MSc

The present investigation was undertaken to elicit information on the
physiological, morphological, biochemical, biometric and anatomical changes
during in vitro propagule multiplication and ex vitro establishment in tissue
cultured plantlets of orchids. Dendrobium hybrid Sonia 17 was the variety
used for the study. The rooting media was supplemented with different levels
of sucrose. At the time of planting the plantlets were subjected to triazole
treatment. During the process of hardening the plantlets were maintained in
hardening chambers with different levels of light and humidity.
Among growth parameters the crop growth rate (CGR), net assimilation rate
(NAR) and relative growth rate (RGR) were found to be high at 40 gIl of
sucrose concentration. The CGR of these plantlets were on par with normal
green house grown plantlets. The photosynthetic rate was found to increase
and the transpiration rate was found to decrease at 40 gIl of sucrose
. concentration.
The maximum survival percentage of the in vitro plantlets occur when 40 g11
of sucrose incorporated in the rooting medium. This may be attributed to the
influence of the sucrose concentration on morphological characters studied viz.
plant height, number of leaves per shoot and number of roots per shoot. With
regard to photosynthetic pigments an increase in the content of total
chlorophyll, chlorophyll a chlorophyll b and carotenoids occurred in plantlets

treated with 40 g/1 of sucrose. Also the protein content and carbohydrate
content was maximum at the above sucrose level. The leaf area, root length,
total fresh weight and dry weight of the plantIets maintained at 40 gIl of
sucrose level were higher than other treatments. These effects ultimately lead
to better survival percentage.
Triazole treatment of plantIets during planting out helped in better survival
percentage. With regard to the growth parameters the effect was distinct
towards the later stage of growth. CG~ NAR and RGR were maximum on the
triazole treated (5 mgll) plantIets and was comparable to normal green house
grown plants. There was marked increase in the photosynthetic rate and
decrease in transpiration rate of plantIets treated with 5 mgll of triazole.
However the photosynthetic rate of normal green house grown plants were
much more than the tissue cultured plantIets.
Regarding morphological characters, increasing concentration of triazole
had negative influence on plant height. However plant height of normal green
house grown plants were distinctively higher than tissue cultured plantIets.
With regard to number of leaves per shoot, triazole treatment showed
significant effect only after 30 days of planting out, whereas the number of
roots per shoot was very much influenced by triazole. The maximum number
of roots was produced at 5mgll of triazole treatment and these effects in turn
influenced higher survival percentage of plantIets.

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Regarding the biochemical aspects total chlorophyll, chlorophyll a,
chlorophyll b and carotenoids contents of plantlets treated with triazole (5 mg/l)
were higher and found to be on par with that of green house grown normal
plants towards the later stage. The protein content was also positively
influenced by triazole treatment (5 mgll) and the value was comparable to that
of normal plantlets. In the case of carbohydrate content the treatment becomes
statistically significant and the normal green house grown plants exhibited
distinctively higher value. The plantlets treated with 5 mgll of triazole were
found to have higher leaf area, root length, root: shoot ratio, total fresh weight
and dry weight than other treatments. However the root shoot ratio and total
fresh weight of treated plantlets were on par with the normal green house
grown plants towards the later stage viz, 45DAP.
The physiological, morphological, biochemical and biometric characters of
the plantlets were also found to be influenced by different levels of light
intensity and humidity maintained in the hardening chamber. Among the
different treatments, plantlets grown at 50 percent light intensity and 70 to 90
percent relative humidity exhibited higher CG~ NAR and RGR. Also these
plantlets exhibited a marked increase in photosynthetic rate and decrease in
transpiration rate. However CGR and NAR of green house grown normal
plants were distinctively higher during the later period. However the normal
plants had distinctively higher CG~ RGR and photosynthetic and very less

transpiration rate than other treatments. The plantlets grown under 50 percent
light intensity and 70 to 90 percent humidity produced maximum plant height,
number of leaves and number of roots per shoot. The survival percent of the
tissue cultured plantlets in the field condition (green house) was found to be
superior under appropriate environment of light and humidity (50 percent light
intensity and 70 to 90 percent relative humidity)
There was not much significant difference among the treatments of light and
humidity in the case of pigment content. However the plantlets grown in the
hardening chamber maintained at 50 percent light intensity and 70 to 90 percent
of relative humidity recorded maximum value of total chlorophyll, chlorophyll
a, chlorophyll b and carotenoids among treatments and the normal green house
grown plants exhibited distinctively higher pigment content. The normal
plantlets recorded marked increase in protein content and carbohydrate content
than the tissue cultured plantlets. In the present study, an environment of 50
percent light intensity and 70 to 90 percent relative humidity was found to be
superior and could favour enhanced leaf area, total fresh weight, total dry
weight and root length of plantlets which ultimately resulted in better survival
percentage.
The in vitro plantlets observed to have anatomical characters as compared to
the normal green house grown plants and hardened plantlets. The stomata
remained open and less number of stomata per unit area of leaf was observed as
the in vitro leaves were concerned. Another peculiarity of the in vitro plantlets

was the absence of cuticle layer. The mesophyll layers were also found to be
less compared to normal plantlets. These anatomical characters were one of the
severe limitations of the micropropagated orchid plantlets during ex vitro
establishment which ultimately resulted in high rate of field mortality.