PhD

For refining and establishing micropropagation system for Anthurium
andreanum cv. Dragon's Tongue, studies were carried out, to solve many of the
problems faced in the micropropagation of the variety, such as, improper foliage
development, prolonged period for callus induction and multiplication.
Investigations were conducted also to explore alternative explants for
micropropagation, induction of somatic embryogenesis and development of
artificial seeds, crop improvement through induction of somaclonal variation and
radiation breeding in vitro. The study was carried out during 1995-98 at the
Plant Tissue Culture Laboratory, Kerala Horticulture Development Programme
(R & D), Kerala Agricultural University, Vellanikkara.
Surface disinfestation with 70 per cent ethyl alcohol wipe followed by
treatment with 0.1 per cent HgCl2 for eight minutes recorded maximum survival
(99.74%) of cultures in the case of leaf explants. For spadix explants, ethyl
alcohol wipe (70%) + emisan (0.1 %) dip for three minutes followed by HgCI2
(0.1 %) for 10 minutes recorded maximum survival (87.56%) of the cultures.
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Among the different explants tried (in vivo and in vitro derived explants) callus
induction was maximum in the case of in vivo leaf explants. Callus was initiated
(86.50%) within 51 days compared to 90 days reported earlier, when cultured
In darkness on to the culture media, Nitsch-white (NW) + kinetin 0.5mgl-1 +
2.4-0 0.3 rngl-1 + sucrose 20 gl-1+ glucose 10 gl-1 + agar 6.0 gl-1. Spadix
explants and in vitro derived explants (leaves, nodes, petiole and roots) showed






good response to callus induction treatments. Among the in vitro derived
explants, root explants recorded the maximum callus multiplication.
Callus induction from spadix explants was better (59.85%) in half strength
MS basal medium supplemented with 2,4-0 0.3 mgl-1 + kinetin 0.5 mgl-1
+Sucrose 30 gl-1 + agar 6 gl-1. Maximum shoot regeneration (92%) was observed
after 46 days in Nitsch media supplemented with BAP 0.5 mgl-1 in the case of
leaf callus. In the case of callus derived from spadix explants, half strength MS
media supplemented with BAP 2.0 mgl-1 + kinetin 2.0 mgl-1 recorded maximum
response (71.42). Multiplication rate (22 per culture vessel) of the shoots and
growth and development of the leaves and shoots were better in conical flasks
(200ml size). For quicker establishment of the plantlets, mud pots gave the best
results (85.33%). Incorporation of vesicular -arbuscular mycorrhizae (VAM)
Glomus so., into the potting mix improved the growth of the plantlets ex vitro.
Among the explants tried for somatic embryogenesis, in vitro derived
leaves (53%) and petiole (18.90%) and immature seeds (8.33%) showed
positive response. Induction of somatic embryoids was observed in the media,
Nitsch-White (NW) supplemented with 2,4-0 1.5 mgl-1 + kinetin 0.15 mgl-1 +
sucrose 20 gl-1 + glucose 10 gl-1 + glutamine 200 mgl-1 + agar 6 gl-1 in explants
derived from in vitro leaves and petiole. For immature seeds, response was
observed in Nitsch media supplemented with 2,4-02.0 mgl-1 + kinetin 0.3 mgl-1
+ sucrose 20 gl-1 + glucose10 gl-1 + glutamine 200 mgl-1. Germination of the
somatic embryoids was highest in half strength MS media supplemented with
BAP 0.1 mgl-1 + glutamine 200 mgl-1 . Viability of the somatic embryoids was

observed to be very low (5-10 days). Encapsulation of somatic embryoids was
achieved with calcium chloride at 50 ~M and sodium alginate at 3 per cent level
• "j
After encapsulation, somatic embryoids can be stored up to 20 days without
much loss in capacity for germination (15.50%). Germination per cent of
encapsulated somatic embryos was improved (39%) after a low temperature
storage (4 0C) for 20 days.
Rudimentary leaves were observed in plantlets regenerated from ninth
and tenth subculture. The colour of the leaves in such plantlets was observed
to be pale green. After transplanting also, the plants exhibited poor leaf growth.
In such plants the leaf area remained smaller than other plants. But, chlorophyll
development was normal.
For radiation breeding using y-rays, the irradiation doses above 150 Gy
were found to be lethal to callus as well as for shoot tips. Maximum response
in terms of plant height, plant spread and leaf area was recorded at lower dose
of 50 Gy. Most significant variation observed in in vitro regenerated plantlets
compared to mother plants was the reduction in leaf area of mutants
regenerated from explants irradiated with 150 Gy. Height of the plant was also
less at higher doses of ƛ-irradiation.
The plants obtained from the trial on induction of somaclonal variation
and radiation breeding were screened for possible mutants and somacional
variants using morphological characters, biochemical markers and cytological
technique. Morphological characters were recorded for observing possible

variability, at periodical intervals after transplanting. Dwarf mutants were
observed at higher doses of y-irradiation.
The biochemical studies using isozymes revealed no difference among
the plants regenerated from different subcultures and different doses of
irradiation. But, difference was observed between the non-irradiated and the
irradiated plants for the number of bands produced. Peroxidase isoenzyme was
found to be the most stable and was expressed in plants regenerated from
different subcultures as well as those from irradiated cultures. Five bands were
resolved in the case of plants from the repeated subculturing and two bands
were resolved in the case of those regenerated from irradiated cultures.
Cytological study showed no alteration in the somatic chromosome
number, which remained uniform at 2n=30+2B, in all the plants regenerated from
the different subcultures and the irradiated cultures.