Primary Somatic Embryos Research Articles

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl−1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA3), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO4·7H2O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.

Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.).

While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Key message: Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation.

Virus Detection and Elimination in Cocoa ( Theobroma cacao L.) Through Somatic Embryogenesis

Cacao swollen shoot virus (CSSV) is a major pathogen that has seriously constrained cocoa production in West Africa, particularly Ghana and Nigeria. The objective of this study was to assess the efficacy of cocoa somatic embryogenesis to produce virus-free clonal propagation material both for replanting and to facilitate the safe international exchange of germplasm. Polymerase Chain Reaction (PCR)-based screening, is employed in this study because of its capacity for CSSV detection prior to the appearance of visual symptoms. Degenerate PCR primers were developed in order to improve the CSSV-strain dependence of earlier tests. The degenerate primers were capable of detecting 37 out of a putative 56 CSSV strains, four more than the sequence specific primers. For tissue culture studies, cocoa staminodes cultures were established from flowers of CSSV-infected cocoa genotypes CL 19/10 strain 1A and Amelonado Plant 2 to produce callus, primary and secondary somatic embryos, with genotype AMAZ 15 used as a virus-free control. PCR-based CSSV detection proved that virus could be detected at callus, primary somatic embryos and secondary somatic embryo stages, indicating that the progress of the virus was progressively impeded. These findings support the use of somatic embryogenesis as a mean of improving CSSV-free clonal propagation of cocoa. Somatic embryogenesis is indeed effective for virus elimination in cocoa and it has been demonstrated to function for a range of cocoa genotypes. This also means that a likely mechanism for the interruption of CSSV movement has been identified.

Embriogénesis somática secundaria en el genotipo de cacao (Theobroma cacao L.) inifap 1 y su descripción histológica

La embriogénesis somática aplicada para la propagación clonal de plantas de cacao, aun es de baja eficiencia, por lo que el objetivo de esta investigación fue evaluar la respuesta de los cotiledones procedentes de embriones somáticos primarios como fuente de explante para inducir embriogénesis somática secundaria (ESS) en presencia de fitohormonas y sin estas en el genotipo de cacao inifap 1, asi como describir los eventos de la ESS desde los 30 hasta los 240 días de cultivo. El uso de las fitohormonas ácido 2,4-Diclorofenoxiácetico (2,4-D) y thidiazuron (TDZ) son importantes para inducir la embriogénesis somática (ES) primaria en cacao. Adicionalmente para generar la ESS se han utilizado fitohormonas. En la presente investigación, fueron establecidos cuatro tratamientos con fregmentos de cotiledón, con 2,4-D y TDZ en forma combinada e independiente, incluyendo un testigo sin fitohormonas. Los resultados mostraron, que los fragmentos de cotiledón cultivados sin fitohormonas favorece la ESS, obteniendo un promedio de seis embriones somáticos secundarios (ESs) a los 210 días de cultivo contra uno para el tratamiento suplementado con TDZ. El meristemo apical y radicular se presentó en un periodo de 240 días, se observaron embriones somáticos con estructuras externas como: cotiledones, ápices y raíces. Los resultados mostraron que la exclusión del 2,4-D y el thidiazuron no son importantes para inducir la ESS. El mayor número de ESs se encontró en un medio desprovisto de fitohormonas por lo que esta información es relevante, debido a que los explantes de cotiledones procedentes de embriones somáticos primarios, generan mayor número de ESs en esta especie. Estos resultados constituyen el primer reporte de la inducción de la ESs sin fitohormonas.

Use of amino acids for a highly efficient somatic embryogenesis in grapevine 'Crimson Seedless'

Somatic embryogenesis influenced by growth regulators and amino acids was studied in in vitro leaves of grapevine 'Crimson Seedless'. In vitro leaves of the cultivar were collected from multiple shoot cultures maintained on Murashige and Skoog’s (MS) basal medium supplemented with 9 μM N 6 -benzyladenine (BA). Among the growth regulators used, BA at 4.5 μM induced higher embryogenic response producing more number of somatic embryos per explant. This response was increased with the addition of 5 μM naphthoxy-1-acetic acid (NOA) to ½ MS containing 4.5 μM BA. Further, supplementation of amino acids in the callus induction medium significantly improved the embryogenic response of in vitro leaves. The higher number of explants showing somatic embryo production (55.3 %) and higher number of somatic embryos per explant (15.5 per explant) were recorded with the supplementation of 5 mM phenylalanine to callus induction medium. Primary somatic embryos showed repetitive embryogenesis on ½ MS medium devoid of growth regulators. Plantlets derived from somatic embryos were transferred to soil-sand-peat mixture (1:1:1 v/v) and hardened plantlets were established in greenhouse with 90 % survival. This somatic embryogenesis system has been successfully used for Agrobacterium -mediated transformation studies in 'Crimson Seedless' in our laboratory. To our knowledge, this is the first report on the use of amino acids for the high efficient somatic embryogenesis in grapevine.

High-efficiency direct somatic embryogenesis and plant regeneration from leaf base explants of turmeric (Curcuma longa L.)

This study describes for the first time a simple and quick direct somatic embryogenesis and plant regeneration protocol for turmeric (Curcuma longa L.) using leaf base explants. Preincubation of explants was carried out on solid Murashige and Skoog (MS) medium containing 4.49 μM 2,4-dichlorophenoxy acetic acid for 20 days under continuous dark condition. Subsequently transfer of the explants for 2 weeks to MS liquid medium containing 1.32 μM 6-benzyladenine (BA) under illuminated condition resulted in the highest percentage of somatic embryo (91.1 %) induction. Secondary somatic embryos which originated from primary somatic embryos were also observed in the same medium supplemented with 2.20 and 2.64 μM BA. Thereafter, mature somatic embryos germinated readily and the maximal plantlet development (87 %) was achieved on 1/2-strength MS medium containing gibberellic acid (GA3) under dark condition. Histological analysis revealed that the proembryogenic masses were initiated directly from the area near the vascular tissues of the leaf base explants. Presence of clear protoderm in the globular structure and procambial strands in the matured stage of somatic embryos confirmed that these structures were true somatic embryos. Somatic embryos were successfully acclimatized ex vitro at a survival rate of 72.9 % and they were normal phenotypes.

Plantlet Regeneration of Somatic embryos from Leaf Explants of Mentha arvensis(L.) A medicinally important Plant

The present study was conducted with the aim of evaluating some of the factors that influence induction and plantlet regeneration of somatic embryos in Mentha arvensis ( L) var piperascens Holmes (menthol or Japanese mint)since there are no available reports on Somatic embryogenesis and regeneration of plantlet in this medicinally important plant species. Leaves from plants growing under temporary shed were cultured on Murashige and Skoog medium fortified with (0.5-5.0mg/L) Napthlene acetic acid (NAA)+(0.5 mg/L)Thidizuron(TDZ). High frequency of somatic embryo formation was found at (2.5mg/L) NAA+ (0.5mg/L) TDZ in leaf explants respectively, Secondary somatic embryogenesis was also observed when primary somatic embryos were sub cultured same somatic embryo induction medium well developed cotyledonary stage embryos were germinated on MS medium supplemented with (0.5mg/L) (NAA) + (0.5-5.0mg/L)TDZ maximum 80% of somatic embryos germination and plant let formation was found at (2.5 mg/L) NAA+ (0.5 mg/L) (TDZ). The post translation survival rate of plants was 80% plants and flowers formation were morphological similar to the mother plants.

English

An attempt was made to study the somatic embryogenesis and plant regeneration from the in vitro leaf explants of Rumex vesicarius L. a renowned medicinal plant, which belongs to polygonaceae family. Effective in vitro regeneration of R. vesicarius was achieved via young leaf derived somatic embryo cultures. Embryogenic callus was induced from leaf explants on Schenk and Hildebrandt (SH) medium supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5 to 3.0 mg/l) along with Kinetin (Kn) (0.5 mg/l). High frequency of somatic embryogenesis was effective on SH medium with 2, 4-D (2.5 mg/l) + Kn (0.5 mg/l) from leaf explants. Secondary somatic embryogenesis was also observed when primary somatic embryos were subculture on the same somatic embryo induction medium. Well developed cotyledonary shaped embryos regenerate 80% of shoots on media containing 2,4-D 0.5 mg/l + 2.0 mg/l BA. The regenerated shoots transferred to rooting media containing Indole- 3- butyric acid (IBA). Efficient rooting of 90% was noted on SH media with 1.0 mg/l IBA. Finally, these in vitro regenerated plantlets were hardened, acclimatized and successfully transferred to the field. The post transplantation survival rate of these regenerated plants was 65 to 70%. The in vitro regenerated plants and flowers were similar to mother plants. This protocol will be useful for genetic transformation experiments in R. vesicarius L. Keywords: Rumex vesicarius L, 2,4-dichlorophenoxy acetic acid (2,4-D), kinetin (Kn), Benzyl adenine (BA), Indole- 3- butyric acid (IBA). African Journal of Biotechnology , Vol 13(45) 4268-4274

Cassava (Manihot esculenta Crantz).

Genetic transformation of plants is an indispensable technique used for fundamental research and crop improvement. Recent advances in cassava (Manihot esculenta Crantz) transformation have facilitated the effective generation of stably transformed cassava plants with favorable traits. Agrobacterium-mediated transformation of friable, embryogenic callus has evolved to become the most widely used approach and has been adopted by research laboratories in Africa. This procedure utilizes axillary meristem tissue (buds) to produce primary and secondary somatic embryos and subsequently friable, embryogenic callus. Agrobacterium harboring a binary expression cassette is used to transform this tissue, which is regenerated via cotyledons and shoot organogenesis to produce rooted in vitro plantlets. This chapter details each step of the procedure using the model cultivar 60444 and provides supplementary notes to successfully produce transgenic cassava.

Pembentukan Embrio Endospermik Sekunder Mangga (Mangifera Indica L.) Gedong Gincu Klon 289

ABSTRACT The improvement of Mangifera indica L. by conventional breeding approaches has been confounded by the long generation cycle, low fruit set, single seed per fruit and high degree of cross pollination. Biotechnology complements conventional breeding and expedite the mango improvement programs. Endosperm culture is a direct method to produce triploid plants. This study aimed to obtain embryo from endosperm culture. The system of secondary somatic embriogenesis in mango described here represents a source of embryogenic material may be used for mass propagation and genetic manipulation of this crop. The method consisted of induction, proliferation, maturation, germination, and histological analysis of the obtaimed embryos. A protocol for plantlet regeneration was developed for Gedong Gincu mango clone 289 through secondary somatic embryogenesis. Primary somatic embryos (proembryo and cotyledonary embryos) were cultured in induction medium to induce the secondary somatic embryos. The best proliferation rate was 0.22 in medium with 1 g L-1 Poly Vinyl Pyrrolidone (PVP) for multiplication of secondary somatic embryos. Maturation of inoculum derived from the proliferation medium supplemented with 2 g L-1 of activated charcoal on medium containing 0.4 mg L-1 BAP provides the average 2.39 embryo formation of cotyledonari phase. The highest germination frequency (20%) was obtained in media with GA3 1.5 mg L-1. Keywords: endosperm, Gedong Gincu, Mangifera indica L, secondary endospermic embrio

Microarray analysis of siberian ginseng cyclic somatic embryogenesis culture systems provides insight into molecular mechanisms of embryogenic cell cluster generation.

Four systems of cyclic somatic embryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters.

Control of Morphological Aberrations in Somatic Embryogenesis of Commiphora wightii (Arnott) Bhandari (Family: Bursaraceae) Through Secondary Somatic Embryogenesis

Somatic embryogenesis in Commiphora wightii was obtained using 5-8 mm long embryos from fruits (65-75 days old) collected during January to June. The embryos gave rise to embryogenic mass on Murashige and Skoog medium supplemented with 2,4-D (4.4 lM), sucrose (3.0 % w/v) and agar (0.6 % w/v), when incubated under dark conditions within 55-65 days. Further, these embryo- genic masses induced somatic embryos (1.00-9.00 mm long) within 40-45 days after transferring them to light on media devoid of 2,4-D. The somatic embryos were asyn- chronous with certain levels of abnormalities. The abnor- malities observed were different sizes of globular embryos, single cotyledonary and fasciated embryos. In order to cir- cumvent these abnormalities and to achieve synchronized induction, studies were carried out to induce secondary somatic embryos. The induction of Secondary Somatic Embryos was achieved on Murashige, Skoog and Schenk and Hildebrand media from primary cotyledonary somatic embryos. These were induced from the edge of cotyledon and the tip of hypocotyl, and were 1.00-3.00 mm in length. A total of 74 % of the two cotyledonary embryos were induced from secondary embryogenesis system against 24 % induced from primary somatic embryogenesis system.

Regeneration in Tetrapleura tetraptera via Indirect Somatic Embryogenesis from Isolated Axillary Meristems

To facilitate the use of biotechnological techniques for conservation, germplasm exchange and improvement of Tetrapleura tetraptera, the study investigated in vitro plant regeneration via somatic embryogenesis from isolated axillary meristem of the plant. Indirect primary somatic embryos were induced on MS basal medium supplemented with nine different concentrations of 2,4-D. Pre-embryogenic calli were made to mature on MS basal medium containing BAP alone or with four concentrations of NAA. Secondary somatic embryos were induced on MS basal medium supplemented with nine different concentrations of 2,4-D. Maturation of the secondary pre-embryogenic calli took place on MS medium fortified with eight concentrations of BAP. Medium fortified with 6.0 mg/l 2, 4-D had the highest frequency of primary embryogenic callus (16.4%) and average number of primary immature embryo per callus (2.3). Culture media supplemented with 0.6 mg/l BAP and 0.1 mg/l NAA recorded the highest primary embryogenesis (24.5%) and average number of matured primary embryo per explant (2.8 embryos/explants). Medium fortified with 1.2 mg/l BAP gave the highest secondary embryogenesis (72.4%) and average number of matured embryo per callus (3.1 embryos per callus). Explants in medium receiving 1.0 mg/l BAP and 0.7 mg/l IBA recorded largest number of shoot-bud (27.6%) and average number of shoot-bud per explant (2.4/explant) with vigorous and green appearance. Shoot elongation was greatest in medium fortified with 1.0 mg/l BAP and 15.0 mg/l IBA with vigorous appearance. The largest number of roots (4.3/plantlet) were formed by plantlets grown in medium supplemented with 1.0 mg/l BAP and 15.0 mg/l IBA with normal appearance. The study concluded that indirect somatic embryos obtained from isolated axillary meristem with 2,4- D, BAP, NAA were capable of regeneration into normal vigorous plants.

Erratum to: Primary and secondary somatic embryogenesis in Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’ and assessment of ploidy stability of somatic embryogenesis process by flow cytometry

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from petal explant of Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’. Somatic embryogenesis was induced from petal explants on the Murashige and Skoog (MS) medium supplemented with 1.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 3.0 mg l−1 6-benzyladenine (BA), yielding the highest mean number of embryos (56.3) per explant after 5 weeks of culture. We evaluated the effects of basal medium and various concentrations of sucrose on the proliferation of secondary somatic embryos. MS medium was observed to be more effective in promoting the proliferation of somatic embryos than half-strength Murashige and Skoog (1/2MS). In addition, 1 % sucrose was also found to be the best in induction of secondary embryogenesis. The highest germination rate (70 %) of the somatic embryos was observed on the MS medium containing 0.2 mg l−1 α-naphthalene acetic acid and 1 g l−1 activated charcoal (AC). Shoots elongated rapidly and roots developed well on hormone-free MS medium with 1 g l−1 AC and successfully acclimated in the greenhouse. Flow cytometric analysis of the primary somatic embryos, secondary somatic embryos, and the somatic embryo-obtained plants along with the parent grown in the greenhouse showed that they all had same identical peaks, indicating that there was no variation of ploidy level during the regeneration process. We expect that our report would be useful for micropropagation and Agrobacterium-mediated genetic transformation studies of this cultivar.

Influence of Type and Age of Primary Somatic Embryo on Secondary and Cyclic Somatic Embryogenesis of Cassava (Manihot esculenta Crantz)

This study investigated the influence of age of the cotyledons, cut from primary somatic embryos (PSE) developed from shoot meristems (SM) or immature leaf lobes (LL), on secondary somatic (SSE) and cyclic (CSE) embryogenesis of two cassava cultivars at th e Central Biotech Laboratory, International Institute of Tropical Agriculture (IITA), Ibadan, between 2006 and 2010. A completely randomized design with three replicates was used for the study. Only PSE at the age of 4 weeks recorded significant (P<0.05) d ifferences in SSE frequency and efficiency between the SM and LL sources. CSE production was highest using 0 to 4 weeks old SSE cotyledons, and significant (P<0.05) differences were only recorded between the SM and LL sources when the age of the SSE cotyle dons was older than 6 weeks. The CSE frequencies from the SM source were significantly greater than that from the LL source when 8 and 10 week -old SSE cotyledons were used. The OriginalResearch Article

Manifestation of embryogenic potential in culture of zygotic embryos of Quercus robur L.

For the initiation of somatic embryogenesis early cotyledonary stage of zygotic embryo explants (from 15th July until late August) was suitable. The highest frequency of differentiation of somatic embryos was obtained on cotyledons of zygotic embryos cultured on basal modified medium MS (with 1/2 concentration macronutrients) or WPM medium containing 500 mg•l&lt;sup&gt;-1&lt;/sup&gt; glutamine, proline and casein hydrolysate and supplemented with 2,4-D (1,0-2,0 mg•l&lt;sup&gt;-1&lt;/sup&gt;) and BAP (0,5-1,0 mg•l&lt;sup&gt;-1&lt;/sup&gt;). The development of somatic embryos was direct and indirect and the process was continuous over a long period. Primary somatic embryos were able to produce secondary embryos. Repetitive somatic embryogenesis led to the proliferation of a large number of new somatic embryos on their cotyledons, hypocotyl or radicula. The process of embryo differentation is asynchronous - various stages of somatic embryos could be observed in embryogenic culture. A somatic embryo conversion was rare on tested media. Embryo germination occured on medium containing BAP (0,1 mg•l&lt;sup&gt;-1&lt;/sup&gt;) or on medium with ABA and GA&lt;sub&gt;3&lt;/sub&gt; (each 0,2 mg•l&lt;sup&gt;-1&lt;/sup&gt;) after a previous culture on WPM medium without plant growth regulators supplemented with sorbitol (6%). The embryo germination occurred also on WPM medium with 0.2 mg•l&lt;sup&gt;-1&lt;/sup&gt; BAP when cultures were mantained at 2&lt;sup&gt;o&lt;/sup&gt;C for 4 weeks. Only 8 somatic embryos developed into plantlets. Their transplantation to &lt;em&gt;in vivo&lt;/em&gt; conditions was unsuccessful.

INDUCTION OF DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE EMBRYOS OF BRASSICA OLERACEA VAR. SABAUDA L.

An efficient plant propagation system through somatic embryogenesis by using immature zygotic embryos was established in Savoy cabbage (Brassica oleracea var. sabauda L.). The effects of zygotic embryo age, presence of 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) (B5-D) in the culture medium, and the pH (5.0 and 5.8) of the B5 plant growth regulator (PGR)-free (B5-0) induction media were investigated. It appeared that the developmental stage of the immature zygotic embryos used as explants was the most important factor for somatic embryogenesis. Highest frequency of somatic embryogenesis (86.67%) was achieved directly on zygotic embryos at cotyledonary stage (1.8 mm long) of development that cultured on B5-0 medium. Optimal pH of the medium was 5.0. Mean number of primary somatic embryos per explant on B5-0 pH 5.0 medium was 7.2. The inclusion of 2,4-D into the induction medium significantly reduced both frequency of somatic embryogenesis (to 53.34 %) and mean number of embryos per explant (to 2.62) in cotyledonary zygotic embryos. After transferring to PGR-free Murashige and Skoog (MS-0) medium 69.69% of the primary somatic embryos of the Savoy cabbage produced secondary somatic embryos (SSEs) Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of Savoy cabbage.

Repetitive somatic embryogenesis from leaves of the medicinal plant Petiveria alliacea L.

Leaf segments from in vitro-grown shoot cultures of Petiveria alliacea were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations of zeatin, thidiazuron, 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). Direct somatic embryogenesis was induced in response to all tested concentrations of 2,4-D and PIC. Primary somatic embryos displayed highly repetitive embryogenesis, both on the induction medium and in liquid hormone-free MS medium. Plantlets were obtained from these secondary embryos at an estimated frequency of 5 %, after 180 days of culture on half-strength MS medium gelled with 0.2 % Phytagel. Simultaneous development of friable non-embryogenic callus was also observed on media containing PIC or 2,4-D at different concentrations. Cell suspension cultures initiated from these callus tissues did not show an increase in biomass. The embryogenic portions formed at the surface of the explants in response to 20.0 μM PIC were inoculated in hormone-free full-or half-strength liquid MS medium (MS0) and showed high rates of secondary embryogenesis, resulting in the production of a mean of 35 embryos for each embryo inoculated at the culture initiation. Embryos that started the conversion process in the liquid MS0 medium originated whole plants at a frequency of 100 % when transferred to MS0 medium solidified with 0.7 % agar. Acclimatization was achieved in 90 % of the converted plantlets, with the production of phenotypically normal plants. This system is potentially useful for the micropropagation of this species, as well as for the production of substances with pharmacological interest, such as dibenzyl trisulfide.

Maturation of Anthurium andraeanum cv. Eidibel somatic embryos from nodal segments

Maturation of somatic embryos of Anthurium andraeanum cv. Eidibel from embryogenic callus was evaluated. Following induction of embryogenic calli from nodal segments, tissues were transferred to 125-mL Erlenmeyer flasks containing 25 mL liquid medium, with 0, 4.52, or 9.05 μM 2,4-dichlorophenoxyacetic acid and 0, 0.47, or 2.32 μM kinetin. Callus cultures were maintained in a dark growth room at 25 ± 2°C. At 45 d, the mass of embryogenic calli, number of primary and secondary somatic embryos, and percentage browning were evaluated. Nonparametric tests were used to evaluate color, texture, and somatic embryo development. The highest yield of somatic embryos was in the medium with 0.47 μM kinetin. Calli were friable, with a lower yield of secondary somatic embryos, and have minimal browning. Histology revealed polar globular somatic embryos and mature somatic embryos with defined apical and root meristematic zones, axillary buds, and primary leaves. These are important features for converting somatic embryos into plantlets.

In Vitro Zygotic Embryo Germination and Somatic Embryogenesis through Cotyledonary Explants of Paeonia lactiflora Pall.

A simple and efficient protocol was developed for somatic embryogenesis from the cotyledon explant of Paeonia lactiflora Pall. Seeds of peony obtained from fieldgrown plants were disinfested and zygotic embryos were excised. For germination, excised embryos were cultured on the Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose, 0.8% (w/v) agar, and different concentrations of N 6 benzyl-adenine (BA) and gibberellic acid (GA₃). The greatest germination percentage (95%) was observed when embryos were cultured on the MS medium with 1.0 mg·L -1 BA and 0.5 mg·L -1 GA₃, and maintained at 25 ± 2°C under a 16 h photoperiod. Thirty days old cotyledon explants were cultured on the MS medium supplemented with different concentrations and combinations of plant growth regulators viz., BA, GA₃, 2,4-dichlorophenoxyacetic acid (2,4-D), and a-naphthalene acetic acid (NAA). After 90 days, the globular embryos were directly formed on the surface of explants. The highest frequency of somatic embryo induction (72.5) was obtained on the MS medium with 3.0 mg·L -1 BA, 1.0 mg·L -1 NAA, 1.0 mg·L -1 GA₃, and 0.1% (w/v) activated charcoal (AC), with a mean number of 14 embryos per explant. Maturation of globular embryos into heart- and torpedo-shape was observed on the same medium. When the torpedo-shaped embryos were transferred onto the same MS medium supplemented with 3.0 mg·L -1 BA, 1.0 mg·L -1 NAA, 1.0 mg·L -1 GA₃, and 0.1% (w/v) AC, secondary somatic embryos were observed on the surface of primary somatic embryos. When the embryos were transferred to the MS medium supplemented with 1.0mg·L -1 each of BA and GA₃, all of them converted into plantlets, but their growth was very slow.