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Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Plant Sciences".

Deadline for manuscript submissions: closed (30 April 2023) | Viewed by 16953

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Special Issue Editors

Dr. Elena Corredoira

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Guest Editor

Misión Biológica de Galicia (MBG-CSIC), Sede Santiago de Compostela, 15782 Santiago de Compostela, Spain
Interests: plant biotechnology; in vitro culture; micropropagation; somatic embryogenesis; woody plants
Special Issues, Collections and Topics in MDPI journals

Dr. Mª Teresa Martínez

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Dear Colleagues,

Somatic embryogenesis is considered a fascinating example of the cellular totipotency concept. During this regeneration process, one or a few somatic cells are subjected to favorable in vitro experimental conditions, and undergo morphological and biochemical changes that induce them to form bipolar structures, which are known as somatic embryos. Although gamete fusion does not take place, somatic embryos resemble zygotic embryos and have the ability to germinate and develop into somatic seedlings. In addition to the clonal propagation application, embryogenic cultures represent excellent target materials for the insertion of isolated gene sequences into somatic cells and the subsequent production of genetically modified plants. This technology is expected to be replaced by the emerging field of plant genome editing, which will most likely require embryogenic cultures as a means of regenerating improved plants. In addition, the application of synthetic seed technology to somatic embryos is being considered with regard to storage and handling qualities. Somatic embryogenic lines can be subjected to the well-developed cryopreservation techniques for the long-term conservation of plant diversity or conservation during the field testing of regenerated plants and subsequent retrieval of superior lines from liquid nitrogen for their exploitation. Finally, the somatic embryogenesis process is an ideal system to study the molecular mechanisms that regulate developmental plasticity in plants. All of these potential applications have made somatic embryogenesis a subject of great scientific interest.

We welcome submissions related to the overall topic of somatic embryogenesis, including the following subtopics:

- Factors affecting the somatic embryogenesis induction step: stress stimuli, endogenous content of auxins and cytokinins, role of explant type and its physiological state, and role of exogenous plant growth regulators;

- Anatomical and histological analysis of somatic embryogenesis process;

- Androgenesis;

- Cryopreservation somatic embryos;

- Genetic transformation somatic embryos;

- Genome editing somatic embryos;

- Synthetic seeds;

- Liquid culture: embryogenic suspensions and bioreactors;

- Gene expression during somatic embryogenesis process;

- Epigenetic modifications: histone modification and DNA methylation.

Dr. Elena Corredoira
Dr. Mª Teresa Martínez
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

cryopreservation
embryo competence
gene expression
genetic transformation
plasticity
somatic embryo
totipotency

Published Papers (9 papers)

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Research

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20 pages, 4101 KiB

Open AccessArticle

Opposite Auxin Dynamics Determine the Gametophytic and Embryogenic Fates of the Microspore

by Yolanda Pérez-Pérez, María Teresa Solís, Alfonso Albacete and Pilar S. Testillano

Int. J. Mol. Sci. 2023, 24(13), 11177; https://doi.org/10.3390/ijms241311177 - 6 Jul 2023

Viewed by 1232

Abstract

The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, in Brassica napus. We analyzed auxin concentration, cellular accumulation, the expression of the TAA1 auxin biosynthesis gene, and the PIN1-like efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and TAA1 and PIN1-like expression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis, TAA1 and PIN1-like genes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

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21 pages, 4330 KiB

Open AccessArticle

Heat-Priming during Somatic Embryogenesis Increased Resilience to Drought Stress in the Generated Maritime Pine (Pinus pinaster) Plants

by María Amparo Pérez-Oliver, María del Carmen González-Mas, Begoña Renau-Morata, Isabel Arrillaga and Ester Sales

Int. J. Mol. Sci. 2023, 24(11), 9299; https://doi.org/10.3390/ijms24119299 - 26 May 2023

Cited by 3 | Viewed by 1378

Abstract

Drought stress is becoming the most important factor of global warming in forests, hampering the production of reproductive material with improved resilience. Previously, we reported that heat-priming maritime pine (Pinus pinaster) megagametophytes during SE produced epigenetic changes that generated plants better adapted to subsequent heat stress. In this work, we tested, in an experiment performed under greenhouse conditions, whether heat-priming will produce cross-tolerance to mild drought stress (30 days) in 3-year-old priming-derived plants. We found that they maintain constitutive physiological differences as compared to controls, such as higher proline, abscisic acid, starch, and reduced glutathione and total protein contents, as well as higher ΦPSII yield. Primed plants also displayed a constitutive upregulation of the WRKY transcription factor and the Responsive to Dehydration 22 (RD22) genes, as well as of those coding for antioxidant enzymes (APX, SOD, and GST) and for proteins that avoid cell damage (HSP70 and DHNs). Furthermore, osmoprotectants as total soluble sugars and proteins were early accumulated in primed plants during the stress. Prolongated water withdrawal increased ABA accumulation and negatively affected photosynthesis in all plants but primed-derived plants recovered faster than controls. We concluded that high temperature pulses during somatic embryogenesis resulted in transcriptomic and physiological changes in maritime pine plants that can increase their resilience to drought stress, since heat-primed plants exhibit permanent activation of mechanisms for cell protection and overexpression of stress pathways that pre-adapt them to respond more efficiently to soil water deficit. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

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25 pages, 3274 KiB

Open AccessArticle

Proteomic and Metabolic Analysis of Pinus halepensis Mill. Embryonal Masses Induced under Heat Stress

by Cátia Pereira, Ander Castander-Olarieta, Itziar A. Montalbán, Vera M. Mendes, Sandra Correia, Ana Pedrosa, Bruno Manadas, Paloma Moncaleán and Jorge Canhoto

Int. J. Mol. Sci. 2023, 24(8), 7211; https://doi.org/10.3390/ijms24087211 - 13 Apr 2023

Cited by 1 | Viewed by 1325

Abstract

Understanding the physiological and molecular adjustments occurring during tree stress response is of great importance for forest management and breeding programs. Somatic embryogenesis has been used as a model system to analyze various processes occurring during embryo development, including stress response mechanisms. In addition, “priming” plants with heat stress during somatic embryogenesis seems to favor the acquisition of plant resilience to extreme temperature conditions. In this sense, Pinus halepensis somatic embryogenesis was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) and its effects on the proteome and the relative concentration of soluble sugars, sugar alcohols and amino acids of the embryonal masses obtained were assessed. Heat severely affected the production of proteins, and 27 proteins related to heat stress response were identified; the majority of the proteins with increased amounts in embryonal masses induced at higher temperatures consisted of enzymes involved in the regulation of metabolism (glycolysis, the tricarboxylic acid cycle, amino acid biosynthesis and flavonoids formation), DNA binding, cell division, transcription regulation and the life-cycle of proteins. Finally, significant differences in the concentrations of sucrose and amino acids, such as glutamine, glycine and cysteine, were found. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

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14 pages, 5687 KiB

Open AccessArticle

Genome-Wide Identification of Homeodomain Leucine Zipper (HD-ZIP) Transcription Factor, Expression Analysis, and Protein Interaction of HD-ZIP IV in Oil Palm Somatic Embryogenesis

by Kamolwan Khianchaikhan, Suvichark Aroonluk, Supachai Vuttipongchaikij and Chatchawan Jantasuriyarat

Int. J. Mol. Sci. 2023, 24(5), 5000; https://doi.org/10.3390/ijms24055000 - 5 Mar 2023

Cited by 2 | Viewed by 1532

Abstract

Understanding the molecular mechanisms underlying somatic embryogenesis is essential for resolving the problems related to the long duration of the process and a low rate of somatic embryo induction in oil palm tissue culture. In this study, we conducted genome-wide identification of the oil palm homeodomain leucine zipper (EgHD-ZIP) family, which is one of the plant-specific transcription factors reported to be involved in embryogenesis. EgHD-ZIP proteins can be divided into four subfamilies, which have similarities in gene structure and protein-conserved motifs within a group. In silico expression analysis showed that the expression of EgHD-ZIP gene members in the EgHD-ZIP I and II families, as well as most members in the EgHD-ZIP IV family, were up-regulated during the zygotic and somatic embryo developmental stages. In contrast, the expression of EgHD-ZIP gene members in the EgHD-ZIP III family was down-regulated during zygotic embryo development. Moreover, the expression of EgHD-ZIP IV genes was validated in the oil palm callus and at the somatic embryo stages (globular, torpedo, and cotyledon). The results revealed that EgHD-ZIP IV genes were up-regulated at the late stages of somatic embryogenesis (torpedo and cotyledon). While BABY BOOM (BBM) gene was up-regulated at the early stage of somatic embryogenesis (globular). In addition, the Yeast-two hybrid assay revealed the direct binding between all members of the oil palm HD-ZIP IV subfamily (EgROC2, EgROC3, EgROC5, EgROC8, and EgBBM). Our findings suggested that the EgHD-ZIP IV subfamily and EgBBM work together to regulate somatic embryogenesis in oil palms. This process is important because it is widely used in plant biotechnology to produce large quantities of genetically identical plants, which can be used for oil palm tissue culture improvement. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

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13 pages, 313 KiB

Open AccessArticle

Reducing Pre- and Post-Treatments in Cryopreservation Protocol and Testing Storage at −80 °C for Norway Spruce Embryogenic Cultures

by Saila A. Varis, Susanna Virta, Itziar A. Montalbán and Tuija Aronen

Int. J. Mol. Sci. 2022, 23(24), 15516; https://doi.org/10.3390/ijms232415516 - 8 Dec 2022

Cited by 4 | Viewed by 1067

Abstract

Somatic embryogenesis (SE) is considered the most effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a storage method that is able to handle large quantities of embryogenic tissues (ETs) reliably and at a low cost is required. The aim of the present study was to compare freezing at −80 °C in a freezer to cryopreservation using liquid nitrogen (LN) as a method for storing Norway spruce ETs. The possibility of simplifying both the pre-treatment and thawing processes in cryopreservation was also studied. The addition of abscisic acid (ABA) to the pre-treatment media and using polyethylene glycol PEG4000 instead of PEG6000 in a cryoprotectant solution were tested. Both the pre-and post-treatments on semi-solid media could be simplified by reducing the number of media, without any loss of genotype or embryo production capacity of ETs. On the contrary, the storage of ETs in a freezer at −80 °C instead of using LN was not possible, and the addition of ABA to the pre-treatment media did not provide benefits but increased costs. The lower regeneration rate after using PEG4000 instead of PEG6000 in a cryoprotectant solution in cryovials was unexpected and unwanted. The simplified pre-and post-treatment protocol will remarkably reduce the workload and costs in the mass-cryopreservation of future forest regeneration materials and in thawing the samples for mass propagations, respectively. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

11 pages, 2436 KiB

Open AccessArticle

Embryogenic Stem Cell Identity after Protoplast Isolation from Daucus carota and Recovery of Regeneration Ability through Protoplast Culture

by Jong-Eun Han, Han-Sol Lee, Hyoshin Lee, Hyunwoo Cho and So-Young Park

Int. J. Mol. Sci. 2022, 23(19), 11556; https://doi.org/10.3390/ijms231911556 - 30 Sep 2022

Cited by 3 | Viewed by 2110

Abstract

Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 10⁶ protoplasts·g⁻¹ FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 10⁴ protoplasts·g⁻¹ FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6–6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

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Review

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32 pages, 7455 KiB

Open AccessReview

A Common Molecular Signature Indicates the Pre-Meristematic State of Plant Calli

by Attila Fehér

Int. J. Mol. Sci. 2023, 24(17), 13122; https://doi.org/10.3390/ijms241713122 - 23 Aug 2023

Cited by 1 | Viewed by 1424

Abstract

In response to different degrees of mechanical injury, certain plant cells re-enter the division cycle to provide cells for tissue replenishment, tissue rejoining, de novo organ formation, and/or wound healing. The intermediate tissue formed by the dividing cells is called a callus. Callus formation can also be induced artificially in vitro by wounding and/or hormone (auxin and cytokinin) treatments. The callus tissue can be maintained in culture, providing starting material for de novo organ or embryo regeneration and thus serving as the basis for many plant biotechnology applications. Due to the biotechnological importance of callus cultures and the scientific interest in the developmental flexibility of somatic plant cells, the initial molecular steps of callus formation have been studied in detail. It was revealed that callus initiation can follow various ways, depending on the organ from which it develops and the inducer, but they converge on a seemingly identical tissue. It is not known, however, if callus is indeed a special tissue with a defined gene expression signature, whether it is a malformed meristem, or a mass of so-called “undifferentiated” cells, as is mostly believed. In this paper, I review the various mechanisms of plant regeneration that may converge on callus initiation. I discuss the role of plant hormones in the detour of callus formation from normal development. Finally, I compare various Arabidopsis gene expression datasets obtained a few days, two weeks, or several years after callus induction and identify 21 genes, including genes of key transcription factors controlling cell division and differentiation in meristematic regions, which were upregulated in all investigated callus samples. I summarize the information available on all 21 genes that point to the pre-meristematic nature of callus tissues underlying their wide regeneration potential. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

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16 pages, 720 KiB

Open AccessReview

Advances in Somatic Embryogenesis of Banana

by Mark Adero, Jaindra Nath Tripathi and Leena Tripathi

Int. J. Mol. Sci. 2023, 24(13), 10999; https://doi.org/10.3390/ijms241310999 - 1 Jul 2023

Cited by 3 | Viewed by 2956

Abstract

The cultivation of bananas and plantains (Musa spp.) holds significant global economic importance, but faces numerous challenges, which may include diverse abiotic and biotic factors such as drought and various diseases caused by fungi, viruses, and bacteria. The genetic and asexual nature of cultivated banana cultivars makes them unattractive for improvement via traditional breeding. To overcome these constraints, modern biotechnological approaches like genetic modification and genome editing have become essential for banana improvement. However, these techniques rely on somatic embryogenesis, which has only been successfully achieved in a limited number of banana cultivars. Therefore, developing new strategies for improving somatic embryogenesis in banana is crucial. This review article focuses on advancements in banana somatic embryogenesis, highlighting the progress, the various stages of regeneration, cryopreservation techniques, and the molecular mechanisms underlying the process. Furthermore, this article discusses the factors that could influence somatic embryogenesis and explores the prospects for improving the process, especially in recalcitrant banana cultivars. By addressing these challenges and exploring potential solutions, researchers aim to unlock the full potential of somatic embryogenesis as a tool for banana improvement, ultimately benefiting the global banana industry. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

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30 pages, 8117 KiB

Open AccessReview

Induction of Somatic Embryogenesis in Plants: Different Players and Focus on WUSCHEL and WUS-RELATED HOMEOBOX (WOX) Transcription Factors

by Marco Fambrini, Gabriele Usai and Claudio Pugliesi

Int. J. Mol. Sci. 2022, 23(24), 15950; https://doi.org/10.3390/ijms232415950 - 15 Dec 2022

Cited by 6 | Viewed by 2851

Abstract

In plants, other cells can express totipotency in addition to the zygote, thus resulting in embryo differentiation; this appears evident in apomictic and epiphyllous plants. According to Haberlandt’s theory, all plant cells can regenerate a complete plant if the nucleus and the membrane system are intact. In fact, under in vitro conditions, ectopic embryos and adventitious shoots can develop from many organs of the mature plant body. We are beginning to understand how determination processes are regulated and how cell specialization occurs. However, we still need to unravel the mechanisms whereby a cell interprets its position, decides its fate, and communicates it to others. The induction of somatic embryogenesis might be based on a plant growth regulator signal (auxin) to determine an appropriate cellular environment and other factors, including stress and ectopic expression of embryo or meristem identity transcription factors (TFs). Still, we are far from having a complete view of the regulatory genes, their target genes, and their action hierarchy. As in animals, epigenetic reprogramming also plays an essential role in re-establishing the competence of differentiated cells to undergo somatic embryogenesis. Herein, we describe the functions of WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors in regulating the differentiation–dedifferentiation cell process and in the developmental phase of in vitro regenerated adventitious structures. Full article

(This article belongs to the Special Issue Recent Advances in Plant Somatic Embryogenesis: Where We Stand and Where to Go?)

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