In this study, a rooting protocol was developed for macadamia plantlets with healthy roots and enhanced growth performance, along with enhanced photosynthetic capability. In vitro-grown shoots rooted in vented vessels containing vermiculite as the supporting material exhibited 100% frequency of root induction, whereas when shoots were grown in non-vented vessels containing a solidified Murashige and Skoog (MS) medium, the frequency of root induction was less than 30%. The formation of root with callus, hyperhydricity, and leaf necrosis was observed in this photomixotrophic closed system. The modification of the vented photoautotrophic system with different concentrations of CO 2 and sucrose were investigated using vermiculite as the supporter. The number of roots, root length, root surface area, fresh weight, and dry weight were significantly higher in plantlets grown in CO 2 -enriched (1,000 lmol CO 2 mol -1 ) photoautotrophic conditions. The water content in both root and shoot tissues of plantlets cultured under photoautotrophic conditions was maximized. In addition, shoot and leaf performances were enhanced in plantlets cultured under CO 2 -enriched photoautotrophic conditions. The supplementation of sucrose (29-88 mM) to culture media in both ambient and elevated CO 2 conditions affected a reduction in the shoot and root performance of in vitro plantlets. Chlorophyll a, chlorophyll b, and total carotenoids in the leaf tissues of plantlets acclimatized in CO 2 -enriched photoautotrophic conditions were enriched, leading to increasing photosynthetic abilities, including chlorophyll fluorescence and net photosynthetic rate. From this investigation, a root induction protocol was established and the production of healthy macadamia plantlets was successfully implemented using CO 2 -enriched photoautotrophic conditions.