Blue sky’s the limit? Somatic embryogenesis as a means of propagating recalcitrant blue spruce (Picea pungens) cultivar Hoopsii

AbstractThe ‘triple-blue’ cultivar of blue spruce (Picea pungens Hoopsii) is notably recalcitrant towards the realm of traditional vegetative propagation methods. Its ability to naturally proliferate is limited by ovule and embryo abortion during the growing season, leading to low viable seed yield. In this study, we established a protocol using somatic embryogenesis (SE) as a means of propagating this popular ornamental cultivar. We collected cones from Hoopsii trees at seven different timepoints throughout the growing season (mid-June to late July in Ottawa (Plant Hardiness Zone 5A)). Female megagametophytes were harvested following each collection and immature zygotic embryos were plated onto induction media. Early somatic embryos began developing from the embryonic tissue (ET) three to five weeks following induction. The highest ET initiation frequency occurred from embryos collected June 20–July 10, suggesting that developmental stage of the embryo was a significant factor in SE induction. The conversion of mature somatic embryos into plantlets (emblings) was completed in eight–ten weeks at a rate of 92.8%. In this study, we demonstrate that in vitro somatic embryogenesis using our optimized protocol is a fast and prolific method for the mass propagation of Hoopsii blue spruce. This is the first report on the production of somatic Hoopsii emblings.

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

Forests ◽

10.3390/f11111210 ◽

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%.

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Somatic Embryogenesis using Immature Zygotic Embryos from Developing Fruits of Papaya (Carica papaya)

HortScience ◽

10.21273/hortsci.30.4.783e ◽

1995 ◽

Vol 30 (4) ◽

pp. 783E-783

Author(s):

S.K. Dhir ◽

U.L. Yadava

Keyword(s):

Somatic Embryogenesis ◽

Somatic Embryos ◽

Carica Papaya ◽

Induction Medium ◽

Zygotic Embryos ◽

Synthetic Seed ◽

Factors Affecting ◽

Secondary Embryos ◽

Potential Use

An efficient protocol has been developed for the in vitro multiplication of papaya (Carica papaya L.) through somatic embryogenesis utilizing immature zgotic embryos. Somatic embryos were initiated on MS basel media supplemented with 5 mg·liter–1 2,4-D, 400 mg·liter–1 glutamine, and 6% sucrose. After culturing for 2 months, 65% of the explants became highly embryogenic. Each explant produced 50 to 80 embryos in 4 months on culture induction medium. Frequency of embryogenesis was increased (75 to 150 somatic embryos on 80% explants) upon supplementing medium with 4% maltose as a carbon source and 100 mg·liter–1 L-asparagine. The embryogenic callus appeared yellow and embryos at different stages of development were well-organized. On regular subculturing, these cultures continued to produce secondary embryos. Following their transfer to the hormone-free medium supplemented with 4% maltose, these embryos germinated. The somatic embryogenesis system is rapid, repetitive, and highly proliferative. Thus, this system may have a potential use in the development of synthetic seed and transgenic papaya plants. Details of important factors affecting somatic embryogenesis will be discussed.

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Somatic embryogenesis in tissue cultures of Podophyllum hexandrum

Canadian Journal of Botany ◽

10.1139/b90-065 ◽

1990 ◽

Vol 68 (3) ◽

pp. 487-491 ◽

Cited By ~ 35

Author(s):

N. Arumugam ◽

Sant S. Bhojwani

Keyword(s):

Somatic Embryogenesis ◽

Somatic Embryos ◽

Basal Medium ◽

Podophyllum Hexandrum ◽

Zygotic Embryos ◽

Tissue Cultures ◽

Globular Embryos ◽

Further Development ◽

Sucrose Level

In vitro multiplication of Podophyllum hexandrum Royle (Podophyllaceae) via somatic embryogenesis is reported. The callus derived from zygotic embryos on Murashige and Skoog medium containing 2 μM BA and 0.5μM IAA differentiated globular embryos. On this medium the globular embryos continued to multiply but failed to mature. Further development of the embryos occurred if the sucrose level in the basal medium was raised to 6% or the medium was supplemented with 1–10 μM NAA. Light and temperatures higher than 25 °C suppressed embryogenesis. Embryogenic potential of the callus has been maintained for over 20 months through subcultures. The somatic embryos developed into plantlets on the basal medium. Key words: endangered species, podophyllotoxin, Podophyllum, somatic embryogenesis.

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Somatic embryogenesis from zygotic embryos of Euterpe oleracea Mart.

Revista Brasileira de Fruticultura ◽

10.1590/s0100-29452002000300004 ◽

2002 ◽

Vol 24 (3) ◽

pp. 601-603 ◽

Cited By ~ 12

Author(s):

Ana da Silva Ledo ◽

Osmar Alves Lameira ◽

Abdellatif Kemaleddine Benbadis ◽

Ilmarina Campos de Menezes ◽

Maria do Socorro Padilha de Oliveira ◽

...

Keyword(s):

Somatic Embryogenesis ◽

Somatic Embryos ◽

Zygotic Embryos ◽

Ms Medium ◽

Euterpe Oleracea ◽

Mineral Salts ◽

Vegetable Material ◽

Morphogenetic Responses ◽

Euterpe Oleracea Mart

The aim of this work was to study the morphogenetic responses of zygotic embryos of açai palm (Euterpe oleracea Mart.) submitted to several conditions of in vitro culture. Several research experiments were conducted, in laboratory, using vegetable material collected from açai palm plants at Embrapa Amazon Oriental, Belém-PA, Brazil. It was possible to verify the expression of a direct, repetitive and no-synchronized model of somatic embryogenesis in mature zygotic embryos cultivated in primary MS medium supplemented with 2,4-D (339.36 muM) and transferred to a secondary MS medium in the presence of NAA (0.537 muM) and 2iP (12.30 muM). The conversion of somatic embryos into seedlings was reached after 210 days with the transfer of the cultures to a third medium with sucrose and mineral salts concentrations reduced to a half, without growth regulators.

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Evaluation of somatic embryogenesis for clonal propagation of western white pine

Canadian Journal of Forest Research ◽

10.1139/x00-115 ◽

2000 ◽

Vol 30 (12) ◽

pp. 1867-1876 ◽

Cited By ~ 32

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.

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Somatic embryogenesis from immature and mature zygotic embryos and from cotyledons and needles of somatic plantlets of Larix

Canadian Journal of Forest Research ◽

10.1139/x94-015 ◽

1994 ◽

Vol 24 (1) ◽

pp. 100-106 ◽

Cited By ~ 32

Author(s):

M.A. Lelu ◽

K. Klimaszewska ◽

P.J. Charest

Keyword(s):

Somatic Embryogenesis ◽

Developmental Stage ◽

Somatic Embryos ◽

Zygotic Embryos ◽

Mature Tree ◽

Immature Zygotic Embryos ◽

Specific Combination ◽

Induction Frequency ◽

Initiation Frequency ◽

First Time

Induction of embryonal masses was achieved from full-sib immature zygotic embryos of Larixdecidua Mill., Larix × eurolepis A. Henry, and Larix × leptoeuropaea, and it was found to be affected by the developmental stage of the embryos. Furthermore, the expiant response depended on a specific combination of parental trees used for production of seeds. For the first time, induction was successful in L. decidua with mature zygotic embryos. These embryos were isolated from one of the three seedlots tested and induction frequency was 5%. Mature somatic embryos of L. × leptoeuropaea expressed an initiation frequency of 83%. Cotyledons and needles of plantlets derived from somatic embryos of L. × leptoeuropaea were used as expiants to induce somatic embryogenesis at a frequency of 8 and 3%, respectively. A cytokinin pretreatment seemed to enhance the frequency of induction. Mature somatic embryos were obtained from embryonal masses derived from expiants of different ontogenic age. Future opportunities of research into the induction of embryonal masses from mature tree tissues are discussed.

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Somatic embryogenesis in mature zygotic embryos of Picea pungens

Scientific Reports ◽

10.1038/s41598-021-98511-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jing Tao ◽

Shigang Chen ◽

Caiyun Qin ◽

Qingmei Li ◽

Jufeng Cai ◽

...

Keyword(s):

Somatic Embryogenesis ◽

Average Rate ◽

Zygotic Embryos ◽

Mature Embryos ◽

Picea Pungens ◽

Average Survival ◽

Maturation Medium ◽

Initiation Frequency ◽

Somatic Embryogenic ◽

Embryogenic Tissues

AbstractThis study developed somatic embryogenesis protocols for Picea pungens (Engelm), an important ornamental species, including initiation, proliferation, maturation, germination, and acclimation. Somatic embryogenic tissues were induced from mature zygotic embryos of five families, with a frequency of $$\ge $$ ≥ 22% for each. Embryogenic tissues (ET) from 13 clones of three families were proliferated for one week, achieving an average rate of 179.1%. The ET of 38 clones of three families were cultured in maturation medium for six weeks; 188 mature embryos on average were counted per gram ET cultured, of which $$\ge $$ ≥ 81.1% appeared normal, and each clone developed at least 28 normally matured embryos. A total of 69.9% or more of cotyledonary somatic embryos germinated normally and developed into normal emblings. The experiment of transplanting the emblings into a greenhouse had an average survival rate of 68.5%. Considerable variation among and within families during initiation and proliferation was observed, but this variation decreased in the maturation and germination. Changing the concentration of plant growth regulator of the initiation medium did not significantly change the initiation frequency. We recommend incorporating these protocols into the current Picea pungens practical programs, although further research is essential to increase efficiencies and reduce cost.

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Somatic embryogenesis in conifers

Canadian Journal of Botany ◽

10.1139/b91-237 ◽

1991 ◽

Vol 69 (9) ◽

pp. 1873-1899 ◽

Cited By ~ 154

Author(s):

T. E. Tautorus ◽

L. C. Fowke ◽

D. I. Dunstan

Keyword(s):

Somatic Embryogenesis ◽

Picea Glauca ◽

Somatic Embryos ◽

Zygotic Embryos ◽

Zygotic Embryogenesis ◽

Direct Gene Transfer ◽

Plantlet Production ◽

Substantial Progress ◽

Liquid Suspensions

Substantial progress has been made towards the development of systems for in vitro embryogenesis in conifers. Since the first report of somatic embryogenesis from zygotic embryos of Picea abies in 1985, cultured explants of at least 18 different coniferous species have been induced to produce somatic embryos. Somatic embryos have been cryopreserved, grown in liquid suspensions, and matured into plants. In addition, plantlets have been regenerated from protoplasts isolated from embryogenic suspensions of Picea glauca and Larix ×eurolepis, permitting studies into direct gene transfer and somatic hybridization. Currently however, it is only possible to obtain somatic embryogenesis from embryonic and juvenile explants. Furthermore, for most species the efficiency of plantlet production from somatic embryos is poor and remains a problem for the commercial utilization of this technology. Biochemical, cytological, and physiological studies of conifer somatic embryogenesis have resulted in improved knowledge concerning the origin of somatic embryos, storage product accumulation during embryo development, and similarities with zygotic embryos. Furthermore, the technique of indirect immunofluorescence microscopy has permitted investigations of the cytoskeleton in conifer cells and protoplasts, providing insights into cell division and morphogenesis. In this review, emphasis is placed on the more fundamental aspects of conifer somatic embryogenesis. Where possible, comparisons between zygotic and somatic embryogenesis are made. Key words: somatic embryogenesis, zygotic embryogenesis, conifers, review.

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Somatic embryogenesis in goiabeira serrana: genotype response, auxinic shock and synthetic seeds

Revista Brasileira de Fisiologia Vegetal ◽

10.1590/s0103-31312001000200001 ◽

2001 ◽

Vol 13 (2) ◽

pp. 117-128 ◽

Cited By ~ 17

Author(s):

MIGUEL P. GUERRA ◽

LIRIO L. DAL VESCO ◽

JEAN PIERRE H.J. DUCROQUET ◽

RUBENS O. NODARI ◽

MAURÍCIO S. DOS REIS

Keyword(s):

Somatic Embryogenesis ◽

Low Temperatures ◽

Somatic Embryos ◽

Synthetic Seeds ◽

Zygotic Embryos ◽

Embryogenic Competence ◽

Number Of Factors ◽

And Control ◽

Genotype Response

The induction and control of in vitro somatic embryogenesis is dependent of a number of factors. The genotype of the mother plant donor of explants and the kind of auxin play essential roles in conferring embryogenic competence. Once obtained somatic embryos may be encapsulated in alginate originating synthetic seeds that can be stored at low temperatures. Considering this the present work aimed at the assessment of five different genotypes, five periods of 2,4-D shock (20 muM for 1, 2, 4, 8 and 16 weeks) (), and different substances supplemented to sodium alginate to obtain synthetic seeds. Zygotic embryos of Feijoa sellowiana were inoculated in basal LP medium (von Arnold and Erikson, 1981) supplemented with Morel’s vitamins, sucrose (3%) and phytagel (0.2%). The highest rates of somatic embryogenesis induction were observed in the genotypes 50-4 and 101. Shock of two weeks in 2,4-D conferred embryogenic competence in the same levels obtained with the shock of 8 weeks. Histological evaluations revealed the direct origin of the somatic embryos from the epidermic surface of cotyledons. Synthetic seeds containing MS salts and sucrose resulted in higher rates of contamination than capsules free of these substances. KNO3 (100 mM) resulted in the opening of 81.2% of the capsules as compared to 0% of opening in the treatment with water.

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In vitro germination and embryogenic competence acquisition of Euterpe edulis Martius immature zygotic embryos

Cropp Breeding and Applied Biotechnology ◽

10.1590/s1984-70332012000300002 ◽

2012 ◽

Vol 12 (3) ◽

pp. 171-178 ◽

Cited By ~ 3

Author(s):

Cleber Witt Saldanha ◽

Maisa Pimentel Martins-Corder

Keyword(s):

Somatic Embryogenesis ◽

Calcium Chloride ◽

Somatic Embryos ◽

Sucrose Concentration ◽

Zygotic Embryos ◽

Palm Tree ◽

In Vitro Germination ◽

Embryogenic Competence ◽

Immature Zygotic Embryos

This study evaluated different aspects of in vitro germination and embryogenic competence of immature zygotic embryos of E. edulis. The embryos germinated on full or half-strength MS (MS or MS/2) medium combined with sucrose (20, 30 and 40 g L-1). The effect of calcium chloride concentrations (0, 2, 4, 8, and 12 mM) on the induction of somatic embryogenesis was tested. The embryos were germinated on MS or MS/2. Germination of zygotic embryos and the number of roots per plantlet were not affected by the culture medium and sucrose concentration. Plantlet height and fresh weight were influenced by both; the difference was greatest in MS medium with 40 g L-1 sucrose. The induction of somatic embryogenesis was not influenced by doses of calcium chloride, whereas the number of somatic embryos formed was affected. The germination capacity of somatic embryos of heart-of-palm tree was not influenced by the media tested.

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