The effect of different closure types, light, and sucrose concentrations on carbon isotope composition and growth of Gardenia jasminoides plantlets during micropropagation and subsequent acclimation ex vitro

The growth of Gardenia jasminoides Ellis plantlets and the development of photoautotrophy during two successive culture stages (shoot multiplication and root induction) in vitro was analyzed. We examined the effects of changes in growth conditions (type of tube closure, light, and sugar levels) on the development of photoautotrophy and growth during micropropagation and sought to establish whether they affected later acclimation to conditions ex vitro. During the two stages in vitro, plantlets were grown in tubes under two different PPFD (50 and 110 micromole m-2 s-1), in media with three different sucrose concentrations (0, 1.5, and 3.0%, w/v) and with two different CO2 levels inside the tubes (controlled by either tightly closed caps or loosely sealed caps, and with an external CO2 concentration of 750 micromole mol -1). The development of photoautotrophy was assessed by determining the difference between the stable carbon isotope composition (delta 13C) of sugar cane sucrose used as a heterotrophic carbon source and that of leaflets grown in vitro. Plantlets from the root-induction stage showed a more highly developed photoautotrophy than those from the shoot- multiplication stage. At both stages, utilization of closed caps was the treatment which most stimulated development of photoautotrophy in plantlets. Also, lowering PPFD or sucrose concentration induced a greater degree of photoautotrophic development, the strongest effect being observed in plantlets cultured inside loosely sealed tubes. During acclimation ex vitro, plantlets taken from loosely sealed tubes in vitro performed better than those cultured inside tightly sealed tubes. The former, as well as recording a larger increase in fresh weight during this stage, also showed more negative delta 13C in the newly developed leaves, which would seem to indicate a better water status during acclimation. Present results validate the usefulness of delta 13C analysis of leaflets as a simple technique in assessing the development of photoautotrophy during culture in vitro. In addition, delta 13C analysis can be extended to evaluate growth conditions during acclimation to ex vitro conditions.