192 W. A. VENDRAME AND A. A. KHODDAMZADEH
more capable to induce PLBs than purine type cytokinins, such as BA
or KIN, even at low concentrations. TDZ has been reported to promote
induction of PLBs from leaf explants ofPhalaenopsis amabilis(Chen
and Chang 2006). TDZ used alone at low concentrations was more effi-
cient for orchids, such asPhalaenopsis(Chen and Piluek 1995; Ernst
1994). When combined with NAA, TDZ allowed the directin vitroshoot
induction and multiplication inDoritaenopsisPurple Gem “Ching Hua”
(Vendrame et al. 2007a). InOncidium, TDZ was also effective in induc-
ing direct PLBs from young leaf explants in half strength MS medium
supplemented with 0.3–3 mg L−^1 TDZ (Chen et al. 1999; Chen and
Chang 2001). Also, TDZ has been reported to promote direct PLBs
induction from the epidermal cells from leaf explants ofPhalaenop-
sis amabilis(Chen and Chang 2006). The ratio of auxin and cytokinin
required for PLB formation is different among various orchid species
(Arditti and Ernst 1993). This is likely due to the large diversity within
the family. For example, a combination of BA and NAA improved pro-
liferation of shootsin vitrofromCyrtopodium saintlegerianumwhen
using BA alone or combined with lower levels of NAA. Subsequent
application of gibberellic acid (GA 3 ) promoted elongation of shoots,
although affected acclimatization negatively (Rodrigues et al. 2015).
Zeng et al. (2015) reviewed various protocols for different species of
Paphiopedilum. The combination of NAA with either BA or TDZ at var-
ious concentrations showed to be the most efficient forin vitropropaga-
tion of most species ofPaphiopedilumused in the study, although mul-
tiple shoots were induced from stem nodal and single shoot explants of
P. rothschildianumin^1 / 2 MS medium without any plant growth regula-
tors. In contrast, PLBs are obtained inSpathoglottis plicatawith either
kinetin or BA, without the addition of auxins (Stella et al. 2015).
In addition, PLB induction is dependent on the species studied and
rate of proliferation can differ based on the composition of the media.
In Phalaenopsis,growth and organogenesis of PLBs are affected by
sucrose concentration (Tanaka 1987). Similarly, sucrose concentration
and the addition of organic supplements, such as banana pulp, coconut
water, and potato extract were effective for PLB proliferation in sev-
eral species, includingCymbidium,Doritaenopsis,Neofinetia,andPha-
laenopsis(Ichihashi and Islam 1999; Kusumoto 1978, 1980).
As far as explant sources, PLBs and plantlets have been reported to
form on cut surfaces of root, stems, flower stalk, nodal buds and leaf
sections ofPhalaenopsis(Pieper and Zimmer 1976), cultured flower
stalk internodes ofPhalaenopsis(Lin 1986), seeds (Chen and Chang
2004a), and leaf explants (Chen and Chang 2006). Once successfully
induced, it is important to maximize the proliferation and utilization