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Norway Spruce Picea abies (L.) Karst

  • Saila Varis
Chapter
Part of the Forestry Sciences book series (FOSC, volume 84)

Abstract

The increasing use of wood as a source of bioenergy, bio-products and conservation of more natural (old) forests with high biodiversity, compel us to find means to increase forest productivity. Using the best quality regeneration material can increase the economic gain obtained from future silvicultured forests. Norway spruce is an important raw material in the European forest industry and it is the most-planted tree species in Finland. However, there is periodically a lack of high-quality Norway spruce seed due to irregular flowering of the species, as well as pests and pathogens which can lower the productivity of seed orchards. To ensure availability of good-quality forest regeneration material, effective vegetative propagation methods like somatic embryogenesis (SE) can be introduced. SE has become the method of choice for vegetative propagation of conifers (Sutton in Ann For Sci 59:657–661, 2002) due to its high multiplication rate and the maintenance of juvenility via cryopreservation that allows long-term field testing of materials.

1 Introduction

The increasing use of wood as a source of bioenergy, bio-products and conservation of more natural (old) forests with high biodiversity, compel us to find means to increase forest productivity. Using the best quality regeneration material can increase the economic gain obtained from future silvicultured forests. Norway spruce is an important raw material in the European forest industry and it is the most-planted tree species in Finland. However, there is periodically a lack of high-quality Norway spruce seed due to irregular flowering of the species, as well as pests and pathogens which can lower the productivity of seed orchards. To ensure availability of good-quality forest regeneration material, effective vegetative propagation methods like somatic embryogenesis (SE) can be introduced. SE has become the method of choice for vegetative propagation of conifers (Sutton 2002) due to its high multiplication rate and the maintenance of juvenility via cryopreservation that allows long-term field testing of materials.

Research of SE methods in Norway spruce started over 30 years ago (Chalupa 1985; Hakman and von Arnold 1985; Jain et al. 1988) and since then protocols have been developed in different laboratories (Vagner et al. 2005; Högberg and Varis 2016). In vitro regeneration is dependent on genotype, physiological state of tissue, and culture conditions. For example the frequency of successful initiations of Norway spruce zygotic embryos varies between crossings (Högberg and Varis 2016), immature zygotic embryos grow embryogenic tissue more often than mature, and half-strength basal medium enhanced embryogenic callus formation in Norway spruce mature zygotic embryos compared with full-strength medium (Jain et al. 1988). However, conditions and handling throughout the cultivation process affect the final result of SE production, many critical phases can be identified from the production process and all of them cumulatively add to the production costs of SE-plants if they cannot be controlled. Somatic embryo development from proembryogenic masses through maturation phases to mature embryo is well described (Filonova et al. 2000), and in recent studies attention has been paid for genetic regulation of the SE development (von Arnold et al. 2016). Research effort has also been put to automatization of the Norway spruce SE production, field testing, and integration of the SE production to breeding programs using emblings as donors for the field test material (Högberg and Varis 2016; Tikkinen et al. 2017).

In this chapter some critical phases are pointed out and improved protocol for Norway spruce SE plant production is described.

2 Protocol of Somatic Embryogenesis in Norway Spruce

2.1 Culture Medium

The most commonly used media for culture of Norway spruce embryogenic tissue (ET) are based on either MS (Murashige and Skoog 1962) or LM (Litvay et al. 1985), which different laboratories have more or less modified (von Arnold and Eriksson 1981; Gupta and Durzan 1986; Jain et al. 1988; Klimaszewska et al. 2001). Here the modified LM (mLM) medium (Klimaszewska et al. 2001) is presented with some modifications based on recent research. The basal composition of mLM medium is listed in Table 1 and compounds whose concentrations vary between proliferation, maturation or germination media are listed in Table 2. Easy-to-use stock solutions can be made from combinations of macrosalts, microsalts and vitamins, or they can be prepared in smaller lots to help traceability in problem situations (Tables 3 and 4). For the same reason stock and media lots are identified with a running number (Table 5). Stocks can be stored in −20 °C up to six months, and thawed stocks in +4 °C for two weeks.
Table 1

Basic compounds of modified LM medium (Klimaszewska et al. 2001) for Norway Spruce based on Litvay et al. (1985)

Compound

mg/l

NH4NO3

825

KNO3

950

MgSO4 × 7H2O

925

KH2PO4

340

CaCl2 × 2H2O

22

H3BO3

31

MnSO4 × 2H2O

21

ZnSO4 × 7H2O

43

Na2MoO4 × 2H2O

1.25

CuSO4 × 5H2O

0.50

CoCl2 × 6H2O

0.125

KJ

4.15

FeSO4 × 7H2O

27.80

Na2EDTA × 2H2O

37.30

Myo-inositol

100

Nicotinic acid

0.5

Pyridoxine HCl

0.1

Thiamine HCl

0.1

Casein hydrolysate

1000

L-glutamine

500

Table 2

Variable compounds in mLM medium for Norway Spruce

Compound

Initiation and proliferation

Maturation

Germination

NH4NO3 (mg/l)

825

825

 

Sucrose (g/l)

10

60

20

Phytagel (g/l)

4

6

6

2,4-D

10 μM

  

BA

5 μM

  

ABA

 

30 μM

 
Table 3

Example of the compositions of stocks for mLM medium

Stock

Ingredients

g/l

LM-NO3

KNO3

95

LM-NO3

NH4NO3

82.5

LM-SO4

MnSO4 × H2O

2.1

LM-SO4

ZnSO4 × 7H2O

4.3

LM-SO4

CuSO4 × 5H2O

0.05

LM-J + Cl2− ½− CaCl2

KJ

0.415

LM-J + Cl2− ½− CaCl2

CoCl2 × 6H2O

0.0125

LM-J + Cl2− ½− CaCl2

CaCl2 × 2H2O

1.1

LM-P + B + Mo− ½KH2PO4

KH2PO4

17

LM-P + B + Mo− ½KH2PO4

H3BO3

3.1

LM-P + B + Mo− ½KH2PO4

Na2MoO4 × 2H2O

0.125

LM-Vitamins

Tiamiini HCl (B1)

0.01

LM-Vitamins

Nikotiinihappo

0.05

LM-Vitamins

Pyridoksiini (B6)

0.01

LM-Vitamins

Tiamiini HCl (B1)

0.01

Myo-inositol

Myo-inositol

40

NaFe-EDTA

NaFe-EDTA

4

Table 4

Hormone stocks for mLM medium

Stock

Solvent

mg/10 ml

2,4-D

EtOH/1 N NaOH

221.0

BA

1 N NaOH

225.3

ABA

1 N NaOH

264.3

Dilute in small amount of EtOH or NaOH and adjust volume with water

Table 5

Example of the “Cook Book Sheet” for mLM proliferation medium

mLM proliferation medium, ½ macroelements

Lot number

Stock

Amount/l

Amount/medium

LM-NO3 stock

10 ml

LM-SO4 stock

10 ml

LM-J + Cl2− ½− CaCl2 stock

10 ml

LM-P + B + Mo− ½KH2PO4 stock

10 ml

LM-Vitamins stock

10 ml

Myo-inositol stock

2.5 ml

NaFe-EDTA stock

10 ml

 

MgSO4 × 7 H2O

0.925 g

 

Casein hydrolysate (N-Z-Amine®A)

1 g

2,4-D stock

100 µl

BA stock

50 µl

 

Sucrose

10 g

 

pH

5.8

 

Ad. maxim water

1000 ml

 

Phytagel

4 g

Add after autoclaving:

 

L-glutamine (0,2 μm filter)

500 mg

 

Solubility 1 g/40 ml

  

Prepare medium without L-glutamine. Take appr. 20 ml of medium, add L-glutamine into it and use 20 ml syringe with 0.2 um filter to add it into autoclaved medium after cooling it to 60 °C. Dosing 20–25 ml of medium per petridish

The same mLM medium containing 2, 4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) as plant growth regulators is applicable both for the induction and proliferation of embryogenic cultures of Norway spruce. The medium is solidified with gellan gum (Phytagel) but can be used in liquid form, for example, in temporary immersion system (TIS) bioreactors. Usually 90 mm petri dishes are used in cultivation in semi-solid medium, especially when automated petri dish filler is utilized, but also cheaper material like freezer boxes or reusable Magenta vessels are able to be used. Automatization helps when numerous petri dishes have to be filled.

For development of somatic embryos 2, 4-D and BA are replaced by (+)-abscisic acid (ABA). The amount of ABA varies in different recipes from 20 to 60 µM (Högberg et al. 2001; Klimaszewska et al. 2001; Vondráková et al. 2014) being 60 µM in original mLM. Because ABA also acts as an inhibitor of plant growth, the high concentration or long contact with it may negatively affect germination and further growth of Norway spruce somatic embryos (Högberg et al. 2001). On the other hand, using a low concentration may require moving of embryos to fresh media during their development (Vondráková et al. 2014). In our experiments embryos developed in a 30 µM ABA concentration without moving to fresh media, and mature embryos had higher germination rates than embryos maturated in a 60 µM ABA concentration (Tikkinen et al. 2018a). High sucrose concentration, and in some cases polyethylene glycol 4000 (PEG), are common in maturation media. Both substances induce osmotic pressure which results in an increased amount of mature embryos (Tremblay and Tremblay 1995). However, PEG has had negative effects on embryo germination and root growth (Bozhkov and von Arnold 1998) and is therefore not included in the Norway spruce medium presented here. For maturation in TIS bioreactors, the media presented here requires further optimization (Salonen et al. 2017).

Mature embryos are germinated on medium without any plant regulators. The chemical form of nitrogen influences root:shoot ratio in growing seedlings of Norway spruce in a way that the organic nitrogen source seems to enhance root growth when compared to seedlings cultivated on an inorganic nitrogen source (Gruffman et al. 2012). Increasing the ratio of organic nitrogen by not including NH4NO3 in the mLM germination medium resulted in increased root length and higher root: shoot ratio (Tikkinen et al. 2018b in press).

2.2 Explant Preparation and Proliferation of Embryogenic Culture

Both mature and immature zygotic embryos can be used as explants in the initiation of Norway spruce embryogenic cultures, although the initiation frequencies are lower with mature embryos compared to immature ones. Following the methodology developed for the induction of ET from primordial shoots of mature trees, successfully applied in White spruce (Klimaszewska et al. 2011), induction of SE and regeneration of emblings has recently been achieved also in Norway spruce (Varis et al. 2018 in preparation). However, the frequency of initiation from Norway spruce shoots has been low.

Immature cones are collected in summer when the heat sum is around 800 degree days and they can be stored in +4 °C for one week (Varis et al. 2018, in press). Cones are cleaned with 70% ethanol, seeds are dissected and placed in sterile water and a drop of dishwashing soap is added to clean the seeds. After one or two rinse cycles in sterile water, seeds are surface sterilized in 70% ethanol for 5 min and rinsed three times in sterilized water. Washing and rinsing can be done either in a petri dish or with the help of a tea ball. Megagametophytes are carefully opened with a scalpel under a stereo microscope and zygotic embryos are placed on the medium. Usually ten zygotic embryos are placed on each Petri dish and each embryo is systematically labeled which may be part of or all of the future line number. Embryos are kept in the dark at 24 °C on the same medium until embryogenic tissue (ET) has grown 5–10 mm and is ready to be excised from the zygotic embryo to establish an embryogenic line (Fig. 1).
Fig. 1

Initiation and proliferation of embryogenic tissue from Norway spruce immature zygotic embryos. a Immature seeds are dissected from the cone using knife for loosening scales and forceps for taking seed carefully out; b tea ball is handful when seeds are surface-sterilized in ethanol and washed in sterilized water; c zygotic embryos dissected from seed are placed on medium. Crossing, embryo numbers and initiation date are market on the lid; d embryogenic tissue grows in 3–8 weeks

Established ETs are subcultured at 12–14-day intervals. ET can be proliferated on semi-solid media in clumps, or spread on the filter like in the maturation phase (see below). Using suspension cultures in continuous rotation seems to be too rough a treatment for most of the cell lines. As an alternative to suspension cultures, Norway spruce ET proliferates well when it is only temporarily immersed into liquid media.

Good looking Norway spruce ET is bright white, with brown spots sometimes forming quite soon after subculturing. Typically the brown parts are avoided, as the newest bright tissue is preferred in subculturing. However, brown spots or brownish coloring in the old growth has no effect on embryo production or viability of cryopreserved ET when the tissue used in maturation and cryopreservation is the newest growth. Maturating and cryopreserving of the Norway spruce tissue should be done as soon after initiation as possible using the newest tissue from cultures to ensure the best embryo production and recovery (Varis et al. 2018, in press).

The protocol of initiation from primordial shoots is presented here according to the findings of Klimaszewska et al. (2011) and applied for Norway spruce. Shoot buds can be collected either in the spring before the growing season or after it in the autumn. Lateral buds can be stored in 2–4 °C for up to 5 days. The basal scales of buds are removed, and buds of each genotype are divided into two 50 ml plastic tubes. The outer resin is washed away by adding 94% ethanol and shaking the tubes for one minute. Washing and disinfecting continues with six minutes of shaking in tap water including a small amount of Tween-20, two minutes of shaking in 70% ethanol, and eight minutes of shaking in 10% (v/v) hydrogen peroxide. After rinsing three times in sterile water, buds are placed on a Petri dish with 100 mg l−1 polyvinylpyrrolidone (PVP) solution. For dissection a smaller amount of buds is placed in a new Petri dish on a filter paper soaked with PVP solution. Buds are cut in two lengthwise, primordial shoots are excised and depending on the size they are recut into two or three parts. Sections of the primordial shoots are placed with their cut surface towards the semi-solid mLM proliferation media. Bud explants are kept in the dark at 24 °C on the same medium and treated like zygotic embryos.

2.3 Maturation and Germination of Somatic Embryos

For induction of somatic embryo maturation about 180 (±20) mg of ET, five to seven days from the last subculture, is mixed in 3 ml liquid media without plant growth regulators (PGR). The suspension is poured onto a paper filter (Whatman #2) which is placed in a Büchner funnel. The liquid is drained by suction and the filter is placed on mLM maturation medium. Developing embryos are kept in the dark at 24 °C on the same medium for eight weeks.

Mature somatic embryos of Norway spruce can be partially desiccated to lower endogenous ABA levels and thus prevent after effects in embryo germination. Shortly: mature embryos are placed on filter paper in a petri dish which is placed on a larger vessel together with a petri dish containing sterile water. The whole package is sealed with parafilm and incubated in the dark for 16–24 days (Bozhkov and von Arnold 1998; Högberg et al. 2001). This procedure has increased germination frequency, but the same effect is also reached when mature embryos are cold stored on maturation media for one to six months (Varis et al. 2018, in press). Embryos should be stored at a stable temperature of 2–4 °C to avoid formation of condensation water inside the petri dish.

Mature Norway spruce embryos should be shortly germinated in vitro on PGR-free medium before acclimatization to greenhouse conditions (Fig. 2). It seems that the traditional five-week germination period (Klimaszewska et al. 2001) does not yield the best result regarding the later life of the embryo. For example, in recent studies (Tikkinen et al. 2018b in press) showed that the survival rate of Norway spruce SE emblings after the first growing season was higher when embryos were germinated only one week compared to five or three weeks germination. In addition to more vital plants, increased height growth was achieved with a shorter germination treatment.
Fig. 2

Germination and acclimatization of Norway spruce somatic embryos a good mature embryos (left side) have well developed apical and root meristems, and at least four cotyledons; b germinated embryos after two weeks on hormone free media; c growing emblings in propagators placed under LED light system

Mature embryos are placed on petri dishes, ten to fifteen embryos per dish, in a way that the cotyledons face up when the petri dishes are placed in a slightly tilted position. Instead of germination on semi-solid media, embryos could be put on a metal mesh placed over liquid media which allows root development in liquid (Högberg and Varis 2016).

The light intensity can be low (5 µmol/m−2/s−1) at the beginning of the germination period and increase towards the end to 130–150 µmol/m−2/s−1. Intensity should be high enough, but the heating effect of the lightning system should be taken into account. The temperature in the germination room should be adjusted so that the target temperature for the germinating embryos inside the petri dishes or other vessels stays within a tolerable range. Attention has to be paid also to the spectrum of the lightning system; fluorescent lamps usually emit much more white wavelengths favorable to the human eye than the red and blue wavelengths that plants use. The photoperiod of the 18 h day/6 h night should be introduced at least for northern genotypes, while with southern genotypes 14–16 h day/10–8 h night may be enough.

2.4 Acclimatization and Field Transfer

Acclimatization of germinated emblings from in vitro to ex vitro conditions is usually the most stressful stage in the emblings’ lives, no matter how careful you are and how optimized conditions are. Acclimatization can be done in a special greenhouse, funnels, or in commercial—either reusable or disposable—propagators, in which temperature and relative humidity can be controlled (Fig. 3). Propagators can be handy when sustaining high humidity is not possible or growing space is limited.
Fig. 3

Norway spruce emblings in the acclimatization and field a plastic lids have been removed when emblings have been acclimatized three weeks in propagators; b acclimatization in the funnel; c embling planted in the field to the spot which has been mounded

Attention has to be paid during every step when handling embryos, starting from the transferring procedure when drying must be avoided by keeping embryos out of media or peat for as short a time as possible and avoiding heating lamps. The temperature during acclimatization should be close to the germination temperature. The relative humidity should be high (85–90%) at the beginning of the growth ex vitro. After a critical period of two to three weeks it should be gradually decreased to the same level used in the greenhouse where seedlings are growing. The light intensity and day length should be at least the same as during germination to avoid the formation of an apical bud.

Growth media is often peat mixed with perlite or vermiculite or sand to increase water and nutrient retention and to aerate the peat. Also, commercially available mini-plugs can be used, especially in propagators. Watering and fertilizing should be done by carefully optimizing the concentration and amount of the liquid. Drying and the need for fertilizer liquid can be evaluated with the help of scaling.

For transferring germinated embryos to the peat the “pricking out” method (Landis et al. 2010) can be introduced: forceps and a modified screwdriver are used for making a small hole in the peat, transferring emblings and straightening the roots. After transferring, the peat should be gently compressed around the emblings to ascertain sufficient soil contact for the developing root (Tikkinen et al. 2018b in press).

2.5 Preservation of Culture and Embryos

Under continuous in vitro culture, embryogenic tissues may decline or even lose their somatic embryo differentiation ability. For maintenance of somatic embryo production capacity of selected lines, as well as for storage of these lines for future use, a reliable cryopreservation method is needed. A cryopreservation protocol based on cryoprotectants added in a suspension culture of Norway spruce and freezing with slow-cooling (Norgaard et al. 1993) is used in many laboratories. More recently, a cryopreservation protocol based on drying of embryogenic tissue of one cell line and direct immersion in liquid nitrogen (LN) without the use of cryoprotectants have also been described (Hazubska-Przybyl et al. 2013).

In recent studies by Varis et al. (2018, in press) different sets of classical pre-treatment with cryoprotectants and cooling methods were tested using a large set of genotypic material. The physiological condition of the tissues, pre-treatment and cryoprotectant applied, as well as the slow-cooling device used were found to affect the recovery of Norway spruce ET. The most reliable option was to select fresh growth from young ETs as samples, and pretreat them on semi-solid medium with increasing sucrose concentration (0.1 M for 24 h; 0.2 M for another 24 h). About 200 mg ET was placed in sterile cryovials (six to eight clumps in each 2 ml cryovial) containing 400 µl liquid mLM medium with 0.4 M sucrose concentration but without plant growth regulators, gelling agent or glutamine. Cryovials were placed on thermoconductive racks which were precooled in −20 °C. Into each cryovial 400 µl pre-chilled cryoprotectant PGD solution (composed of polyethylene glycol 6000, glucose, and Me2SO 10% w/v each) was added in 200 µl aliquots within the time period of 30 min. Cryovials were then incubated for 30 min in thermoconductive racks. Freezing of the samples should be done with the slow-cooling method using a programmable cooling device at the rate of 0.17 °C/min to −38 °C (Fig. 4).
Fig. 4

Cryopreservation of pretreated samples. a Programmable freezer for slow cooling; b in the freezer cryovials are in the metal rack and should be moved to cryoboxes quickly, e.g. by placing them in styrox box with liquid nitrogen at the bottom of it; c cryoboxes are moved to liquid nitrogen containers as soon as possible

When samples are taken from LN they should be immediately thawed in a water bath +37 °C for 2 min. Cryovials should be wiped with 70% ethanol and the content of the tube is poured onto the sterilized paper filter (Whatman #2) and placed in the Büchner funnel. The cryostorage liquid is drained off by suction and the tissue is washed with the same liquid mLM medium which was used in the pretreatment process. Samples are placed on medium with a sucrose content of 0.2 M, and transferred every 24 h on medium with decreasing sucrose concentration (0.1 and 0.03 M). All filters with tissue are transferred to new media every two weeks. With this method, on average 87% of the genotypes were recovered without any effect on their genetic fidelity, as shown by microsatellite markers and embryo production capacity (Varis et al. 2018, in press).

In Nordic tree species transfer of somatic plants from the laboratory to nursery for further growth is not possible during Northern winter conditions. Plant production using heated greenhouses with additional lights is possible, but not cost-efficient. Thus, storing mature embryos in cold enables production of somatic embryos in the laboratory throughout the year, and allows synchronization of their development and then germination for further cultivation at the proper time, i.e. spring. Even circa one-year on storage, using the method described earlier, resulted in higher germination percentages compared to fresh ones, however, part of the embryos had started to germinate during storage, and the amount of good-looking embryos was reduced (Varis et al. 2018, in press).

3 Research Prospects

For practical application of Norway spruce SE the most important aspect is to improve the cost efficiency of the whole process. Currently the SE-process described here and used in many laboratories is based on manual labor, which is time consuming and expensive. Automatization of the SE-process has been the focus of the research in some laboratories or companies for some time; developments include the application of bioreactors, mainly Temporary Immersion Systems (TIS), automated embryo selection and artificial seed production.

There are several commercially available TIS models in which liquid medium is applied at intervals to plant material that is located in a compartment that is separate from the medium. The advantages of the TIS systems in embryo germination and plant production have been documented with several commercially important species like coffee, and benefits have been shown to include the reduction in workload and thus in cost of the produced plant (Etienne and Berthouly 2002). However, only limited data are available on the effect of the different culture parameters, such as the immersion frequencies, on ET culture during the proliferation and maturation stages. In Norway spruce SE, the proliferation and maturation of ETs in TIS bioreactors has been achieved, although especially the maturation phase could still be further optimized to increase propagation efficiency (Salonen et al. 2017). Optimizing may be challenging and time consuming, with all the culture process steps having to be optimized for a wide variety of genetic materials. Besides productivity of the cultures, the cost-efficiency and easiness of sterilization and aseptic handling of TIS during the culturing process is important. TIS models commercially available at the moment are designed for growing plants, thus they are higher than needed for tissue growth and one vessel takes too much space in the autoclave.

Automated embryo selection and planting of the germinated embryos into peat could remarkably reduce the manual work. Many kinds of modern seed planting machines are available, but applications for somatic embryos are rare.

Despite recent breakthroughs (Klimaszewska et al. 2011) the problem of tissues of mature conifers being generally recalcitrant for SE induction requires further research. The possibility to propagate mature conifer trees with known characters via SE in combination with cost-efficient mass propagation would have an enormous impact on forestry with subsequently increased productivity and/or production of tailored raw materials for special end-users.

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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Natural Resources Institute FinlandPunkaharjuFinland

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