PhD

Standardisation of in vitro techniques for mass multiplication of
Aranthera and Dendrobium orchid varieties was attempted. The studies were
carried out at the Plant Tissue Culture Laboratory, Department of Horticulture,
College of Agriculture, Vellayani, during 1992-96.
Attempts for the in vitro propagation via enhanced release of axillary
buds, somatic organogenesis and somatic embryogenesis were made. One
variety Annie Black of the monopodial orchid Aranthera and five varieties of
the sympodial orchid Dendrobium were subjected to the initial response studies
Sonia-17 (Dendrobium) and Annie Black (Aranthera) varieties were selected
for detailed studies. Explants like shoot apices, leaf segments, root segments,
keikis, inflorescence stalk were used. Mercuric chloride 0.1 per cent for ten
minutes was identified as an effective surface sterilant. The lowest rate of
microbial contamination was observed from January to May.
The effect of culture medium (basal medium, mode of culture, strength
of MS basal medium, major nutrient element, plant growth substances, casein
hydrolysate, peptones, glutamine, sucrose, vitamins, yeast and malt extracts,
fruit juices, coconut water, ethylene inhibitors) and culture conditions on
in vitro shoot proliferation via enhanced release of axillary buds were studied.


Among the various explants tried culture establishment via enhanced
release of axillary buds could be induced only from shoot apex explants of all
the varieties. Culture establishment could be best induced in Vacin and Went
basal medium. Sonia-17 recorded 90.0 per cent bud initiation in VW media
supplemented with NAA 1.5 mg/l, BA 1.0 mg/l, Sucrose 30.0 g/l, Coconut
water 15.0 ml/l, agar 6.0 g/l within 14 days. Sonia-17 was used for shoot
proliferation studies.
Maximum shoot proliferation (35.33 shoots per culture) from shoot
apex explants of Sonia-17 could be induced in half-strength MS basal medium
supplemented with BA 2.5 mg/l, NAA 1.0 mg/l, Sucrose 30.0 g/l, boiled and
filtered coconut water 150.0 m/l and agar 6.0 g/l.
Among the Dendrobium varieties only Sonia-17 responded to direct
organogenesis. Of the various explants tried only the leaf base (from culture)
could initiate direct organogenesis. The best treatment identified was half-
strength MS basal medium (solid) supplemented with NAA 1.5 mg/l, BA 1.0
mg/l, sucrose 30.0 g/l, Coconut Water 150.0 ml/l, agar 6.0 g/l activated
charcoal 1.0 g/l, under darkness. This treatment recorded 9.17 shoot of 4.92
cm length having 3.50 leaves per shoot. Maximum proliferation .of shoot
(3.33 shoots/culture) obtained via direct organogenesis could be achieved in
half-strength MS basal medium (solid) supplemented with BA 2.5 mg/l, NAA
1.0 mg/l, sucrose 30.0 g/l, agar 6.0 g/l and coconut water 150.0 ml/l.
Direct organogenesis from the leaf base of Arantliera var. Annie Black
could be initiated in half-strength MS basal medium supplemented with BA


3.0 mg/l, NAA 2.0 mg/l, sucrose 30.0 g/l, coconut water 150.0 ml/l, agar 6.0
g/l in the presence of light. Only one shoot per culture could be induced.
Half-strength MS basal medium supplemented with BA 2.5 mg/l, NAA 0.1
mg/l, ascorbic acid + citric acid (150 ppm) supplemented with coconut water
150 ml/l, agar 6.0 g/l and sucrose 30.0 g/l was the treatment identified as best
for shoot proliferation in Annie Black.
Half-strength MS basal medium supplemented with BA 3.0 mg/l NAA
2.0 mg/l, Sucrose 30.0 g/l, agar 6.0 g/l and coconut water 150 ml/l could
induce the development of protocorm-like Bodies (PLB's) on the shoot apex
explants of Annie Black in 50.0 per cent of the cultures.
Attempts to standardise in vitro propagation via callus-mediated
somatic organogenesis and somatic embryogenesis were not successful.
In vitro flowering in Sonia-17 was observed when half strength MS
basal medium supplemented with BA 2.5 mg/l and sucrose 30.0 g/l, coconut
water 150.0 ml/l and agar 6.0 g/l was left unsubcultured for 3-4 months.
In vitro regeneration of roots in Sonia-17 could be best obtained in
half-strength MS basal medium (solid) supplemented with NAA 1.0 mg/l,
sucrose 30.0 g/l, agar 6.0 g/l in the presence of light. This treatment could
produce 20.17 roots of 5.08 cm length. An increase of 2-8 roots/shoot could
be observed after a period of one and a half-month.

Ex . vitro rooting studies were not successful. All the plantlets survived
in the different containers used. Coconut husk was found to be the best
medium/container. Hardening the in vitro plantlets in a green house with
misting facility recorded cent per cent survival. Survival of plantlets irrigated
with nutrient solution as well as hormone spray solution was poor. An increase
in shoot length, leaf number, leaf length, leaf width, root number and root
length by 2.72 cm, 1.06, 4.25 cm, 1.32 cm, 6.40 and 5.0 cm respectively
could be observed after four months of planting out.
The mean number of stomata per unit area, total chlorophyll,
chlorophyll a were less in the leaves of in -vitro grown plantlets. Rate of
water loss through leaves was greatest through the leaves of in vitro plantlets.
The cost of production of a single orchid plantlet was found to be



Rs. 3.68.