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

Forests ◽  
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.

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

2009 ◽  
Vol 55 (No. 2) ◽  
pp. 75-80 ◽  
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 (<I>Picea abies</I> [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.

scholarly journals Pinus canariensis plant regeneration through somatic embryogenesis

2020 ◽  
Vol 29 (1) ◽  
pp. eSC05
Author(s):  
Ander Castander-Olarrieta ◽  
Paloma Moncaleán ◽  
Itziar A. Montalbán
Keyword(s):  
Somatic Embryogenesis ◽  
Cell Lines ◽  
Somatic Embryos ◽  
Embryogenic Tissue ◽  
Established Cell Line ◽  
Zygotic Embryos ◽  
Pinus Canariensis ◽  
Embryogenic Cell ◽  
Embryogenic Cell Lines ◽  
Established Cell

Aim of the study: To develop an efficient method to regenerate plants through somatic embryogenesis of an ecologically relevant tree species such as Pinus canariensis.Area of study: The study was conducted in the research laboratories of Neiker-Tecnalia (Arkaute, Spain).Material and methods: Green cones of Pinus canariensis from two collection dates were processed and the resulting immature zygotic embryos were cultured on three basal media. The initiated embryogenic tissues were proliferated testing two subculture frequencies, and the obtained embryogenic cell lines were subjected to maturation. Germination of the produced somatic embryos was conducted and acclimatization was carried out in a greenhouse under controlled conditions.Main results: Actively proliferating embryogenic cell lines were obtained and well-formed somatic embryos that successfully germinated were acclimatized in the greenhouse showing a proper growth.Research highlights: This is the first report on Pinus canariensis somatic embryogenesis, opening the way for a powerful biotechnological tool for both research purposes and massive vegetative propagation of this species.Keywords: acclimatization; Canary Island pine; micropropagation; embryogenic tissue; somatic embryo.Abbreviations used: embryogenic tissue (ET); established cell line (ECL);  somatic embryogenesis (SE); somatic embryos (Se’s).

scholarly journals Norway spruce (Picea abies) genetic transformation with modified Cry3A gene of Bacillus thuringiensis.

2013 ◽  
Vol 60 (3) ◽  
Author(s):  
Jindřich Bříza ◽  
Daniela Pavingerová ◽  
Josef Vlasák ◽  
Hana Niedermeierová
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Bacillus Thuringiensis ◽  
Picea Abies ◽  
Norway Spruce ◽  
Somatic Embryos ◽  
Marker Gene ◽  
Selection Marker ◽  
Embryogenic Tissue ◽  
35S Promoter ◽  
Rt Pcr

Modified versions of the Cry3A gene of Bacillus thuringiensis (Bt) were transferred into Norway spruce (Picea abies). Both the biolistic approach and Agrobacterium tumefaciens mediated procedure were employed for transformation of embryogenic tissue (ET) cultures. The latter method proved to be more efficient yielding 70 transgenic embryogenic tissue lines compared with 18 lines obtained by biolistics. The modified Cry3A genes were driven by a 35S promoter and the nptII screenable selection marker gene was used in all vectors. The transgenic ETs were molecularly characterized and converted into mature somatic embryos. Germinating embryos formed plantlets which were finally planted into perlite and their Cry3A gene transcription activities were demonstrated by RT-PCR.

Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce

2000 ◽  
Vol 113 (24) ◽  
pp. 4399-4411 ◽  
Author(s):  
L.H. Filonova ◽  
P.V. Bozhkov ◽  
V.B. Brukhin ◽  
G. Daniel ◽  
B. Zhivotovsky ◽  
...  
Keyword(s):  
Cell Death ◽  
Somatic Embryogenesis ◽  
Programmed Cell Death ◽  
Norway Spruce ◽  
Somatic Embryos ◽  
Nuclear Dna ◽  
Second Phase ◽  
Embryo Suspensor ◽  
Large Central Vacuole ◽  
Suspensor Cells

In the animal life cycle, the earliest manifestations of programmed cell death (PCD) can already be seen during embryogenesis. The aim of this work was to determine if PCD is also involved in the elimination of certain cells during plant embryogenesis. We used a model system of Norway spruce somatic embryogenesis, which represents a multistep developmental pathway with two broad phases. The first phase is represented by proliferating proembryogenic masses (PEMs). The second phase encompasses development of somatic embryos, which arise from PEMs and proceed through the same sequence of stages as described for their zygotic counterparts. Here we demonstrate two successive waves of PCD, which are implicated in the transition from PEMs to somatic embryos and in correct embryonic pattern formation, respectively. The first wave of PCD is responsible for the degradation of PEMs when they give rise to somatic embryos. We show that PCD in PEM cells and embryo formation are closely interlinked processes, both stimulated upon withdrawal or partial depletion of auxins and cytokinins. The second wave of PCD eliminates terminally differentiated embryo-suspensor cells during early embryogeny. During the dismantling phase of PCD, PEM and embryo-suspensor cells exhibit progressive autolysis, resulting in the formation of a large central vacuole. Autolytic degradation of the cytoplasm is accompanied by lobing and budding-like segmentation of the nucleus. Nuclear DNA undergoes fragmentation into both large fragments of about 50 kb and multiples of approximately 180 bp. The tonoplast rupture is delayed until lysis of the cytoplasm and organelles, including the nucleus, is almost complete. The protoplasm then disappears, leaving a cellular corpse represented by only the cell wall. This pathway of cell dismantling suggests overlapping of apoptotic and autophagic types of PCD during somatic embryogenesis in Norway spruce.

Plant regeneration by somatic embryogenesis in Pinus thunbergii resistant to the pine wood nematode

2019 ◽  
Vol 49 (12) ◽  
pp. 1604-1612
Author(s):  
Tingyu Sun ◽  
Yanli Wang ◽  
Lihua Zhu ◽  
Xiaoqin Wu ◽  
Jianren Ye
Keyword(s):  
Somatic Embryogenesis ◽  
Plant Regeneration ◽  
Somatic Embryo ◽  
Cell Line ◽  
Somatic Embryos ◽  
Zygotic Embryo ◽  
Pinus Thunbergii ◽  
Embryogenic Tissue ◽  
Embryo Production ◽  
Somatic Embryo Production

Pine wilt disease (PWD) is a severe threat to pine forests in East Asia. Screening and breeding of resistant varieties is a very effective way to prevent and control PWD; however, no reliable somatic embryogenesis system has yet been developed for the elite nematode-resistant Pinus thunbergii Parl. line. In this study, we studied the plant regeneration via somatic embryogenesis of nematode-resistant P. thunbergii. Initiation of embryogenic tissue was significantly affected by seed family (p = 0.017), immature zygotic embryo stage (p = 0.032), and initiation medium (p = 0.004). Seed family 37 was the most favorable female parent for initiation of P. thunbergii. Furthermore, the initiation rate increased from the pre-embryonic stage to the cleavage polyembryonic stage. The optimal medium was I2, containing 2,4-dichlorophenoxyacetic acid (9 μmol·L−1) and 6-benzyladenine (4.4 μmol·L−1). A statistically significant interaction between cell line and subculture time (24 months) was observed in the influence on proliferation rate, somatic embryo production, and percentage germination (p < 0.001). In this study, the highest somatic embryo production was achieved using cell line 37-1 (1983 somatic embryos per gram fresh mass), with approximately 83.5% of somatic embryos germinating after transferring to germination medium, of which 77.6% converted into plantlets.

scholarly journals Stable Agrobacterium-mediated transformation of Norway spruce embryogenic tissues using somatic embryo explants

2011 ◽  
Vol 57 (No. 7) ◽  
pp. 277-280 ◽  
Author(s):  
D. Pavingerová ◽  
J. Bříza ◽  
H. Niedermeierová ◽  
J. Vlasák
Keyword(s):  
Norway Spruce ◽  
Somatic Embryos ◽  
Plant Material ◽  
Southern Hybridization ◽  
Embryogenic Tissue ◽  
Embryogenic Cell ◽  
Reproductive Cycles ◽  
Embryogenic Tissues ◽  
Transgenic Callus ◽  
Embryogenic Lines

In conifers and other plants with long reproductive cycles, transformed embryogenic tissues can serve as a convenient source of plant material for the testing of insecticidal or fungicidal transgene efficiency. In this report, transgenic embryogenic tissue was obtained after the transformation of somatic embryos of Norway spruce (Picea abies (L.) Karst.) by Agrobacterium tumefaciens with the gus-intron chimeric gene. The stable integration of transgenes was confirmed by PCR and Southern hybridization. The transformation was successful only in a suitable embryogenic cell line sensitive to Agrobacterium. Out of the nine embryogenic lines tested only one gave transgenic callus.

scholarly journals Differential success of somatic embryogenesis in random gene pool of Norway spruce

2008 ◽  
Vol 53 (No. 2) ◽  
pp. 74-87
Author(s):  
M. Mauleová ◽  
J. Vítámvás
Keyword(s):  
Somatic Embryogenesis ◽  
Norway Spruce ◽  
Somatic Embryos ◽  
Significant Variation ◽  
Open Pollination ◽  
Embryogenic Cultures ◽  
Peg 4000 ◽  
Partial Desiccation ◽  
Somatic Embryogenic ◽  
Mature Seeds

Somatic embryogenic cultures were established from proembryonal suspensor masses (PEMs) derived from mature seeds of Norway spruce. In this study we used more than 4,300 seeds of <i>Picea abies</i> from randomly collected commercial seed lot (originated from open-pollination). Most of the studies are focused on selected genotypes known for higher response to propagation protocols. As indicated in this study, there is a significant variation in success rate of somatic embryogenesis in randomly selected seed lot of Norway spruce. Nutrient GD (1 to 4), LP (1 to 5) media and different level of plant grow regulators (BA, NAA, kinetin and 2,4D) were used for initiation and proliferation of embryogenic cultures. Transfer of embryogenic callus onto medium containing abscisic acid stimulated development of early-established individual embryos. Media GD (5 and 6) and LP (9 to 11) supplemented with ABA (7.5; 20; 38 &mu;M) and PEG 4000 (2%), were used for stadium of maturation. Conversion of somatic embryos to plantlets was stimulated by partial desiccation treatment (HRH-treatment) and by medium changes. On these media plantlets started to regenerate within three weeks.

Evaluation of somatic embryogenesis for clonal propagation of western white pine

2000 ◽  
Vol 30 (12) ◽  
pp. 1867-1876 ◽  
Author(s):  
R E Percy ◽  
K Klimaszewska ◽  
D R Cyr
Keyword(s):  
Somatic Embryogenesis ◽  
Somatic Embryos ◽  
Clonal Propagation ◽  
Embryogenic Tissue ◽  
Zygotic Embryos ◽  
Pinus Monticola ◽  
White Pine ◽  
Western White Pine ◽  
Embryogenic Lines

A multiyear program was undertaken to develop a somatic embryogenesis system for clonal propagation of western white pine (Pinus monticola Dougl.). Developing seeds were used to initiate embryogenic lines from families used in blister-rust (Cronartium ribicola J.C. Fisch.) resistance breeding programs in British Columbia. The most responsive seeds contained zygotic embryos ranging in development from late cleavage polyembryony to the early dominance stage. Overall, 14 of 15 open-pollinated families produced embryogenic lines. The best results (0.8-6.7% initiation) were obtained using modified Litvay medium with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) at 2.25 µM. Proliferation of embryogenic tissue was enhanced by culturing tissue as a thin layer on filter paper supports. Approximately 300 lines representing 18 open- and control-pollinated families were cryopreserved. The highest number of mature somatic embryos was obtained on maturation medium containing 120 µM abscisic acid, 180 mM sucrose, and 1.0% gellan gum. Of 61 lines tested on this medium, 77% produced mature somatic embryos. In vitro germination and early growth occurred at a high frequency (90-95%), and plants from 45 genotypes were subsequently transferred to a greenhouse.

scholarly journals Physiological and Structural Aspects of In Vitro Somatic Embryogenesis in Abies alba Mill

Forests ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1210
Author(s):  
Terezia Salaj ◽  
Katarina Klubicová ◽  
Bart Panis ◽  
Rony Swennen ◽  
Jan Salaj
Keyword(s):  
Somatic Embryogenesis ◽  
Cell Lines ◽  
Somatic Embryos ◽  
Abies Alba ◽  
Embryogenic Tissue ◽  
Zygotic Embryos ◽  
Peg 4000 ◽  
Initiation Frequency ◽  
Embryogenic Tissues

Initiation of somatic embryogenesis from immature zygotic embryos, long-term maintenance of embryogenic tissue in vitro or by cryopreservation, as well as maturation, of somatic embryos of Abies alba Mill. are reported in this study. For the initiation of embryogenic tissues, a DCR medium containing different types of cytokinins (1 mg.L−1) were tested. During three consecutive years, 61 cell lines were initiated out of 1308 explants, with initiation frequencies ranging between 0.83 and 13.33%. The type of cytokinin had no profound effect on the initiation frequency within one given year. Microscopic observations revealed presence of bipolar somatic embryos in all initiated embryogenic tissues. Besides the typical bipolar somatic embryos, huge polyembryonal complexes, as well as “twin” embryos, were observed. Maturation of somatic embryos occurred on a DCR medium supplemented by abscisic acid (10 mg.L−1), polyethylene glycol (PEG-4000, 7.5%) and 3% maltose. The maturation capacity was cell-line dependent. All of the four tested cell lines produced cotyledonary somatic embryos, though at different quantities, of 16 to 252 per g of fresh weight. After germination, seedlings developed, but their further growth soon stopped after the formation of a resting bud. Altogether, seven cell lines were cryopreserved, using the slow-freezing technique. After rewarming, all tested cell lines showed regrowth rates between 81.8 and 100%.

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

2009 ◽  
Vol 55 (No. 4) ◽  
pp. 156-161 ◽  
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 (<I>Picea abies</I> [L.] Karst.) transgenic clones by means of <I>Agrobacterium</I>. 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 <I>Agrobacterium</I> 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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