Number Of Somatic Embryos Research Articles

Somatic embryogenesis to overcome low seed viability and conserve wild banana (Ensete superbum (Roxb.) Cheesman)

The seed viability, ex vitro germination, and percentage of in vitro zygotic embryo germination were found to be very low in Ensete superbum (Roxb.) Cheesman. Only 33.33% of seeds were viable, and the ex vitro germination percentage was only 5%, while the percentage of in vitro zygotic embryo germination was 33%. Somatic embryogenesis experiments produced competent callus on Murashige and Skoog (MS) medium supplemented with 2.5 mg L−1 2,4-D and 3 mg L−1 BAP from inflorescence explants. The embryogenic callus produced the maximum number of somatic embryos on MS basal medium kept in a dark chamber for 15 wk. Half-strength MS medium supplemented with 500 mg L−1 glutamine was optimal for somatic embryo germination and development of plantlets. Regenerated plants had 80 to 90% survival rate. Therefore, somatic embryogenesis can be considered as an efficient method to overcome a drastic reduction in population and to achieve germplasm conservation.

Application of Adaptive Neuro-Fuzzy Inference System-Non-dominated Sorting Genetic Algorithm-II (ANFIS-NSGAII) for Modeling and Optimizing Somatic Embryogenesis of Chrysanthemum

A hybrid artificial intelligence model and optimization algorithm could be a powerful approach for modeling and optimizing plant tissue culture procedures. The aim of this study was introducing an Adaptive Neuro-Fuzzy Inference System- Non-dominated Sorting Genetic Algorithm-II (ANFIS-NSGAII) as a powerful computational methodology for somatic embryogenesis of chrysanthemum, as a case study. ANFIS was used for modeling three outputs including callogenesis frequency (CF), embryogenesis frequency (EF), and the number of somatic embryo (NSE) based on different variables including 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), sucrose, glucose, fructose, and light quality. Subsequently, models were linked to NSGAII for optimizing the process, and the importance of each input was evaluated by sensitivity analysis. Results showed that all of the R2 of training and testing sets were over 92%, indicating the efficiency and accuracy of ANFIS on the modeling of the embryogenesis. Also, according to ANFIS-NSGAII, optimal EF (99.1%), and NSE (13.1) can be obtained from a medium containing 1.53 mg/L 2,4-D, 1.67 mg/L BAP, 13.74 g/L sucrose, 57.20 g/L glucose, and 0.39 g/L fructose under red light. The results of the sensitivity analysis showed that embryogenesis was more sensitive to 2,4-D, and less sensitive to fructose. Generally, the hybrid ANFIS-NSGAII can be recognized as a powerful computational tool for modeling and optimizing in plant tissue culture.

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.

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.&#x0D; Place and Duration of Study: Department of Biotechnology, Kakatiya university, Warangal. Telangana, India, 3 years.&#x0D; 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) .&#x0D; 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.&#x0D; 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.

Radiosensitivitas Kalus Embriogenik Kopi Robusta Bp 436 Terhadap Iradiasi Sinar Gamma

&lt;p&gt;&lt;em&gt;High genetic diversity is one factor that determines the success of plant breeding. Mutation induction by gamma ray irradiation is one method to improve plant genetic diversity. This study aimed to 1) obtain growth regulators composition suitable in inducing embryogenic callus, 2) determine the effect of gamma ray irradiation on the growth and development of somatic embryos, and 3) obtain lethal dose (LD) &lt;sub&gt;20&lt;/sub&gt; and &lt;sub&gt;50&lt;/sub&gt; values in Robusta coffee BP 436. The study was conducted at the Tissue Culture Laboratory, Industrial and Beverage Crops Research Institute, from May 2017 to December 2018. Explants used were young leaves of Robusta coffee BP 436. Callus induction used ½ MS media with 2,4-D (4.52 μM) and 2-iP (0.00; 4.93; 9.86; 14.79; and 19.72 μM) treatment. Mutation induction was performed using gamma radiation dosed at 0, 10, 20, 30, 40, and 50 Gy treatments. The regeneration media was ½ MS containing GA&lt;sub&gt;3&lt;/sub&gt; (0 and 1 mg/l). The study used a complete randomized design with 10 replications. The results showed the best combination of plant growth regulator to induce the callus was 2.4-D 4.52 μM + 2-iP 19.72 μM. The fresh weight of cultures was inhibited above 30 Gy, whereas the number of somatic embryos decreased at doses above 10 Gy. Addition of GA&lt;sub&gt;3&lt;/sub&gt; 1 mg/l in regeneration media increased the number of somatic embryos in torpedo phase, but not in gamma irradiation exposed calluses. The LD&lt;sub&gt;20&lt;/sub&gt; and LD&lt;sub&gt;50&lt;/sub&gt; of Robusta coffee BP 436 are 16.81 and 28.52 Gy, respectively.&lt;/em&gt;&lt;/p&gt;

Effect of Thermal Stress on Tissue Ultrastructure and Metabolite Profiles During Initiation of Radiata Pine Somatic Embryogenesis.

Climate change will inevitably lead to environmental variations, thus plant drought tolerance will be a determinant factor in the success of plantations and natural forestry recovery. Some metabolites, such as soluble carbohydrates and amino acids, have been described as being the key to both embryogenesis efficiency and abiotic stress response, contributing to phenotypic plasticity and the adaptive capacity of plants. For this reason, our main objectives were to evaluate if the temperature during embryonal mass initiation in radiata pine was critical to the success of somatic embryogenesis, to alter the morphological and ultrastructural organization of embryonal masses at cellular level and to modify the carbohydrate, protein, or amino acid contents. The first SE initiation experiments were carried out at moderate and high temperatures for periods of different durations prior to transfer to the control temperature of 23°C. Cultures initiated at moderate temperatures (30°C, 4 weeks and 40°C, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50°C, 5 min). No significant differences were observed either for the percentage of embryogenic cell lines that produced somatic embryos, or for the number of somatic embryos per gram of embryonal mass. Based on the results from the first experiments, initiation was carried out at 40°C 4 h; 50°C, 30 min; and a pulse treatment of 60°C, 5 min. No significant differences were found for the initiation or number of established lines or for the maturation of somatic embryos. However, large morphological differences were observed in the mature somatic embryos. At the same time, changes observed at cellular level suggested that strong heat shock treatments may trigger the programmed cell death of embryogenic cells, leading to an early loss of embryogenic potential, and the formation of supernumerary suspensor cells. Finally, among all the differences observed in the metabolic profile, it is worth highlighting the accumulation of tyrosine and isoleucine, both amino acids involved in the synthesis of abiotic stress response-related secondary metabolites.

Influence of Silver Nitrate on Somatic Embryogenesis Induction in Arabica Coffee (Coffea arabica L.)

Plant somatic embryogenesis (SE) has been defined as the formation of embryos from a single or group of haploid or somatic cells [1, 2]. Low frequency (LFSE) and high frequency somatic embryogenesis (HFSE) have been described. In the first type, somatic embryos are induced directly from pro-embryogenic cells of explants, while in the second, they originate from embryogenic callus [1]. It has been suggested that in LFSE the origin of somatic embryos is unicellular, whereas in HFSE has been described as unicellular or multicellular [3]. SE is a powerful biotechnological tool used to propagate elite plants or to conserve important genotypes [4]. Moreover, SE offers an efficient in vitro regeneration approach as a fundamental step in plant genetic improvement for studying basic aspects of ontogenesis of somatic embryos [5]. In Coffea spp., the first studies of SE have been reported at the beginning of 1970 [6]. Since then, a large quantity of LFSE and HFSE protocols have been optimized demonstrating that coffee is not a recalcitrant species for SE [4]. In the LFSE the somatic embryos are obtained faster (approximately 70 days) using only one medium meanwhile in HFSE several media are used and somatic embryo formation takes 9-10 months [4]. Although, in LFSE small number of somatic embryos are obtained (around 10 per explant) compared to hundreds of somatic embryos obtained per gram of embryogenic calli [4], the unicellular origin of somatic embryos in LFSE represents an advantage for the chemical and physical mutagenesis, genetic transformation and genetic editing, since prevents or reduces the appearance of chimeras [7]. In C. arabica and C. canephora many factors (such as genotype, explant type, the physiological state, age and growth conditions of the donor plants, the season of collection, nutrient composition of the medium, the volume of dissolved CO2 or O2 in the culture flask, and plant growth regulators) that affect LFSE induction have been studied [3, 8, 9, 10, 11, 12, 13]. However, few studies reported the effect of silver nitrate on LFSE using leaf explants of C. arabica L. and to the best of our knowledge it has not been analyzed using Caturra and Catuai, which are two economic important producer cultivars in Costa Rica. Since SE is genotype dependent, the culture medium need to be modified for the different genotypes [7].Therefore, the objective of this study was to determine the influence of the benzyladenine (BAP), indole-3-acetic acid (IAA), and silver nitrate (AgNO3) on low frequency somatic embryogenesis using leaf explants of Coffea arabica L. cultivars Caturra and Catuai.

Effect of Water Stress Induced by Polyethylene Glycol 6000 on Somatic Embryogenesis in Cocoa (&amp;lt;i&amp;gt;Theobroma cacao&amp;lt;/i&amp;gt; L.)

Climate change, coupled with insufficient and irregular rains, led to a decline in the productivity of Ivorian cocoa production. The present study aimed to screen three cocoa (Theobroma cacao L.) genotypes in order to evaluate their response to water stress at callus and somatic embryos induction and proliferation stages. Staminode and petal explants of the genotypes C1, C14 and C16 from the collection of National Center of Agronomic Research (CNRA) of Ivory Coast, were placed on medium DKW (Driver & Kuniyuki Walnut) in the presence of different concentrations of Polyethylene glycol (PEG) 6000 (0, 25, 50, 75, 100 and 125 g/l). This polymer was used as osmoticum to stimulate water stress. Data were recorded for callus induction frequency, callus fresh weight, embryogenic callus percentage and average number of somatic embryos. The results obtained showed that intensity of callus proliferation decreased with increasing concentration of PEG 6000. In all genotypes tested, only the petal explants underwent somatic embryogenesis. The induction rates and average number of somatic embryos per explant were reduced with the increase in the concentration of PEG 6000. Among the tested genotypes, C1 produced somatic embryos with all concentrations of PEG 6000 and expressed highest frequencies of induction (62%) and average number of somatic embryos per explant (6.22). This genotype would be the most tolerant to water stress. Somatic embryos obtained with high concentrations of PEG 6000 in this study provide an important basis for the selection and further production of water stress-tolerant varieties of cocoa.

Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos.

Somatic embryogenesis (SE) provides us a potent biotechnological tool to manipulate the physical and chemical conditions (water availability) along the process and to study their effect in the final success in terms of quantity of somatic embryos produced. In the last years, our research team has been focused on the study of different aspects of the SE in Pinus spp. One of the main aspects affecting SE is the composition of culture media; in this sense, phytohormones play one of the most crucial roles in this propagation system. Many studies in conifers have shown that different stages of SE and somatic embryo development are correlated with distinct endogenous phytohormone profiles under the stress conditions needed for the process (i.e., cytokinins play a regulatory role in stress signaling, which it is essential for radiata pine SE). Based on this knowledge, the aim of this study was to test the effect of different temperatures (18, 23, and 28°C) and gelling agent concentrations (8, 9, and 10 gL-1) during the maturation stage of Pinus radiata SE in maturation and germination rates. Parallel, phytohormone profile of somatic embryos developed was evaluated. In this sense, the highest gellan gum concentration led to significantly lower water availability. At this gellan gum concentration and 23°C a significantly higher number of somatic embryos was obtained and the overall success of the process increased with respect to other treatments assayed. The somatic embryos produced in these conditions showed the highest concentration of iP-type cytokinins and total ribosides. Although, the different conditions applied during maturation of somatic embryos led to different hormonal profiles, they did not affect the ex vitro survival of the resulting somatic plants, where no significant differences were observed.

Influence of auxins on somatic embryogenesis in Haworthia retusa Duval

An efficient in vitro plant regeneration method through somatic embryogenesis has been established in Haworthia retusa. Somatic embryos were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and α-naphthaleneacetic acid (NAA) either alone or in combination with 4 μM thidiazuron (TDZ). Of the four auxins studied, IBA was found to be the most promising in terms of somatic embryo induction, followed in decreasing frequency by 2,4-D, IAA, and NAA. The highest somatic embryo induction (60.7%), with a mean of 20.7 embryos per leaf explant, was observed on MS medium amended with 20 μM IBA. The inclusion of 4 μM TDZ to the auxin-containing medium significantly (p < 0.0001) increased the somatic embryo induction frequency as well as the number of somatic embryos. The best combination for somatic embryogenesis was IBA + TDZ. The highest incidence of somatic embryo induction (100%), with a mean of 55.8 somatic embryos, was obtained on a culture medium containing 16 μM IBA + 4 μM TDZ. Somatic embryos germinated best on MS medium supplemented with 2 μM gibberellic acid. Morphological variations were observed among the regenerated plantlets. Well-developed plantlets obtained from germination media were acclimatized in the greenhouse.

Somatic Embryogenesis and Plant Regeneration From Primordial Shoot Explants of Picea abies (L.) H. Karst. Somatic Trees.

The recalcitrance of adult conifer tissues has prevented vegetative propagation of trees with known and desired characteristics. Somatic embryogenesis (SE) initiation protocol, recently developed for white spruce (Picea glauca, Klimaszewska et al., 2011), was applied in order to examine the feasibility, frequency and timing of SE induction from primordial shoots (PS) of Norway spruce (P. abies). In total, 39 genotypes were screened from 2015 to 2017 using 4–6 years old trees of SE origin as explant donors. Two genotypes responded: 11Pa3794 produced six proliferating embryonal mass (EM) sublines and 11Pa4066 produced 23 EM sublines. SE initiations occurred at the beginning of April, when the temperature sum (d.d.) started to accumulate, and at the end of October or beginning of November when the chilling unit (ch.u.) sum was over 500. EM sublines from both genotypes contained numerous early somatic embryos as detected by acetocarmine staining. The sublines of 11Pa4066 produced the mean of 78.6 ± 12.8 cotyledonary somatic embryos /g FW, but 11Pa3794 produced only a few cotyledonary somatic embryos that were able to germinate. The original EM lines (from which the trees were regenerated) had produced the same number of somatic embryos in 2011 maturations, which was approximately 120 somatic embryos /g FW. Microsatellite analyses conducted with both responsive genotypes confirmed the genetic stability of the EM sublines compared with the donor trees growing in the field. SE protocol developed for white spruce PS explants was also suitable for PS of Norway spruce if the explants were in the responsive developmental stage.

Production of bioactive cyclotides in somatic embryos of Viola odorata

Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1 mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation.

Somatic embryogenesis induction of litchi (Litchi chinensis Sonn.) from leaves of mature trees

Induction and development somatic embryos of litchi (Litchi chinensis Sonn.) from leaflet explants obtained from mature litchi trees were investigated. Somatic embryogenesis was significantly influenced by the cultivar, plant growth regulators, position of leaflet and orientation of leaf explants. Adaxial-face up orientation of leaf explants significantly enhanced embryogenesis in comparison with adaxial-face down orientation. Maximum number of somatic embryos were obtained using median position from basal leaflet with adaxial-face up orientation cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 2, 4 dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) (37.67). Two types of salts media (MS and Gamborg’s B5) as well as different sucrose concentrations were examined for development and maturation of somatic embryos. MS salts medium with 45g/l sucrose was effective on size of mature somatic embryos (8.00 &gt; 0.5 cm SE and 6.33 &lt; 0.5 cm SE) compared with B5 salts medium (4.33 &gt; 0.5 cm SE and 4.66 &lt; 0.5 cm SE) on the same concentration of sucrose. Development of somatic embryos occurred on the media without growth regulators, heart and torpedo stage somatic embryos were obtained. Somatic embryos enlarged more and turned to milky opaque in color and finally reached to mature milky cotyledonary stage. Scanning electron microscopy revealed different morphological development of somatic embryos. In addition, Anatomical observation showed differences in the embryogenic and non-embryogenic cells. Furthermore, anatomical observation for development stages of somatic embryos.

Preparation of nitric oxide-releasing photo-crosslinked electrospun chitosan nanofibrous scaffolds for bone tissue engineering

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50–500 μM and 10–50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.

16S and 18S metagenomics analysis of fouling residues from aquaculture

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50–500 μM and 10–50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.

A novel energy-efficient version of the Calvin cycle

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50–500 μM and 10–50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.

Microbial electrochemical technology (MET) platform for turning carbon dioxide into a suitable substrate for a chain-elongation fermenter

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50–500 μM and 10–50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.

Somatic Polyembriogenesis of Larix sibirica in Embryogenic in vitro Culture

The initiation of somatic embryos and their propagation in the long-term proliferating embryonal suspensor mass of Larix sibirica were studied. It was found that the increase in the number of somatic embryos in the embryogenic culture occurred as a result of cleavage of the globules of the somatic embryo and the suspensor; it less often occurred as the result of budding of the suspensor and the separation of the embryonal tubes of the suspensor. In the case of long-term proliferating cell lines (more than 8 years), the rate of cleavage did not weaken. A conclusion on the identity of morphogenic processes underlying the development of zygotic and somatic embryos of conifers is made, which is confirmed by the concept of T.B. Batygina (1999) on the parallelism of their development in vivo and in vitro.

Direct somatic embryogenesis of potato [Solanum tuberosum (L.)] cultivar ‘Kufri Chipsona 2’

Direct somatic embryo induction was achieved from leaf and internodal explants of Solanum tuberosum (L.) cultivar ‘Kufri Chipsona 2’ on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 10.0 µM silver nitrate (MS1 medium) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2.5 µM) and 6-benzyladenine (BA; 1.0 µΜ). It was observed that in absence of AgNO3, friable callus was induced from cut ends of the explants, which does not develop into any kind of organised structure; thus highlighting the requirement of AgNO3 for somatic embryogenesis in potato. Furthermore, the effect of medium strength, sucrose concentration and heat shock treatment on somatic embryogenic potential of explants was also investigated. When the strength of basal medium was reduced to half, the frequency of internodal segments differentiating somatic embryos was almost double in comparison to full strength MS medium. Sucrose concentration and heat shock treatment were found to have interactive effect on somatic embryo induction. Explants subcultured on medium containing 174 mM sucrose and subjected to heat shock (1 h; 50 °C) showed maximum somatic embryo differentiation. Although, the percent explants differentiating somatic embryos decreased sharply with increase in sucrose concentration (> 174 mM), yet the number of somatic embryos differentiated per explant were found to increase with further increase in sucrose concentration. Histological observations revealed that somatic embryos directly developed from epidermis of leaf explant and cut ends of internodal segments progressed from globular to cotyledonary stage after passing through intermediate embryogenic stages (heart shaped and torpedo shaped). Conversion of somatic embryos into plantlets (92%) was achieved on MS1 medium supplemented with BA (10.0 µM) and gibberellic acid (15.0 µM) and all regenerated plants were found to be phenotypically alike.

Effects of copper and arsenic stress on the development of Norway spruce somatic embryos and their visualization with the environmental scanning electron microscope

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50–500 μM and 10–50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.