This article reports the effect of growing conditions on the anatomical and ultrastructural changes associated with the development of photoautotrophy in Gardenia jasminoides Ellis plantlets during shoot multiplication in vitro. Two photosynthetic photon flux densities (PPFD) (50 and 100 µmol m-2 s-1 PPFD: L50 and L100, respectively) and two sucrose concentrations in the culture medium (5 and 30 g L-1: S5 and S30, respectively) were assayed. An increase in PPFD stimulated the development of photosynthetic tissues and led to higher photosynthesis and dark respiration regardless of the sucrose level assayed. However, the effect of sucrose in the medium depended on the PPFD. For the high-PPFD treatment, a low sucrose concentration in the medium stimulated the development of photosynthetic tissues, whereas the opposite effect of sucrose was observed at low PPFD. This study demonstrated that an increase in light intensity to moderate values such as L100 has a beneficial effect on the development of structural changes associated with photoautotrophy. Such an effect is stimulated by low sucrose (S5) levels in the medium. These modifications of usual growing conditions (such as L50 combined with S30) for micropropagation may prove to be useful in mass propagation of gardenia. However, the use of a low sucrose level in addition to conventional growing PPFD may be counterproductive.