Non-embryogenic Tissues Research Articles

Initiation of somatic embryogenesis in Pinus caribaea var. hondurensis using mature female gametophytes as explants

Due to the difficulties of harvesting immature cones in genetic breeding orchards, such as high costs and the considerable time required, this study aimed to evaluate the somatic embryogenesis (SE) initiation of Pinus caribaea var. hondurensis using the mature female gametophyte as the initial explant. The basic medium for embryogenesis initiation was composed of QL (Quoirin and Lepoivre, 1977) nutrients, sucrose, myo-inositol, glutamine, casein hydrolisate, thiamine, 2,4-D, BAP and agar. This medium was compared with DCR (Gupta and Durzan, 1985) and WV5 (Coke, 1996) media, supplemented or not with 0.1 g L−1 folic acid (FA). Afterwards, three concentrations of 2,4-D (10, 20 and 30 μM) combined or not with BAP (2, 4 and 8 μM) were tested. After 90 days the percentages of explants with embryogenic tissue (ET), embryo-like structures and oxidation were evaluated. We observed somatic tissue initiation` on mature female gametophytes. On QL medium supplemented with FA, the initiation was observed on up to 42% of female gametophytes. The combination of 10 μM 2,4-D and 4 μM BAP resulted in the highest initiation rate. The formation of ET was confirmed in all treatments by a histochemical test. Embryo-like structures developed on QL medium supplemented with FA, were first transferred to basic medium supplemented with 1.5 g L−1 AC for 30 days and finally to the same medium supplemented with 10% polyethylene glycol 3350 and 120 µM abscisic acid. The expression of SERK-1 and GER-1 genes was analyzed. SERK-1 was expressed only in the ET, while GER-1 was expressed in both the embryogenic and non-embryogenic tissues (non-ET). However, the somatic embryos studied through morphological and histochemical tests proved to be abnormal and did not develop further.

Induction of Somatic Embryogenesis in Tamarillo (Solanum betaceum Cav.) Involves Increases in the Endogenous Auxin Indole-3-Acetic Acid.

Somatic embryogenesis (SE) is a complex biological process regulated by several factors, such as the action of plant growth regulators, namely auxins, of which the most physiologically relevant is indole-3-acetic acid (IAA). In tamarillo, an optimized system for induction of SE creates, after an induction process, embryogenic (EC) and non-embryogenic callus (NEC). In this work the endogenous levels of auxin along the induction phase and in the calli samples were investigated using chemical quantifications by colorimetric reactions and HPLC as well as immunohistochemistry approaches. Differential gene expression (IAA 11, IAA 14, IAA 17, TIR 1, and AFB3) analysis during the induction phase was also carried out. The results showed that the endogenous IAA content is considerably higher in embryogenic than in non-embryogenic calli, with a tendency to increase as the dedifferentiation of the original explant (leaf segments) evolves. Furthermore, the degradation rates of IAA seem to be related to these levels, as non-embryogenic tissue presents a higher degradation rate. The immunohistochemical results support the quantifications made, with higher observable labeling on embryogenic tissue that tends to increase along the induction phase. Differential gene expression also suggests a distinct molecular response between EC and NEC.

Optimization of Different Factors for Initiation of Somatic Embryogenesis in Suspension Cultures in Sandalwood (Santalum album L.)

Santalum album (L.) is a prized tropical tree species of high therapeutic and industrial importance. The wood of these naturally grown plants is extensively harvested to acquire therapeutically important metabolite santalol and be used for additional functions such as in wood statuette industries. Due to high demand, it is crucial to maintain a sufficient plant population. An easy protocol for establishing cell suspension culture initiated from the loose embryogenic callus mass of sandalwood was realized by shifting 6–8-week-old morphogenic calli acquired from the mature embryonic axis and cotyledon explant cultures in fluid media. The asynchronous embryogenic cultures were sloughed with clumps of flourishing cell clumps and embryos of various progressive phases along with diffident non-embryogenic tissues. The frequency of embryo proliferation was evidenced to determinethe expansion pace of embryogenic masses under diverse conditions. The intonation of initiation and creation of cell suspension was under the directive of the influence of exogenous plant growth regulators amended in the nutrient medium at different concentrations and combinations. Maximum relative growth rate (386%) and clumps/embryoids in elevated integers (321.44) were accomplished on MS nutrient medium fortified with 2.0 mg L−1 2,4-D in association with 0.5 mg L−1 BA and 30.0 g L−1 sucrose raised from mature embryonic axis-derived calli. Plantlet regeneration in higher frequency (84.43%) was evidenced on MS medium amended with 1.0 mg L−1 each of TDZ and GA3 in conjunction with 0.5 mg L−1 NAA and 20.0 g L−1 sucrose. Mature embryonic axis-derived calli were found to be constantly better than mature cotyledon-derived calli for raising profitable and reproducible cell suspension cultures. Regenerants displayed normal growth and morphology and were founded successfully in the external environment after hardening.

Profiles of endogenous ABA, bioactive GAs, IAA and their metabolites in Medicago truncatula Gaertn. non-embryogenic and embryogenic tissues during induction phase in relation to somatic embryo formation

Main conclusionDuring the 3-week-long induction phase, when M. truncatula cells leaf explants from non-embryogenic genotype (M9) and embryogenic variant (M9-10a) were forming the callus, biosynthesis and degradation of ABA, Gas and IAA proceeded at different levels. Induction of embryo formation is related to a lower ABA content, compared to the content of IAA and that of total bioactive GAs.Endogenous phytohormones are involved in the regulation of zygotic embryogenesis, but their role, especially of ABA, a plant growth inhibitor, in inducing somatic embryogenesis (SE) in angiosperms is still incompletely known. To arrive a better understanding of the ABA role in the process, we analyzed simultaneously and in detail changes in the contents of both ABA and five bioactive GAs (GA4, GA7, GA1, GA3, GA6) and IAA in M. truncatula non-embryogenic M9 (NE) and embryogenic M9-10a (E) genotypes. The initial leaf explants of both genotypes, and particularly NE, contained many times more ABA compared to the total bioactive GAs or IAA. In tissues during the entire 21-day induction all the hormones mentioned and their metabolites or conjugates were present; however, their contents were found to differ between the lines tested. The ABA level in primary explants of NE genotype was more than two times higher than that in E genotype. An even larger difference in the ABA content was found on the last day (day 21) of the induction phase (IP); the ABA content in E callus was over six times lower than in NE callus. In contrast, the IAA and GAs contents in primary explants of both genotypes in relation to ABA were low, but the contents of IAA and GAs exceeded that of ABA in the M9-10a tissues on the last day of IP. It is shown for the first time that endogenous ABA together with endogenous bioactive GAs and IAA is involved in acquisition of embryogenic competence in Medicago truncatula leaf somatic cells. These findings have a strong functional implication as they allow to improve the SE induction protocol.

Development-Related miRNA Expression and Target Regulation during Staggered In Vitro Plant Regeneration of Tuxpeño VS-535 Maize Cultivar.

In vitro plant regeneration addresses basic questions of molecular reprogramming in the absence of embryonic positional cues. The process is highly dependent on the genotype and explant characteristics. However, the regulatory mechanisms operating during organ differentiation from in vitro cultures remain largely unknown. Recently, miRNAs have emerged as key regulators during embryogenic callus induction, plant differentiation, auxin responses and totipotency. Here, we explored how development-related miRNA switches the impact on their target regulation depending on physiological and molecular events taking place during maize Tuxpeño VS-535 in vitro plant regeneration. Three callus types with distinctive regeneration potential were characterized by microscopy and histological preparations. The embryogenic calli (EC) showed higher miRNA levels than non-embryogenic tissues (NEC). An inverse correlation for miR160 and miR166 targets was found during EC callus induction, whereas miR156, miR164 and miR394 displayed similar to their targets RNA accumulation levels. Most miRNA accumulation switches took place early at regenerative spots coincident with shoot apical meristem (SAM) establishment, whereas miR156, miR160 and miR166 increased at further differentiation stages. Our data uncover particular miRNA-mediated regulation operating for maize embryogenic tissues, supporting their regulatory role in early SAM establishment and basipetala growth during the in vitro regeneration process.

Expression analysis of two SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes during in vitro morphogenesis in Spanish cedar (Cedrela odorata L.).

Somatic embryogenesis (SE) is one of the most important steps during regeneration, but the molecular mechanism of SE remains unclear for Cedrela odorata. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) is one of the genes associated with induction of SE and is considered a marker of cells competent to form somatic embryos. Our objective was to clone and characterize the SERK1 and SERK2 gene homologues and analyze their expression patterns during in vitro morphogenesis in Spanish cedar. CoSERK1 and CoSERK2 were isolated from cedar, both share domains characteristic of the SERK family, including leucine-rich repeats, a proline-rich motif, a transmembrane domain, and kinase domains. Embryogenic cultures were established from callus cultures induced on medium supplemented with 1mg/L dicamba. Histological sections were studied to determine the embryogenic nature of the samples. The CoSERK1 gene was highly expressed during the acquisition of embryogenic competence. The expression level of SERK1 was lower in non-embryogenic tissues and organs than in embryogenic calli, and it was higher in 3-week old embryogenic calli. CoSERK2 gene was highly expressed in leaves and shoots but no difference in expression was obtained between somatic and embryogenic tissues. These results suggest that the expression of CoSERK1 is associated with somatic embryogenesis induction and could be used as a potential marker to monitor the transition from competent to embryogenic cells and tissues in Spanish cedar.

Cloning and characterization of Somatic Embryogenesis Receptor Kinase I (EgSERK I) and its association with callus initiation in oil palm

The somatic embryogenesis receptor kinase (SERK) gene has been extensively studied in many plant species due to its role in conferring embryogenic competence to somatic cells. The oil palm (Elaeis guineensis Jacq.) full-length SERK I (EgSERK I) cDNA was first isolated from cell suspension culture using RACE-PCR. Total length of EgSERK I cDNA was 2378 bp in length with a 5’UTR region (358 bp) longer than 3’UTR region (130 bp) and the ORF was 1890 bp (629aa). The deduced amino acid sequence of EgSERK I contained protein domains commonly present in reported SERK proteins, including the hallmark proline-rich region and C-terminal domains. EgSERK I was most highly expressed in leaf explants and also detected in all tested tissues, including vegetative tissues, reproductive tissues, embryogenic tissues, and non-embryogenic tissues, suggesting that it may have a broad role in plant growth and development. Expression of EgSERK I in leaf explant was upregulated by minimal auxin concentration at the initial 6 h of incubation in callus induction media. EgSERK I mRNA was detected in the adjacent cells of the vascular tissues in the midvein region of leaf explants which serves as the callus initiation point of callogenesis in oil palm. Collectively, our findings suggest that the EgSERK I gene is involved in the callus initiation stage of oil palm somatic embryogenesis by transducing the signal to switch on the dedifferentiation process, triggering cellular reprogramming to form callus.

Insights into the early stage of Pinus nigra Arn. somatic embryogenesis using discovery proteomics

The somatic embryogenesis in conifers represents a suitable model of plant regeneration system facilitating studies of fundamental aspects of an early development as well as in vitro micropropagation. The aim of our study was to deeper understand the somatic embryogenesis in the conifer tree Pinus nigra Arn. Comparative proteomic analysis based on 2D-PAGE in 1) proliferating embryogenic tissues (E) initiated from immature zygotic embryos, 2) non-embryogenic calli (NEC) initiated from cotyledons of somatic seedlings of the same genotypes, 3) embryogenic tissues that lost the maturation capacity (E-L) of two cell lines (E362, E366). Investigated pine tissues showed distinct structural features. The 24 protein spots were altered in both cell lines in comparison of embryogenic and non-embryogenic tissues. These proteins are involved in disease and defence mechanism, energy metabolism and biosynthesis of cell wall components. Two of three protein spots detected only in embryogenic form of both cell lines are similar to water deficit inducible protein LP3, the third remains uncharacterised. The loss of the maturation capacity was accompanied by changes in 35 and 38 protein spots in 362 and 366 cell lines, respectively. Only two of them were altered in both cell lines, suggesting non-uniform process of ageing. Biological significanceSomatic embryogenesis in conifers represents an experimental system for the study of early plant development as well as a biotechnological tool for large-scale micropropagation. The obtained results give a new insight into the process of somatic embryogenesis of a conifer Pinus nigra Arn. by revealing differences at proteomic levels among in vitro cultured tissues characterised by different embryogenic potential. Microscopic investigations have also shown differences in the structural organisation of studied tissues.

Physiological and biochemical features of embryogenic and non-embryogenic peach palm (Bactris gasipaes Kunth) cultures

Both embryogenic and non-embryogenic peach palm (Bactris gasipaes Kunth) cultures arise during somatic embryogenesis induction, and both tissue types are often observed growing side-by-side from the same explant. To better understand why this occurs, samples from each tissue type were analyzed for their endogenous concentrations of indole-3-acetic acid (IAA), abscisic acid (ABA), polyamines, and amino acids with high-performance liquid chromatography and for total phenolics with spectrophotometry. Embryogenic cultures contained significantly higher concentrations of IAA, ABA, and total amino acids, whereas non-embryogenic tissue contained more total polyamines and phenolics. The greater IAA concentrations in embryogenic cultures supported the role of that hormone as a marker of embryogenic potential. Putrescine was especially prevalent in non-embryogenic cultures; however, the decreased putrescine/spermine + spermidine ratio in embryogenic cultures added support to the conclusions of previous studies in other species that this can serve as a marker of embryogenic competence. Though embryogenic cultures contained higher total amino acids, each culture type had different concentrations of specific amino acids.

Embryogenic cell suspension culture induction and plantlet regeneration ofRauvolfia serpentina(L.) Benth.: influence of different plant growth regulator concentrations and combinations

Establishment of embryogenic cell suspension culture from the embryogenic callus culture of Rauvolfia serpentina (L.) was attempted by transferring 4–6 weeks-old asynchronous embryogenic calli raised from mature embryo and cotyledon explants cultures in liquid media. The cultures obtained were inundated mostly with clumps of proliferating globular embryos with modest non-embryogenic tissues. The number and size of somatic embryos/clumps was recorded to calculate growth of embryogenic tissues under various conditions. Initiation and proliferation of embryogenic suspension culture was influenced by various exogenous plant growth regulators fortified to the culture medium at variable magnitude. For the establishment of suspension cultures, MS medium fortified with 2.0 mg l−12,4-D with 0.5 mg l−1 BAP was found to be the most effective. For subsequent subculturing, the reduced level of 2,4-D (1.0 mg l−1) in combination with 0.5 mg l−1 BAP promoted somatic embryogenesis at a faster rate. Frequent and efficient plantlet regeneration occurred on MS medium supplemented with 0.5 mg l−1, each of BAP, TDZ and NAA. Higher in vitro rooting response (root proliferating efficiency, number of roots and mean root length) was exhibited by MS rooting medium amended with 0.1 mg l−1 IBA. A combination of 65% relative humidity and 28°C temperature regime exhibited higher survival of regenerated plantlets (∼95%) followed by 60% RH and 30°C (∼90%). Later approximately 85% plants survived after transplantation in the field.

Molecular characterization and evolution studies of a SERK like gene transcriptionally induced during somatic embryogenesis in Phoenix Dactylifera L v Deglet Nour

A somatic embryogenesis receptor kinase like (SERKL) cDNA, designated PhSERKL, was isolated from date palm (Phoenix Dactylifera L) using RACE PCR. PhSERKL protein shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. Phylogenetic analyses using PHYLIP and Notung 2.7 programs suggest that the SERK proteins of some plant species resulted from relatively ancient duplication events. We predict an ancestor protein of monocots and dicots SERK using FASTML program. Somatic embryogenic cultures of date palm were established following transfer of callus cultures to medium containing 2, 4-dichlorophenoxyacetic acid. The role of PhSERKL gene during establishment of somatic embryogenesis in culture was investigated using quantitative real-time PCR. PhSERKL gene was highly expressed during embryogenic competence acquisition and globular embryo formation in culture. Overall, levels of expression of PhSERKL gene were lower in nonembryogenic tissues and organs than in embryogenic callus.

Transcriptome analysis of callus from Picea balfouriana.

BackgroundPicea likiangensis var. balfouriana (Rehd. et Wils.) Hillier ex Slavin (also known as Picea balfouriana) is an ecologically and economically important conifer that grows rapidly under optimum conditions and produces high-quality wood. It has a wide geographic distribution and is prevalent in southwest and eastern regions of China. Under suboptimal conditions, P. balfouriana grows slowly, which restricts its cultivation. Somatic embryogenesis has been used in the mass propagation of commercial species. However, low initiation rates are a common problem and the mechanisms involved in the induction of somatic embryogenesis are not fully understood. To understand the molecular mechanisms regulating somatic embryogenesis in P. balfouriana, high-throughput RNA-seq technology was used to investigate the transcriptomes of embryogenic and non-embryogenic tissues from three P. balfouriana genotypes. We compared the genes expressed in these tissues to identify molecular markers with embryogenic potential.ResultsA total of 55,078,846 nucleotide sequence reads were obtained for the embryogenic and non-embryogenic tissues of P. balfouriana, and 49.56% of them uniquely matched 22,295 (84.3%) of the 26,437 genes in the Picea abies genome database (Nature 497: 579-584, 2013). Differential gene expression analysis identified 1,418 differentially expressed genes (false discovery rate <0.0001; fold change ≥2) in the embryogenic tissues relative to the non-embryogenic tissues, including 431 significantly upregulated and 987 significantly downregulated genes. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that the most significantly altered genes were involved in plant hormone signal transduction, metabolic pathways (starch and sucrose metabolism), and phenylalanine metabolism.ConclusionsWe found that the initiation of embryogenic tissues affected gene expression in many KEGG pathways, but predominantly in plant hormone signal transduction, plant-pathogen interaction, and starch and sucrose metabolism. The changes in multiple pathways related to induction in the P. balfouriana embryogenic tissues described here, will contribute to a more comprehensive understanding of the mechanisms involved in the initiation of somatic embryogenesis. Additionally, we found that somatic embryogenesis receptor kinase (SERK), arabinogalactan proteins, and members of the WUS-related homeobox protein family may play important roles and could act as molecular markers in the early stage of somatic embryogenesis, as reported previously.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-553) contains supplementary material, which is available to authorized users.

Morphological and Physiological Characterization of Embryogenic and Non-embryogenic Tissues of Centella asiatica

Plant Tissue Cult. &amp; Biotech. 24(1): 125-129, 2014 (June)D. O. I. http://dx.doi.org/10.3329/ptcb.v24i1.19257

ANTIOXIDANT ENZYMES ACTIVITY IN EMBRYOGENIC AND NON-EMBRYOGENIC TISSUES IN SUGARCANE

The objective of this work was to induce direct somatic embryogenesis from segments of immature leaves of the RB872552 variety of sugarcane and to correlate this morphogenic event with oxidative stress. Two previously described protocols were utilized for the induction of somatic embryogenesis in sugarcane with different supplementations of the culture medium and different incubation conditions. For the conversion of embryos into plants was used MS medium without phytoregulators. Histological analyses and activity of antioxidant enzymes were also conducted for the embryogenic and non-embryogenic tissues. The formation of somatic embryos was obtained in 81 % of the explants with the combination of regulators 2,4-D (2,4-dichlorophenoxyacetic acid)and BAP (6-benzylaminopurine) when incubated under 16 h photoperiod. With regards to the antioxidant enzymes, there was increased activity of peroxidase and an increase in the soluble protein content in embryogenic tissues, whereas lower activities of polyphenol oxidase and catalase appeared in these tissues compared to nonembryogenic tissues. It could be inferred that oxidative stress plays an important role in the induction of somatic embryogenesis in sugarcane.

Comparative Proteomic Analysis of Auxin-Induced Embryogenic and Nonembryogenic Tissues of the Solanaceous Tree Cyphomandra betacea (Tamarillo)

Cyphomandra betacea (tamarillo) is a tree that produces edible, highly nutritional fruits. In tamarillo, somatic embryogenesis (SE) is achieved through a two-step process starting with the formation of an embryogenic tissue on an auxin-rich medium and further development of embryos, following tissue transfer to an auxin-free medium. During the induction stage, both embryogenic (EC) and nonembryogenic calli (NEC) arise from the same explant (immature leaves or mature zygotic embryos) in the presence of either picloram or 2,4-dichlorophenoxyacetic acid. In an attempt to find somatic embryogenic-specific proteins, a comparative analysis of the proteome of tamarillo's EC and NEC was performed. Analysis of 2-DE gels revealed ca. 150 differentially expressed proteins, from which 22 have been identified by LC-MS/MS. Proteins exclusively or predominantly expressed in EC included metabolism-related proteins, such as enolases or treonine synthases, and also heat-shock and ribosomal proteins. Pathogenesis-related proteins were found mainly in NEC. A number of additional differentially expressed proteins involved in various functional categories were also identified. A quantitative real time PCR (qPCR) analysis revealed no significant differences at the mRNA level for 11 differentially expressed proteins, with exception of the pathogenesis-related proteins that were up-regulated in NEC. This seems to indicate that a posttranscriptional control might be responsible for the proteomic differences detected.

Cloning and Molecular Characterization of a SERK Gene Transcriptionally Induced During Somatic Embryogenesis in Ananas comosus cv. Shenwan

A somatic embryogenesis receptor-like kinase (SERK) gene, designated as AcSERK1, was isolated from pineapple (Ananas comosus cv. Shenwan). AcSERK1 shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, transmembrane domain, and kinase domains. Somatic embryogenic cultures of pineapple were established following transfer of callus cultures to Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid. The role of AcSERK1 during establishment of somatic embryogenesis in culture was investigated. The AcSERK1 was highly expressed during embryogenic competence acquisition and global embryo formation in culture. These findings were obtained along with morphological changes in callus cultures exhibiting embryogenic potential. Overall, levels of expression of AcSERK1 were lower in nonembryogenic tissues and organs than in embryogenic callus. In situ hybridization analysis revealed that AcSERK1 expression was detected in embryogenic tissues, including single competent cells, meristematic centers wherein embryogenic structures are formed, and global embryos. These results suggested that AcSERK1 expression was associated with induction of somatic embryogenesis and that it could be used as a potential marker gene to monitor the transition of pineapple callus tissues into competent and embryogenic cells and tissues.

Role of enzymes and identification of stage-specific proteins in developing somatic embryos of chickpea (Cicer arietinum L.)

Accumulation of proline, activities of peroxidase (POX), catalase (CAT), phenylalanine ammonia lyase (PAL) and malate dehydrogenase (MDH) were studied during different developmental stages of somatic embryos in chickpea. Callus cultures that did not form somatic embryos served as control. While increased levels of proline and POX activity were noticed in globular stages of embryos, CAT activity increased during early and late heart-shaped embryo formation indicating tissue-specific activation of these enzymes. The activity of PAL reached a peak during torpedo and cotyledonary stages of embryo development. On the other hand, MDH activity enhanced during the germination of somatic embryos inferring more requirement of energy during this stage. Electrophoretic (sodium dodecyl sulfate polyacrylamide gel electrophoresis) pattern of proteins revealed that ten bands are associated with non-embryogenic tissues, whereas 11 bands with globular, heart, torpedo and cotyledonary stages of embryo development and nine bands during the germination stage of embryos. Two extra stage-specific protein bands with molecular masses of 16 and 18 kDa appeared during globular, heart, torpedo, and cotyledonary stages. But, these bands disappeared during germination of embryos and are absent in non-embryogenic cultures. This study thus may help in the identification of proteins and the role of above enzymes during different developmental stages of somatic embryo induction and their maturation in a recalcitrant leguminous crop plant chickpea.

Sequence and Expression Analysis of EgSAPK, a Putative Member of the Serine/Threonine Protein Kinases in Oil Palm (Elaeis guineensis Jacq.)

In present study, EgSAPK (EU805512), an oil palm transcript coding for a putative SAPK protein kinase, have been molecular characterized. The cDNA for EgSAPK isolated from an oil palm cell suspension culture is 1470 bp in length with a longest Open Reading Frame (ORF) of 963 bp. No translation start codon could be identified so EgSAPK cDNA sequence is lacking the 5’-end. The deduced protein sequence shares 89% identity with the serine/threonine protein kinase SAPK9 from rice (AB125310.1). Real time PCR results showed that the expression levels of EgSAPK varied in different oil palm tissues and the EgSAPK gene shares a similar expression pattern with the SAPK gene of rice. Furthermore, the transcription of the EgSAPK gene in green embryo, white embryo and embryogenic calli tissues were higher than in non-embryogenic calli tissues. Southern blot analysis showed that the EgSAPK gene might be present as a single copy gene in the oil palm genome. These results suggest that EgSAPK may have a similar function as the SAPK gene of rice and thus can be a candidate marker for oil palm somatic embryogenesis.

Detection of a SERK-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos

Somatic embryogenesis involves different molecular events including differential gene expression and various signal transduction pathways. One of the genes identified in early somatic embryogenesis is S OMATIC E MBRYOGENESIS R ECEPTOR-like K INASE (SERK). Cocos nucifera (L.) is one of the most recalcitrant species for in vitro regeneration, achieved so far only through somatic embryogenesis, although just a few embryos could be obtained from a single explant. In order to increase efficiency of this process we need to understand it better. Therefore, the purpose of the present work was to determine if an ortholog of the SERK gene is present in the coconut genome, isolate it and analyze its expression during somatic embryogenesis. The results showed the occurrence of a SERK ortholog referred to as CnSERK. Predicted sequence analysis showed that CnSERK encodes a SERK protein with the domains reported in the SERK proteins in other species. These domains consist of a signal peptide, a leucine zipper domain, five LRR, the Serine-Proline-Proline domain, which is a distinctive domain of the SERK proteins, a single transmembrane domain, the kinase domain with 11 subdomains and the C terminal region. Analysis of its expression showed that it could be detected in embryogenic tissues before embryo development could be observed. In contrast it was not detected or at lower levels in non-embryogenic tissues, thus suggesting that CnSERK expression is associated with induction of somatic embryogenesis and that it could be a potential marker of cells competent to form somatic embryos in coconut tissues cultured in vitro.

Extracellular localization of napin in the embryogenic tissues of Brassica napus spp. oleifera

Napin, a storage protein, has been reported to be transcribed abundantly during the pre-embryogenic stage and associated with the induction of Brassica napus secondary embryogenesis. In this study, we studied the distribution pattern of napin in the winter oilseed rape embryogenic tissue in comparison to that of the non-embryogenic tissue using the indirect immunofluorescence localisation coupled with the ultrastructural immunogold labelling techniques. Immunolocalisation studies revealed that the extracellular matrix layer outside the outer epidermal cell wall of B. napus embryogenic tissues contained napin. This is the first study to report the extracellular localisation of napin. In addition, we have also further characterised the expression pattern of Eg1 that encodes for napin in the B. napus embryogenic tissue.