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The present investigation entitled “Seed characterization and standardization of in vitro
germination protocol in Vanda tessellata (Roxb.) Hook. ex G. Don” was carried out with the
objective of standardization of in vitro seed germination protocol in Vanda tessellata along with
morphological and morphometrical characterization of seeds. The study was conducted at
Saraswathy Thangavelu Extension Centre of JNTBGRI, Puthenthope, Thiruvananthapuram and at
College of Agriculture, Vellayani, Thiruvananthapuram during 2021-22.
The research work included 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 its evaluation. All experiments were carried out in five replicates and mean
was calculated.
For experiment I & II, V. tessellata germplasm repository maintained at Saraswathy
Thangavelu Extension Centre of JNTBGRI, Puthenthope was used for morphological
characterization. The study revealed that V. tessellata is an epiphytic, monopodial orchid with yearround flowering. Inflorescence is axillary, erect, and simple raceme, bearing an average of seven
flowers. Flowers are bilaterally symmetric, resupinate, having green pedicel, greyish green calyx
and corolla with dark brown tessellation, labellum white speckled with violet tinge inside and mid
lobe lilac- blue. Time of anthesis was from morning to evening in a day (6am to 6pm) and under
natural conditions, longevity for an unfertilized flower was around 22 days. Stigma receptivity
remained throughout the day after anthesis and continue till the withering of flower.
The male and female reproductive parts are fused together and become the gynostemium
or column. Pollen grains are packed together as a pollinium with 2 waxy, globular pollinia with an
average 7,30,000 pollen production per pollinium. Pollen was ovate to obovoid, smooth, exine was
smooth and thick and pollinia were mostly in tetrad stage. Intine was not so prominent or absent.
Almost all pollen were viable with a fertility percentage of 98.85 and a pollen germination
percentage of 78.2.
After pollination, flowers started to senesce, greening and swelling of ovary occurred and
it took one week for pod setting. The ripening of a capsule took 120-135 days. The capsule
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contained millions of seeds, where 76% of them had well filled embryo and 70.6% among them
were viable. Mature seeds have spathulate shape and seed mass have yellow colour with a prolatespheroid, light violet coloured embryo and a reticulated testa. V. tessellata seeds had a lower
percentage of air space (31.69%) and SV/EV ratio of 1.5 shown that they are adapted to restricted
or localized distribution.
In experiment III, four different basal nutrient media (solid and liquid) viz. MS, Half MS,
Knudson C and Mitra, supplemented with various growth regulators were examined for asymbiotic
seed germination. pH of the medium was adjusted to 5.8 with either 1.0 N sodium hydroxide
(NaOH) or 1.0 N hydrochloric acid (HCl) before being solidified with 8 gL−1 agar in case of solid
media. Thereafter, 10 ml or 50 ml of the medium was dispensed into culture tubes/bottles and
autoclaved.
Under experiment IV, matured seeds of 120-135 days old were cultured on four different
media viz. MS, Half MS, Knudson C and Mitra, both solid and liquid with and without plant growth
regulators. The enlargement of the embryo and rupturing of seed coat was the first sign of seed
germination followed by greening and emergence of embryo out of the seed coat and later
developed into protocorm and differentiated into shoot meristem and rhizoids in opposite
directions. Leaves were produced from green protocorm. Real roots were formed later. Apart from
these developmental stages, some seeds developed into multiple protocorm like bodies which on
sub-culturing produced multiple shoots with small leaves and roots.
From the present investigation it can be concluded that the best media for seed germination
was Knudson C (liquid) media followed by Mitra (liquid) media, for further protocorm
development and leaf formation was MS media with 0.5 mgL-1 BAP (liquid), for leaf and root
development was MS media with 0.5 mgL-1 BAP and 0.5 mgL-1 NAA (solid) and for multiple shoot
production with leaves and roots was MS media 1 mgL-1 BAP + 1 mgL-1 NAA (solid).
Vanda tessellata is an endangered and valuable wild orchid species so its conservation and
utilization for genetic improvement is worthful and therefore it can be multiplied on large scale by
adopting proper in vitro propagation method.