1 Introduction

Radiata pine ( Pinus radiata D. Don) is one of the most widely planted exotic pine species in rainfall environments of the Southern hemisphere (Yan et al. 2006). Its fast growth has stimulated an exhaustive study of wood production, and the development of breeding programs (Espinel et al. 1995; Codesido and Fernández-López 2009). Although utility of in vitro organogenesis has been proven for clonal propagation of this species (Aitken-Christie et al. 1985), a limitation of this method is the high cost of the process for mass production commercially. Other systems to achieve in vitro propagation of Pinus radiata adult trees have been developed (Montalbán et al. 2013), but changes in the attributes of resulting plants have sometimes been observed and rejuvenation of the material has been transitory under in vitro conditions. Somatic embryogenesis (SE) has been the most important development for plant tissue culture , not only for mass propagation but also for enabling the implementation of biotechnological tools that can be used to increase the productivity and wood quality of plantation forestry . Therefore, many efforts have been made in the last years to develop and optimize SE systems that can be used in the breeding programs.

Somatic embryogenesis in P. radiata was first described by Smith et al. (1994) followed by improved protocols of different aspects of SE such as initiation (Hargreaves et al. 2009; Montalbán et al. 2012), maturation (Montalbán et al. 2010), cryopreservation (Hargreaves et al. 2002) and expression of genes (Aquea and Arce-Johnson 2008; García-Mendiguren et al. 2015). Modifications of the tissue culture media are likely to influence the success of SE initiation (Montalbán et al. 2012). However, few studies have focused on the impact of temperature (Kvaalen and Johnsen 2007). It is known that modifications in water availability, either by solute-imposed water stress or by physical restriction, will impact the development of embryonal masses (EMs) (Klimaszewska et al. 2000). Although the effect of different concentrations of gellan gum at maturation has been studied (Teyssier et al. 2011; Morel et al. 2014), the combination of different temperatures and water availability has not been previously tested at the initial stages of SE in conifers. As reviewed by Von Aderkas and Bonga (2000) and Neilson et al. (2010), it is clear that stress has the potential to induce or improve embryogenesis in species that have been considered recalcitrant.

Long periods of proliferation of the EMs can produce losses by contamination, somaclonal variation, or a decrease in their ability to generate embryos together with the high maintenance costs (Breton et al. 2006). One way to overcome this bottleneck is the cryopreservation of EMs; EMs are kept in liquid nitrogen because these low temperatures induce the synthesis of proteins that favours the conservation and subsequent viability of the EMs (Kong and von Aderkas 2011). However, this method presents some drawbacks such as: it is a complex technique comprising several stages (Gale et al. 2007); – it is an expensive process from the economic and technical point of view (Bomal and Tremblay 2000); it is necessary the presence of cryoprotectants that prevent the formation of ice crystals (Salaj et al. 2012); the most popular cryoprotectant is DMSO but is toxic (Arakawa et al. 1990) and may be the cause of genetic and epigenetic changes in tissues (Krajnakova et al. 2011). Nowadays, efficient and reproducible protocols for EMs cryopreservation have been described in Pinaceae (Lelu-Walter et al. 2008). However, cryopreservation of somatic embryos (Se) has been achieved for periods less than one month (Barra-Jiménez et al. 2015) in Quercus species, which does not guarantee long-term storage . Preliminary studies on P. radiata and other conifers (Hargreaves et al. 2004; Kong and Von Aderkas 2011), suggest that it is possible to develop simple alternative cryopreservation of Se at low temperatures maintaining their viability in the future.

An improved protocol for initiation of EMs, proliferation , somatic embryo maturation and germination as well as low temperature Se storage are presented in this chapter. Furthermore, recent studies focused on SE optimization in Pinus radiata are shown.

2 Initiation of Embryogenic Tissue

Cone collection and embryo stage assessment

One-year-old green female cones , enclosing immature zygotic embryos of Pinus radiata at the precotyledonary stage (Montalbán et al. 2012), are collected and stored at 4 °C until processing. Cones are usually processed within one week, although they can be stored for more than one month with no detriment in SE initiation rates (Montalbán et al. 2015).

Seed sterilization

Intact cones are sprayed with 70% (v/v) ethanol, split into quarters and all immature seeds dissected. Then, immature seeds are surface sterilized in H2O2 10% (v/v) plus two drops of Tween 20® for 8 min and then rinsed three times under sterile distilled H2O in sterile conditions in the laminar flow unit. Seed coats are removed and whole megagametophytes containing immature embryos are excised out aseptically and placed horizontally onto the medium (Fig. 1).

Fig. 1
figure 1

Initiation of embryonal masses from Pinus radiata megagametophytes cultured at 23 °C on EDM (Walter et al. 2005), bar 0.2 cm

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Basal medium preparation

Initiation of embryogenic tissue is usually carried out on EDM basal medium (Walter et al. 2005, Table 1) at 23 °C. The initiation medium contains 30 g L−1 sucrose, 4.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 2.7 µM benzyladenine (BA) and 3 g L−1 gelrite®. The pH is adjusted to 5.7, and the medium is sterilized at 121 °C for 20 min. After autoclaving, filter-sterilized solutions (pH 5.7) of the following amino acids are added to partially cooled medium prior to dispensing into Petri dishes (90 × 9 × 20 mm): 550 mg L−1 L-glutamine, 525 mg L−1 asparagine, 175 mg L−1 arginine, 19.75 mg L−1 L-citrulline, 19 mg L−1 L-ornithine, 13.75 mg L−1 L-lysine, 10 mg L−1 L-alanine and 8.75 mg L−1 L-proline.

Table 1 Constituents of Pinus radiata in vitro culture medium including salt components and organic additives. Note that hormonal supplements, carbohydrate source, activated charcoal or agar concentrations are specified in the text according to culture stage
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Culture conditions and incubations period

Cultures were maintained in the dark at 22 ± 1 °C for 4–8 weeks.

3 Embryonal Masses Evaluation

After 4–8 weeks on initiation medium, the number of initiated embryonal masses (3–5 mm in diameter) per Petri dish are evaluated.

4 Embryogenic Tissue Proliferation

Proliferating tissues are separated from the megagametophytes and subcultured to proliferation medium every 2 weeks. Initiation and proliferation medium only differ in the concentration of Gelrite®, being 3 g L−1 for the first and 4.5 g L−1 for the second. Following four periods of subculturing, actively growing embryogenic tissues are recorded as established cell lines (ECLs). Proliferation is carried out in darkness.

5 Somatic Embryo Maturation

The EMs are suspended in EDM (Table 1) broth (lacking growth regulators) and shaken vigorously by hand for several seconds. A 5 mL aliquot containing 80–90 mg of embryonal fresh mass is transferred to filter paper (Whatman no.2) in a Büchner funnel. A vacuum is applied for 10 s, and the filter paper with the attached tissue is transferred to maturation medium (Montalbán et al. 2010). The maturation medium contained the salt formulation of EDM (Table 1), 9 g L−1 gellan gum , 60 µM abscisic acid , 60 g L−1 sucrose and the amino acid mixture used for the initiation and maintenance of the EMs. Maturation is carried out in darkness.

6 Somatic Embryo Germination

After 15 weeks, Se (Fig. 2) are transferred to germination medium. This medium contains half-strength macronutrients LPm (Quoirin and Lepoivre 1977, as modified by Aitken-Christie et al. 1988) (Table 1) with 2 g L−1 of activated charcoal and 9.5 Difco agar. Petri dishes are tilted at a 45º angle with embryonic root caps pointing downwards and incubated under dim light for 7 days. Cultures are then maintained at a 16-h photoperiod at 100 µmol m−2 s−1 using cool white fluorescent tubes (TFL 58 W/33; Philips, France). Plantlets (Fig. 3) are subcultured onto fresh germination medium every 6 weeks. The whole in vitro SE process is carried out at 23 °C.

Fig. 2
figure 2

Maturation of Pinus radiata somatic embryos cultured at 23 °C on EDM (Walter et al. 2005), bar 1.1 cm

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Fig. 3
figure 3

Germination of Pinus radiata somatic embryos cultured at 23 °C on half-strength macronutrients LP (Quoirin and Lepoivre 1977, as modified by Aitken-Christie et al. 1988), bar 1.4 cm

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7 Somatic Plantlet Acclimatization

After 14–16 weeks on the germination medium, the plantlets are transferred to sterile peat:perlite (2:1) and acclimatized in a greenhouse where the humidity is progressively decreased from 99 to 70% during the first month.

8 Abiotic Stress: A Way to Improve the Somatic Embryogenesis Process

In order to evaluate the effect of different physical and chemical conditions on radiata pine SE and to identify what initial stage of SE has the greatest impact on the success of embryogenesis , initiation was carried out in following the same methodology described in Sect. 2. Different concentrations of gellan gum were added prior to autoclaving to increase or reduce water availability in the medium (2, 3 or 4 g L−1 Gelrite®), and the explants were stored at 18, 23 or 28 °C (Fig. 4). In summary, nine different treatments were applied.

Fig. 4
figure 4

Scheme of the experimental design, cultures were stored at initiation at three different temperatures: 18 °C (circle), 23 °C (square) or 28 °C (hexagon) and at three different agar concentrations (inside circles, squares and hexagons). The rest of the process was carried out under standard conditions

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Statistically significant differences in the percentage of initiation among temperatures and gellan gum concentrations were found (García-Mendiguren et al. 2016).

When considering temperature alone, initiation percentages in explants induced at 28 °C were significantly lower (4%) than those induced at 18 or 23 °C (17–13%, respectively). With respect to gellan gum , megagametophytes cultured on medium containing 4 g L−1 gellan gum showed significantly higher initiation (16%) in comparison to those cultured at 2 and 3 g L−1), which showed initiation values of 9% and 10%, respectively.

At the proliferation stage, statistically significant differences were identified only between temperatures (28 °C resulted in a significantly higher proliferation percentage (65%) when compared to explants initiated at 18 and 23 °C (35%). Regarding the number of Se per gram of EM, statistically significant differences were observed among initiation temperatures. ECLs initiated at 28 °C produced a significantly higher number of Se (486 Se g−1 EM) than those initiated at 23 °C (319 Se g−1 EM) (García-Mendiguren et al. 2016). Our results suggest that the initial conditions of the process positively impact the number of embryos produced several months later. Temperature presumably exerts a selective pressure in the early stages of embryogenesis and results in lower initiation rates but higher rates of proliferation and maturation (Fehér 2015). Although the different gellan gum concentrations tested show significant differences in water availability, this did not induce significant differences in the number of Se produced.

In summary, we observe a marked effect of initiation conditions on Se production, showing differences when that conditions are applied several months before. In light of the conclusions obtained in this study, initiation at 18 °C and 4 g L−1 gellan gum can be used to enhance the number of ECLs and thus enhance diversity within clonal plantations. On the other hand, incubation at 28 °C and the addition of 2 g L−1 gellan gum at initiation increase the efficiency of the process and result in a larger number of clones from a selected cross in a genetic improvement program.

9 New Methods for Storing Pinus radiata Genetic Resources

P. radiata Se are placed onto a sterile Whatman filter (nº 2) and the filter laid on Petri dishes containing EDM (Table 1) (Walter et al. 2005) supplemented with 60 g L−1 sucrose and 9 g L−1 Gelrite®; after autoclaving the amino acid mixture of the EDM medium (Table 1) is added. The Petri dishes are sealed with parafilm and can be stored at 4 °C for 1 year (Fig. 5). The percentage of germination is not affected by storage, improving the rates obtained in Se not conserved in cold (85%) (Fig. 6).

Fig. 5
figure 5

Pinus radiata somatic embryos after 12 months of storage at 4 °C, bar 0.4 cm

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Fig. 6
figure 6

Germination of Pinus radiata somatic embryos after 12 months of storage at 4 °C, bar 0.8 cm

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10 Research Prospects

Forestry productivity can be increased via the planting of high-value trees. Clonal propagation by somatic embryogenesis has the ability to enhance this amplification process and capture the benefits of breeding programs (Pullman et al. 2005) and it should be implemented with other technologies as cryopreservation of the embryonal masses (Park 2002) and/or somatic embryos . Our future researches activities are focused on corroborate the following hypotheses:

  • Extreme environmental conditions during the early stages of somatic embryogenesis in Pinus spp. determine the adaptative characteristics of the somatic plants produced.

  • The adaptive characteristics of the somatic plants of P. radiata are translated into differences in biochemical, molecular and physiological quantifiable characteristics, which could be used as early indicators of stress tolerance.

  • The EMs and Se of P. radiata can be stored at 4, −20 and −80 °C minimizing the costs and use of cryoprotectans.