Effect of Light and Activated Charcoal on Tracheary Element Differentiation in Callus Cultures of Pinus Radiata D. Don

Abstract

Light has been found to increase the proportion of tracheary elements differentiating in callus cultures derived from xylem-parenchyma of Pinus radiata D. Don grown on an induction medium containing activated charcoal but no phytohormones. The differentiation rate increased from 20% when callus was grown in darkness to 45% when callus was grown with a 16 h or 24 h photoperiod. When callus was grown with a 16 h photoperiod, tracheary elements were observed 2 days after transfer of callus to the induction medium, as compared to 5 days when callus was cultured in darkness. The differentiation rate was also influenced by the concentration of activated charcoal added to the induction medium, the optimum concentration being 5 g l )1 . Exclusion of activated charcoal from the induction medium decreased the differentiation rate to 2%. The activities of the lignin-related enzymes L-phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase were significantly higher in cell cultures grown with a 16 h photoperiod as compared to when grown in darkness. The results show that light had a stimulating effect on tracheary element differentiation and the activities of lignin-related enzymes in P. radiata callus cultures. The new growth conditions markedly improve this cell culture system and make it particularly useful for functional gene testing and cell-wall analysis of in vitro grown tracheary elements of coniferous gymnosperms.

Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiata

“…P. radiata callus cultures were maintained and induced to differentiate TEs as described earlier (20). For TE purification, differentiated callus cultures (Ϸ3 g) were resuspended in Mops-KOH buffer (20 mM; pH 6.8; 10 ml), containing 20 mM sodium metabisulfite and homogenized by using a Retsch (Newtown, PA) PM200 ball mill for 20 min (vibration frequency, 1,800 min Ϫ1 ) in Teflon vials (25 ml) containing Teflon beads (2 ϫ 8 mm).…”

Abiotic and Biotic Stresses and Changes in the Lignin Content and Composition in Plants

“…During the differentiation of cultured calli of Pinus radiata into tracheids, it was found a positive effect of light on the activity of the enzymes PAL and CAD, which resulted in an increase in the concentration of lignin when the calli were transferred from dark to a photoperiod of 16 h (Möller et al 2006). Continuous light (16.7 W/m 2 ) increased the activity of anionic and cationic PODs and laccases in mungbean hypocotyls, resulting in an increase in the lignin content (Chen et al 2002).…”

Characterization of the structure, expression and function of Pinus radiata D. Don arabinogalactan-proteins

“…Radiata pine callus culture cells were grown as described previously (Möller et al 2006). Tracheid diVerentiation was induced for 24 h on tracheid induction medium (TIM) supplemented with 5 g/l activated charcoal, after which the cells were transferred to TIM containing 5, 10, 20 and 30 M -GlcY and 30 M -GalY reagent.…”

“…Tracheid diVerentiation was induced for 24 h on tracheid induction medium (TIM) supplemented with 5 g/l activated charcoal, after which the cells were transferred to TIM containing 5, 10, 20 and 30 M -GlcY and 30 M -GalY reagent. Rate of tracheary element diVerentiation was determined as described in Möller et al (2006). Callus cells were gently suspended in 0.5 ml P6-SHv medium (Hotter 1997) containing 1 l 0.1 mg/ml FDA and cell viability was calculated on the basis of more than 200 cells counted under UV-light using a Zeiss Axio microscope.…”