Somatic Embryo Yield and Quality From Norway Spruce Embryogenic Tissue Proliferated in Suspension Culture

Somatic embryogenesis is being piloted for the commercial production of genetically improved Norway spruce (Picea abies L. Karst) forest regeneration material in Finland. The main challenge to making the process commercially relevant is the dependence on time-consuming and highly skilled manual labor. Automation and scaling up are needed to improve cost-effectiveness. Moving from the proliferation of embryogenic tissue on semisolid media to suspension cultures could improve process scalability. In a series of four experiments (overall, with 20 cell lines, 4–9 per experiment), the suitability of proliferation in suspension culture for Norway spruce somatic embryogenesis was evaluated based on the growth rate, indicators of stress conditions, good-quality cotyledonary embryo yield, and embling survival in a greenhouse. The proliferation rate in suspension was found equal to on semisolid media, but with a remarkable genotypic variation. Embryogenic tissue matured directly without pre-treatments from suspension onto semisolid media produced lower numbers of good-quality embryos than tissue matured from semisolid media. Rinsing the suspension-grown tissue with hormone-free liquid media before maturation improved embryo yield, bringing it closer to that of semisolid-grown tissue. Decreasing 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in suspension proliferation media to 0.5 or 0.1 times those in semisolid media did not affect tissue growth and did not improve embryo production. The hydrogen peroxide (H2O2) content and guaiacol peroxidase activity were elevated in suspension cultures compared with semisolid medium, which had the same plant growth regulator content. In one experiment out of four, the greenhouse survival of germinants was lower when proliferation was carried out in full strength suspension than on semisolid media; in other experiments the survival rates were equal.

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Telomere Length in Norway Spruce during Somatic Embryogenesis and Cryopreservation

Plants ◽

10.3390/plants10020416 ◽

2021 ◽

Vol 10 (2) ◽

pp. 416

Author(s):

Tuija Aronen ◽

Susanna Virta ◽

Saila Varis

Keyword(s):

Somatic Embryogenesis ◽

Telomere Length ◽

Norway Spruce ◽

Genotypic Variation ◽

Production Capacity ◽

Stress Factors ◽

Embryo Production ◽

One Year ◽

Plant Telomeres

Telomeres i.e., termini of the eukaryotic chromosomes protect chromosomes during DNA replication. Shortening of telomeres, either due to stress or ageing is related to replicative cellular senescence. There is little information on the effect of biotechnological methods, such as tissue culture via somatic embryogenesis (SE) or cryopreservation on plant telomeres, even if these techniques are widely applied. The aim of the present study was to examine telomeres of Norway spruce (Picea abies (L.) Karst.) during SE initiation, proliferation, embryo maturation, and cryopreservation to reveal potential ageing or stress-related effects that could explain variation observed at SE process. Altogether, 33 genotypes from 25 families were studied. SE initiation containing several stress factors cause telomere shortening in Norway spruce. Following initiation, the telomere length of the embryogenic tissues (ETs) and embryos produced remains unchanged up to one year of culture, with remarkable genotypic variation. Being prolonged in vitro culture can, however, shorten the telomeres and should be avoided. This is achieved by successful cryopreservation treatment preserving telomere length. Somatic embryo production capacity of the ETs was observed to vary a lot not only among the genotypes, but also from one timepoint to another. No connection between embryo production and telomere length was found, so this variation remains unexplained.

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Polyamines during somatic embryo development in Norway spruce (Picea abies [L.])

Journal of Forest Science ◽

10.17221/84/2008-jfs ◽

2009 ◽

Vol 55 (No. 2) ◽

pp. 75-80 ◽

Cited By ~ 9

Author(s):

J. Malá ◽

M. Cvikrová ◽

P. Máchová ◽

O. Martincová

Keyword(s):

Somatic Embryogenesis ◽

Picea Abies ◽

Norway Spruce ◽

Somatic Embryos ◽

Developmental Stages ◽

Embryogenic Tissue ◽

Growth Media ◽

Mature Embryos ◽

Proliferation Medium ◽

Maturation Medium

Contents of free polyamines (putrescine, spermidine and spermine) were determined in different developmental stages of Norway spruce (Picea abies [L.] Karst.) somatic embryos by means of HPLC. Determinations were performed embryogenic tissue after 4 weeks of the growth on proliferation medium, after 2 and 5 weeks of the culturing on maturation medium, and 2 weeks after desiccation. Maturation of somatic embryos (after 5 weeks) was accompanied by increase of concentrations of putrescine (2.3 times) and spermidine (3.2 times). In comparison with above mentioned polyamines, spermine concentrations were significantly lower (4.3 times). Two weeks after desiccation, the concentrations of putrescine decreased 5.4 times and spermidine 2.2 times in comparison with mature embryos. To improve the efficiency of somatic embryogenesis of less responsive genotypes, the supplementation of growth media by polyamines is discussed.

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Tolerance of Norway spruce (Picea abies [L.] Karst.) embryogenic tissue to penicillin, carbapenem and aminoglycoside antibiotics

Journal of Forest Science ◽

10.17221/100/2008-jfs ◽

2009 ◽

Vol 55 (No. 4) ◽

pp. 156-161 ◽

Cited By ~ 2

Author(s):

J. Malá ◽

D. Pavingerová ◽

H. Cvrčková ◽

J. Bříza ◽

J. Dostál ◽

...

Keyword(s):

Somatic Embryogenesis ◽

Picea Abies ◽

Norway Spruce ◽

Aminoglycoside Antibiotic ◽

Embryogenic Tissue ◽

Transformation Protocol ◽

Selective Media ◽

Accelerated Growth ◽

Embryogenic Tissues ◽

Carbapenem Antibiotic

Somatic embryogenesis is conveniently utilized for the preparation of Norway spruce (Picea abies [L.] Karst.) transgenic clones by means of Agrobacterium. The establishment of successful transformation protocol requires to determine the tolerance of growing embryogenic tissue to antibiotics in culture and selective media. In 5 Norway spruce lines (genotypes) differences in the tolerance of embryogenic tissues to penicillin antibiotics (amoxicillin, carbenicillin, and ticarcillin), carbapenem antibiotic (meropenem) used for the Agrobacterium growth prevention, and aminoglycoside antibiotic (kanamycin) used in selective media were determined. Of the penicillin derivatives, amoxicillin was optimally tolerated in all lines and, in addition, its highest concentration accelerated growth in more rapidly growing lines. Ticarcillin was similarly tolerated but no growth acceleration was observed in any line. As regards carbenicillin, only the lowest concentration was observed to be well tolerated by all lines whereas all concentrations of meropenem were well tolerated in all lines except for slowly growing line 28, the growth of which was retarded by the concentration of 20 mg/l. The aminoglycoside antibiotic kanamycin was well tolerated by the embryonic tissue of all lines in the concentration of 10 mg/l and less in the concentration of 25 mg/l. The concentrations of 50 mg/l and 100 mg/l appeared as intolerable in all lines. Toxicity of kanamycin manifested at first in the browning and later in the growth cessation of embryogenic tissue.

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Light Spectra during Somatic Embryogenesis of Norway Spruce—Impact on Growth, Embryo Productivity, and Embling Survival

Forests ◽

10.3390/f12030301 ◽

2021 ◽

Vol 12 (3) ◽

pp. 301

Author(s):

Saila Varis ◽

Mikko Tikkinen ◽

Sakari Välimäki ◽

Tuija Aronen

Keyword(s):

Somatic Embryogenesis ◽

Norway Spruce ◽

Somatic Embryos ◽

Pulse Width Modulation ◽

Production Costs ◽

Embryogenic Tissue ◽

Light Spectrum ◽

Propagation Process ◽

Embryo Survival ◽

Light Spectra

For Norway spruce (Picea abies (L.) Karst.) somatic embryogenesis (SE) culture conditions throughout the propagation process affect the final result. Many critical phases can be identified, and all of them cumulatively increase the production costs of SE plants if they cannot be controlled. In order to determine the best lighting protocol for each SE step, Norway spruce embryogenic tissue (ET) was proliferated, and somatic embryos were matured under different light wavelengths, wavelength combinations, and in the dark. Overall, using low-intensity LED lights during proliferation or at the end of maturation had little effect on the growth of ET, embryo productivity, or embryo survival; on the other hand, major negative effects could not be seen. This is beneficial from a practical point of view, indicating no need for lighting or protection of SE cultures from light during their handling in these steps of the propagation process. When somatic embryos were germinated under different spectra, significant differences in embling shoot and root growth, as well as in the survival of the emblings, were found. The best treatment varied between trials, and the genotype of the SE culture was found to have a stronger effect than the light spectrum, indicating that various light spectra and also intensity adjusted using pulse width modulation (PWM) can be successfully applied to the SE germination phase in Norway spruce.

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