185 1. Introduction Norway spruce [Picea abies (L.) Karst.] is an economi- cally important tree species that plays a key role in natural ecosystems in the boreal zone from Norway to Siberia, and is one of the most valuable European forest tree species. The natural distribution of P. abies in Italy spans the entire Alpine range, where it ascends to an altitude of 2,000 m, and it is also found in part of the northern Apennines. A large portion of the spruce forests are used commercially and managed with varying levels of intensity. In Italy, this species is also the most cultivated for the production of Christmas trees for its conical shape, compact, intense green foliage, and rapid growth. The Christmas trees pres- ent on the Italian market are certified because they derived from 90% cultivations in forest nurseries and the trees are usually obtained by seed. Seeds are collected from selected stands, and the obtained seedlings are cultivated in a nursery for almost four years: two years as “seed- lings” in seedbeds, and two to three years as “transplan- tations” in fields depending on the desired size. Christ- mas tree cultivation is concentrated mainly in Veneto and Tuscany (Arezzo and Pistoia districts) where about 800 hectares are devoted to this crop. Nevertheless, a consider- able amount of the seedlings are imported from Belgium, Denmark, Holland, and Germany for the establishment of Christmas tree plantations. Recently, increased importa- tion from Hungary, Romania and Moldavia has evidenced problems related to the genetic control of propagation ma- terial. Thus, to avoid genetic mixing between autochtho- nous species and those coming from abroad, the imported seedlings carry a special label which certifies their origin from specialized cultivations, nationality, and indication as not intended for reforestation. A sustainable Christmas tree market should aim to in- crease the production of plants from autochthonous species characterized by high levels of germinability and growth, and to produce seedlings of adequate size and quality in a short time for transplantation. The commercial importance of genetic characteristics and origin of the propagation material for plantation quality are well documented. In particular, rates of early growth and morphological and physiological characteristics are among the features that differ between populations. Consequently, the provenance of seeds in relation to the choice of planting site is extremely important. Furthermore, identification of both the best seed provenance and environmental conditions for germination and growth of seedlings could be the first objective to improve nursery production. The influence of provenance has not been widely reported on seed germi- nation of Picea, while it is well documented for seedling growth. At provenance level, strong relationships are gener- Influence of seed provenance on the propagation of Picea abies (L.) Karst L.M.R. Rinaldi ¹, A. Leva ¹ (*), L.P. D’Acqui ² ¹ Istituto per la Valorizzazione del Legno e delle Specie Arboree, Consiglio Nazionale delle Ricerche, Via Madonna del Piano, 10, 50019 Sesto Fiorentino (FI), Italy. ² Istituto per lo Studio degli Ecosistemi, Consiglio Nazionale delle Ricerche, Via Madonna del Piano, 10, 50019 Sesto Fiorentino (FI), Italy. Key words: germination, growth, Picea abies, somatic embryogenesis, substrate. Abstract: Picea abies (L.) Karst. is the most common species cultivated in Italy for sale as Christmas trees. The aim of this study was to identify the best seed provenance to improve plant production. Three Italian provenances - Gran Bosco di Salbertrand, Pezzel e Fochino, and Val di Fiemme - were examined. A part of seeds were sown on benches in a greenhouse after cold stratification, and another part was used for induction of somatic embryogenesis. In the first experiment, two different amounts of organic matter (manure), as components of growing media, were evaluated, while in the second one different levels and combinations of growth regulators in the medium were tested. The seed provenance and composition of the growing medium influenced seed germination and seedling growth. The best performance on both growing media was achieved by the Val di Fiemme provenance, and in general seedling emergence and growth were the most favourable in the growing medium with a lower organic matter amount. Also, the in vitro cultures evidenced differences in the ef- ficiency of somatic embryogenesis among the provenances. Adv. Hort. Sci., 2015 29(4): 185-191 * Corresponding author: leva@ivalsa.cnr.it Received for publication 21 July 2015 Accepted for publication 1 August 2015 186 Adv. Hort. Sci., 2015 29(4): 185-191 ally observed between traits that characterise the duration of the growth period and the degree of lignification. Variation in bud flushing and initiation of shoot growth among plants derived from different Norway spruce seed provenances is assumed to be regulated both by differential responses to the accumulated temperature sum in spring and by conditions during acclimation in the preceding year (Schmidt-Vogt, 1976; Dormling, 1982). Accordingly, trees from with north- ern latitude or high altitude provenances, which are adapted to a short growing season, have inferior growth potential compared with those adapted to a longer growing season. Variation between plants from different provenances has been demonstrated for a number of other traits, such as nu- trient demands, respiratory activity, shade tolerance, and differences in crown shape, and resistance to snow and ice damage (Schmidt-Vogt, 1976). On the other hand, seedling morphological characteristics before planting were found to highly affect seedling growth during the first years after transplantation (Tsakaldimi, 2006). Furthermore, fertilizer application during two years improved survival and increased annual height of Betula pu- bescens and Larix sibirica (O´Skarsson et al., 2006). Many studies have reported the effect of manure on increase di- ameter and height growth of some species of Pinus (Nour- shad and Ghorani, 1990), and biomass (root and shoot dry weight) of potted and bare rooted P. taeda seedlings (Kiani et al., 2005). Moreover, combinations of types of soil in different ratios of nutrients have also influenced seed ger- mination of important forest species (Selivanovskaya and Latypova, 2006). Germination, survival, growth, and bio- mass of Cupressus arizonica and Cupressus sempervirens are enhanced using organic matter and in particular, manure has increased the maximum quality index (Dickson et al., 1960; Ahmadloo et al., 2012). Therefore, the aim of this study was to assess the in- fluence of seed provenance on emergence and seedling growth of Picea and, as an ancillary purpose, to evaluate if an increase of the amount of organic matter (dust manure) in the growing media compared to that commonly used can improve or affect the emergence and seedling growth of Picea seeds of different provenance. Further, consid- ering that somatic embryogenesis is potentially the most promising method for vegetative propagation of conifer- ous genera (Dunstan et al., 1995), the influence of seed provenance has also been evaluated on somatic embryo germination starting from embryogenic tissues. 2. Materials and Methods Seed lots The seeds were provided by the National Centre for the Study and Maintenance of Forest Biodiversity (CNBF) of Peri (Verona, Italy). CNBF is primarily involved in the pro- duction, selection, and conservation of forest seeds (of trees and shrubs). Specifically, the forest seeds produced by the Centre come from forests enrolled in the “Libro Nazionale Boschi da Seme” (L.N.B.S.), and traditional and “special” harvesting methods are used in order to preserve the genetic diversity of the species. All the material is certified and trace- able through Global Positioning System (GPS) technology (http://www3.corpoforestale.it). In this study, mature seeds of Picea abies (L.) Karst. harvested in October 2011 from selected populations of the following three Italian prov- enances were evaluated: Gran Bosco di Salbertrand - GS (L.N.B.S n. 088, CSR n. 034-Pie; 45°N; altitude 1030-1900 m); Pezzel e Fochino - PF (L.N.B.S n. 137 SO 037; 46°N; 1350-1540 m); and Val di Fiemme - VF (L.N.B.S n. 023 Val di Fiemme TN; 46°N; 1600-2000 m). Certification of seed quality (purity, weight of 1000 seeds, germinability, cut test, viability, value crop) in accor- dance with the ISTA (International Seed Testing Associa- tion) was carried out by CNBF and is reported in Table 1. In vivo experiment The seeds from each provenance were stored sepa- rately in plastic bags at 5°C. Later, two sowing beds (each 1.60 m × 5.0 m) were prepared in a greenhouse. A common nursery soil, composed of a mixture of 75% sand, 10% perlite, and 15% organic material (mixture of peat, manure) for a total organic C content of 6.26%, was utilized for the first bed (Substrate 1). The same substrate with a rate higher than one-and-a-half of organic mate- rial (total organic C 9.82% ) was used for the second bed (Substrate 2) (Table 2). Chemical parameters of growing media were determined according to the SISS methods (SISS, 1985), whereas the ones of organic carbon (OC), nitrogen (N), and inorganic carbon (IC) by using NA 1500 CHNS Analyzer, Carlo Erba (Milan, Italy) coupled with the procedure reported by Santi et al. (2006). In March 2012, an average of 20 and 15 g seeds, respectively, for GS, PF, and VF provenances were sown on each sowing bed. Before the trials, the seeds were soaked in water for 12 h. The seeds were sown in seven rows, and a thin layer Table 1 - Certification of seed quality Seed provenance Purity (%) Weight 1000 seeds Germinability (%) Cut Test (%) No. viable seeds/g Value crop (%) Gran Bosco Salbertrand (LNBS n.088 (SR n.054- Pie.) 94.5 7 71 91 96 67.1 Pezzel e Fochino (LNBS n.137 SO037) 92 6.4 74 88 113 68.1 Val di Fiemme (LNBS n.023 Val di Fiemme) 97.2 8.8 92 97 119 89.4 187 Rinaldi et al., Influence of seed provenance on the propagation of Picea abies (L.) Karst of sand was applied to cover the seeds. The sowing beds were watered as necessary. After emergence, the seed- lings were fertilised twice at intervals of 15 days with 50 ppm Flory 4 (Agrochimica). Emergence rates were recorded in March, April, and May. The final stem height (cm) was determined for all seedlings at 90 days, the time of their transfer to pots. In June 2012 the seedlings were transferred to pots (volume 80 ml) filled with the same growing media. Two hundred seedlings per provenance were distributed in five multipot PVC pots of 40 places each. The seedlings were transferred to a shaded (50% light reduction) location outdoors and cultivated until the end of July 2013. Growth was evaluated after 4, 12 and 52 weeks. On the final date, 15 seedlings per provenance were randomly selected for root and shoot biomass mea- surements. The fresh weight (FW) and dry weight (DW), obtained by drying in an oven at 70°C for 48 h, were de- termined. The root-to-shoot DW ratio was also calculated for each seedling. In vitro experiment For induction of embryogenic tissues, the seeds were sterilized in 2% (v/v) sodium hypochlorite solution for 20 min and rinsed three times with sterile distilled water. The zygotic embryo was dissected from each seed and placed on solid BM1 medium (Gupta et al., 1987) supplemented with 1% sucrose, hydrolysed casein (500 mg/l), L-glu- tamine (450 mg/l), and myo-inositol (1g/l), and solidified with 0.3% Phytogel. Five or 10 μM 2,4-dichlorophenoxy- acetic acid (2,4-D) in combination with 2.5 or 5 μM 6-ben- zyladenine (BA) were included in the medium. The trial was carried out in Petri dishes (100 mm × 15 mm) with 40 embryos per provenance, divided into five replicates of eight embryos each. The embryos were incubated in the dark at 20°C. The cultures were assessed after incubation for four weeks. For culture proliferation, the embryogenic tissues were subcultured three times on fresh medium and kept in the dark for development of early-stage somatic embryos. The fresh weight of embryogenic tissues was measured at the end of each subculture. For somatic embryo matu- ration, embryogenic tissues from material derived from each provenance were transferred to medium containing abscisic acid (ABA) to allow maturation of the somatic embryos. The maturation medium was BM1 medium sup- plemented with 20 μM ABA, 2% sucrose, and 7.5% poly- ethylene glycol (PEG-4000). Embryogenic tissue clumps growing vigorously and of about the same size (about 1 cm³ in volume) were selected to monitor the embryo matu- ration process. The cultures were maintained in the dark at 20°C for about five weeks until the somatic embryos reached cotyledonary stage. Thirty randomly selected ma- ture somatic embryos per provenance were transferred to germination BM1 basal medium supplemented with 0.2% activated carbon (AC), 2% sucrose and 0.4% Phytogel. The embryos were cultured under photoperiod (16h/8h, light/dark) for eight weeks, after which time the plantlets were transferred to ex vitro conditions. They were placed in multipot PVC pots filled with a mix of pumice, peat, and organic fertilizer, and they were transferred to green- house conditions. Collection data and statistical analysis With regard to seedling emergence in the greenhouse, the number of seeds that were sown on each seed bed for each provenance was calculated using the proportion: Weight in grams of 1000 seeds:1000=Weight in grams of seeds sown on each bed:x For each provenance, seedling emergences were nor- malized with respect to the number of seeds put to germi- nate on each bed. Therefore, the data on the emergence of seedlings from different provenances were normalized to 100 according to this proportion: Seedling emergence:Total seeds put to germinate x row of seed bed=x:100 Seedling data (emergence and height) were arranged in a completely randomized design, and the statistical sig- nificance of all differences in emergence and height of the seedlings was tested using factorial analysis of variance (ANOVA), with the substrate and provenance as the main factors. Differences among provenances were evaluated using Tukey’s Multiple-Range Tests (Snedecor and Co- chran, 1980) at the 5% level of significance. For seedling growth parameters, means and standard errors were calcu- lated for material derived from each provenance. All statis- tical analyses were performed with STATISTIX software version 8 (Analytical Software, Tallahassee, FL, USA). 3. Results In vivo experiment The three provenances significantly (p<0.05) varied in emergence and seedling height (cm) in the two different Table 2 - Chemical characteristics of growing media Growing media Substrate 1 Substrate 2 C(z) (%) 6.26 9.82 Organic matter %) 10.91 17.25 N (y) (%) 0.2 0.39 C/N 31.3 25.17 IC (x) (%) 2.83 1.47 CEC (w) ( mol c kg-1 ) 14.4±2.7 32.0±3.9 pH 7.3±0.2 7.1±0.2 (z) C= Organic carbon; (y) N= Nitrogen; (x) IC= Inorganic carbon; (w) CEC= Cationic exchange capacity. 188 Adv. Hort. Sci., 2015 29(4): 185-191 substrates used in the seed beds (Table 3). Seed provenance significantly affected all these processes. In addition, the substrate effect was also significant except at 15 days after sowing, and the substrate x provenance interaction effect was also significant. More precisely, seedling emergence in the greenhouse occurred 15 to 30 days after sowing, and thereafter it was very low or non-existent (data not shown). With regard to substrate 1, which contained a lower organic matter content, seedling emergence differed significantly among the three provenances at both 15 and 30 days after sowing. At 15 days, VF showed 58% seed- ling emergence, whereas GS and PF showed 38 and 20% seedling emergence, respectively. After 30 days, the per- centage of seedling emergence for PF increased to 25%, whereas GS and VF (Fig. 1A) showed the highest seedling emergences of 62 and 91%, respectively. As for substrate 2, which contained the highest organic matter content, the emergence percentage ranged from 34% for GS to 36 and 38% for VF and PF, respectively, after 15 days. Subse- quently, a large percentage of seedlings for GS and PF ap- peared flaccid and twisted as if suffering from wilt. These seedlings continued to decline, assumed a dark colour and greasy appearance, and then quickly disintegrated to leave areas of bare soil in the seed bed (Fig. 1B). After 30 days, seedling emergence was reduced to 20%, whereas that for VF increased to 47%. Seeds from the VF provenance generated the most seedlings on both substrates; seeds from PF displayed the worst performance on the same substrates at the end of culture in the greenhouse (Table 3). In contrast, GS prov- enance produced the most seedlings only on substrate 1. The final stem height was determined for all seedlings at 90 days from sowing. The stem height of seedlings ranged from 2.4 to 4.3 cm on substrate 1, and from 2.1 to 3.2 cm on substrate 2. VF seedlings showed the greatest stem height on substrate 1; PF seedlings exhibited the lowest on both substrates. The stem height of GS seedlings was highest on substrate 1. The growth trends were not different for seedlings of the provenances measured after 4, 12 and 52 weeks culti- vation in pots (Fig. 2). At the time of initial measurement (4 weeks), PF (7.9 cm) and GS (7.4 cm) seedlings showed superior stem growth compared to VF seedlings (6.4 cm). At the end of the cultivation period (52 weeks), GS and PF seedlings had mean stem heights of 14.7 and 13.8 cm, respectively, whereas VF seedlings attained a mean height of 10 cm (Fig. 2). The minimum standard defined by the European Economic Community for growth of three-year- old seedlings is 14 cm in height (E.E. C. Minimum Stan- dards) (Magini, 1977). Therefore, the recorded growth in seedlings of 15 months old from GS and PF provenance exceeded the minimum standard. Among the provenances, seedlings grown in pots exhibited changes in early growth, and fresh and dry matter allocation (Table 4). GS seedlings showed significantly higher values in fresh shoot biomass. On the other hand, VF provenance, compared with GS, Table 3 - Seedling emergence and early growth of three provenances of P. abies in two different substrates Growing medium Provenance Seedling emergence (%) Seedling height (Z) (cm)15 days 30 days Substrate 1 Gran Bosco di Salbertrand 38 b 62 b 4.3 a Pezzel e Fochino 20 c 25 cd 2.4 d Val di Fiemme 58 a 91 a 3.7 b Substrate 2 Gran Bosco di Salbertrand 34 bc 20 d 2.6 d Pezzel e Fochino 38 b 20 d 2.1 d Val di Fiemme 36 bc 47 bc 3.2 c Two-way ANOVA (P values) Substrate (S) 0.4152 0.0000 0.0000 Provenance (P) 0.0005 0.0000 0.0000 S x P 0.0001 0.0013 0.0000 Values for each parameter, column, and factor followed by different letters are significantly different (Tukey’s multiple range test, p < 0.05). (z) The final stem height was determined for all seedlings at 90 days from sowing: the average for Gran Bosco di Salbertrand, Pezzel e Fochino and Val di Fiemme was calculated on 200, 123 and 331 seedlings for the substrate 1 and on 867, 257 and 801 seedlings for the substrate 2, respectively. Fig. 1 - Emergence and growth of seedlings on different seed beds at 30 days after sowing: (A) seedlings from the provenance Val di Fiemme on substrate 1; (B) at left, seedlings from the provenance Pezzel and Fochino, showing areas of bare soil, and at right, seed- lings from the provenance Val di Fiemme on substrate 2. 189 Rinaldi et al., Influence of seed provenance on the propagation of Picea abies (L.) Karst showed similar values in dry shoot biomass, root biomass, and root/shoot ratio, but lower values were always found in PF provenance. In vitro experiment Explants from all examined provenances give rise to embryogenic tissues (Table 5). The mature zygotic em- bryos from GS seeds yielded a higher frequency (13.5%) of embryogenic tissue induction, compared with those for PF (8.6%) and VF (5.5%). A higher frequency (13.4%) of embryogenic tissue induction, independent of provenance, was achieved on medium supplemented with low con- centrations of growth regulators (5 μM 2,4-D and 2.5 μM BA) than on medium with higher concentrations of growth regulators (10 μM 2,4-D and 5μ M BA). With regard to the proliferation of embryogenic tissues, only the prov- enance GS showed the highest increase in fresh weight of embryogenic tissue and the capacity for embryo matura- tion in the presence of low concentrations of growth regu- lators. In contrast, a greater increase in embryogenic tissue fresh weight was achieved for all provenances on medium supplemented with the higher concentrations of 2,4-D and BA. These cultures were whitish to translucent, and were characterised by vigorous growth (Fig. 3 A, B); maturation of embryogenic tissues for each provenance is reported in Table 5. Specifically, GS and PF embryogenic tissues showed the highest maturation efficiency (mean of 52 and 73 somatic embryos per clump, respectively), whereas the VF embryogenic tissues produced an average of only 24 somatic embryos per clump. The maturation process up to the cotyledonary stage required about 5 weeks of culture on the maturation medium (Fig. 3 C-E). Over 74% of the somatic embryos germinated rapidly after transfer to the germination medium. The development of plantlets with true needles was observed within 1 to 2 weeks (Fig. 3F). Table 4 - Morphological characteristics of Picea abies seedlings grown in pots outdoors Provenance Mean number Shoot biomass (g) Root biomass (g) Root to shoot ratio Branche Roots Fresh Dry Fresh Dry G S 3.0±0.46 a 9.7±1.34 a 5.15±0.09 a 2.71±0.08 a 1.23±0.12 a 1.06±0.07 a 0.395 a P F 1.3±0.44 a 7.0±1.29 a 2.15±0.18 c 1.88±0.16 b 0.52±0.12 b 0.44±0.07 b 0.257 a V F 1.8±0.63 a 7.0±1.29 a 4.45±0.10 b 2.66±0.09 a 1.16±0.14 a 1.05±0.09 a 0.390 a All parameters of growth were evaluated after 52 weeks of growth in pots. The data were subjected to analysis of variance. Values are the mean of a minimum of 15 seedlings for each provenance. Means within a column followed by a different letter are significantly different (Tukey’s multiple range test, P< 0.05). GS= Gran Bosco di Salbertrand; PF= Pezzel e Fochino; VF= Val di Fiemme. Table 5 - Induction of embryogenic cultures, proliferation, and somatic embryo maturation Provenance Embryogenic cultures (%) Increase of mass embryogenic (g) Embryo maturation 5μM 2.4D+ 2.5μM BA 10μM 2.4D+ 5μM BA No. somatic embryos/clump Total no. embryos/clump Gran Bosco Salbertrand 13.5±2.35 a 1.09±0.60 a (z) 1.51±0.26 a 52.0±15.0 b 619 Pezzel e Fochino 8.6±2.72 a 0.22±0.15 a 1.66±0.39 a 73.0±11.5 a 881 Val di Fiemme 5.5±2.11 a 0.30±0.10 a 1.13±0.48 a 24.0±9.0 c 288 Growth regulators 5μM 2.4D+ 2.5μM BA 13.4±1.57 a 10μM 2.4D+ 5μM BA 5.0±2.72 b (z) Data represent increase of fresh weight over time. The data were subjected to analysis of variance. Values within a column followed by a different letter are significantly different (Tukey’s multiple range test, P < 0.05). Fig. 2 - Stem heights of Picea abies seedlings grown in pots outdoors measured after 4, 12, and 52 weeks of cultivation in pots. Data represent the means (±SE) for seedlings from the Gran Bosco di Salbertrand (1), Pezzel e Fochino (2), and Val di Fiemme (3) provenances. A minimum of 200 seedlings were measured for each provenance. 190 Adv. Hort. Sci., 2015 29(4): 185-191 4. Discussion and Conclusions The efficiency of P. abies propagation from seeds and by somatic embryogenesis was closely related to the provenance of the plant material. Furthermore, the emergence and growth of seedlings propagated in seed beds, and the capacity for somatic embryogenesis were influenced by the composition of the growing medium. With regard to seed propagation, the best performance in terms of production and seedling growth was achieved by the Val di Fiemme provenance on both growing me- dia, whereas the Pezzel e Fochino provenance showed the worst performance. Moreover, the positive results of VF provenance on emergence and growth of seedlings in seed beds are also confirmed by the best values regarding seed quality of this provenance. In addition, the organic matter added to growth medium and its content should be taken into account. Malakouti and Homaei (2004) report- ed that organic matter improved seed germination and seedling growth, providing suitable conditions for seed- ling production. On the contrary, this study shows that the highest content of organic matter seems to induce a detrimental effect on growth and seedling survival. Probably high amounts of organic matter in the nursery environment promotes favourable conditions for the de- velopment of pathogens living in the growing medium. Therefore, the mortality of Gran Bosco di Salbertrand and Pezzel e Fochino seedlings could be due to a disease, likely damping off, as evidenced from areas of bare soil in the seed beds. In contrast, the VF seedlings showed su- perior growth and survival and seem to be more resistant to pathogens. Consequently, control of organic-matter content, humidity level in seed beds, and reduction in the duration of cultivation under greenhouse conditions are important factors to achieve a high percentage of seed- ling emergence and growth. At the end of the cultivation period in pots, the fifteen-month seedlings of GS prov- enance showed a height growth in excess of the minimum standard for three-year-old seedlings. However, the VF and GS provenances showed similar values in dry matter, Fig. 3 - Somatic embryogenesis from mature zygotic embryos of Picea abies; proliferation of embryogenic tissues (A and B); maturation (C-E) and germination (F) of somatic embryos. 191 Rinaldi et al., Influence of seed provenance on the propagation of Picea abies (L.) Karst while lower values were always found in PF. On basis of these results, the production of Christmas trees could be increased by the use of Val di Fiemme seeds, which showed higher germinability and growth of seedlings and by shortening the time for transplanting. To the authors’ knowledge, this is the first report of the application of somatic embryogenesis technology to P. ab- ies seeds from different Italian provenances. Our results demonstrate a low production of embryogenic tissues, and among the provenances a different efficiency was evident with regard to the proliferation and maturation of somatic embryos. Only the GS provenance was able to develop somatic embryos capable of germination on both prolif- eration media; the other provenances gave rise only to so- matic embryos on medium with a high concentration of growth regulators. Specifically, GS and PF provenances showed the highest maturation efficiency in terms of the number of embryos per embryogenic clump. These results obtained in vitro are in line with previous studies conduct- ed on conifers. Chen et al. (2010) reported low frequency of embryo initiation and genetic specificity of explants as serious problems associated with embryogenesis induc- tion. Moreover, the induction of somatic embryogenesis varies greatly among different genotypes under identical culture protocols and proliferation and maturation levels have been identified as major constraints to somatic em- bryogenesis (Tang et al., 2001). In order to improve the efficiency of propagation from seeds and by somatic embryogenesis, further studies will be finalized to manipulate the composition of the growing medium to improve the emergence and growth of seed- lings and frequency of embryogenic tissues. Acknowledgements The Norway spruce seed material was kindly supplied by the National Centre for the Study and Maintenance of the Forest Biodiversity of Peri (Verona, Italy). The authors are grateful to the” Istituto per l’Agricoltura ed Ambiente” of Firenze for providing the planting site for the trials, Prof. G. Coppola for the helpful suggestions, and P. Pestelli for his support in seedling production in greenhouse. References AHMADLOO F., TABARI M., HAMED Y., KOOCH Y., AH- MAD R., 2012 - Effects of soil nutrient on seedling perfor- mance of arizona cypress and medite cypress. - Annals of Biological Research, 3(3): 1369-1380. CHEN S., CHEN S., CHEN F., WU T., WANG Y., SHANJUN Y., 2010 - Somatic embryogenesis in mature zygotic embryos of Picea likiangensis. - Biologia, 65(5): 853-858. DICKSON A., LEAF A.L., HOSNER J.F., 1960 - Quality ap- praisal of white spruce and white pine seedling stock in nurs- eries. - The Forestry Chronicle, 36(1): 10-13. DORMLING I., 1982 - Frost resistance during bud flushing and shoot elongation in Picea abies. - Silva Fennica, 16: 167-177. DUNSTAN D.I., TAUTORUS T.E., THORPE T.A., 1995 - So- matic embryogenesis in woody plants, pp. 471-538. - In: THORPE T.A. (ed.) In vitro embryogenesis in plants. Klu- wer Academic Publishers, Dordrecht, The Netherlands, pp. 566. GUPTA P.K., DURZAN D.J., FINKLE B.J., 1987 - Somatic polyembryogenesis in embryogenic cell masses of Picea ab- ies (Norway spruce ) and Pinus taeda (loblolly pine) after thawing from liquid nitrogen. - Canadian Journal of Forest Research, 17: 1130-1134. ITALIAN SOCIETY FOR SOIL SCIENCE, 1985 - Metodi Nor- malizzati di Analisi del Suolo. - Edagricole, Bologna, Italy. KIANI B., SHARAJI ROSTAMII T., TAHERI F., 2005 - Study- ing ability rhizogenesis Pinus taeda seedlings in bare root and potting condition. - Iranian Journal Natural Resources, 58(2): 333-338. MAGINI E., 1977 - Appunti di vivaistica forestale. - Clusf Co- operativa Editrice Universitaria. MALAKOUTI M.J., HOMAEI M., 2004 - Soil fertility of arid and semi-arid regions (Difficulties and Solutions). - Tarbiat Modares University Press, Tehran, Iran, pp. 482. NOURSHAD M., GHORANI M., 1990 - Research projects se- lection the best soil mixture in order to container seedlings production. - Afforestation and Park Bureau Press, Mazan- daran, pp. 57. O´SKARSSON H., SIGURGEIRSSON A., RAULUND-RAS- MUSSEN K., 2006 - Survival, growth, and nutrition of tree seedlings fertilized at planting on Andisol soils in Iceland: Six-year results. - Forest Ecology and Management, 229(1- 3): 88-97. SANTI C.A., CERTINI G., D’ACQUI L.P., 2006 - Direct deter- mination of organic carbon by dry combustion in soils with carbonates. - Communications in Soil Science and Plant Analysis, 37: 155-162. SCHMIDT-VOGT H., 1976 - Die Fichte. Band I. Taxomomie, Verbreitung, Morphologie, Okologie, Waldgesellschaften. - Verlag Paul Parey, Hamburg and Berlin. SELIVANOVSKAYA S.Y.U., LATYPOVA V.Z., 2006 - Effects of composted sewage sludge on microbial biomass, activity and pine seedlings in nursery forest. - Waste Management, 26(11): 1253-1258. SNEDECOR G.W., COCHRAN W.G., 1980 - Statistical meth- ods. - Iowa University Press, Ames, Iowa, USA. TANG W., WHETTEN R., SEDEROFF R., 2001 - Genotypic control of high-frequency adventitious shoot regeneration via somatic organogenesis in loblolly pine. - Plant Science, 161: 267-272. TSAKALDIMI M., 2006 - Kenaf (Hibiscus cannabinus L.) core and rice hulls as components of container media for grow- ing (P. halepensis M.) seedlings. - Bioresource Technology, 97(14): 1631-1639.