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Bioscience Methods 2012, Vol.3, No.9, 55
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57
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rate is still the key factor seriously restricting the
improvement of crops through genetic transformation
(Popelka and Alpeter, 2003). Tissue culture is a
powerful tool for studying and solving, problems in
plant biotechnology. A tissue culture system provides
considerable quantities of target tissue.
To check the response of fresh orange juice and dry
milk on in vitro regeneration, we used selected media
combination that we were reported already (Ijaz et al.,
2012). This media combination consisted of CIM3
(Callus induction medium 3), genotype; S
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2003
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us
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359, Callus age; 21 days old calli and RM3
(Regeneration medium 3). CIM3 contained 3 mg/L
2, 4
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D, 40 g/L
sucrose and basal MS salt. RM3
contained 0.1 mg/L 2, 4
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D, 0.25 mg/L BAP, 40 g/L
sucrose and basal MS salt. We just modified the RM3
in this media combination by adding orange juice
RM3 (a) and dry milk RM3 (b). Calli of genotype
S
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2003
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us
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359 were induced on CIM3 and 21 days
old calli were transferred to RM3 (control), RM3 (a)
(modified regeneration medium by adding orange
juice) and RM3 (b) (modified regeneration medium by
adding dry milk) (Figure 1).
Figure 1 RM3 (b) medium modified by adding dry milk
The analysis of variance (ANOVA) table depicts the
significant effect of regeneration media on
in vitro
regeneration (Table 1). This comparative study rev-
ealed that the RM3 (a) (modified RM3 by adding
orange juice) gave maximum number of plants (Figure
2) the mean value 166.33 shoots per explant. This
response of RM3 (a) was statistically significantly
different from control (RM3) and RM3 (b); modified
RM3 by adding dry milk. But RM3 and RM3 (b) are
statistically similar with mean value of 134.67 and 137.77
shoots per explant respectively (Table 2, Figure 3).
Table 1 Analysis of variance (ANOVA) table for Regeneration
Source of
variation
Degrees of
freedom
Sum of
squares
Mean
squares
F-value
RM
2
1944.22
972.111 5.38*
Error
6
1084.00
180.667
Total
8
3028.22
Note: * = Significant (P<0.05)
Figure 2 Comparative study of RM3(a)
Note: A: Regeneration of shoots on RM3 (a); B: Plant lets of
RM3 (a) on root induction media; C: Plants of RM3 (a) in
green house condition
Table 2 Means of regeneration media ± SE
Treatment
Mean ± SE
RM3
134.67±7.86 B
RM3(a)
166.33±8.76 A
RM3(b)
137.77±6.09 B
Note: Means sharing similar letters are statistically
non-significant (P>0.05)
Figure 3 Regeneration response of genotype S
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2003
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us
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359
on RM3 (control)
Note: RM3 (a): RM3 medium modified by adding orange juice;
RM3 (b): RM3 medium modified by adding dry milk
Plant juices and liquid endosperms have been shown
to possess stimulatory properties. The inclusion of
liquid endosperm from immature corn (Netien et al,