The method of plant tissue culture in photoautotrophic conditions has several advantages. Increasing survival rate of transferring in vitro plants to soil and decreasing of probability of fungal and bacterial contaminations are examples. However, steady injection of CO2 to the system is a major limitation, the use of containers surrounding by accumulated CO2 in growth chamber instead of CO2 enrichment removes the limitation. Actively growing shoot tips of micro propagated River Red Gum (Eucalyptus cammaldulensis) were used as explants, and sugar-and hormone - free MS medium at three levels (quarter, half and full) and Enshi medium, were used as media treatments in this study. Four types of supporting material were used: 2.0 gl-1 Gelrite, Foam material, 8.Og Vermiculite per culture vessel and 2.0 gl-1 Gelrite plus 6.0 9 vermiculite. All cultures were maintained for 26 days in the two growth chambers, using indirect CO2-enriched (3000±100ppm) (photoautotrophic) and CO2-nonenriched (semi photoautotrophic). The comparison between two conditions showed that, photoautotrophic condition was significantly superior in fresh and dry weight of aerial parts and left area. Stem length was significantly superior in semi photoautotrophic condition when using appropriate supporting materials. The result showed that CO2 –enriched system was a key factor in increasing quality and quantity of in vitro plantlet production. As an alternative accumulated CO2 in surrounding of containers in growth chamber could be used and the way for improvement of conventional micro propagation, when providing gaseous exchange system is not available.