International Journal of Horticulture, 2017, Vol.7, No.18, 146-153
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Analysis of variance (ANOVA) was constructed and Duncan’s multiple range test (DMRT) was calculated at 5%
probability level among various treatments (Steel and Torrie, 1986).
2.2 Phytochemical analysis
Total Phenolic Contents:
The total phenolic contents of shoots of intact plant and in vitro micropropagated shoots
were determined by Folin-Ciocalteu method (Jahan et al., 2011). Gallic acid was used as standard to compare the
results. Various concentrations (0.01 to 0.10 mg/ml) of Gallic acid were prepared. Aliquot (1 ml) of these were
mixed with Folin-Ciocalteu reagent (5 ml) and solution of Na
2
CO
3
(4 ml, 20%). The blue colored was developed
and incubate for one hour. Then, absorbance was noted with spectrophotometer at 765 nm. A blank solution
(without extract), shoots of intact plant and in vitro micropropagated shoots were also run in similar way. Total
amount of polyphenolic were calculated by the following formula and results were expressed as mg of gallic acid
equivalent per gram of fresh weight of plant material.
T = C × V/M
Where
T = Total phenolic contents in mg GAE/g of plant extract.
C = Unknown concentration of plant extract calculated from standard curve in mg/ml.
V = Volume of extracts taken in ml.
M = Weight of plants extracts taken in grams.
Detection of Alkaloids: Alkaloids were detected by using method described by Tamilselvi et al. (2012). The small
portion extracts mixed separately with 0.5ml of dilute hydrochloric acid and filtered. The filtrate was divided into
two portions and tested with various alkaloidal agents.
i) Mayer’s test: The Mayer’s reagent (a drop or two) was added in the one portion of filtrate along the sides of
the test tube. A white creamy precipitate indicated the test as positive.
ii) Dragendorffs reagent test: A drop or two of Dragendorff’s reagent was added to the above filtrate by the
sides of the test tube. An Orange brown precipitate indicated the test as positive.
2.3 Total flavonide contents total flavonide contents
Plant extracts (1.5 ml) was mixed with 2% ALCL
3
(2%) and shake vigorously. After ten minutes the absorbance
was noted at 367 nm. Total flavonide contents were determined with quercetin standard curve, and represented as
quercetin equivalent (QE) mg/g of fresh weight of plants.
3 Results
The effect of eight different media for micropropagation of cv. PRI-Red and FD 69-1 was compared. The
in vitro
micropropagated shoots of best micropropagation medium were assessed for the production of polyphenoles.
There was a clear correlation between NAA and BAP levels on micropropagation response. Media with BAP lone
were showing excellent response for micropropagation and promoted maximum number of shoots per explant
with 1.75 or 2.75 mg/l BAP as contained in media MPm6 and MPm7 (Figure 1A). While media with NAA lone
showed less or even no shoot induction (Figure 2) as MPm3 medium promoted callus induction rather shoot
development. Medium with both BAP and NAA strongly inhibited micropropagation response (Figure 3).
Significant variation among media, genotypes and even in their interaction was revealed in analysis of variance
table (Table 2).
Among these media, MPm 7 showed an excellent response by promoting maximum number of shoots per explant.
Medium, MPm7 gave 7.5 shoots per explant from cv. PRI-Red and 3 shoots per explant from genotype, FD 69-1
(Table 3; Figure 4). This was followed by MPM 6 which gave 5 shoots per explant and 1.8 shoots per explant
from PRI-Red and FD 69-1 respectively (Table 3; Figure 4). The remaining media gave comparatively poor
response for shoot induction. Minimum number of shoots per explant was observed in PRI-Red on MPm1 and in
