Differentiation Of Somatic Embryos Research Articles

Direct somatic embryogenesis of Malaxis densiflora (A. Rich.) Kuntze.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant of Malaxis densiflora has been developed for the first time. In the present study, in vitro seed derived protocorm explants were cultured on half strength Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram and Dicamba individually and in combination with cytokinins BAP, TDZ and Kn for its effectiveness to induce the differentiation of somatic embryos. The best response was observed in protocorms cultured half strength MS medium supplemented with 2,4-D at 3.39 μM and TDZ at 6.80 μM. Both epidermal and sub epidermal cells were involved in the formation of embryos. The proembryos developed into globular stage and subsequently developed into protocoms. Complete plantlets were formed after 60 days of culture. The plantlets were acclimatized in plastic pots containing sterilized vermiculite. The survival rate was 76%.

Plant regeneration through direct somatic embryogenesis, antioxidant properties, and metabolite profiles of Swertia corymbosa (Griseb.) Wight ex C.B. Clarke

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant of Swertia corymbosa has been developed for the first time. In the present study, in vitro derived leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) individually and in combination with cytokinins for its effectiveness to induce the differentiation of somatic embryos. The antioxidant activities of methanolic extracts from non-embryonic callus (NEC), pre-embryoid masses (PEM), somatic embryos at globular stage (SEG), somatic embryos at heart-shaped stage (SEHS), and cotyledonary embryos (SEC) of S. corymbosa were evaluated using three in vitro assays: scavenging of free radicals (DPPH and ABTS) and ferric reducing antioxidant test. In all cases, the methanolic extract from SEG demonstrated better antioxidant activity than those taken from other tested samples. Higher amounts of swertianin (1), methylswertianin (2), and 1,2- dihydroxy-6-methoxyxanthone-8-O-β-D-xylopyranosyl (3) were found in SEG stage followed by SEHS and PEM when compared to NEC and SEC.

Clonal propagation of Lomandra longifolia by somatic embryogenesis

A plant regeneration system based on somatic embryogenesis was developed for the efficient clonal propagation of Lomandra longifolia. Cultured leaf-bases, immature inflorescences and immature ovaries of L. longifolia formed embryogenic calli, with subsequent somatic embryo induction upon subculture to Murashige and Skoog (MS) agar medium supplemented with 2.0mgL−1 2,4-diclorophenoxyacetic acid (2,4-d), 500mgL−1 casein hydrolysate, 100mgL−1 myo-inositol, and 30gL−1 sucrose. Of the three types of explants, immature inflorescences and immature ovaries produced only embryogenic calli, whereas leaf bases produced both embryogenic and non-embryogenic calli. Root tips cultured on 2,4-d-containing media formed a tissue that did not form somatic embryos, but instead differentiated into shoot-buds. Somatic embryo differentiation and plantlet regeneration occurred best from embryogenic calli on 2,4-d-free basal medium or MS basal medium containing 1.0mgL−1 NAA and 0.1mgL−1 BAP; and the resultant in vitro-formed plantlets were successfully transferred to soil. Morphological and anatomical data describing development of calli and somatic embryos provided evidence for plant regeneration via somatic embryogenesis and, most likely, single cell origin of somatic embryos.

Partial resistance of carrot to Alternaria dauci correlates with in vitro cultured carrot cell resistance to fungal exudates.

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.

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<sup>-1</sup> glutamine, proline and casein hydrolysate and supplemented with 2,4-D (1,0-2,0 mg•l<sup>-1</sup>) and BAP (0,5-1,0 mg•l<sup>-1</sup>). 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<sup>-1</sup>) or on medium with ABA and GA<sub>3</sub> (each 0,2 mg•l<sup>-1</sup>) 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<sup>-1</sup> BAP when cultures were mantained at 2<sup>o</sup>C for 4 weeks. Only 8 somatic embryos developed into plantlets. Their transplantation to <em>in vivo</em> conditions was unsuccessful.

Changes in ATP, glucose-6-phosphate and NAD(P)H cellular levels during the proliferation and maturation phases of Abies alba Mill. embryogenic cultures

The aim of the present study was to evaluate the adenosine triphospate (ATP), glucose-6-phosphate (glu-6P) and reduced form of nicotinamide adenine dinucleotide phosphate (NAD(P)H) cellular levels during the proliferation and maturation phases of Abies alba Mill. somatic embryos. For a better understanding of the dynamics of these parameters during the proliferation cycle, four embryonic cell lines were tested. During the maturation period, three independent experiments were conducted, focused on the effects of PEG-4000 (5 or 10% (w/v)) and abscisic acid (16, 32 or 64 μM) applied together (Experiments A and B) or with addition of gibberellic acid (Experiment C) on the dynamics of bio-energetic molecules and on the mean number of cotyledonary somatic embryos. Our results demonstrated that the cellular levels of bio-energetic molecules strongly depended on the composition of maturation media. Generally, the higher the number of cotyledonary embryos produced, the higher the level of ATP observed after a 2-week maturation period. The cellular level of ATP, glu-6P and NAD(P)H increased, particularly after the transition from the proliferation to the maturation phase when the differentiation and growth of somatic embryos occurred.

Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane

Differences in competence acquisition and subsequent embryo maturation in embryogenic and non-embryogenic callus of sugarcane var. SP79-1011 were evaluated using histomorphological analysis, growth curves, numbers of somatic embryos, and polyamine contents. Embryogenic callus was formed by cells with embryogenic characteristics such as a rounded shape, prominent nuclei, a high nucleus: cytoplasm ratio, small vacuoles and organized globular structures. However, non-embryogenic callus presented dispersed, elongated and vacuolated cells with a low nucleus: cytoplasm ratio; these characteristics did not allow for the development of somatic embryos even upon exposure to a maturation stimulus. These results suggest that non-embryogenic callus does not acquire embryogenic competence during induction and that maturation treatment is not sufficient to promote somatic embryo differentiation. The use of activated charcoal (AC; 1.5 g L−1) resulted in a higher somatic embryo maturation rate in embryogenic callus but did not yield success in non-embryogenic callus. Embryogenic callus incubated with control (10 μM 2,4-dichlorophenoxyacetic acid) and maturation (1.5 g L−1 AC) treatments for 28 days showed similar patterns of total free polyamines; these results differed from the results observed with non-embryogenic callus, suggesting that embryogenic callus already exhibits a characteristic pattern of endogenous polyamine levels. At 28 days of culture with maturation treatment, embryogenic callus exhibited significantly higher levels of free Spm than embryogenic callus incubated with control treatment and non-embryogenic callus incubated with both treatments. This result suggests that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var. SP79-1011.

Regeneration and growth of peony (Paeonia spp.) in vitro

Vegetative propagation of peony is usually carried out by grafting onto herbaceous peony roots (tree peony) or by dividing tuberous root clumps (herbaceous peony). However, conventional propagation is time-consuming and too slow for a large production and therefore <i>in vitro<i/> methods have been developed. The regeneration ability of different organs of herbaceous and tree peony has been reported in this review. The role of exogenous and endogenous growth regulators in the differentiation and growth of shoots, roots and somatic embryos is presented. The problem of dormancy in the somatic embryos and in the shoots or plants propagated <i>in vitro<i/> are also discussed.

Differential responses to somatic embryogenesis of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.)

To assess the potential of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.) to somatic embryogenesis and somatic embryo proliferation, mature zygotic embryos of nine commercial genotypes of E. guineensis (BRSC2001, BRSC2328, BRSC2301, BRSC3701, BRSCM1115, BRSC7201, BRSC2528, BRSC2501, and BRSCN1637) were used. Explants were incubated on Murashige and Skoog (MS) supplemented with 450 μM picloram, 3.0 % sucrose, 500 mg l−1 glutamine, and 2.5 g l−1 activated charcoal, and gelled with 2.5 g l−1 Phytagel. After induction, for differentiation and maturation, the embryogenic calli (ECs) were transferred into fresh medium supplemented with 0.6 μM naphthaleneacetic acid (NAA) and 12.30 μM 2-isopentenyladenine (2iP) or 40 μM picloram in combination with 0.3 g l−1 activated charcoal, and 500 mg l−1 glutamine. Somatic embryos were converted into plants on MS medium with macro- and micro-nutrients at half strength, 2 % sucrose, and 2.5 g l−1 activated charcoal, and gelled with 2.5 g l−1 Phytagel. In general, zygotic embryos swelled after 14 days. Primary calli, which were observed in all the genotypes after 45–60 days of culture, eventually progressed to ECs at 90 days. At this time, scanning electron microscopy (SEM) analysis showed cellular differences between compact and friable calli. After 150 days in the induction phase, the ECs with proembryos that were transferred to the medium for differentiation and maturation, differentiated asynchronically into somatic embryos at globular and torpedo stages. The results showed that BRSC2328 and BRSCM1115 had the highest potential for EC formation (90–100 %) and somatic embryo differentiation (40.7 and 52.5 somatic embryos per callus, respectively) when compared to other genotypes. After approximately 90 days of culture on MS basal medium without growth regulators, protrusion of the leaf primordia was observed, characterizing the onset of germination of the somatic embryos into plants.

Changes in protein pattern during different developmental stages of somatic embryos in chickpea

Mature embryonic axes were used for chickpea (Cicer arietinum L.) regeneration via somatic embryogenesis. Qualitative and quantitative estimation of protein profile during somatic embryogenesis by SDS-PAGE and densitometric analysis showed differential expression of various storage proteins at different stages of somatic embryo development, which was compared with the profile of developing seeds. Total protein content in somatic embryos of chickpea increased from globular stage [2.9 μg mg-1(f.m.)] to cotyledonary stage [4.8 μg mg-1(f.m.)] and then started decreasing during onset of maturation and germination [up to 1.5 μg mg-1(f.m.)]. Differential expression of seed storage proteins, late embryogenesis abundant (LEA) proteins and proteins related with stress response were documented at different stages of somatic embryogenesis. Germinating somatic embryos showed degradation products of several seed storage proteins and the appearance of new polypeptides (76.8, 67.6, 49.9 and 34.2 kDa), which were absent during differentiation of somatic embryos. A low molecular mass (17.7 kDa) polypeptide was uniformly present during all stages of somatic embryogenesis and it may belong to a group of stress-related proteins. This study describes the expression of true seed storage proteins like legumin, vicilin, convicilin and their subunits at different stages of somatic embryogenesis, which may serve as excellent markers for embryogenic pathway of regeneration in chickpea.

Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death

Somatic embryogenesis is a useful tool to propagate conifers vegetatively. However, a major limitation in many pine species is the low quality of cotyledonary somatic embryos. The aim of this study has been to elucidate the developmental pathway of somatic embryos in Scots pine (Pinus sylvestris), to identify deviations from the normal pathway and to identify processes that might disturb normal development. Initially we compared the developmental pathway of somatic embryogenesis in representative cell lines yielding cotyledonary embryos with normal and abnormal morphology. Early embryos carrying suspensor cells in excess of the normal number (supernumerary) were more frequent in cell lines giving rise to abnormal cotyledonary embryos. In this study we show that the frequency of early somatic embryos with supernumerary suspensor cells increased after treatment with the auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Furthermore, the yield of developing embryos increased significantly after treatment with the antiauxin 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB), but the morphology of the embryos was not affected. The number of cells undergoing PCD was analyzed using a TUNEL-assay. The frequency of TUNEL-positive cells was high both in proliferating cultures and during differentiation of early somatic embryos. However, the pattern of TUNEL-positive cells was similar in normal somatic embryos and in embryos with supernumerary suspensor cells. Together our results suggest that the presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development.

Diluição celular, características do meio de cultura e biorreatores de imersão temporária na diferenciação e regeneração de células em suspensão de bananeira

Altos custos de produção geralmente limitam o uso comercial da micropropagação. O uso de meios de cultura líquidos é considerado uma solução para a automação e redução de custos. Entretanto, dependendo da cultivar de bananeira, esse processo pode mostrar diferentes níveis de dificuldade, e adaptações nos protocolos são necessárias. Neste estudo, experimentos de diferenciação celular e regeneração de plantas foram desenvolvidos em células em suspensão de banana pela avaliação da densidade inicial de células, meios de cultura e sistemas de imersão temporária. Para tanto, uma sequência de três experimentos foi realizada: o primeiro avaliou os efeitos da densidade celular (0,5; 1 e 2 mL), meios de cultura (M1: 1/2MS, 0,1 g.L-1 de ácido ascórbico, 0,1 g.L-1 de L-prolina, 30 g.L-1 de sacarose e 10 µM de 2iP; M2: MS, 30 g.L-1 de sacarose, 2,2 µM de BAP e 11,4 µM de AIA) e períodos de diferenciação celular (40 e 130 dias); o segundo experimento analisou o efeito do tamanho dos propágulos diferenciados em meio líquido (aprox. 2,5; 5 e 10 mm em diâmetro) na formação de embriões somáticos ou na regeneração de plantas; finalmente, um terceiro experimento avaliou o efeito de sistemas de cultivo com papel-filtro cobrindo o meio de cultura semissólido e sistemas de imersão temporários na diferenciação dos propágulos e na regeneração de plantas. Não foram observadas diferenças significativas entre os meios de diferenciação, porém as melhores diluições de células para a diferenciação foram de 1 e 2 mL/30mL de meio de cultura, enquanto diluições de 0,5 mL/30 mL de meio aumentaram a oxidação celular. A extensão do período de diferenciação de 40 para 130 dias foi importante para produzir maior número de calos/propágulos uniformes e com pelo menos de 10 mm de diâmetro, que puderam ser usados em sistemas de imersão temporária (biorreatores) para a diferenciação de embriões somáticos e regeneração de plantas. Considerando todos os sistemas de regeneração, verificou-se que o uso de meios de regeneração de consistência semissólida com papel-filtro na superfície do meio é o mais responsivo para a diferenciação de embriões somáticos e regeneração de plantas.

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

The aim of this study has been to identify important processes that regulate early stages of embryo development in conifers. Somatic embryogenesis in Picea abies has become a model system for studying embryology in conifers, providing a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages. We have used this model to analyze global changes in gene expression during early stages of embryo development by generating an expression profile of 12,536 complementary DNA clones. This has allowed us to identify molecular events regulating putative processes associated with pattern formation during the earliest stages of embryogenesis which have not been identified on the molecular level in conifers before. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm, and programmed cell death.

Establishiment of molecular markers for early selection of embryogenic cultures with high embryogenic potential in brazilian pine (Araucaria angustifolia(BERT) O. KTZE)

Background Brazilian pine (Araucaria angustifolia (Bert) O. Ktze) is the only native conifer species with economic importance in Brazil. Recently, due to intensive exploitation Brazilian pine was included in the official list of endangered Brazilian plants. Biotechnology tools, like somatic embryogenesis (SE), may become a potentially useful tool for mass clonal propagation and ex situ conservation of commercial and endangered plant species, especially conifers. SE involves the coordinated execution of four steps (embryogenic culture (EC) induction, proliferation, maturation, and plant regeneration). As observed for other conifers, the presence of well-developed early somatic embryos (SE) in EC of Brazilian pine can be considered the pre-requisite for embryo maturation in a medium supplemented with abscisic acid (ABA) and osmotic agents. However, in some genotypes even the presence of bipolar SE does not guarantee embryo maturation. Since SE morphology cannot be used as the only factor for EC selection, the development of molecular markers for early detection of embryogenic cultures responsive to maturation promoters (ABA and osmotic agents) is highly desirable. Polyamines (putrescine (Put), spermidine (Spd), and spermine (Spm)) have been classified as plant growth regulators and hormonal second-messengers playing a critical role in various growth and developmental processes in plants, such as the differentiation and development of somatic embryos. The relation Put/Spd has demonstrated the best answers for predicting embryogenic potential in different plant species. Apart from polyamines quantification, the analysis of gene expression has been used to detect the expression of embryogenesis regulating genes like somatic embryogenesis receptor kinase (SERK), wuschel-related WOX (WOX), and ABA insensitive-1 (ABI1) during conifer somatic embryogenesis. In order to develop molecular markers for early detection of EC with high embryogenic potential in Brazilian pine, we measured the polyamine content (free and conjugate) and the expression of three embryogenesis-regulating genes (SERK, WOX and ABI1) during the proliferation phase of ECs with different maturation capabilities.

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (Passiflora edulis Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.

LEAFY COTYLEDON1, FUSCA3 expression and auxin treatment in relation to somatic embryogenesis induction in Arabidopsis

The expression pattern of the LEC1 and FUS3 genes during somatic embryogenesis in Arabidopsis explants (immature zygotic embryos) induced in vitro was analysed, using Real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC1 but not FUS3 within a 30 day time course of somatic embryo development, and a significant auxin-dependent upregulation of LEC1 was found over the time course. In contrast to embryogenic culture, the level of LEC1 and FUS3 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. In addition, the expression profile of LEC1 and FUS3 was followed in the embryogenic culture derived from 35S::LEC2-GR explants. A significant increase of LEC1 but not FUS3 activity was observed under LEC2 overexpression induced in auxin-treated explants. The work provides further experimental evidence on LEC gene involvement in the embryogenic response in Arabidopsis somatic cells, and also implicates LEC1 function in more advanced stages of SE culture in relation to somatic embryo differentiation and development.

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

The aim of this study has been to identify important processes that regulate early stages of embryo development in conifers. Somatic embryogenesis in Picea abies has become a model system for studying embryology in conifers, providing a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages. We have used this model to analyze global changes in gene expression during early stages of embryo development by generating an expression profile of 12,536 complementary DNA clones. This has allowed us to identify molecular events regulating putative processes associated with pattern formation during the earliest stages of embryogenesis which have not been identified on the molecular level in conifers before. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm, and programmed cell death.

Sulphur depletion altered somatic embryogenesis in Theobroma cacao L. Biochemical difference related to sulphur metabolism between embryogenic and non embryogenic calli

Somatic embryogenesis is a useful tool for Theobroma cacao improvement and propagation. Depending on culture medium composition, different morphogenetic structures (including somatic embryo) occur in response to alteration of genes expression patterns and biochemical changes. The effect of SO 4 2 - ion deficiency in culture media on somatic embryogenesis was studied through sequential replacement of MgSO 4 and K 2 SO 4 by MgCl 2 and KCl, respectively, at different steps of somatic embryogenesis. It appears that explants gradually lost their embryogenic competence as the period of exposition to sulphur free medium increases. These results suggest that, sulphur availability and the duration to sulphur exposition might modulate the expression of genes involved in somatic embryo differentiation in T. cacao . Cysteine, glutathione, reducing sugars, cysteine synthase and cysteine desulfurase activities were analysed in different morphogenetic structures obtained in vitro . Cysteine and reducing sugars contents appeared to be higher in embryogenic calli than their nonembryogenic homologues, whereas glutathione content appears to be lower in embryogenic calli. Cysteine synthase activities also discriminate the embryogenic calli from non embryogenic calli. In the embryogenic calli, the ratio cysteine synthase/cysteine desulfurase activities were above unit. The assimilation of exogenous sulphur (sulphate) for the synthesis of cysteine might hence be crucial for somatic embryogenesis in T. cacao . This explains the reduction and the absence of somatic embryo response observed during sulphur depletion in culture media. Sulphur nutrition is therefore critical in cacao somatic embryogenesis. Keyswords: Cacao, embryo, sulphate, cysteine synthase, glutathione, deficiency African Journal of Biotechnology Vol. 9(35), pp. 5665-5675, 30 August, 2010

Somatic embryogenesis and plant regeneration of Lilium ledebourii (Baker) Boiss., an endangered species

A somatic embryogenesis (SE) protocol was established for the regeneration of Lilium ledebourii (Baker) Boiss. whole plants using new vegetative bulblet microscales and transverse thin cell layers (tTCLs) of young bulblet roots as the explant sources. Bulblets were induced from bulb scale explants cultured for at least 3 months in the dark on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar, and different concentrations of α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and thidiazuron. Embryo-like structures were obtained from tTCL explants of 3-month-old bulblets (excised from bulb scale explants) following culture on solid MS medium containing 3% sucrose and various concentrations of NAA and BA for 3 months in the dark. Both the explant source and the type of plant growth regulators affected the differentiation of somatic embryos. The highest percentage (65.55%) of embryogenesis was obtained from bulblet microscale tTCLs cultured on solid MS medium containing 0.54 μM NAA and 0.44 μM BA. Plants with normal shoots and roots were obtained following a 3-month culture of embryos on growth regulator-free MS medium at 25 ± 1°C under a 16/8-h light/dark photoperiod (light intensity 40 μmol m−2 s−1, cool-white fluorescent light). The plants were successfully acclimatized in the growth chamber.

Abscisic acid and methyl jasmonate as regulators of ethylene biosynthesis during somatic embryogenesis of Medicago sativa L.

Effects of abscisic acid (ABA) and methyl jasmonate (MeJA) on ethylene production, ACC oxidase (ACO) activity, and content of 1-aminocyclopropane-1-carboxylic acid (ACC) during indirect somatic embryogenesis (SE) of Medicago sativa L. were studied. ABA and MeJA, at 50 μM, were applied during the induction, proliferation, or differentiation phase. ABA decreased ethylene production at the beginning of callus and SE induction and during the differentiation of somatic embryos. The hormone inhibited ACO activity in explants with overgrowing callus during the first two weeks of induction, in embryogenic suspension and also in differentiating embryos. The ACC content was reduced by ABA in callus at the end of SE induction, in embryogenic suspension and in globular embryos, but elevated in cotyledonary embryos. MeJA had no significant effect on ethylene production during M. sativa SE, despite the fact, that it inhibited ACO activity during the first two weeks of induction and in torpedo and cotyledonary embryos. The ACC content was increased by MeJA in 14-day-old callus and embryogenic suspension but was inhibited in globular embryos. Both ABA and MeJA seem to be involved in the regulation of ethylene biosynthesis during distinct phases of SE in M. sativa. It might be considered that exogenous ABA, more probably than MeJA, exerts its inhibitory effect on M. sativa somatic embryo formation by modifying ethylene production.