Induction Of Somatic Embryogenesis Research Articles

Effect of 2, 4-D and Picloram on Somatic Embryogenesis in Carica papaya var. P-7-9

Immature zygotic embryos of Carica papaya L. var. P-7-9 were inoculated on half-strength MS with different concentrations of 2,4-D and picloram alone and their combinations. The highest induction (80%) of somatic embryogenesis was MS supplemented with 2,4-D (7.0 mg/l) followed by 2,4-D (4.0 mg/l) + picloram (1.0 mg/l) but no embryogenesis was observed in auxin-free medium. The addition of osmoticum such as PEG and ABA significantly increased the maturation of somatic embryos. BAP, TDZ and NAA were used for shoot induction. Combination of 2.0 mg/l BAP and 0.5 mg/l NAA was found significantly better for shoot bud proliferation and 70% multiplication. The percentage of germinating embryos was enhanced significantly when the MS fortified with GA3 (0.5 mg/l). However, the high frequency of embryo germination and plantlets formation were obtained with 2,4-D (4.0 mg/l) + picloram (1.0 mg/l).
 Plant Tissue Cult. & Biotech. 29(1): 25-32, 2019 (June)

Plant Regeneration in Buckwheat (Fagopyrum esculentum Moench.) via Somatic Embryogenesis and Induction of Meristemoids in Abnormal Embryos

An efficient in vitro regeneration protocol is reported for common buckwheat. A combination of 0.5 mg/l 2,4-D and 0.2 mg/l BAP with sucrose showed highest induction of somatic embryogenesis from cotyledon and hypocotyl explants. More than 35% of normal somatic embryos matured on MS. MS with 2% sucrose were found best for germination and conversion of somatic embryos to plantlets. In tissue culture, abnormal somatic embryos usually occur. In this report, abnormal embryos are also used to induce shoot organogenesis, adding to the number of final regenerants and ensuring full utilization of regenerative propagules. A treatment of 0.2 mg/l BAP induced meristemoids in 60% of underdeveloped embryos and a combination of 0.5 mg/l BAP and 0.5 mg/l AgNO3 led browning and senescence-free progression of shoot buds to well developed shoots, which were subsequently rooted in half strength MS containing 2% sucrose and 0.25 mg/l IBA. The regenerated plants survived acclimatization, flowered and set seeds.
 Plant Tissue Cult. & Biotech. 29(1): 33-47, 2019 (June)

Induksi Embriogenesis Somatik Pada Anggrek Vanda Sumatrana Schltr. dengan Penambahan Beberapa Konsentrasi Asam 2,4-Diklorofenoksiasetat (2,4-D)

The research about induction of somatic embryogenesis of Orchid Vanda sumatrana Schltr. by giving 2,4-Diclorophenoxiacetic acid (2,4-D), was conducted from May to July 2016 in plant Physiology and tissue Culture Laboratory, Biology Departement, Matematics and Natural Science Faculty, Andalas University. The aim of this study was found the concentration of 2,4-D to induce somatic embryogenesis of Vanda sumatrana. The research used Completely Randomized Resign (CDR) with 6 treatments and 4 replication. The treatments were : without 2,4-D (control); 1 mg/L; 2 mg/L; 3 mg/L; 4 mg/L; 5 mg/L. The result showed that 2 mg/L 2,4-D and 3 mg/L 2,4-D were concentrations to induct somatic embryogenesis.

Induction of somatic embryogenesis in two cultivars of anthurium analysed by scanning electron microscopy

Somatic embryogenesis is an advantageous tool in the commercial production of micropropagated anthurium plantlets. As such, the aim of this study was to establish a protocol for the induction of somatic embryogenesis in Jureia and Luau cultivars. Defoliated nodal segments, 1.0 cm in length and containing one bud, were used as explants. The experimental design was completely randomised, in a 2 x 3 x 5 factorial scheme (cultivar: Jureia and Luau x auxin: 2,4-D, NAA and Picloram x concentration: 0, 2.5, 5.0, 7.5, and 10.0 μM), with 30 treatments in a scheme of plots split over time (15, 30, 45, 60, 75 and 90 days). The anatomy and percentage of embryogenic callus formation were analysed. The structures formed, analysed by scanning electron microscopy, corresponded to embryogenic calli. The Luau cultivar was superior in forming embryogenic calli. For the two cultivars, among the auxins under study, NAA demonstrated a greater induction potential for somatic embryogenesis, with the concentration of 7.5 μM giving the highest mean values. The 90-day evaluation period showed the maximum formation of embryogenic calli; however, mean values were fairly similar to the 75-day evaluation period. To induce embryogenic calli, therefore, it is suggested that the nodal segments be inoculated into a culture medium with added NAA growth regulator at a concentration of 7.5 μM, and that the explants remain in this medium for 75 days after inoculation.

Somatic Embryogenesis in Wheat and Barley Calli in vitro Is Determined by the Level of Indoleacetic and Abscisic Acids

The effect of a number of phytohormones (IAA, ABA, and cytokinines) on the induction of in vitro somatic embryogenesis in callus cultures of wheat, cultivar Bashkirskaya 26, barley, cultivar Steptoe, and its ABA-deficient mutant AZ34 has been studied. It was shown that the ability or inability for somatic embryogenesis in callus tissue of both wheat and barley is determined by the IAA : ABA ratio. However, the level of cytokinines was similar in both embryogenic and nonembryogenic calli of each object studied. The analysis of callus tissue of the ABA-deficient mutant AZ34 revealed the stimulating role of ABA not only in the induction of somatic embryogenesis but also in the formation of normal embryos.

Effect of calcium, BAP and putrescine on somatic embryo induction in juvenile explants of Eucalyptus grandis × E. urophylla hybrids

Considering the constant improvement of Eucalyptus cloning and the search for new technologies to produce plantlets of this species, somatic embryogenesis has attracted interest from research groups and forestry companies that use advanced genetic breeding and cloning programs. The objective of the present study was to verify the effect of concentrations and sources of calcium, concentrations and effect time of cytokinin BAP and polyamine putrescine on the induction and development of somatic embryos in juvenile explants of Eucalyptus grandis x E. urophylla. Cotyledonary explants were inoculated into culture medium containing calcium chloride (MS medium) or calcium nitrate (JADS medium) as source of calcium. Different concentrations of calcium were also used, for MS medium containing: 4.40 gL-1 (control-Ca), 6.60 gL-1 (50% increase over control - Ca 50) and 8.80 gL-1 (increase of 100% over control – Ca 100) of calcium nitrate; and for JADS medium containing: 11.81 gL-1 (Ca), 17.72 gL-1 (Ca50) and 23.62 gL-1 (Ca100) of calcium chloride. Cotyledon explants were inoculated into the primary induction medium (PIM) containing 20.71 μM picloram as growth regulator. At 10, 20 and 30 days of primary induction, the explants were transferred to the secondary induction medium (SIM) containing 20.71 μM picloram and 11.10 μM BAP or 28.36 μM putrescine. The culture medium containing calcium nitrate provided higher callogenesis when compared to the medium containing calcium chloride. The increase in calcium concentration in the media did not provide higher percentage of induction of somatic pro-embryos. However, the addition of 28.36 μM putrescine to the culture medium provided a higher percentage of induction of somatic embryogenesis. The number of somatic pro-embryos formed per explant was higher when BAP and putrescine were added to the culture medium when compared to medium containing only picloram. To obtain a greater number of somatic pro-embryos of Eucalyptus grandis × E. urophylla, the JADS culture medium containing 28.36 μM putrecine should be used.

Plant Regeneration via Somatic Embryogenesis in Solanum nigrum L. (Black nightshade) (Solanaceae)

Aim: To study the effect of various plant growth regulators (PGRs) for induction of somatic embryogenesis and plantlet formation from cotyledon and leaflet explants in S. nigrum (night shade) an important medicinal plant used in treatment of digestive problems and skin infections.
 Place and Duration of Study: Department of Biotechnology, Kakatiya university, Warangal. Telangana, India, 3 years.
 Methodology: Cotyledon (0.8 cm2) and leaflet explants (0.8-1.0 cm2) from 3 week and 4 week old were cultured on MS medium supplemented with 30 g/L sucrose along with different concentrations of 0.5 mg/L BAP+NAA (0.5 – 6.0 mg/L) .
 Results: Maximum percentage of somatic embryogenesis was observed in cotyledon(89%) and leaf (98%) explants on MS medium augmented with 0.5mg/L BAP in combination with 2.0 mg/L NAA whereas the highest number of somatic embryos per explant (86 ± 0.19) was formed in leaflet explant.
 Conclusion: Somatic embryogenesis was induced from both cotyledon and leaf explants. Since it is threatened and medicinally important species S. nigrum, the present protocol can be used for its conservation and genetic transformation experiments.

NEP-TC a rRNA Methyltransferase Involved on Somatic Embryogenesis of Tamarillo (Solanum betaceum Cav.).

Somatic embryogenesis (SE) is an important biotechnological tool for large-scale clonal propagation and for embryogenesis research. Moreover, genetic transformation and cryopreservation procedures in many species rely on efficient SE protocols. We have been studying different aspects related to SE induction and somatic embryo development in tamarillo (Solanum betaceum Cav.), a small tree from the Solanaceae family. Previous proteomic analyses identified a protein (NEP-TC, 26.5 kDa) consistently present in non-embryogenic calluses of tamarillo, but absent in the embryogenic ones. In this work, the role of NEP-TC during SE was assessed by gene expression analysis and immunolocalization. The results obtained demonstrated that NEP-TC is a putative member of the SpoU rRNA methylase family. This protein, present in the cytoplasm and nucleus, is expressed in non-embryogenic cells and not expressed in embryogenic cells. Slightly enhanced SE induction levels in tamarillo plants with NEP-TC down-regulated levels also supports the role of this protein on SE induction. Heterologous expression was used to confirm NEP-TC rRNA methyltransferase activity, with enhanced activity levels when rRNA was used as a substrate. These data relate a putative member of the SpoU methylase family with plant morphogenesis, in particular with SE induction.

Somatic Embryogenesis Induction in Woody Species: The Future After OMICs Data Assessment.

Very early somatic embryogenesis has been recognized as a powerful method to propagate plants in vitro. For some woody species and in particular for some coniferous trees, somatic embryogenesis induction has become a routine procedure. For the majority, the application of this technology presents yet many limitations especially due to the genotype, the induction conditions, the number of embryos produced, maturation, and conversion, among other factors that compromise the systematic use of somatic embryogenesis for commercial purposes especially of woody species and forest trees in particular. The advancements obtained on somatic embryogenesis in Arabidopsis and the development of OMIC technologies allowed the characterization of genes and the corresponding proteins that are conserved in woody species. This knowledge will help in understanding the molecular mechanisms underlying the complex regulatory networks that control somatic embryogenesis in woody plants. In this revision, we report on developments of OMICs (genomics, transcriptomics, metabolomics, and proteomics) applied to somatic embryogenesis induction and its contribution for understanding the change of fate giving rise to the expression of somatic embryogenesis competence.

Development of embryogenic dogwood cultures and the regeneration of plants.

Somatic embryogenesis in flowering dogwood (Cornus florida) has been achieved, but not the regeneration of plants with active shoot growth. To improve plant regeneration, eight media treatments were tested for induction of somatic embryogenesis from immature zygotic embryos. Somatic embryogenesis was obtained on three media containing the plant growth regulators (PGRs) 0.1 mg l-1 picloram, 2.0 mg l-1 2,4-dichlorophenoxyacetic acid, or 0.1 mg l-1 indole-3-butyric acid (IBA). Somatic embryogenesis was also induced on Woody Plant Medium without PGRs. Overall, 92% of the somatic embryos examined converted into plants with active root and shoot growth. This is the first report of somatic embryo-derived plants of C. florida that have active shoot growth and that could be transferred to soil. Embryogenic suspensions were established from IBA-treated cultures that could serve as a target for C. florida bioengineering.

Somatic Embryogenesis in the Medicago truncatula Model: Cellular and Molecular Mechanisms.

Medicago truncatula is now widely regarded as a legume model where there is an increasing range of genomic resources. Highly regenerable lines have been developed from the wild-type Jemalong cultivar, most likely due to epigenetic changes. These lines with high rates of somatic embryogenesis (SE) can be compared with wild-type where SE is rare. Much of the research has been with the high SE genotype Jemalong 2HA (2HA). SE can be induced from leaf tissue explants or isolated mesophyll protoplasts. In 2HA, the exogenous phytohormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) are central to SE. However, there are interactions with ethylene, abscisic acid (ABA), and gibberellic acid (GA) which produce maximum SE. In the main, somatic embryos are derived from dedifferentiated cells, undergo organellar changes, and produce stem-like cells. There is evidence that the SE is induced as a result of a stress and hormone interaction and this is discussed. In M. truncatula, there are connections between stress and specific up-regulated genes and specific hormones and up-regulated genes during the SE induction phase. Some of the transcription factors have been knocked down using RNAi to show they are critical for SE induction (MtWUSCHEL, MtSERF1). SE research in M. truncatula has utilized high throughput transcriptomic and proteomic studies and the more detailed investigation of some individual genes. In this review, these studies are integrated to suggest a framework and timeline for some of the key events of SE induction in M. truncatula.

Signaling Overview of Plant Somatic Embryogenesis.

Somatic embryogenesis (SE) is a means by which plants can regenerate bipolar structures from a somatic cell. During the process of cell differentiation, the explant responds to endogenous stimuli, which trigger the induction of a signaling response and, consequently, modify the gene program of the cell. SE is probably the most studied plant regeneration model, but to date it is the least understood due to the unclear mechanisms that occur at a cellular level. In this review, the authors seek to emphasize the importance of signaling on plant SE, highlighting the interactions between the different plant growth regulators (PGR), mainly auxins, cytokinins (CKs), ethylene and abscisic acid (ABA), during the induction of SE. The role of signaling is examined from the start of cell differentiation through the early steps on the embryogenic pathway, as well as its relation to a plant’s tolerance of different types of stress. Furthermore, the role of genes encoded to transcription factors (TFs) during the embryogenic process such as the LEAFY COTYLEDON (LEC), WUSCHEL (WUS), BABY BOOM (BBM) and CLAVATA (CLV) genes, Arabinogalactan-proteins (AGPs), APETALA 2 (AP2) and epigenetic factors is discussed.

Differential protein profiles in interspecific hybrids between Elaeis oleifera and E. guineensis with contrasting responses to somatic embryogenesis competence acquisition

Understanding the molecular events that initiate somatic embryogenesis (SE) may help optimize clonal propagation protocols in oil palm. The objective of this work was to identify differentially abundant proteins during the induction of SE in two interspecific F1 hybrids of Elaeis oleifera × Elaeis guineensis with contrasting responses (responsive and non-responsive) to the SE process. Leaf explants were obtained and submitted to SE induction medium for up to 180 days. Explants were collected at 0, 14, 90 and 150 days of induction (doi). Proteins were extracted and analyzed by two-dimensional electrophoresis. Analyses were focused at 14 and 150 doi, representing the initial and late stages of embryogenic competence acquisition, respectively. The results indicate that at 14 doi a high amount of stress is present which results in cellular dedifferentiation. At 90 doi, cells (of the responsive genotype) seem adapted and have kept stress under control, allowing the use of energy for cellular proliferation that occurs at 150 doi. Therefore, the control of stress and oxidation seem to be crucial for callus development. We highlight proteins associated to oxidative stress, protein processing, energy metabolism and development as potentially involved in embryogenic competence acquisition.

Non-Zygotic Embryogenesis in Hardwood Species

Hardwood species are valuable biological resources that have an important role in the economy and ecology of ecosystems worldwide. Non-zygotic or somatic embryogenesis (SE) is a powerful tool in plant biotechnology as it is a form of clonal propagation, amenable to cryopreservation of valuable germplasm and genetic transformation including gene editing. The SE process involves five steps and includes somatic embryo induction, proliferation, maturation, plantlet conversion, and subsequent plant acclimatization. This review aims to provide a general overview of these steps in different SE systems developed for hardwood species. Factors that influence the induction stage such as the age of the donor plant, genotype and culture media are discussed. The role of different explant types, i.e. zygotic embryos and non-zygotic tissues, such as roots, flower tissues, nodes, internodes, leaves or shoot apices, in SE induction are especially emphasized. Histological studies of the origin of somatic embryos and the sequence of events leading to their development from initial explants are assessed. Maintenance of embryogenic capacity carried out by subculture of embryogenic inocula on semisolid or liquid media through cell suspension cultures or by temporary immersion systems is described. At present, the main concerns associated with the application of SE for large-scale propagation of elite hardwoods are related to the embryo maturation, germination, and plantlet conversion steps, and these are highlighted in this review. Finally, molecular aspects associated with somatic embryo induction and development are also described. Attempts to overcome the hurdles identified in the embryogenic process, and future lines of research are proposed.

Somatic embryos from Phalaenopsis classic Spotted Pink (Orchidaceae) protocorms

ABSTRACT: The aim of the present study was to induce the formation of somatic embryos in protocorms from Phalaenopsis Classic Spotted Pink hybrids at two physiological maturation stages, namely: 80 and 120 days after seed inoculation (DASI). Protocorms were inoculated in ½ MS medium supplemented with 0.1 mg L-1 ANA and 3 mg L-1 TDZ. Protocorms were inoculated 120 days after sowing were more developed at the 15th cultivation day due to the formation of pro-embryogenic structures. It was possible seeing the formation of globular- and torpedo-stage somatic embryos at the 30th day of cultivation in somatic embryogenesis (SE) induction medium. The protocorms inoculated at the 80th DASI did not formed somatic embryos; they oxidized 20 days after cultivation in SE-induction medium. The formation of somatic embryos happened directly on the explant, thus characterizing a direct somatic embryogenesis. The embryos converted into plants when the somatic embryos were transferred to the nutrient medium containing no growth regulator. Therefore, it was concluded that the somatic embryos induction from protocorms 120 days after sowing was positive, since the embryos were able to become plants and presented vegetative organs with morphological traits similar to those of the matrix plant.

The effect of sucrose concentration on growth and induction of somatic embryogenesis in Catharanthus roseus callus

Abstract. Mohammed NMI, Said AGE. 2018. Date palm (Phoenix dactylifera) seeds germination. Cell Biol Dev 2: 63-68. This study aimed to examine if pre-germination treatments could effectively promote date palm (Phoenix dactylifera L.) seed germination in the Nursery of the Horticultural Sector, Ministry of Agriculture and Irrigation, Khartoum, Sudan. Seeds were soaked in water for 0, 2, 4, 6, 8, 10, and 12 days before being planted in one of the following media: sand:clay (1:1), sand:clay (1:2), sand:clay (1:0), sand:clay (0:1), and sand:clay (2:1). The factorial completely randomized block design was employed in this investigation. The 6-day soaking time resulted in the highest percentage of seeds germinating; as the soaking time increased, the percentage of seeds germinating decreased. The 6-day soaking time also improved seed germination, allowing for a 50 % final germination percentage in the shortest time. Sowing seeds in a 2:1 soil mix decreased germination days, resulting in a substantial increase in germination percentage compared to other soil type mixes evaluated, regardless of soaking duration. In addition, seeds soaked for 6 days and planted in a 2:1 soil mix produced the tallest seedlings.

Content and immunohistochemical localization of hormones during in vitro somatic embryogenesis in long-term proliferating Larix sibirica cultures

We carried out immunoassay of plant hormones [indoleacetic acid (IAA), cytokinins and abscisic acid (ABA)] in the extracts from explants (megagametophytes and embryos) and from four long-term proliferating embryogenic cell lines of Larix sibirica. Immunohistochemical detection of hormones has also been performed at the stage of initiation of somatic embryogenesis and in embryogenic cultures during the process of somatic embryo morphogenesis. During induction of somatic embryogenesis in tissue culture, the first stage of cell development is manifested in their elongation and polarization. Immunohistochemical analysis of hormones showed localization of IAA at one pole of elongated cells of L. sibirica. The polarity of embryogenic structure development persisted later on. The difference has been shown in the content of hormones between the explants and embryogenic cultures. High level of hormones was detected in the cell line (Cl4), distinguished by great number of globular embryos and high capacity for their maturation. Hybrid line Cl5 (L. sibirica × L. sukaszewii) formed very short suspensor, accumulated higher amount of ABA in the head of somatic embryos (SEs) than did other lines, while SEs of this cell line did not maturate. Cl6 was characterized by low plant hormones content in proliferating embryogenic cultures and low yield of mature somatic embryos.

Plant regeneration, developmental pattern and genetic fidelity of somatic embryogenesis derived Musa spp.

Multiplication of banana cvs. Grand Naine (Musa AAA, Cavendish-sub group) and Rasthali (Musa AAB, Silk-sub group) were attempted through somatic embryogenesis. The influence of position of male flower buds, amino acid supplements in the induction of somatic embryogenesis and field performance of embryogenic cell suspension (ECS) derived banana plants were studied. Differentiated immature male flower buds positioned at 6–8 th bract whorl as explants showed better callus induction and somatic embryogenesis. Supplementation with glutamine at 400 mg L−1 along with 20:20 g L−1sucrose: maltose in maturation media induced a 10-fold increase in somatic embryo formation compared to control. Cotyledonary stage somatic embryos desiccated for 2 h showed higher germination compared to non-desiccated embryos. The plantlets generated were hardened, and the genetic fidelity of the plantlets was confirmed using ISSR marker. To check the field performance of ECS derived plants, plantlets were hardened and planted in the field along with meristem and sucker. During the field growth, these ECS derived plants were morphologically similar to those of control plants. In this experiment, it was observed that ECS derived banana plants displayed normal phenotype as that of plants grown from meristem and sucker. The protocol developed could be useful highly for large-scale micropropagation or genetic manipulation studies in these commercially important banana cultivars.

Somatic Embryogenesis of Melon (Cucumis melo L.) As Affected by Culture Media and Composition of Plant Growth Regulators

Conventional production of melon hybrid seeds requires a long time. Propagation through plant tissue culture can be an alternative method to produce hybrid melon seedlings in order to fulfill the high demand for uniform seedlings. Our current study was aimed to determine the type of propagation media and the best concentration of picloram for the induction of somatic embryogenesis in melon from young seed explants. The study was expanded to examine the effective concentrations of two of auxins, 2-4 D and NAA, combined with BAP to induce somatic embryogenesis in melon using hypocotyl explants. The experiment was conducted at Plant Tissue Culture Laboratory, Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University. The experiment was arranged in a randomized complete block design with three replications. The first experiment tested three types of planting media (MS, B5, and WPM) and four levels of picloram concentration (0, 0.5, 1.0, 1.5 mg.L-1). The second experiment tested auxin (2,4-D and NAA) concentrations of 0, 1, 2, 3, 4, 5 mg.L-1 and two BAP concentrations, 0 and 1 mg.L-1. The first study showed that no somatic embryos were formed with the media types and picloram concentration tested; the seeds, however, germinated and formed callus. In the second study, there was an interaction between concentration levels of auxin (2,4-D and NAA) and BAP on induction of somatic embryos using hypocotyl explants. Somatic embryo formation can be induced with treatment of 1 mg.L-1 NAA + 0.1 mg.L-1 BAP and 2 mg.L-1 NAA + 0.1 mg.L-1 BAP. The highest yield of embryos formation was with the treatment of 1 mg.L-1 NAA + 0.1 mg.L-1 BAP at 0.53 embryos per explant. Keywords: embryogenesis, tissue culture media, melon, Picloram, 2,4 D, BAP

The effects of an osmoregulator, carbohydrates and polyol on maturation and germination of 'Golden THB' papaya somatic embryos.

This study evaluated the effect of osmoregulators and carbohydrates on the maturation and germination of somatic embryos of papaya 'Golden THB'. Cotyledon explants from papaya seedlings germinated in vitro on basal MS medium were cultured on somatic embryogenesis induction medium (IM) containing MS salts, myo-inositol, sucrose, agar and p-chlorophenoxyacetic acid. After 50 days, embryogenic calli were transferred onto maturation media (MM) for 45 additional days. For experiment 1, a MS-based medium supplemented with abscisic acid, activated charcoal and concentrations of PEG 6000 (0; 40; 50; 60 and 70 g L-1) was used, whereas for experiment 2 malt extract concentrations (0; 0.1; 0.2; 0.3 and 0.4 g L-1) were assessed. The normal cotyledonary somatic embryos produced in experiment 2 were transferred to the germination medium (GM). The GM consisted of full-strength MS medium, sucrose, agar and was supplemented with myo-inositol at varying concentrations (0; 0.275; 0.55 and 0.825 mM). The PEG concentrations tested impaired the maturation of 'Golden THB' papaya somatic embryos. The MM, supplemented with malt extract at 0.153 g L-1, promoted the greatest development of normal somatic embryos (18.28 SE calli-1), that is, two cotyledonary leaves produced 36.56 SE calli-1. The supplementation with 0.45 mM myo-inositol provided the highest germination percentage (47.42%) and conversion to emblings.