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which developed from induced callus (Kane et al. 1994). Organogenesis
usually occurs in three steps: (1) the action of synthetic growth regula-
tors on explants; (2) the removal of this stimulus; and (3) stimulation
of the meristemoids or sphaeroblast to develop into adventitious buds
and roots (Durzan 1982).
The types of explants, concentrations, and combination of plant
growth regulators play a remarkable role duringin vitropropagation of
different orchids (Arditti and Ernst 1993). Plant growth regulators are
active in low concentration with the ability to modify plant growth. The
most important plant growth regulators are auxins and cytokinins.
Auxins are known to promote cell expansion and division, growth of
tissues, and root development in plants (Mockaitis and Estelle 2008).
Some of the commercial synthetic auxins commonly used in tissue cul-
ture are 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid
(IBA), and 1- naphthaleneacetic acid (NAA). Despite the fact that the
most synthetic auxins have phytotoxic effect on plant growth at high
concentrations, they are useful for promoting growth of PLBs at low
concentrations. Auxins, especially NAA affects the process of regener-
ation in monopodial epiphytic orchid species promoting the formation
of PLBs (Arditti and Ernst 1993). The first study using auxin was per-
formed forin vitroregeneration of PLBs inRhynchostylis giganteaand a
low concentration of 0.1 mg L−^1 NAA was used (Vajrabhaya and Vajrab-
haya 1970). InPhalaenopsis bellina, 2,4-D at 0.8μm has been reported
to affect PLB induction, resulting in 53% of PLB formation (Mahmood
and Chew 2008). In contrast, Fonnesbech (1972) reported that 2,4-D
was not convenient forin vitropropagation ofCymbidium cygnus“Ing-
hams” since it resulted in abnormal growth and disturbed chlorophyll
synthesis. In another study, Kuo et al. (2005) reported that PLBs that
were induced by 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) onPha-
laenopsishad their frequency of formation retarded by 2,4-D. The use
of 2,4-D has also been implicated to result in high somaclonal variation
(Saieed et al. 1994), which could result in undesirable changes in flower
color or even loss of fragrance, such as inPhalaenopsis bellina.
Cytokinins are involved in cell division and the most common
cytokinins used in orchid micropropagation are 6-benzylaminopurine
(BA), kinetin (KIN), and TDZ. Cytokinins are usually used in combi-
nation with auxins. The balance between these two plant growth regu-
lators is usually required to initiate growth or differentiation in tissue
culture (George et al. 2008). Subculture procedure with medium includ-
ing cytokinin can cause the cells to divide simultaneously after a delay
period, thus inducing multiplication (Jouanneau 1975). When compar-
ing cytokinins, Huetteman and Preece (1993) reported that TDZ was