Abstract
Experiments were conducted in the Department of Crop Botany, BAU, to develop a
protocol on Protocorm like bodies multiplication and PLB-derived plantlets
regeneration of Mokara orange orchid in vitro. As treatment effects of carbohydrates
(sucrose, maltose, fructose and glucose), PGRs (IAA, BA and GA3) organic extracts
(banana, potato and rice extracts and coconut water) and different light intensities (0,
12, 24, 48, 96 µmol m-2s-1) were used. Culture was done in MS medium and
maintained in 100 ml Erlenmeyer flasks at 25±1°C in growth room. Treatments were
arranged following CRD where each treatment replicated 5 times. Among the
carbohydrates sucrose 20 g L-1 was the best for better growth and development of
PLBs and PLB-derived plantlets. As energy source sucrose supported the better PLBS
regeneration (121.0 flask-¹) and plantlets production (19.00 flask-1) after 3 months of
culture which was significantly higher compared to maltose, fructose and glucose. IAA
1.5 mg L-1 was the best among PGRs supplemented into medium for enhancing
growth and development of PLBs in vitro. The highest number (142.5 flask-¹) and
weight (0.960 g flask-¹) of PLBs and PLB-derived plantlets were obtained in medium
supplemented with 1.5 mg L-1 IAA. But growth of PLB-derived plantlets was
enhanced the best with 1.5 mg L-1 of BA after 3, 6, and 9 months of culture. Almost all
growth parameters of plantlets were higher in medium supplemented with BA
compared to those for IAA and GA3. Among the organic extracts 50 ml L-1 of banana
was the best for supporting the growth and development of PLBs. Banana extract
supplemented at 50 ml L-1 into the medium regenerated the highest number of PLBs
flask-1 (314.60). But growth and development of PLB-derived plantlets were enhanced
with 100 ml L-1 of potato extract supplemented into the culture medium. The growth
and development of plantlet in vitro, such as the number of leaves (5.51 plantlet-¹),
length (4.01 cm), width of leaf (0.560 cm), length of shoot (4.20 cm), weight of shoots
(0.524 g) and weight per plantlet (0.660 g), were the highest at 100 ml L-1 potato extract
supplement into medium after 9 months of culture. Thus potato extract might be
recommended for the better growth and development of plantlets in vitro. Better
growth and development of PLBs and PLB-derived plantlets were supported the best
at 24.0 µmol m-2s-¹ light intensity compared to those were at higher or lower light
intensities. Gross anatomy of PLBs revealed those all cells of PLBs formed with thin
walled parenchymatous tissues and after 2 months of culture there were few areas of
meristematic cells having dense cytoplasms. There were several somatic embryos
regenerated at the same time in the different parts of a PLB in spite of the presence of
a shoot apical meristem. Developing vascular tissues were observed in transverse
section of PLB of three months old. The anatomy of aerial roots revealed a primary
characteristic structure of dicotyledonous plant but stem shows the characteristics of
a monocotyledonous plant. An amphystomatic homogeneous mesophyll with aerial
spaces were seen in the leaf structure, which may be considered as an adaptive
feature to adverse environmental condition for the plant.