Effect of ex vitro conditions on growth and acquisition of autotrophic behaviour during the acclimatisation of chestnut regenerated in vitro L. Carvalho, S. Ama ˆncio * DBEB/CBAA, Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, Portugal Accepted 28 February 2002 Abstract This study reports the effects of light and CO 2 availability during the acclimatisation phase on micropropagated plantlets of a chestnut hybrid (Castanea sativa  Castanea crenata). After in vitro phases at 50 mmol m 2 s 1 , the plantlets were transferred directly from in vitro root induction to ex vitro acclimatisation under four different treatments corresponding to irradiances of 150 and 300 mmol m 2 s 1 each one in association with CO 2 concentrations of 350 and 700 mll 1 . For all treatments, the survival rates were above 80%. CO 2 concentration at 700 mll 1 promoted growth measured as relative growth rate and net assimilation rate. Leaf area was also promoted by high CO 2 , but without the corresponding increase in dry weight. A biomass increase of 100% was obtained when high CO 2 was combined with high irradiance. Total chlorophyll content of in vitro (persistent) and ex vitro (new) formed leaves was mainly affected by the irradiance. Chlorophyll a/chlorophyll b ratio of new leaves at the end of all the acclimatisation treatments attained the value of full autotrophic leaves. Unlike starch, the concentration of soluble sugars was significantly down- regulated by CO 2 concentration. Sucrose was exported from persistent leaves independently of the treatments. As growth proceeded, new expanded leaves became the carbohydrate source and exported sucrose to the growing apices and other new leaves. The effect of high irradiance (300 mmol m 2 s 1 ) combined with high CO 2 concentration (700 mll 1 ) on the acquisition of autotrophic behaviour by the plants is discussed. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Acclimatisation; (Castanea sativa  Castanea crenata); Chlorophyll; CO 2 ; Growth analysis; Irradiance; Soluble sugars; Starch Scientia Horticulturae 95 (2002) 151–164 * Corresponding author. Tel.: þ351-213-653-418; fax: þ351-213-635-031. E-mail address: samport@isa.utl.pt (S. Ama ˆncio). 0304-4238/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0304-4238(02)00037-7