Carla Ragonezi, Mário Rui Castro, Mónica Lima, Maria Amely Zavattieri Laboratório de Melhoramento e Biotecnologia Vegetal, ICAM, Universidade de Évora; Apartado 94, 7002-554 Évora Codex. (e-mail: cazi04@hotmail.com) Materials and Methods The basal medium used for all rooting experiments was WPM (Lloyd & McCown 1981) with half macronutrient, containing Difco Bacto-Agar (0.65%) and a carbon source (see below), and adjusted to pH 5.8 using NaOH (1N) before autoclaving. For the induction phase (two weeks) this medium was supplemented with NAA (Naphthalene acetic acid) 10.7μM (2mg/L) as in Oliveira (2003) and contained 0.117M (23.185 g/L) of glucose (WPMRI). It was then placed in a growing chamber for one week at 19 ºC in total darkness and one week in 16 h photoperiod, 19 ºC day/night temperatures. The induction was followed by the expression phase on WPM basal medium, also with half the macronutrients, without growth regulators and 0.0584 M (11,59 g/L) of glucose (WPMRE), 16 h photoperiod, 24/19 ºC day/night temperatures. The root emergence was studied during six weeks. Introduction Micropropagation of conifers has been shown to be feasible but some species, like Pinus pinea L., are recalcitrant to standard treatments. The main problem of micropropagation of these pines for large production of good clones is the rooting of micro cuttings, a problem that remains unsolved. Attempts at improving the process, by various research groups, have met with limited success, but were unsuitable for the transition to acclimation. In particular, our group has obtained a significant advance by changing carbon source and temperature during the induction phase of in vitro rooting (Potes et al. 1999, 2001). Even then, these results are not always consistent, various clones remain recalcitrant, and there is a precocious halt in root development that impairs an efficient acclimation phase. The results presented here represented one year of work to improve the previous established protocol for the induction and expression phase of rooting and is a part of an ongoing Project on “Analysis and Mastering of Root Growth Signalling by Ectomycorrhizal Fungi on Pinus pinea Microshoot Cultures” financed by Portuguese Government - FCT Project 71437/2006. Main Conclusions Results Fig. 3 – Elongation phase of the roots Fig. 2 – Expression phase of the rooting process Fig. 4– Acclimatation phase in mixed substrate Fig. 6 – Rooting percentage per clone tested in essay 9 after 6 weeks Fig. 5 – Rooting percentage per essay at the end of the expression phase Fig. 7 –Estimated root expression per week, considering all the essays presented in fig. 5. estimated average per week and accumulated Table 1 – Media composition and physical conditions of the essays included in fig. 6 WPMRI: WPM induction medium; WPMRE: WPM expression media Essays Induction phase medium Induction phase – physical conditions Expression phase medium Expression phase 8 WPMRI (with 2mg/L of NAA) 1 st week in the dark, at 19º C, follow by one week 19º /16ºC with 16h photoperiod WPMRE 19º /16ºC with 16h photoperiod 9 WPMRI (with ½ of macro and 2mg/L of NAA) 1 st week in the dark, at 19º C, follow by one week 19º /16ºC with 16h photoperiod WPMRE (½ of macro) 19º /16ºC with 16h photoperiod 10 WPMRI (with ½ of macro and 2mg/L of NAA) 1 st week in the dark, at 16º C, follow by one week 19º /16ºC with 16h photoperiod WPMRE (½ of macro) 19º /16ºC with 16h photoperiod 11 WPMRI (with ½ of macro and 2mg/L of NAA) 4 days in the dark, at 16º C, follow by 10 days 19º /16ºC, with 16h photoperiod WPMRE (½ of macro) 19º /16ºC with 16h photoperiod 12 WPMRI (with ½ of macro and 1,5 mg/L of NAA) 1 st week in the dark, at 19º C, follow by one week 19º /16ºC with 16h photoperiod WPMRE (½ of macro) 19º /16ºC with 16h photoperiod 13 4 days in bi – distiled esteril water and WPMRI (with ½ of macro and 2mg/L of NAA) 1 st week 19º /16ºC 16h photoperiod follow by one week 19º /16ºC with 16h photoperiod WPMRE (½ of macro) 19º /16ºC with 16h photoperiod 15 WPMRI (with ½ of macro and 2mg/L of NAA macros) 1 st week in the dark 19º /16ºC 16h/8h, 24/19ºC follow by one week 24º /19ºC with 16h photoperiod WPMRE (½ of macro) 19º /16ºC with 16h photoperiod Fig. 1 – Growth of microshoots in activated charcoal 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 0 2 4 6 Percentage of Rooting Week Real rooting percentage per essay per week E8 E9 E10 E11 E12 E13 E15 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% 90,00% PAC5 P1M23 P1M25 P1M30 P1M37 P1M38 Percentage of Rooting Clone Rooting percentage per clone 0,00% 20,00% 40,00% 60,00% 80,00% 100,00% 0 1 2 3 4 5 6 Percentage of Rooting Week Estimated rooting per week 1 –The combination of high glucose concentration, 2 mg l -1 of NAA, half macronutrients composition (WPMRI 1/2) and 19ºC in the induction phase of the rooting process was the most effective combination for most of the clones tested (essay 9; fig.7). The reduction of macronutrients composition enhanced the rooting percentage in more than 20% over the same conditions comparatively with complete macro nutrients composition. Nevertheless, few clones remained recalcitrant to the induction treatment. It will be necessary to study the reason of this behaviors. 2 – With the analysis of all essays, it was possible to established that a peak of rooting occurs always in the second week of the expression phase (fig.9). This peak represent approx. 60% of the expected root expression (fig. 8). Based in that, it’s possible to estimate the total number of microshoot s per clone that will have roots at the end of 6 weeks, before transplanting to substrate mixture.