The novel low xylan content transgenic cottonwood (P. trichocarpa) was used to elucidate recalcitrance of enzymatic saccharification with or without four different pretreatment methods. The xylan contents of two transgenic samples (8Di3 and 8Di5) were 11.4% and 11.7%, respectively, as compared with the wild type (16.0%). Contrarily, the lignin contents of two transgenic samples were 23.1% and 24.5%, respectively, as compared with the wild type (20.8%). The four pretreatments were dilute acid (0.1% sulfuric acid, 185 degrees C, 30 min), green liquor (6% total titratable alkali (TTA), 25% sulfidity based on TTA, 185 degrees C and 15 min), auto hydrolysis (185 degrees C, 30 min) and ozone delignification (25 degrees C, 30 mm). Following the pretreatment, enzymatic saccharification was carried out using three enzyme charges of 10, 20 and 30 FPU per gram of substrate. The removal of lignin and hemicelluloses varied with the type of pretreatment and with the lignin content of the transgenic trees. High lignin content implied low enzymatic saccharification. Low xylan content native substrates lead to high enzymatic saccharification. High S to V (sryingaldehyde to vanillin) ratio substrates had high delignification during pretreatment. Compared to the wild type, the transgenics were better choice as feed stocks due to higher enzymatic saccharification without pretreatment which mean low the cost of bio-ethanol. Compared to three pretreatment methods, the green liquor pretreatment greatly improves the conversion of polysaccharides in general. (C) 2011 Elsevier Ltd. All rights reserved.