In the last years, efforts have been carried out to propagate high-value trees through somatic embryogenesis (SE) in order to increase forest productivity. SE can capture the benefits of breeding or genetic engineering programs by multiplying trees with improved characteristics ( Pullman & Bucalo, 2014 ).

In this sense, Aleppo pine ( Pinus halepensis Miller), a species native to the Mediterranean basin, can thrive under semi-arid climatic conditions in a wide variety of soils ( Maestre & Cortina, 2004 ) and is susceptible to be used in large afforestations in the future scenario of climate change ( Jeddi et al ., 2009 ).

It is generally known that adjustments of tissue culture media can result in higher success of SE initiation and proliferation frequencies ( Montalbán et al ., 2012 ). However, few studies have focused on the impact of temperature ( Kvaalen & Johnsen, 2008 ), and the effect of different concentrations of agar in the culture medium has been studied mostly at maturation stage ( Teyssier et al ., 2011 ; Morel et al ., 2014 ). In this sense, in our laboratory García-Mendiguren et al . (2016) described in Pinus radiata (D. Don) that different temperatures and water availability conditions at initiation affected subsequent phases of SE; however, this effect on subsequent phases of the process disappeared when different environmental conditions were applied at proliferation. Then, Pereira et al . (2016) found out that different environmental conditions at initiation did not influence maturation and germination phases in P. halepensis . Taking all this information into account, our aim was to analyze the effect of physical and chemical conditions at proliferation stage of P. halepensis SE on the final success of the process including the obtention of somatic plants.

One-year-old cones of Pinus halepensis from five open-pollinated trees located in Berantevilla (Spain) were used. The cones were stored following Montalbán et al ., (2015) . Initiation of embryonal masses (EMs, Fig. 1a) was performed as described in Montalbán et al . (2013) . Ten embryogenic cell lines (ECLs) were randomly selected and four EMs with an approximate diameter of 16 mm from each ECL were transferred to different proliferation conditions (Fig. 2). Embryonal masses were subcultured after two weeks to the same proliferation condition. After one month in the different proliferation conditions described in Fig. 2, EMs were subjected to maturation. Maturation and germination were carried out according to Pereira et al . (2016) . At maturation, four replicates per ten ECLs and nine proliferation treatments (a total of 360 Petri dishes) were performed. At germination, three Petri dishes (with 20 somatic embryos per Petri dish) per each ECL and proliferation treatment were maintained under 16 h photoperiod at 100 µmol/(m ² s) provided by cool white fluorescent tubes (TFL 58 W/33; Philips, France). After 16 weeks on germination medium, the plantlets were transferred to sterile peat:perlite (70:30, v/v) and acclimatized in a controlled greenhouse, progressively decreasing the humidity from 99 to 70 %.

The water availability of the proliferation culture media was determined following Garcia-Mendiguren et al . (2016), performing five replicates per culture condition.

At the end of maturation stage, the percentages of maturation were calculated. Logistic regression and the corresponding analysis of deviance were conducted to assess the effect of the considered variables on maturation and germination rates. A heteroscedastic linear mixed model (and its analysis of variance) was conducted with the square root of the number of somatic embryos per gram of EM as the response variable. Temperature and agar concentration were considered as fixed effects and the ECL as a random effect.

A two-way analysis of variance was carried out to assess the effect of temperature and agar concentration on the water availability of the media and on ex vitro survival of somatic plantlets.

All EMs grew properly in the different environmental conditions assayed. The medium solidified with 5.5 g/L agar showed significantly lower water availability than the media solidified with 4.5 g/L and 3.5 g/L agar. Neither the temperature nor the interaction between the temperature and the agar concentration were statistically significant. These results are in agreement with previous studies on initiation stage ( Pereira et al ., 2016 ) where only significant differences were found for agar concentration.

As almost all ECLs and treatments produced somatic embryos (89 out of 90, Fig. 1b), no statistically significant differences in the percentages of maturation were found among temperatures nor among agar concentrations nor double interactions between the variables. With respect to the number of somatic embryos per gram of EM, statistically significant differences were found for the interaction between the temperature and the agar concentration. However, these differences were only observed between EMs proliferated at 23ºC in a culture medium containing 3.5 g/L agar (860 somatic embryos per gram of EM) and those proliferated at 28ºC with 3.5 g/L agar (1212 somatic embryos per gram of EM). Our results are in agreement with results obtained by Kvaalen & Johnsen (2008) in Picea abies , and opposite to findings reported by García-Mendiguren et al . (2016) in P. radiata where they did not find differences among proliferation treatments for somatic embryo production. Furthermore, different environmental conditions at initiation stage of P. halepensis SE, did not lead to significant differences in the number of somatic embryos obtained ( Pereira et al ., 2016 ). So, for this species, it appears that different environmental conditions have a greater influence when applied during proliferation than during initiation.

Statistical analysis of germination rates showed a significant interaction between the temperature and the agar. The highest germination rate (74%) was achieved in somatic embryos coming from EMs proliferated at 28ºC in a culture media supplemented with 5.5 g/L agar (Fig. 1c). However, embryos generated from EMs proliferated at lower temperatures (18ºC-5.5 g/L agar and 23ºC-4.5 g/L agar) did not show statistical differences in terms of germination with the abovementioned treatment (Fig. 3). The plantlets from EMs proliferated at 18ºC (69.5%) showed a significantly higher ex vitro survival than those proliferated at 28ºC (49.8%); plantlets from standard proliferation temperature (23ºC) did not show significant differences with the abovementioned temperatures (54.2%). The agar concentration at proliferation did not have a significant effect on ex vitro survival.

In summary, this work shows that manipulation of environmental conditions (temperature and water availability) during proliferation of EMs provokes an effect after several months in the production somatic plants. The best results are obtained proliferating the EMs at the lowest temperature assayed (18ºC) and reducing the water availability of the culture medium.