Fig. 3 Development of new PLBs from culturing a protocorm
(a) on ½ MS medium without PGRs, (b) on ½ MS
medium containing 1 mg/l BA and 0.5 mg/l NAA without
AC, and (c) with 0.2% AC.
Table 2 Effects of PGRs and AC on PLB formation and
proliferation after 10 weeks of culture.
Medium AC PLB for- No. of PLBs PLB prolif-
(%) mation (%) per explant eration (%)
PGR-free ½ MS 0.0 21.7b 2.91d 6.7c
0.2 28.3b 4.63c 21.7b
½ MS+BA+ NAA 0.0 46.7a 5.84b 11.7bc
0.2 63.3a 6.87a 46.7a
F-test p 6 0.01 p 6 0.01 p 6 0.01
C.V. (%) 36.4 12.0 39.1
Means followed by different letters within a column are
significantly different by DMRT.
PLB induction, proliferation, and plant
regeneration
The highest percentage of PLB formation (63%) was
obtained on ½ MS medium supplemented with 1 mg/l
BA in combination with 0.5 mg/l NAA, 15% CW,
0.2% AC and 0.7% agar. Statistically significant
difference was observed among treatments. Culture
media supplemented with AC seemed to induce PLB
proliferation better than culture medium without AC
(Fig. 3). AC adsorbed the inhibitory phenolic and
carboxylic compounds produced by the tissues in culture19,
so the medium without AC showed browning
of PLBs (Fig. 3b) as a result of low proliferation rate
of PLBs (Table 2). Half-strength MS medium without
PGRs was suitable for plantlet regeneration which
gave the number of shoots (> 3 cm long) at 6.67
shoots. The medium supplemented with BA promoted
the PLB proliferation resulted in an inferior growth
Fig. 4 Shoot development from PLBs cultured on ½ MS
medium without PGRs and ½ MS containing 1 mg/l BA,
0.5 mg/l NAA and 0.2% AC after 3 months of culture.
Fig. 5 (a) Shoot development from PLBs cultured on ½ MS
medium without PGRs (left) and ½ MS containing 1 mg/l
BA, 0.5 mg/l NAA, and 0.2% AC (right) after 3 months
of culture and (b) survival of plantlets after 1 month of
acclimatization.
of plantlets (Fig. 4). Similar results were obtained by
Abbas et al17 who found that the best culture medium
suitable for seedling development of G. scriptum was
½ MS medium supplemented with 15% CW. The
PLBs can be regenerated into complete plantlets with
vigorous shoot and root formation after 3 months of
culture (Fig. 5a).
Greenhouse acclimatization
After 6 months of culture on ½ MS medium without
PGRs and supplemented with 2% sucrose and 0.2%
AC, complete plantlets were formed. Fully developed
plantlets with vigorous shoots and roots were successfully
acclimatized and grown under the greenhouse
with about 60% shading and 80% relative humidity.
A total of 100 plantlets were planted. After 1 month