Indirect Embryogenesis Research Articles

An Overview: Somatic Embryogenesis Through Thin Cell Layer (TCL) Technique

Somatic embryogenesis (SE) has been described as the most efficient pathway to obtaining plants. Somatic embryogenesis can be divided into direct and indirect embryogenesis. Direct SE occurs when embryos are started directly from explant tissues (preembryogenic cells) creating an identical clone, while indirect SE occurs from unorganized tissues (calli) which are further developed into embryos. While different explants have been tested, e.g., shoot tips, adventitious buds, leaf primordia, zygotic embryos, inflorescences, and transverse thin cell layers (TCLs) (consisting of a few cell layers, usually 0.5–2.0 mm thick). TCLs have been excised from different plant organs and successfully used as explants for SE. Thin cell layers (TCLs) can be prepared from almost any plant organ. These, when cut longitudinally, are referred to as lTCLs, and when cut transversally, are referred to as tTCLs. This article provides an overview of the concept of TCL as applied to induce SE in different plant species and remarks the factor that affecting this technique. This review will certainly revitalize this important technology so that it can be used effectively for successful mass propagation.

Indirect somatic embryogenesis in sweet orange cv. “Mosambi”

This study was carried out for standardization of a reliable protocol by using different explants (epicotyl,cotyledon, and root), plant bioregulators (PBRs), and carbon sources on indirect embryogenesis during 2019-2022. Among the treatments tested, T4 (MS + 2,4-D (1.5 mg L-1) + BAP (1.0 mg L-1) + ME (500 mg L-1)) proved bestfor callusing (90.89 %), callus fresh weight (0.83 g), turgid weight (0.84 g) and dry weight (0.08 g) as comparedto others. In terms of callusing (83.72%), callus fresh weight (0.67 g), turgid weight (0.68 g), dry weight (0.05g) and callus water content (11.28 %), explants epicotyl (E) showed its superiority over others. Similarly, thetreatment combination T4 × E surpassed others in callusing potential. Embryogenesis (59.09%) and germination(33.61%) were best in T7 (MS + BAP (2 mg L-1) + NAA (0.1 mg L-1) + ME (500 mg L-1)). Among the different carbonsources, 5% glycerol supplemented with T7 proved best in inducing the highest number of somatic embryos/callus mass (73.26), embryogenesis (65.27%), and plantlet formation (68.77%). The standardized protocol canbe used for indirect embryogenesis for different genotypes of sweet orange.

Overview of Somatic Embryogenesis.

Somatic embryogenesis is a natural phenomenon through which somatic embryos are produced from somatic cells although. It is considered the most efficient morphogenic pathways for plant multiplication. One of the key features of somatic embryogenesis is the use of cellular totipotency, where dedifferentiation is induced to foster cell proliferation, followed by the induction of differentiation using plant growth regulators to produce new plants. There is a cell group with the potential to undergo the somatic embryogenesis pathway through adequate stimulation (plant growth regulators, incubation conditions, and supplementation of the culture medium). There are two somatic embryogenesis pathways in plants: direct and indirect embryogenesis. Direct somatic embryogenesis consists of the formation of embryos directly from isolated cells, without the formation of "callous" tissue. Indirect somatic embryogenesis is characterized by the formation of a callus as a stage that precedes the formation of somatic embryos. It should be stressed that not all plant cells have this morphogenic capacity; consequently, determining the type of factors that drive this type of response has been challenging. This book provides the reader with updated available information on the techniques, relevant protocols, and tools to perform somatic embryogenesis in different plant species for economic purposes.

Distribution of polyprenol and dolichol in oil palms from Pisifera parents and mature plants from tissue culture propagation

Abstract. Kartika EBA, Siregar LAM, Basyuni M. 2021. Distribution of polyprenol and dolichol in oil palms from Pisifera parents and mature plants from tissue culture propagation. Biodiversitas 22: 3423-3436. Oil palm tissue culture is carried out through indirect embryogenesis, which causes somaclonal diversity to occur at the in vitro propagation stage, especially in the callus growth phase. In the cells of all living organisms can be found a group of polyisoprenoid compounds. This study aims to determine variations in oil palm plants resulting from tissue culture based on the presence of polyisoprenoid compounds. Oil palm leaf samples (Elaeis guineensis Jacq.) were collected from the plantation of PT. Socfin Indonesia, North Sumatra, Indonesia. Sample extraction, saponification, isolation of polyisoprenoid compounds, and two-dimensional thin-layer chromatography analysis were carried out to obtain data related to total lipids, polyprenol and dolichol profile, and Carbon (C) chain length polyprenol and dolichol from oil palm leaves of Pisifera parents and their propagated derivatives by indirect embryogenesis. The results showed that the amount of polyisoprenoid mother S24 was 2.41 mg/g higher than that of the parentS8 at 2.17 mg/g dry weight. Total polyisoprenoid fromS8 plants in vitro ranged from 0.71 - 8.53 mg/g dry weight, with the lowest total polyisoprenoid found atS894/22 and the highest was found atS893/2. While for total polyisoprenoid from plant tissue culture of Pisifera S24 ranged from 0.73 - 8.05 mg/g dry weight, with the lowest total polyisoprenoid found at S24H14 and the highest was found at S24H16. The parent plants of Pisifera S8 and S24, as well as plants resulting from tissue culture, were categorized as having lipid pattern type II, which showed a balanced distribution of polyprenol with dolichol. The longest carbon chain was found in vitro plantsS8 93/4 ranged from C50-C110, while the shortest was found in plants produced in vitro S24H7 starting from C45-C55. There were variations in the carbon chain length of polyprenol and dolichol in the leaf samples derived from in vitro propagation of the Pisifera S8 and S24 parents.

Embriogénesis y organogénesis de aguacate criollo mexicano

La variedad Drymifolia de aguacate (Persea americana Mill.) es usada generalmente como portainjerto por lo que es importante implementar las técnicas de embriogénesis somática y la organogénesis in vitro para su mejora genética, conservación y propagación clonal. En este trabajo se evaluaron embriogénesis somática directa, indirecta y organogénesis de la variedad Drymifolia. La germinación de los embriones somáticos maduros se indujo con 0.5 mg L-1 de 6-N-bencil amino purina (BAP) y 1 mg L-1 de ácido giberélico (GA3). De las seis accesiones evaluadas, Celaya 79, Comonfort 53, San Miguel, BG24, BG181 y Zutano, solo San Miguel respondió al proceso de embriogénesis. En la embriogénesis directa la eficiencia más alta de regeneración se obtuvo con 0.2 mg L-1 de picloram (46%) y 10 mg L-1 de ANA (40%). En la embriogénesis indirecta la accesión San Miguel formó callos con 0.2 mg L-1 de picloram y tuvo una eficiencia de regeneración de 45% conservando su potencial de regeneración hasta por seis meses. Con respecto a la organogénesis, se cultivaron embriones cigóticos inmaduros decapitados en medio con reguladores de crecimiento o sin ellos y las seis accesiones respondieron positivamente a ambas condiciones. La accesión Comonfort 53 tuvo mayor eficiencia de regeneración (54%) con reguladores de crecimiento. Este estudio realizado del 2016 a 2018, proporciona un enfoque nuevo y prometedor para la regeneración y multiplicación de plantas de P. americana var. Drymifolia a través de embriones cigóticos.

CG Demethylation Leads to Sequence Mutations in an Anther Culture of Barley Due to the Presence of Cu, Ag Ions in the Medium and Culture Time.

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations (‘Cu*Ag’) acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of ‘Cu*Ag’, and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

ABSTRACTAn efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L−1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).

Enhanced Indirect Somatic Embryogenesis of Date Palm Using Low Levels of Seawater.

Date palm tolerates salinity, drought, and high temperatures. Arid and semiarid zones, especially the Middle East region, need a huge number of date palms for cultivation. To meet this demand, tissue culture techniques have great potential for mass production of plantlets, especially using the indirect embryogenesis technique; any improvement of these techniques is a worthy objective. Low levels of salinity can enhance growth and development of tolerant plants. A low level of seawater, a natural source of salinity, reduces the time required for micropropagation processes of date palm cv. Malkaby when added to MS medium. Medium containing seawater at 500ppm total dissolved solid (TDS) (12.2mL/L) improves callus proliferation, whereas 1500ppm (36.59mL/L) enhances plant regeneration including multiplication of secondary embryos, embryo germination, and rooting.

Cell Viability, Mitotic Index and Callus Morphology of Byrsonima verbascifolia (Malpighiaceae)

The combined analysis of cell viability, mitotic index and callus morphology of species of commercial interest may favor the identification of cell masses with embryogenic characteristics at early stages of cultivation, allowing the development or improvement of protocols for in vitro plant regeneration through indirect embryogenesis. Byrsonima verbascifolia L. (DC.) (Malpighiaceae) is a Brazilian medicinal species native to the Cerrado, which has a low seed germination rate due to tegument dormancy. This study aimed to identify cells with embryogenic characteristics during growth of callus of B. verbascifolia, by assessing cell viability, mitotic index and cell morphology. Callus was induced from leaf explants and inoculated on MS medium with 4.52 μM 2,4-D and 4.44 μM benzylaminopurine (BAP). Friable calli were obtained at four distinct growth phases: lag (until the 30th), exponential (from 31st to the 70th day), linear (from 71st to the 100th day) and decline (from 101st to the 120th day). Calli with 20 and 30 days showed cells with embryogenic characteristics, that is, isodiametric shape, juxtaposed, with small vacuoles, thin cell wall and presence of amyloplasts. After 120 days, calli were mostly elongated cells with lower cell viability and mitotic index.

Establishment of Plant Production System through Direct and Indirect Embryogenesis from Anther Culture of Carrot

Establishment of Plant Production System through Direct and Indirect Embryogenesis from Anther Culture of Carrot

Micropropagation of Bunium Persicum (Boiss.) B. Fedtsch. via Indirect Somatic Embryogenesis

Bunium persicum (Boiss.) B. Fedtsch., a member of the Apiaceae family, is one of the oldest and most economically important species. Because of seed dormancy and lack of hypocotyl growth when cotyledon emerged, In vivo cultivation of Bunium persicum has not succeeded. In order to investigate the effects of some plant growth regulators on somatic embryogenesis of Bunium persicum in tissue cultured condition, we have used indirect embryogenesis technique. Explant harvested from root, hypocotyl and cotyledon (leaf) were investigated on MS media combined with 2, 4-D, BAP, NAA (each of those with 6 concentration levels: 0, 0.5, 1, 2, 3 and 4 mg/l) and furthermore Kin (using 3 concentration levels: 0, 0.2 and 0.5 mg/l). Calli developed into different media and after 3 or 4 weeks some of them regenerated embryos. The best indirect somatic embryogenesis was obtained in a medium treated with root explants and the highest indirect embryogenesis was resulted from media treated with 2 mg/l 2, 4-D, 0.5 mg/l BAP or 1 mg/l BAP + 0.2 Kin. Also the highest percentage of callogenesis was observed on MS media containing 0.5 mg/l NAA+0.5 mg/l Kin, 4mg/l NAA or 2 mg/l 2, 4-D + 0.5 mg/l Kin. Keyword- Bunium persicum (Boiss.) B. Fedtsch.; Somatic Embryogenesis; Callus; Hypocotyls

Comparison of direct and indirect embryogenesis protocols, biolistic gene transfer and selection parameters for efficient genetic transformation of sugarcane

The comparison of direct and indirect somatic embryogenesis (DSE and ISE) for biolistic transformation of sugarcane by minimal expression cassettes indicated the highest transformation efficiency with ISE (2.2 independent transgenic plants per shot) and the most rapid production of transgenic plants with DSE (12 weeks from explant to plants in soil). Microprojectiles of 0.3 μm diameter produced 5 times more transgenic lines than 1 μm microprojectiles when used at the same weight basis per shot. A significant reduction of the number of hybridization signals in the Southern blot was also observed with 0.3 μm microprojectiles when compared to 1.0 μm microprojectiles. This suggests that the lower DNA carrying capacity and greater number of the smaller microprojectiles contributes to more transgenic lines with less complex transgene integration. When geneticin sulfate, was used for selection following DSE, significantly more (4.8 times) transgenic plants were produced than with paromomycin sulfate and an equal number of non-expressing plants were produced with both selection agents. In conclusion, optimization of two alternative morphogenic routes for regeneration (DSE and ISE), biolistic and selection parameters generated rooted, transgenic plants of a commercially important sugarcane cultivar with simple transgene integration and within 12 or 19 weeks of culture initiation, respectively. Reducing the complexity of the transgene integration and minimizing the time in tissue culture will likely enhance the performance of the transgenic events.

Induction of somatic embryogenesis from different explants of shoot cultures derived from young Quercus alba trees

The induction of somatic embryogenesis from shoot apices and leaf explants of shoot cultures derived from 6- to 7-year-old white oak (Quercus alba L.) trees is reported in this study. Embryogenic response was obtained in two out of the three genotypes evaluated with embryo induction frequencies up to 50.7% for WOQ-1 and 3.4% for WOQ-5 genotypes. The embryogenic explants formed translucent nodular structures and cotyledonary-stage somatic embryos, which developed from callus tissue, indicating an indirect embryogenesis process. An efficient procedure was developed for WOQ-1 material on the basis of the most appropriate leaf developmental stage. Growing leaves excised from two nodes below the shoot apex showed the highest embryogenic induction index. These leaves contain cells in an undifferentiated state, as shown by the presence of precursor cells of stomata, absence of intercellular spaces and low starch content in the mesophyll cells. Nodular structures and/or somatic embryos began to arise 7–8 weeks after culture initiation, although most emerged after 9–12 weeks in culture. The sequence of application of media for somatic embryo induction was optimized with a two-step procedure consisting of culturing the explants in medium supplemented with 21.48 μM NAA and 2.22 μM BA for 8 weeks and transfer of explants into plant growth regulator-free medium for another 12 weeks. Clonal embryogenic lines were established and maintained by secondary embryogenesis. Embryo germination (30%) and plantlet conversion (16.6%) were achieved after cold storage for 2 months.

Shoot apex explants for induction of somatic embryogenesis in mature Quercus robur L. trees

A procedure for inducing somatic embryos in shoot apex explants (2 mm) excised from shoot proliferation cultures established from adult oak trees (Quercus robur) was investigated. Embryogenesis was induced in shoot tip as well as leaf explants in three out of the five genotypes evaluated. Somatic embryos were formed by culture in induction medium supplemented with 21.48 muM naphthalene acetic acid and 2.22 muM benzyladenine for 8 weeks, and successive transfer of explants to expression media with a low concentration of growth regulators and without them. Both types of explants formed callus tissue from which somatic embryos developed, indicating indirect embryogenesis. Although the embryogenic frequencies were lower than 12%, it did not prevent the establishment of clonal embryogenic lines maintained by repetitive embryogenesis. Histological study confirmed an indirect somatic embryogenesis process from shoot tip explants, in which leaf primordia and the corresponding axial zones were involved in generating callus, whereas the apical meristem itself did not proliferate. The origin of embryogenic cells appeared to be associated with dedifferentiation of certain parenchymal cells in callus regions after transfer of explants to expression media without auxin. Division of embryogenic cells gave rise to proembryo aggregates of unicellular origin, although a multicellular origin from bulging embryogenic areas would also seem possible. Further development led to the formation of cotyledonary-stage somatic embryos and nodular embryogenic structures that may be considered as anomalous embryos with no clear bipolarity. Inducement of somatic embryos from explants isolated from shoot cultures ensures plant material all year round, thus providing a significant advantage over the use of leaf explants from field-grown trees.

Genetic transformation of the euploid Saccharum officinarum via direct and indirect embryogenesis

The genetic nature of foreign gene transfer and integration into commercial sugarcane hybrids with complex genetic backgrounds is unclear and unpredictable. A pure genetic background in genetic engineering approaches might lead to more predictable results within Saccharum officinarum than in a hybrid genome. S. officinarum plants (Badila and Black Cheribon genotypes) expressing a reporter gene was regenerated from microprojectile-bombarded leaf disks and callus. The concentration of auxin added to the basal media, both prior to bombardment and during regeneration, significantly affected callus induction and somatic embryogenesis. A ten-fold decrease in 2,4-D concentration resulted in somatic embryos forming directly from sugarcane leaf disks. Somatic embryos were produced via direct embryogenesis from 7 days old Black Cheribon leaf disks on 0.5 mg/l 2,4-D. A callus interface initiated on 3 mg/l 2,4-D from Badila leaf disks was preferred for indirect embryogenesis to produce the maximum number of transgenic Badila plants. These approaches lead to the successful establishment of genetic transformation systems for two S. officinarum genotypes that could be used in the future for gene function studies and breeding purposes.

INFLUENCE OF POLYAMINE ON INDUCTION OF ADVENTIVE EMBRYONY IN PAPAYA (CARICA PAPAYA L.)

Somatic embryogenesis has been reported in different papaya cultivars across the world. However, all report suggests indirect embryogenesis in papaya with minimum of 3-6 months required for embryo formation. We have investigated role of polyamines (spermidine, spermine and putriscine) on direct embryogenesis on immature zygotic embryos excised from papaya cultivar Pusa Delicious. A brief exposure (2-4 weeks) of putriscine on induction medium (100 μM) augmented direct embryogenesis on explant. However, prolonged exposure of putriscine was found detrimental.

In Vitro Micropropagation of Freesia hybrida and the Assessment of Genetic and Epigenetic Stability in Regenerated Plantlets

Freesia hybrida is an important worldwide cut flower, especially in America and Europe. For efficient regeneration of this flower from young inflorescence and rachillae in tetraploid, we developed a simple in vitro micropropagation protocol. Explants of Freesia hybrida can regenerate plantlets through somatic embryogenesis via two kinds of pathways, that is, directly from the epidermal cells or indirectly from an embryonic callus, depending on the exogenous plant growth regulators (PGRs) used in the culture media. In direct embryogenesis, when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 11.43 μM indole acetic acid (IAA) and 4.44 μM 6-benzylaminopurine (6-BA), the induction rate was 84% for young inflorescence and 100% for rachillae. After the multishoots were subcultured on the rooting MS medium containing 1.08 μM α-naphthalene acetic acid (NAA), the rooting rate was close to 100%. In indirect embryogenesis, embryonic calluses were formed when the culture medium contained 22.22 μM 6-BA and 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), and the induction rate was 92.4% for young inflorescence and 100% for rachillae. After the embryonic calluses were transferred to the medium supplemented with 11.43 μM IAA and 13.33 μM 6-BA, they could develop into plantlets with roots. In assessing the two regeneration pathways in terms of genetic and epigenetic fidelity of the regenerants, two kinds of molecular markers [amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP)] were employed. The AFLP analysis used 20 primer pairs that yielded 916 scorable bands among the donor plant and 11 regenerants from direct embryogenesis, of which 8 (0.87%) were polymorphic. The regenerants from indirect embryogenesis had 1075 clear bands of which 3 (0.27%) were polymorphic scorable bands from 18 primer pairs. Moreover, the variant band patterns included two types, that is, loss-of-original and gain-of-novel bands. MSAP analysis revealed that tissue culturing of the flower induced DNA cytosine methylation alterations in both CG and CNG levels and patterns compared with the donor plant. The variation rate was 1.1 and 1.3% for the direct and indirect embryogenesis pathways, respectively. The findings show that tissue culture of flowering plants is a form of stress which can induce some heritable epigenetic variations and should be considered in future long-term genotype preservation programs of Freesia hybrida.

Ferula gummosa Boiss. Embryogenic Culture and Karyological Changes

Ferula gummosa Boiss. a highly valuable medicinal plant which naturally propagates in very limited areas of the Middle East with specific environmental conditions. The production of Ferula gummosa somatic embryos and the karyological analysis of somatic seedlings were the purpose of this study. High frequency indirect embryogenesis was induced in callus derived from zygotic embryonic axes. Embryogenesis was obtained when callus tissues were placed onto an agar induction Murashige and Skoog medium with 1-naphthalene acetic acid and after the transfer of the cultures in a thermoperiod regime of 16 h, 19 degrees C/8 h, 7 degrees C under photoperiod of 16 h light/8h dark. Embryogenic callus tissues were maintained by subculture on induction medium. Globular proliferation was achieved with suspension culture in the Murashige and Skoog medium added with 1-naphthalene acetic acid or 2,4-dichlorophenoxyacetic acid for two weeks. Maturation of embryos and development of plantlets arose on the induction agar medium, but was better after transfer into the hormone free Murashige and Skoog medium. However, the level of abnormal embryos was high. Direct embryogenesis was obtained from somatic seedlings. The best results were obtained from hypocotyl explants. Embryo induction was achieved by two week culture of the explants in 2,4-dichlorophenoxyacetic acid liquid medium; somatic embryo growth and maturation was recovered on the hormone free medium. High level of abnormalities was recorded in the culture. Karyological analysis showed a high incidence level of cytochimerism in somatic seedlings with chromosome stickiness, polypoidy and aneuploidy in metaphase cells of the same root tip. The frequency of these karyological changes varied with the type of somatic embryos with regard to morphological abnormalities. Normal and abnormal rooted somatic seedlings were able to grow until production of the first leaf and then entered dormancy in the same manner as zygotic plantlets.

Responses of Five Locally Adapted Sweet Potato (Ipomoea batatas (L.) Lam)Cultivars to in vitro Plant Regeneration via Direct and Indirect Embryogenesis

Responses of Five Locally Adapted Sweet Potato (Ipomoea batatas (L.) Lam)Cultivars to in vitro Plant Regeneration via Direct and Indirect Embryogenesis

Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers

In this work, some of the different in vitro developmental pathways into which tomato microspores or microsporocytes can be deviated experimentally were explored. The two principal ones are direct embryogenesis from isolated microspores and callus formation from meiocyte-containing anthers. By means of light and electron microscopy, the process of early embryogenesis from isolated microspores and the disruption of normal meiotic development and change of developmental fate towards callus proliferation, morphogenesis, and plant regeneration have been shown. From microspores isolated at the vacuolate stage, embryos can be directly induced, thus avoiding non-androgenic products. In contrast, several different morphogenic events can be triggered in cultures of microsporocyte-containing anthers under adequate conditions, including indirect embryogenesis, adventitious organogenesis, and plant regeneration. Both callus and regenerated plants may be haploid, diploid, and mostly mixoploid. The results demonstrate that both gametophytic and sporophytic calli occur in cultured tomato anthers, and point to an in vitro-induced disturbance of cytokinesis and subsequent fusion of daughter nuclei as a putative cause for mixoploidy and genome doubling during both tetrad compartmentalization and callus proliferation. The potential implications of the different alternative pathways are discussed in the context of their application to the production of doubled-haploid plants in tomato, which is still very poorly developed.