Single-Base Resolution Methylomes of Somatic Embryogenesis in Theobroma Cacao L. Reveal Epigenome Modifications Associated With Somatic Embryo Abnormalities

Abstract Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.

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Pengaruh Sitokinin, Jenis Eksplan, dan Genotipe terhadap Embriogenesis Somatik Kakao

Jurnal Tanaman Industri dan Penyegar ◽

10.21082/jtidp.v3n2.2016.p71-82 ◽

2016 ◽

Vol 3 (2) ◽

pp. 71

Author(s):

Nur Ajijah ◽

RR. Sri Hartati

Keyword(s):

Tissue Culture ◽

Somatic Embryogenesis ◽

Somatic Embryo ◽

Theobroma Cacao ◽

Somatic Embryos ◽

Interaction Effects ◽

Embryo Formation ◽

Primary Somatic Embryogenesis ◽

Primary Somatic Embryos

Information on the effect of cytokinins on cacao (Theobroma cacao L.) primary somatic embryogenesis and its interaction with explant types and genotypes is not yet known. This study aimed to evaluate the effect of cytokinins and its interaction with explant types and genotypes on cacao somatic embryogenesis. The study was conducted at tissue culture laboratory of IAARD, Bogor from April until December 2012 and October 2014 until February 2016. Three types of cytokinins i.e. kinetin (0.58, 1.16, and 2.32 μM), thidiazuron (0.01, 0.02, and 0.04 μM) and benzylaminopurine (0.55, 1.11, and 2.22 μM) in combination with 9 μM 2,4-D were tested for their effectiveness in inducing somatic embryogenesis from petals and staminoid explants of Cimanggu 1 genotype. Furthermore, three levels of kinetin (0.58, 1.16, and 2.32 μM) also in combination with 9 μM 2,4-D were evaluated for their influences on the somatic embryogenesis from petals and staminoid explants of three cacao genotypes i.e. Sulawesi 02, ICCRI 04 and Cimanggu 3. The result demonstrated that 2.32 μM kinetin and staminoids explant were more effective to induce cacao somatic embryogenesis of Cimanggu 1 genotype (7%, 0.23 embryos/explant). Additionally, there were interaction effects between the level of kinetin with explant types and genotype on the percentage of explants forming embryo at 12 weeks after culture. The highest percentage of somatic embryo formation was shown by ICCRI 04 genotype with the use of petals explant and a kinetin level of 1.16 μM (31.85%), but not significantly different from the level of kinetin 2.23 μM (25.55%). The formation of primary somatic embryos of cacao is largely determined by the type and level of cytokinins, type of explant, and genotype.

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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 and In vitro Plant Regeneration in Vigna trilobata (L.) Verd. - A Potential Pasture Legume

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v19i1.4990 ◽

1970 ◽

Vol 19 (1) ◽

pp. 89-99

Author(s):

K. Choudhary ◽

M. Singh ◽

M. S. Rathore ◽

N. S. Shekhawat

Keyword(s):

Somatic Embryogenesis ◽

Growth Regulators ◽

Somatic Embryos ◽

Basal Medium ◽

Long Term Study ◽

Continuous Application ◽

First Time ◽

Pasture Legume

This long term study demonstrates for the first time that it is possible to propagate embryogenic Vigna trilobata and to subsequently initiate the differentiation of embryos into complete plantlets. Initiation of callus was possible on 2,4-D. Somatic embryos differentiated on modified MS basal nutrient medium with 1.0 mg/l of 2,4-D and 0.5 mg/l of Kn. Sustained cell division resulted in globular and heart shape stages of somatic embryos. Transfer of embryos on to a fresh modified MS basal medium with 0.5 mg/l of Kn and 0.5 mg/l of GA3 helped them to attain maturation and germination. However, the propagation of cells, as well as the differentiation of embryos, were inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking these growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in Vigna species are discussed. Key words: Pasture legume, Vigna trilobata, Globular, Heart shape, somatic embryogenesis D.O.I. 10.3329/ptcb.v19i1.4990 Plant Tissue Cult. & Biotech. 19(1): 89-99, 2009 (June)

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EFFECT OF 2,4-D AND NAA ON SOMATIC EMBRYOGENESIS FROM APICAL PORTION OF GROUNDNUT (ARACHIS HYPOGAEA L.)

International journal of multidisciplinary advanced scientific research and innovation ◽

10.53633/ijmasri.2021.1.7.01 ◽

2021 ◽

Vol 1 (7) ◽

pp. 119-124

Author(s):

Muniappan V ◽

Manivel P ◽

Prabakaran V ◽

Palanivel S ◽

Parvathi S

Keyword(s):

Somatic Embryogenesis ◽

Somatic Embryo ◽

Arachis Hypogaea ◽

Somatic Embryos ◽

Mature Embryo ◽

Induction Medium ◽

Embryogenic Cultures ◽

Apical Portion ◽

Arachis Hypogaea L ◽

Somatic Embryo Induction

Somatic embryogenesis was carried out epicotyl portion of the mature embryo/apical portion. The somatic embryo induction medium containing 2,4-D or NAA (10.0 to 50.0 mg/l). Of the two concentrations tested 2,4-D (30.0mg/l) recorded the highest percentage of response followed by NAA (30.0mg/l). But the highest number of somatic embryo were recorded in 30.0mg/l of 2,4-D followed by NAA. The apical portion of the mature embryo formed direct embryos without any intervention of callus. The maximum percentage of embryogenic cultures were noticed in 30.0mg/l of 2,4-D followed by NAA at 30.0mg/l. for the differentiation of somatic embryos, the embryogenic masses were transferred to medium without any growth regulator. The maximum number of somatic embryos per culture was recorded in 30 mg/l of 2,4-D followed by 30.0 mg/l of NAA. Keywords: Arachis hypogaea L.,Somatic Embryogenesis, 2,4-D and NAA

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Induction of Somatic Embryogenesis in Vulnerable Medicinal Plant Hedychium spicatum Buch-Ham ex Smith

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v23i2.17506 ◽

2014 ◽

Vol 23 (2) ◽

pp. 147-155 ◽

Cited By ~ 1

Author(s):

Dinesh Giri ◽

Sushma Tamta

Keyword(s):

Somatic Embryogenesis ◽

Medicinal Plant ◽

Somatic Embryos ◽

Synthetic Seeds ◽

Ex Situ ◽

Secondary Embryogenesis ◽

Zygotic Embryos ◽

High Concentration ◽

Cotyledon Explants ◽

Short Period

This protocol has been developed for somatic embryogenesis in Hedychium spicatum. Simultaneously, a method has also been developed for the production of synthetic seeds by using somatic embryos. Direct somatic embryos were developed on cotyledon explants of zygotic embryos on MS supplemented with high concentration of NAA (20.0 µM). Induction of secondary embryogenesis was best in 2,4-D supplemented medium fortified with activated charcoal. Germination of somatic embryos was enhanced by using GA3. Besides this, round and semi-hard beads of somatic embryos (synthetic seeds) could be produced by using 2% Na-alginate and 100 mM calcium chloride and more than 30% germination of synthetic seeds was achieved in MS. Well acclimated plants produced via somatic embryogenesis and/or synthetic seeds were transferred to field where more than 60% survived. This simple study enabled us to obtain a number of plantlets throughout the year each cycle requiring a short period of time. Besides propagation, this study provided an ex situ method for conservation of this vulnerable Himalayan species.D. O. I.http://dx.doi.org/10.3329/ptcb.v23i2.17506Plant Tissue Cult. & Biotech. 23(2): 147-155, 2013 (December)

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Can Ethylene Inhibitors Enhance the Success of Olive Somatic Embryogenesis?

Plants ◽

10.3390/plants11020168 ◽

2022 ◽

Vol 11 (2) ◽

pp. 168

Author(s):

Muhammad Ajmal Bashir ◽

Cristian Silvestri ◽

Amelia Salimonti ◽

Eddo Rugini ◽

Valerio Cristofori ◽

...

Keyword(s):

Somatic Embryogenesis ◽

Salicylic Acid ◽

Silver Nitrate ◽

Embryogenic Callus ◽

Somatic Embryos ◽

Cobalt Chloride ◽

Zygotic Embryos ◽

Ethylene Inhibitors ◽

Embryogenic Calli ◽

Stimulatory Action

An efficient in vitro morphogenesis, specifically through somatic embryogenesis, is considered to be a crucial step for the application of modern biotechnological tools for genetic improvement in olive (Olea europaea L.). The effects of different ethylene inhibitors, i.e., cobalt chloride (CoCl2), salicylic acid (SA), and silver nitrate (AgNO3), were reported in the cyclic somatic embryogenesis of olive. Embryogenic callus derived from the olive immature zygotic embryos of the cultivar Leccino, was transferred to the expression ECO medium, supplemented with the ethylene inhibitors at 20 and 40 µM concentrations. Among these, the maximum number of somatic embryos (18.6) was obtained in media containing silver nitrate (40 µM), followed by cobalt chloride (12.2 somatic embryos @ 40 µM) and salicylic acid (40 µM), which produced 8.5 somatic embryos. These compounds interfered on callus traits: white friable embryogenic calli were formed in a medium supplemented with 40 µM cobalt chloride and salicylic acid; in addition, a yellow-compact embryogenic callus appeared at 20 µM of all the tested ethylene inhibitors. The resulting stimulatory action of silver nitrate among all the tested ethylene inhibitors on somatic embryogenesis, clearly demonstrates that our approach can efficiently contribute to the improvement of the current SE protocols for olive.

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Pinus canariensis plant regeneration through somatic embryogenesis

Forest Systems ◽

10.5424/fs/2020291-16136 ◽

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).

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Efficient Plant Regeneration from Cotyledon and Midrib Derived Callus in Eggplant (Solanum melongena L.)

Journal of Bio-Science ◽

10.3329/jbs.v14i0.439 ◽

1970 ◽

Vol 14 ◽

pp. 31-38 ◽

Cited By ~ 3

Author(s):

M Rahman ◽

M Asaduzzaman ◽

N Nahar ◽

MA Bari

Keyword(s):

Somatic Embryogenesis ◽

Plant Regeneration ◽

Key Words ◽

Somatic Embryo ◽

Callus Induction ◽

Somatic Embryos ◽

Solanum Melongena ◽

Induction Medium ◽

Root Induction ◽

Ms Medium

Somatic embryos were obtained from cotyledon and midrib explants of Solanum melongena L., cultivar Loda. For callus induction, medium was supplemented with different concentrations of auxin singly or in combination with BAP. The best callusing 83-85% was obtained from both of the explants cultured on MS medium containing 2.0 mgl-1NAA + 0.05 mgl-1BAP. Somatic embryogenesis and shoot regeneration was achieved after transferring the calli to MS medium supplemented with BAP, GA3, NAA and Zeatin. Cotyledon derived calli showed better performance (87%) for regeneration than that of midrib (82%) when sub cultured on MS medium having 2.0 mgl-1 Zeatin + 1.0 mgl-1 BAP. For root induction, MS + 3.0 mgl-1 IBA was proved to be better treatment for average number (14-15) and mean length (12 cm) of roots than those of other treatments. Key words: Eggplant; cotyledon; midrib; callus induction; somatic embryo J. bio-sci. 14: 1-9, 2006

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Two alternative pathways of somatic embryo origin from polyembryonic mature stored seeds of Pinus koraiensis Sieb et Zucc.

Canadian Journal of Botany ◽

10.1139/b97-055 ◽

1997 ◽

Vol 75 (3) ◽

pp. 509-512 ◽

Cited By ~ 16

Author(s):

P. V. Bozhkov ◽

I. S. Ahn ◽

Y. G. Park

Keyword(s):

Somatic Embryo ◽

Somatic Embryos ◽

Zygotic Embryo ◽

Pinus Koraiensis ◽

Zygotic Embryos ◽

Dichlorophenoxyacetic Acid ◽

Strong Negative Correlation ◽

Alternative Pathways ◽

2,4 Dichlorophenoxyacetic Acid ◽

Embryo Origin

Individual mature stored seeds of Pinus koraiensis sometimes contain several viable zygotic embryos originated through the processes of simple and cleavage polyembryony. To induce the embryonic process, isolated zygotic embryos were cultured on five different media all supplemented with 10 μM 2,4-dichlorophenoxyacetic acid and 5 μM 6-benzyladenine. Two alternative pathways of somatic embryo origin were revealed. The first pathway was associated with the production of a friable, translucent callus in the hypocotyls–cotyledon region of the dominant zygotic embryo. The second pathway was related to the proliferation of a translucent, moist, and mucilaginous tissue (termed embryonal–suspensor mass) in the suspensor region of the dominant zygotic embryo. Both types of tissues contained early somatic embryos. Regression analysis has shown a strong negative correlation between the frequencies of formation of embryogenic callus and embryonal–suspensor mass both at 3 and 8 weeks of culture (r = − 0.85; p = 0.07 and r = −0.71; p = 0.17, respectively). Key words: Pinus koraiensis; polyembryonal seeds; somatic embryogenesis; embryogénie callus; embryonal–suspensor mass.

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