
both genotypes were scored on the basis of callus
proliferation rate. Analysis of variance of data revealed
significant variability between genotypes as well as
among CFM. Interaction between genotypes and CFM
was also highly significant (Table 1). Data over five
weeks of culturing, revealed that callus formation
response of both genotypes was good at all 2,4- D
levels, alone or in combination with kinetin, but
less response was observed when BAP was used
in combination with different levels of 2,4-D. In
genotype, S-2003- us-127, when BAP was used in
combination with 2,4- D no callus mass formation was
observed but swallowing of ex-plant was noted
(Figure 1). But in genotype S- 2003-us- 371, BAP in
combination with 2,4-D gave very little callus
formation with mean value of 1.00.
Figure 1 Ex-plant swelling in genotype S- 2003-us- 127
Note: In this genotype, explant swelling was observed and no
callus formation was observed when 2,4- D was used in
combination with BAP
Genotype S -2003-us- 127 showed an excellent callus
mass proliferation by scoring the mean value of 5.00
on CFM3, CFM4, CFM11 and CFM12. Genotype
S-2003 -us-371 gave highest mass of calli on CFM3,
CFM11 and CFM12 with mean value 4.00. Comparison
among callus formation media (CFM) revealed that
the response of CFM3, CFM11 and CFM12 was the
best with mean of 4.50, followed by the response of
CFM4 and CFM10 by scoring the mean value of 3.50.
As for as, genotypes are concerned, the response of
genotype S- 2003-us- 127 was overall good with mean
score of 2.67 followed by the genotype S-2003-us-371
with mean value of 2.33 (Table 2; Figure 2).
These both genotypes gave embryogenic calli on
callus induction media. Different stages of calli of
both genotypes can be seen in Figure 3 and Figure 4.
1.3 Regeneration Response in sugarcane genotypes
In order to obtain an efficient regeneration, plant
growth regulators (Auxin and cytokinin) as well as
amino acid play a key role for in vitro regeneration.
CFM3 and CFM11 were selected for in vitro regeneration
study, because on these media, both genotypes
Table 1 Analysis of variance (ANOVA): Statistical analysis (ANOVA) for callus formation in different sugarcane genotypes on
different CFM
Source of variation Degrees of freedom Sum of squares Mean squares F-value
Genotype 1 2.040 2.040 15.06**
Callus formation media (CFM) 11 182.520 16.593 122.53**
Genotype x CFM 11 24.840 2.258 16.67**
Error 48 6.500 0.135
Total 71 215.901
Note: **: Highly significant (P<0.01)