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Investigations on "In vitro pollination in kacholam (Kaempferia galanga
L.) for seed set" were carried out at the Department of Plantation Crops and Spices
and Centre for Plant Biotechnology and Molecular Biology, College of
Horticulture, Vellanikkara during 1999 to 2001.
The six kacholam ecotypes under study exhibited variability in yield and
quality characters and these characters remained scattered among the ecotypes.
The flowering season of kacholam ranged from June to August when
planted on 5th May. The practices of staggered planting at monthly intervals from
is" May to is" July and maintaining the crop as biennial, together could extend
the flowering season up to June to October as against June to August in the normal
planting. Increasing the rhizome bit size from 5-10 g to 20-25 g did not confer any
marked advantage for flowering.
The study of floral biology and morphology of kacholam showed that the
ecotypes took 48.0 to 68.5 days for flowering from planting. The inflorescences
produced 4.0 to 11.0 flowers and the blooming period ranged from 9.3 to 14.9
days. The anthesis started by 4.00 am and continued up to 5.00 am. Anther
dehiscence occurred shortly after the anthesis and took place between 4.30 am to
5.15 am. The flowers are characterized by a spiny stigma and a long style of mean
length of 4.52 cm, which passes through the groove present in the only fertile
stamen. The ovary measured a mean length of 4.11 mm and a diameter of 2.68 mm
and recorded a mean ovule number of 20.0. The ovules recorded a mean length of
96.33 urn and breadth of 61.16 urn at the middle.
The mean pollen fertility with acetocarmine stain in the ecotypes was 76.33
per cent. The study for selection of a medium to support pollen germination and

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tube growth resulted in the identification of ME3 medium of pH 6 as the best
medium while Brewbaker and Kwack's medium and the medium standardized by
Rekha (1993) were also found favourable. The mean pollen viability in the ME3
medium was 78.80 per cent.
Seed set and development was obtained in kacholarn through in vitro
pollination, The flowers were collected in the early hours of the day (6.30 to 7.30
am) on the day of anthesis. They were surface sterilized by dipping in
streptocycline solution (500 rngl") and wiping with 70 per cent alcohol followed
by rinsing in mercuric chloride (0.1 %) for 3 minutes in the laminar flow. The
sterilants were completely removed by three washings in sterile distilled water.
Bacterial contamination interfered culture establishment. in vitro sensitivity studies
revealed that copper oxychloride 2500 mg r' could effectively suppress gram
negative white bacteria and gram positive pink and yellow bacteria occurring in the
cultures. Among the various methods of pollination tried, ovary/ovules developed
in intra ovarian pollination, placental pollination and modified placental
pollination. Pollination was done with pollen grains suspended in ME3 medium.
The placental pollination method was found best for ovule development.
The experiments on culture establishment showed that MS medium at half
and full strength 'Supplemented with hormones can support ovary / ovule
development but half MS medium was superior. The cultures varied in their
hormone requirements and four media combinations were identified for ovule
development.
i) 12 MS + 3 % sucrose + 2,4-D 0.2 mg r'
ii) 12 MS + 3 % sucrose + BA 1.0 + kinetin 1.0 mg r'
iii) ~ MS + 3 % sucrose + BA 0.5 + NAA 3.0 mg rl
iv) 12 MS + 3 % sucrose + BA 1.0 + kinetin 3.0 + 2,4-D 0.2 mg r'
The maximum ovule development was observed in the medium of 12 MS +
3 % sucrose + 2,4-D 0.2 mg r'. Addition of dOl~hle the quantity of the vitamin





stock of the MS medium to the medium of ~ MS + 3 % sucrose + BA 0.5 + NAA
3.0 mg r' was good for ovule development. The supplements casein hydrolysate,
coconut water, yeast extract and L-Glutamine did not favour ovule development.
The pollen pistil interaction studies after in vitro. placental pollination
showed that pollen tube growth is sufficient to cover the entire length of the ovule.
Histological examination of placental pollinated ovules at various stages from 2
DAP to 25 DAP showed embryo of increasing size. This confirmed fertilization in
placental pollination. The pollinated ovules developed into dark brown arillate
seeds at 20 DAP. The size of the seeds was more than 10 times the size of the
ovules. The kacholam ovary after intra-ovarian pollination developed into a thick
walled capsule with light brown seeds but the size increase was not substantial.
The small arillate seeds had an outer thick seed coat and an inner thin seed
coat. The seed coat enclosed a cavity, which is typical of monocots. In the cavity,
endosperm with embedded embryo was seen.
The seeds developed after in vitro pollination did not germinate even after
subjecting to various in vivo and in vitro germination treatments.