Culture conditions
A successful in vitro culture process also depends on appropriate culture conditions.
Temperature is a critical factor for callus induction and subsequent rooting culture. In
many cases, 25 ± 2C was always recommended for callus induction [Inagaki et al.
1981; Khunachak et al. 1987; Feng and Wolyn 1991; Chen et al. 1997; Lin et al.
2006a]. However, high temperature (≥25 ± 1C) was not favorable for rooting. The
optimal temperature for rooting was 20 ± 1C [Lin et al. 2006b]. During in vitro culture,
light condition is no less important than temperature as well. It affects formation of
cladophylls which are responsible for photosynthesis. In the initial callus induction
culture, light is not always needed. For instance, Feng and Wolyn [1991], Gonzalez
Castanon [2002] as well as Pontaroli and Camadro [2005] reported their callus induction
cultures were conducted in darkness. On the contrary, some other researchers illustrated
different light intensity was applied during callus induction culture. It usually
ranged from 1000 lx to 5000 lx [Inagaki et al. 1981, Lai et al. 1991, Chen et al. 1997,
Mamiya and Sakamoto 2000]. Photoperiod together with light intensity influences the
efficiency of organ formation, e.g. 16 hours light period was frequently applied in many
plantlet proliferation cultures [Gonzalez Castanon 2002, Bojnauth et al. 2003].