MSc

The present study entitled “Seed characterization and standardization of in vitro seed
germination protocol in Rhynchostylis retusa (L.) Blume.” was carried out in the Department
of Plant Breeding and Genetics, College of Agriculture, Vellayani during the year 2020-22.
The experiment was aimed at seed characterization and standardization of asymbiotic seed
germination protocol in Rhynchostylis retusa (L.) Blume.
Germplasm repository maintained at Saraswathy Thangavelu Extension Centre of
JNTBGRI, Puthenthope, Thiruvananthapuram was utilized to observe the flower characters
and the capsules for the characterization and in vitro seed germination study.
The research work was divided into four experiments. Experiment I - evaluation of
floral characters, experiment II -morphological and morphometric characterization of seeds,
experiment III- preparation of in vitro seed germination media, and experiment IV -in vitro
seed germination in culture media and evaluation. All the experiments were carried out in five
replicates and mean was calculated.
In the first experiment, various floral, pollen and capsule characters were noted and
qualitative characters were expressed descriptively after observing the plants behaviour closely
and quantitative characters were assessed and mean was worked out. Rhynchostylis retusa was
seasonal (June-August) in flowering behaviour. Two types of plants were observed, one with
white flowers having purple spots or tessellation and other with blush of purple on white
background. Anthesis was between late evening and early morning. The stigma was receptive
in young flowers until five days after anthesis and after which the stigma become dried out.
Morning hours until 10.30 am showed maximum receptivity than other times of the day. Under
natural conditions capsule setting was noticed within seven days of flowering.
Morphology of pollinia was observed visually. They were crushed and stained using
acetocarmine to understand the individual pollen structure, number, arrangement, viability etc.
The pollen load per pollinium estimated by haemocytometer averaged to 2,06,000.
Furthermore, almost all the pollen was found to be viable and 73.64% of them germinated in
sucrose solution of 10 per cent strength. The fruit or capsule at maturity was 2.18 cm long and
0.86 cm wide. Undehisced mature capsules of 145-180 days of pollination was found to be best
for in vitro culture.
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In experiment II, seeds of R. retusa was characterized morphologically under
compound light microscope and morphometric data analysed using microscopic software
package Leica LAZ 500. The seeds were fusiform shaped, elongated, with prolate spheroid
embryo of violet colour. The testa cells were yellowish white and reticulated. At maturity 78.74
percent of the seed volume was filled with air space. The viability of seeds used for seed culture
was worked out to be 78.87 percentage. Out of the millions of seeds in a capsule 41.87
percentage of seeds were found to be well filled with large embryo of regular shape.
In experiment III- for the in vitro seed germination studies four different media viz.,
MS, half-strength MS, Knudson C orchid media and WPM including liquid and solid with and
without growth hormones were prepared and used.
The seeds from mature disinfected capsules were scooped out and inoculated under
sterile conditions. Five replicates of each media was inoculated with seeds. Observations were
recorded on the number of days for showing seed greening and chlorophyll embryo, for
protocorm development, for number of protocorms produced and number of protocorms with
shoot apex and multiple shoots, number of days for root and leaf initiation. Half MS medium
(liquid) was observed to be the best among the different media used for asymbiotic seed
germination of Rhynchostylis retusa. Half MS was best media for greening and protocorm
development. If we compare the overall response for different stages of development half MS
with 0.5 mgL-1 BAP (solid) was found best for callusing and shoot proliferation. Half MS with
0.5 mgL-1 BAP and 1 mgL-1 NAA (solid) for multiple shoots, small leaves and root
development. Initiation of germination was seen within two months of culture inoculation. The
grain shaped protocorm needed to be subcultured at proper time and calloid like structures with
multiple shoots could be divided to separate units and each unit developed to separate plantlets.
Seed characterization provides an insight into understanding biology, ecology and
evolutionary background. Mass multiplication by asymbiotic seed culture can be a viable
option for restoring this threatened species and can be utilized for further genetic improvement
programme.