MATERIALS AND METHODSPlant material

MATERIALS AND METHODS
Plant materials
The capsules or pods of G. speciosum orchid were collected
from plants with self-pollination for 150 days
after pollination (DAP). The capsules were harvested
in March 2012 at Suratthani province. The capsules
were brought to the laboratory for disinfection and
culture.
Seed morphology and viability studies
For seed viability analysis, freshly isolated seeds from
capsules were stained with 1% (v/v) 2, 3, 5-triphenyl
tetrazolium chloride (TTC) in darkness overnight under
shaking condition at 5 rpm for 30 min and kept under
static condition at 30 °C for 24 h. The seeds were
then observed under a stereomicroscope. Percentage
of seed viability was calculated by the number of red
colour staining embryos divided by the total number
of seeds examined and multiplied by 100.
Effects of basal media on seed germination
To determine the effects of basal media on seed germination
and subsequent development to protocorms,
capsules at 150 DAP were used as seed explants. The
capsules were disinfected and cut vertically over a
sterile Petri-dish. The seeds were then removed and
placed on the filter paper. The seeds were surface
sterilized again by dipping into 15% (v/v) sodium
hypochlorite (NaOCl) solution containing 0.5% (v/v)
Tween-20 for 10 min, rinsed three times with sterile
distilled water, and placed on half strength Gamborg’s
B-5 (½ B5) or New Dogashima (ND) or half
strength Murashige and Skoog (½ MS) medium supplemented
with 0.1% activated charcoal (AC) and
kept at 251 °C with a 16/8 h photoperiod. All
experiments were performed in three independent
replicates with 10 culture Petri dishes per replication.
In each Petri dish, one gram of seeds (approximately
815 seeds) was used. The germination percentage of
seeds at 30 days was calculated by the number of germinated
seeds or protocorms divided by the number
of seeds cultured and multiplied by 100. The seedderived
protocorms were used in these experiments
related to PLB induction and proliferation.
PLB induction, proliferation, and plantlet
regeneration
For PLB induction, seed-derived protocorms at
3 months of culture (approximately 5 mm in length)
from previous step were transferred to ½ MS medium
supplemented with 1 mg/l of 6-benzylaminopurine
(BA) alone or in combination with 0.5 mg/l of -
naphthalene acetic acid (NAA), with or without 0.2%
AC, 15% coconut water (CW) and 0.7% agar. Ten
explants were put in each culture bottle and six culture
bottles were used for each treatment. The percentage
of PLB formation and the number of PLBs per explant
were recorded after 8 weeks of culture. The proliferation
rate was also calculated after 10 weeks of culture.
For plantlets developed from PLBs, the cluster of
PLBs (3–5 PLBs) were cultured on ½ MS medium
without any plant growth regulators or supplemented
with 1 mg/l BA in combination with 0.5 mg/l NAA,
15% CW, 0.2% AC, and 0.7% agar. The number of
shoots was recorded after 3 months of culture.
All cultures were maintained at 252 °C under a
16 h photoperiod with light at an intensity of 10 μmol
m