International Journal of Horticulture, 2017, Vol.7, No.17, 138-145
140
Experimental layout and statistical analysis
: Experiment was repeated three times and in each repeats 10 test
tubes and each containing single explant were used. The experiment will be conducted in completely randomized
design (CRD). Analysis of variance (ANOVA) will be constructed and All-pair wise comparison test will be
calculated at 5% probability level among various treatments (Steel and Torrie, 1986).
Data recording
: Data were collected in the form of stolon length, number of days for the initiation of
microtuberization, number of microtubers produced per explant, time period for microtubers maturation, skin
color, flesh color, proximate analysis, effect of physiological age of microtubers on sucrose and starch contents
and histoanatomical comparison between microtubers and wild type potato.
Histoanatomy
: As mentioned earlier, in this research study
in vitro
microtuberization in studied potato genotype
was tried to be established using different photomixotrophic conditions. As the results showed that excellent
in
vitro
microtuberization was achieved in TM5. Hence for histoanatomy, microtubers of TM5 were used and
compared with field grown potato tuber of same genotype. For this, thin sectioning of both microtubers and field
grown tubers were performed to prepare slides for microscopy. Staining of tissue was done using Safranin dye
and Fast green dye. After that tissue was fixed by latex and covered with cover slip.
Effect of physiological age on sucrose and starch contents
: The effect of physiological age of tuber on sucrose
and starch contents was also investigated. For this, a comparison was made between microtubers of two different
ages viz., 35 days old and 60 days old. For determining the sucrose and starch contents in microtubers of both age
groups, microtubers were dried in oven at 60°C for 48 hours and subsequently were ground into fine powder.
Then, for preparing sucrose solution 20 mg of this powder were added in 800µl d
3
H
2
O and then vortex briefly
followed by centrifugation at 13200 rpm for 5 minutes. Supernatant was taken into fresh reaction tube and then
stored. Similar way was adopted for preparing starch solution but in this case sample was incubated at 70°C for
90 minutes prior to centrifugation. Then sucrose and starch contents were estimated using UV visible
spectrophotometry. Sucrose and starch contents were quantified by recording the absorbance at 620 nm and 630
nm respectively.
Proximate analysis
: Effect of photomixotrophic conditions on biochemical attributes was also investigated. For
this purpose, proximate analysis of microtubers and field grown tubers of PRI-Red was performed. Thereby, dry
matter, crude proteins, fat, fiber extract and ash contents were estimated. For dry matter,
in vitro
microtubers and
field grown potato tubers were weighed before and after drying in an oven for 48 hours at 60 °C and ground into
fine powder. Crude protein was estimated using Kjeldahl method, while fat was determined by soxhlet extraction
and defat sample taken after fat extraction was used to determine fiber extract after removing protein and
carbohydrates by treating it with H
2
S0
4
and NaOH respectively. However ash content was measured by heating
the sample. For this sample was taken in crucible and placed in pre-heated Muffle Furnace (at 350°C for 24
hours).
3 Results and Discussion
The effect of photomixotrophic conditions on
in vitro
microtuberization in terms of theireffect on morphology,
biochemistry, physiology and anatomy ofmicrotubers was investigated.For this purpose, nine
in vitro
tuberization
media having different photomixotrophic conditions were investigated and data was recorded in the form of
number of microtubers per explant, stolon length, number of days of microtubers, skin color, flesh color,
physiological, biochemical and anatomical characteristics.
In vitro
micropropagated shoots of potato cv.PRI-Red
were cut axenically in laminar air flow hood in such a way that each cutting contained 1-2 buds. These cutting of
genotype to be studied were cultured on media for investigating the response of these media to microtuberization.
The data were recorded in the form of number of microtubers produced per explant. Subsequent to this, data
collected were subjected to the statistical analysis. In this case, Analysis of Variance table (Table 2) revealed
significant variation among
in vitro
tuberization media. All-pair wise comparison test depicts that, tuberization
medium 5 (TM5) was excellent (Table 3; Figure 1) because maximum number of microtubers per explant was
