Somatic Embryo Development Research Articles

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.

Molecular characterization of pine embryogenesis: pursuing the role of a putative non-specific lipid-transfer protein

Background Embryogenesis in Gymnosperms presents unique characteristics when compared to Angiosperm embryogenesis. However, little is known about the molecular regulation of embryo development in Gymnosperms because most studies have been conducted in Angiosperm model species. Due to the economic relevance of some forest species included in this group of plants, namely pines, efforts have been made worldwide for the establishment of efficient clonal propagation methods, of which somatic embryogenesis is an example. A deeper knowledge of the molecular players regulating zygotic embryo development in pines will likely contribute to a better understanding and control over somatic embryo development in vitro. We have previously performed a global gene expression analysis during maritime pine (Pinus pinaster) embryo development [1] and have selected a set of transcripts for further characterization based on their expression profile. One of such transcripts (PpAAILTSS1) putatively encodes a protein of the Alpha-Amylase Inhibitors (AAI)/Lipid Transfer (LT)/Seed Storage (SS) protein family, non-specific lipid-transfer protein (nsLTP)-like subfamily.The AAI-LTSS family of proteins is unique to higher plants and includes cereal-type alpha-amylase inhibitors, lipid transfer proteins, seed storage proteins, and similar proteins. Proteins in this family are known to play important roles, in defending plants from insects and pathogens, lipid transport between intracellular membranes, nutrient storage, as well as in developmental processes. PpAAI-LTSS1 is up regulated in zygotic embryos at a pre-cotyledonary stage of development [2]. The aim of this work was to characterize in detail the expression patterns of PpAAI-LTSS along embryo development and its upstream regulatory sequence.

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.

Gene expression during early somatic embryogenesis in Brazilian pine (Araucaria angustifolia (Bert) O. Ktze)

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, under the “vulnerable” category. Biotechnology tools like somatic embryogenesis (SE) are potentially useful for mass clonal propagation and ex situ conservation strategies of commercial and endangered plant species. In spite of that, numerous obstacles still hamper the full application of SE technology for a wider range of species, including Brazilian pine. To enhance somatic embryogenesis in Brazilian pine and to gain a better understanding of the molecular events associated with somatic embryo development, we analyzed the steady-state transcript levels of genes known to regulate somatic embryogenesis using semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR). These genes included Argonaute (AaAGO), Cup-shaped cotyledon1 (AaCUC), wushel-related WOX (AaWOX), a S-locus lectin protein kinase (AaLecK), Scarecrow-like (AaSCR), Vicilin 7S (AaVIC), Leafy Cotyledon 1 (AaLEC), and a Reversible glycosylated polypeptide (AaRGP). Expression patterns of these selected genes were investigated in embryogenic cultures undergoing different stages of embryogenesis, and all the way to maturation. Up-regulation of AaAGO, AaCUC, AaWOX, AaLecK, and AaVIC was observed during transition of somatic embryos from stage I to stage II. During the maintenance phase of somatic embryogenesis, expression of AaAGO and AaSCR, but not AaRPG and AaLEC genes was influenced by presence/absence of plant growth regulators, both auxins and cytokinins. The results presented here provide new insights on the molecular mechanisms responsible for somatic embryo formation, and how selected genes may be used as molecular markers for Brazilian pine embryogenesis.

Hormone-Induced Indirect Regeneration Protocol for Scutellaria baicalensis Georgi (Huang-qin)

There is no report available to date on somatic embryogenesis or even callus formation and plant regeneration in Scutellaria baiclensis (Huang qin). Ours is the first report on somatic embryo development in Scutellaria baicalensis. Plant growth regulators Thidiazuron [TDZ: N-phenyl N'- (1,2,3-thidiazol-5-ylurea)], 6-benzyladenine (BA), and 2,4-dichlorophenoxyacetic acid (2,4-D) were tested alone or in combination for their capacity to induce indirect somatic embryogenesis from leaf, internode, nodal portion, and root explants of Scutellaria baicalensis. The TDZ (4 mg/l) showed the highest percentage of calli-induction frequency in various explants, viz., leaf (42.2%), nodal stem portion (62.9%), and root (63.4%). The TDZ-derived calli were incubated on MS salt + B5 vitamin medium containing glutamine (1.5mM) (MSE) and fortified with various combinations of TDZ, BA, and 2,4-D for induction of somatic embryos. No somatic embryogenesis was recorded in any culture at this stage. On transferring the above friable calli onto hormone-free MSB medium and after 12 days of culture, several globular and heart-shaped embryos appeared over the surface of green compact callus. The mature somatic embryos were dissected out and cultured on hormone-free MS medium for germination. Plantlets were hardened in a growth chamber and, after 30 days, transferred to greenhouse and subsequently to pots and field. There was no morphological difference in plantlets regenerated by this procedure and normal seed-grown plant.

Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini‐pig (Sus Scrofa)

To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.

Evidence of somatic embryogenesis for plantlet regeneration in Muscari neglectum Guss.

Muscari neglectum Guss. is one of the ornamental species from Liliaceae family and has been used as well as other species of the genus Muscari, for its medicinal properties. The root is anti-inflammatory, antiallergic, aphrodisiac and pectoral stimulant. Somatic embryogenesis in M. neglectum Guss. was induced from bulb explants cultured in Murashige and Skoog (MS) medium plant growth regulator free or supplemented with different concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA). The best results were obtained on plant growth regulator free medium or with lower NAA and BA concentrations and 30 g/l sucrose. Somatic embryo development was observed on 1/2 MS medium plant growth regulator free under continuous illumination with fluorescent light. Matured somatic embryos germinated and converted to plantlets in the same medium or containing 1 mg/l BA after 3-4 months. The plantlets left in the medium especially containing BA produced bulbs after 5 months. Key words: Bulb explant, Muscari neglectum, plantlet regeneration, somatic embryogenesis.

Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50μg/mL vitamin C 15h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

In Vitro Callus Induction and Embryogenesis of Oil Palm (Elaeis guineensis Jacq.) from Leaf Explants

This research was to study in vitro callus induction and somatic embryogenesis in oil palm from leaf explants. Young leaf segments from mature oil palm were cultured on MS medium supplemented with different concentrations of 2,4-D with or without addition of 2 g/l activated charcoal (AC) or 2,4-D and picloram. Embryogenesis induction was done using MS medium containing 2,4-D 450 µM and benziladenine 4.4 µM with 3g/l activated charcoal. The treatment of 2,4-D 15 µM resulted in the highest percentage of callus induction. The treatment of 2,4-D and AC showed that 2,4-D 450 µM and AC led to higher percentage of callus induction than that of 2,4-D 400 µM and 2 g/l AC. Embryogenesis occured in 27 out of 250 clumps of primary callus was occurred after 2-3 times subcultures. Somatic embryo development occurred when the embryogenic callus was transferred on the same basal medium supplemented with casein hydrolysate with 1 µM BA or growth regulator free basal medium with 2 g/l activated charcoal.

A high-frequency cyclic secondary somatic embryogenesis system for Cyclamen persicum Mill

In this study, a high frequency cyclic secondary somatic embryogenesis system was established for Cyclamen persicum. Moreover, the influence of primary somatic embryos (PSEs) at different stages of development, number of passages, abscisic acid (ABA), and sucrose concentrations on secondary somatic embryogenesis were investigated. Primary somatic embryos, at different stages of development, were incubated on an induction medium consisting of half-strength Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962), 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg/l 6-benzyladenine (BA). These initial PSE cultures along with calli were transferred to a plant growth regulator (PGR)-free medium to promote secondary somatic embryo (SSE) development. Embryogenic calli were induced at a frequency >90% from all PSE cultures. Both morphology and size of PSEs influenced embryogenic competence. Large-sized globular embryos (GEs) yielded the highest number of SSEs followed by small- and large-sized torpedo-stage embryos (TEs). Passages of induced calli obtained from large-sized GEs had no effects on frequency of secondary embryogenesis; however, they significantly influenced SSE production. Calli from the first passage exhibited the highest competence for secondary embryogenesis, 100% frequency of SSEs, producing 3,306 and 1,296 SSEs per gram fresh weight callus and embryo, respectively. Incubating embryogenic calli derived from large-sized primary GEs in the first passage on a medium containing 30 g/l sucrose, and supplemented with either 0.5 or 1.0 mg/l abscisic acid (ABA) increased the frequency of TEs, consequently enhancing normal embryo development of SSEs. Transfer of such embryogenic calli to a PGR-free medium containing 60 g/l sucrose was optimal for SSE induction, producing 3,204 SSEs per gram callus and 8% TEs.

English

Callus induction and somatic embryogenesis from hypocotyl explants of some Iranian cottons spp. (Hashem abad, Kerman, Termez and Sepid) were compared with Coker 312 through induction and formation of embryogenic calli on medium of Murashige and Skoog (MS) with Gamborg vitamins (B5) supplemented with the following compositions: MSB1&nbsp;(0.5 mg/l zeatin), MSB2&nbsp;(1 mg/l zeatin), MSB3&nbsp;(0.5 mg/l 2,4-dichlorophenoxyacetic acid, 0.1 mg/l kinetin), MSB4&nbsp;(1 mg/l 2,4-D, 0.5 mg/l kinetin, 0.5 mg/l zeatin) and MSB5&nbsp;(2 mg/l &alpha;-naphtalene-3-acetic acid, 1 mg/l kinetin, 0.75 mg/l MgCl2).&nbsp;The optimum medium for the proliferation of embryogenic calli was MS medium containing B5&nbsp;vitamins, 1 mg/l 2,4-D, 0.5 mg/l kinetin and 0.5 mg/l zeatin and the optimum medium for the development of somatic embryos was MS medium (NH4NO3&nbsp;was removed and KNO3&nbsp;amount doubled) containing B5vitamins, 40 g/l sucrose and without hormone.&nbsp;Media MSB1, MSB2&nbsp;and MSB4&nbsp;gave the highest percentage (100%) of calli induction in Coker 312 but the lowest induction (46.66%) was observed when Hashem abad explants were cultured in the MSB3&nbsp;medium.&nbsp;Embryogenesis percentage of Termez (2.22 to 24.40%), Hashem abad (1.85 to 9.73%) and Sepid (9.06 to 22.28%) genotypes were significantly lower than that of Coker 312 (66.66 to 94.33%). The Kerman genotype did not show embryogenesis. In the histological studies, the different development stages of the embryos (globular, heart, torpedo and cotyledonary) together with callus cells were showed.&nbsp; &nbsp; Key words:&nbsp;Hypocotyl explants, somatic embryo,&nbsp;in vitro&nbsp;regeneration, germination, somatic embryogenesis histology.

Regeneration of pineapple (Ananas comosus L.) plant through somatic embryogenesis

An efficient protocol for a complete plant regeneration by somatic embryogenesis was developed with Smooth Cayenne pineapple (Ananas comosus L.). Previous works showed that this species is responsive to somatic embryogenesis. In the present work the influence of components of culture medium in the induction, development and conversion of somatic embryos was investigate in order to establish a somatic embryogenesis protocol. Nodular callus (83.67%) was initiated from leaf explants of young plants on CIM3 medium. The highest frequency (37.6%) of embryogenic callus induction was obtained from 4-week-old calluses on EIM3 medium supplemented with 3.0 mg/l picloram. The highly organized callus induction and the development of somatic embryos were achieved after the transfer of callus clumps onto EIM3 medium containing 1.0 mg/l BAP + 0.1 mg/l NAA. The frequency of somatic embryo formation was of 39.5 ± 2.45 embryos per callus. Up to 97% of the somatic embryos were converted into complete plants within 4 week on MSB medium with 1.0 mg/l BAP + 0.05 mg/l GA3 + 500 mg/l glutamine. The continuation of the elongation of the shoots occurred on this medium). Shoots obtained from all the above methods were rooted in MSB medium with activated charcoal. Complete plantlets were transferred onto specially made polyethylene bags containing soil mixture and transferred to the greenhouse. Survival rate of the plantlets under ex vitro conditions was 98% and maximum average number of plantlets (80 ± 0.6). The well-developed plantlets were transferred to an open field where the plants produced normal fruits.

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.

Somatic embryogenesis, regeneration and in vitro production of glycyrrhizic acid from root cultures of Taverniera cuneifolia (Roth) Arn.

Taverniera cuneifolia (Roth) Arn. or Indian licorice is considered to be a substitute for Glycyrrhiza glabra L. owing to an equivalent content of glycyrrhizic acid (GA). GA recognized as the main active ingredient of T. cuneifolia, GA imparts several medicinal properties to these plants. However, research on this plant is scanty with no published record on tissue culture studies. Present study demonstrates a method for (1) induction and successful development of somatic embryos from the root culture, (2) regeneration of plants, and (3) GA production by root cultures of T. cuneifolia. Shoot initiation frequency of cultured roots ranged from 52.57% to 97.71%. Plant regeneration frequency (germination) from somatic embryos was 88.66% in 1/4-strength Murashige and Skoog (MS) medium supplemented with 2% sucrose, as against 68% in half 1/2-strength MS containing 2% sucrose. Glycyrrhizic acid of 1.32 mg/gm of dry weight was obtained in full-strength MS medium supplemented with 3% sucrose, through high-performance thin layer chromatography analysis with standard GA in root cultures. The present study provides an efficient method for the mass production of plants from a single mother plant as well as the potential root culture system to study the GA production pathways in T. cuneifolia.

Efficient plant regeneration of Malaysian indica rice MR 219 and 232 via somatic embryogenesis system

Improvement on rice plant regeneration system from an embryogenic callus using two Malaysian indica rice MR 219 and MR 232 was developed in this study. Different stages of rice explants (zygotic embryos) were tested for callus induction and regeneration using various carbon sources and amino acids. The present study shows that dough stage of zygotic embryos was the best stage of explants for the embryogenic callus induction and regeneration of both rice cultivars. The embryogenic callus nature with the compact structure was confirmed by scanning electron microscopy (SEM) analysis. Inclusion of maltose at the concentration between 10 and 20 mg/L shown higher responsive for the development of green somatic embryos and initiation of rice regeneration with an average of 29–37 (87–91%) regenerated plantlets, respectively. All in vitro regenerated rice plantlets were green, morphological and physiologically healthy condition. Rice plantlets were hardened in acclimatization room for 3 weeks and later transferred into soil with 95% survival in both varieties were recorded. Hence, regeneration system from zygotic rice embryos via somatic embryogenesis system was carried out successfully by using 10 g/L of maltose and combinations of glutamine, asparagine and arginine amino acids.

Lauric acid improves the growth of zygotic coconut (Cocos nucifera L.) embryos in vitro

Coconuts (Cocos nucifera L.) germinating in situ make use of both sucrose and fatty acids, notably lauric acid, as sources of energy and carbon, but current tissue culture methods routinely include only sucrose in the culture medium. The aim of the experiments was to establish whether lauric acid could improve the growth and development of zygotic coconut embryos in culture. The culture medium of zygotic embryos was adjusted to give various concentrations of sucrose and lauric acid. The concentration of free lauric acid was increased at specific times of the culture. At the end of the experiments, plantlet growth was measured. Added at day zero, lauric acid inhibited germination. Added at 60 or 75 days, lauric acid (75 μM, unbound concentration) showed a marked stimulation of plantlet growth and development. When 14C-labelled lauric acid was used, radioactivity was incorporated mainly into longer chain fatty acids of complex lipids, notably of the phospholipid fraction. Supplementation at these times may mimic conditions in situ and suggests that the supply of fatty acids may represent a physiological requirement for continued growth. The experiments with radioactive lauric acid confirm that it provides carbon for the synthesis of new structural lipids. The method may provide a means of improving the development of coconut somatic embryos in future.

Comparison of potency between histone deacetylase inhibitors trichostatin A and valproic acid on enhancing in vitro development of porcine somatic cell nuclear transfer embryos

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer (SCNT) embryos. Such modification includes an increase in histone acetylation. Histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA) and valproic acid (VPA), have been known to maintain a high cellular level of histone acetylation. Hence, treatment of nuclear transfer embryos with HDACi may increase the efficiency of cloning. The present study attempted direct comparison of TSA and VPA with regard to the potency of enhancement of in vitro development in porcine SCNT embryos. Reconstructed oocytes using fetal fibroblasts were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 d thereafter without HDACi. The frequency of blastocyst formation was significantly higher (P<0.05) in embryos treated with VPA than the frequencies with TSA and without HDACi (125/306, 40.8% vs. 94/313, 30.2% vs. 80/329, 23.4%). In addition, VPA treatment significantly increased (P<0.05) the number of inner cell mass (ICM) cells compared with the control (15.6 ± 1.7 vs. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control groups (12.9 ± 3.0 vs. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of porcine SCNT embryos, particularly by an increase in blastocyst formation and in the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos.

A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis

Secondary somatic embryogenesis has been postulated to occur during induction of peach palm somatic embryogenesis. In the present study this morphogenetic pathway is described and a protocol for the establishment of cycling cultures using a temporary immersion system (TIS) is presented. Zygotic embryos were used as explants, and induction of somatic embryogenesis and plantlet growth were compared in TIS and solid culture medium. Light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to describe in vitro morphogenesis and accompany morpho-histological alterations during culture. The development of secondary somatic embryos occurs early during the induction of primary somatic embryos. Secondary somatic embryos were observed to develop continually in culture, resulting in non-synchronized development of these somatic embryos. Using these somatic embryos as explants allowed development of cycling cultures. Somatic embryos had high embryogenic potential (65·8 ± 3·0 to 86·2 ± 5·0 %) over the period tested. The use of a TIS greatly improved the number of somatic embryos obtained, as well as subsequent plantlet growth. Histological analyses showed that starch accumulation precedes the development of somatic embryos, and that these cells presented high nucleus/cytoplasm ratios and high mitotic indices, as evidenced by DAPI staining. Morphological and SEM observations revealed clusters of somatic embryos on one part of the explants, while other parts grew further, resulting in callus tissue. A multicellular origin of the secondary somatic embryos is hypothesized. Cells in the vicinity of callus accumulated large amounts of phenolic substances in their vacuoles. TEM revealed that these cells are metabolically very active, with the presence of numerous mitochondria and Golgi apparatuses. Light microscopy and TEM of the embryogenic sector revealed cells with numerous amyloplasts, large nuclei and nucleoli, and numerous plasmodesmata. Plantlets were obtained and after 3 months in culture their growth was significantly better in TIS than on solid culture medium. However, during acclimatization the survival rate of TIS-grown plantlets was lower. The present study confirms the occurrence of secondary somatic embryos in peach palm and describes a feasible protocol for regeneration of peach palm in vitro. Further optimizations include the use of explants obtained from adult palms and improvement of somatic embryo conversion rates.

Effects of different amino acids on somatic embryogenesis of strawberry (Fragaria × ananassa Duch.)

This study was conducted to optimize different types and concentrations of amino acids on somatic embryogenesis induction, development and maturation of leaf explants in strawberry cultivars (‘Camarosa’, ‘Paros’ and ‘Kurdistan’). Calli derived from leaf sections were transferred onto MS medium with 1.0 mg/l 2,4-d + 0.5 mg/l BAP supplemented with 0.0, 50, 100, 150 or 200 mg/l concentrations of proline, alanine and glutamine. Stimulation of embryogenesis and embryo development was strictly dependent on the type and concentration of amino acid in the medium. Proline (100 mg/l) was much more effective than glutamine and alanine, on induction and development of somatic embryogenesis in all cultivars. Cultures grown on amino acid-free medium attained lower somatic embryos than cultures grown on amino acid treated medium. Low concentrations (50 mg/l) and high concentrations (200 mg/l) of amino acids tested were inefficient for embryogenesis induction as well as for somatic embryos development.

Possible Effect From Shear Stress on Maturation of Somatic Embryos of Norway Spruce (Picea abies)

Somatic embryogenesis is the only method with the potential for industrial scale clonal propagation of conifers. Implementation of the method has so far been hampered by the extensive manual labor required for development of the somatic embryos into plants. The utilization of bioreactors is limited since the somatic embryos will not mature and germinate under liquid culture conditions. The negative effect on mature embryo yields from liquid culture conditions has been previously described. We have described the negative effects of shear stress on the development of early stage somatic embryos (proembryogenic masses; PEMs) at shear stresses of 0.086 and 0.14 N/m(2). In the present study, additional flow rates were studied to determine the effects of shear stress at lower rates resembling shear stress in a suspension culture flask. The results showed that shear stress at 0.009, 0.014, and 0.029 N/m(2) inhibited the PEM expansions comparing with the control group without shear stress. This study also provides validation for the cross-correlation method previously developed to show the effect of shear stress on early stage embryo suspensor cell formation and polarization. Furthermore, shear stress was shown to positively affect the uptake of water into the cells. The results indicate that the plasmolyzing effect from macromolecules added to liquid culture medium to stimulate maturation of the embryos are affected by liquid culture conditions and thus can affect the conversion of PEMs into mature somatic embryos.