Embryogenic Cell Suspension Research Articles

Plant regeneration from embryogenic callus and cell suspensions of Brachiaria brizantha

Plant regeneration from embryogenic callus and cell suspensions of Brachiaria brizantha

Morphohistological and molecular profiles during the developmental stages of somatic embryogenesis of Musa acuminata cv. ‘Berangan’ (AAA)

The present study established a protocol for generating embryogenic cell suspension (ECS) cultures from an early stage embryogenic callus of Musa acuminata cv. ‘Berangan’. The necessary baseline data for morphohistological recognition and the selected gene expression for differentiating embryogenic and non-embryogenic cells were defined to enable early selection, as well as for quality assessment, which could aid in scaling up the production of these cells. In this study, male bud-derived callus on Murashige and Skoog (MS) medium containing 23 μM 2,4-dichlorophenoxyacetic acid (2,4-D) was transferred to M1 medium containing different concentrations of 2,4-D and picloram for somatic embryo initiation. ECSs established in liquid MS medium (M2a) containing 4.1 μM biotin, 10 mg/l ascorbic acid, 100 mg/l glutamine, 100 mg/l malt extract, 4.5 μM 2,4-D, 1.0 μM zeatin, and 20 g/l sucrose were used to study embryogenic development. The frequency of embryo development and maturation was influenced by the culture media. The highest frequency of embryo development (96.4 %) and maturation (66.4 %) was achieved on solid MS hormone-free medium. Almost 69 % of embryos were induced to form shoots on the MS medium containing 34 μM 6-benzylaminopurine (BAP). Histological study indicated that only embryos with distinct layer of protoderm having cells with prominent nucleus and dense cytoplasm were able to regenerate into plantlets. Endochitinase and phenylalanine ammonia-lyase (PAL) genes were expressed at all the developmental stages, and their expression level was tightly regulated during embryo development and reached its maximum in germinating embryos. Expression of these genes in embryogenic cells was higher than in non-embryogenic cells. The study findings suggested that these genes play an important role in the developmental stage and regeneration pathway of somatic embryos of banana.

In vitro xylem vessel elements formation from banana embryogenic cells and expression analysis of vessel development-related genes

Tracheary elements formation was observed in banana embryogenic cells cultured in brassinolide-supplemented medium after a period of 10 days. The highest frequency of tracheary elements observed after 10 days was up to 31.88 %. The auxin, 2,4-D was found to be inhibitor of tracheary elements formation in brassinolide-supplemented medium and at a concentration of 1.5 µM of 2,4-D, the frequency of tracheary elements formation was reduced to zero. The appearance of secondary wall in these xylem vessel elements was observed by toluidine blue staining and lignin autofluorescence under fluorescence as well as laser confocal microscopy. Similar to lignin autofluorescence imaging, the calcofluor white staining also revealed development of characteristic secondary wall in these xylem vessel elements indicating deposition of cellulose along with lignin during secondary wall formation. The xylem vessel elements induced comprised of fiber-like elements (FLEs) and tracheary elements with deposition of secondary wall. Expression analysis of lignin biosynthesis pathway genes indicated that important genes like CCR, CCoAOMT and COMT were induced as early as fifth day of brassinolide (BL) treatment. Changes in transcript level of key master regulators of xylogenesis during tracheary elements formation showed that expression of vascular-related NAC domain containing transcription factors (VND) were up-regulated with induction of VND1–3 and VND6–7 expression as early as third day of brassinolide treatment. This study of in vitro tracheary elements formation in embryogenic cell suspension cultures is useful in understanding the genes involved in secondary wall thickening in banana.

Histological and ultrastructural analysis of the Banana cv. Prata-Anã embryogenic calluses and cell suspension

The analysis of the developmental stages during somatic embryogenesis has been essential to elucidate the in vitro plants embryogenesis process and can validate the somatic embryogenesis induction and progression. This work aimed to characterize the embryogenic calluses and cell suspension of Banana cv. Prata-Ana, anatomically and ultrastructurally, respectively. For the calluses induction and anatomical analyses, the male flower were disinfested and inoculated on MA1 solid medium. At intervals of 30 days up to 11 months, calluses were collected, fixed in FAA and prepared for analysis in optical microscopy. For the cell suspension and ultrastructural analyses, calluses4 months old calluses, obtained on the first experiment, were transferred to different liquids cultures mediums: MRT, MGM, CNPMF or MA2 and maintained in aggitation. At intervals of 30 days calluses were collected and prepared for analysis in scanning electron microscope. The male flowers showed, after three months on culture medium, heterogeneous calluses formed by clusters of small isodiametric cells that gave rise to globular structure with meristems tissues, clearly differentiated from other cells around the calluses. The globular structure, after ten months on the culture medium, developed to torpedo stage showing the meristematic tissues protoderm, ground meristem and procambium. The cell suspension cultivated on MRT culture medium showed embryos on globular and torpedo stage.

Electroporation and morphogenic potential of Gentiana kurroo (Royle) embryogenic cell suspension protoplasts

This article presents our further in vitro studies into the morphogenic potential of gentian cells, organs, and tissues after modification of their genome. The objective was to study the effect of electroporation and the introduction of foreign genes on the morphogenic potential of Gentiana kurroo embryogenic cell suspension protoplasts. Protoplasts were electroporated with DNA plasmids carrying nptII and bar genes. The stability of cell membranes, the contents of electroporation buffer, the length of electric pulse, the number of pulses and the strength of the electric field were studied. We determined the highest electroporation efficiency by evaluating the highest protoplast survival rate under specific physical conditions. The best results were achieved in the presence of EB1 electroporation buffer where the viability of protoplasts was 70.1%. Protoplast survival at this higher level required culture temperatures near 0EC, and a 20 μs electric pulse with an electric field of 1.0 kV/cm. After seven days of agarose embedded protoplast culture, a selective agent – kanamycin – was introduced to the medium. The cell transformation effect was improved by a long term culture of callus, regenerated somatic embryos and transformants in the presence of 50 mg/l kanamycin.

A putative BABY BOOM-like gene (CaBBM) is expressed in embryogenic calli and embryogenic cell suspension culture of Coffea arabica L

The acquisition of embryogenic cell suspension (ECS) cultures has been one of the main objectives to maximize clonal propagation of the coffee plant. However, the majority of somatic embryogenesis induction requirements are genotype-dependent. Therefore, molecular markers linked to the embryogenic transition events may be useful. The BABY BOOM (BBM) gene can be considered as one of those markers, as it is related to the embryogenic process and to cell proliferation. BBM homologous sequences were obtained from Expressed Sequence Tags (ESTs) in a databank generated by the Brazilian Coffee Genome Project. We selected EST-contigs that showed similarities with BBM sequence from different species. Two EST-contigs (C2 and C9) were expressed in silico in cellular suspension libraries and embryogenic calli of coffee. Contig C9, defined as BBM-like (CaBBM), presented similarity with BBM genes and showed 2-fold change in expression in ECS relative to embryogenic calli (EC). Contig C2, on the other hand, was related to the ERF-like family. It showed basal expression in non-embryogenic calli (NEC) and approximately 66- and 311-fold less in ECS and EC compared with CaBBM in the same samples, respectively. These data suggest that CaBBM is likely to be a BBM ortholog in Coffea arabica, which has potential for use as a molecular marker to further increase the methodological efficiency of in vitro culture of coffee.

Assessment of RNAi-induced silencing in banana (Musa spp.).

BackgroundIn plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana.ResultsUsing embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6–8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt).ConclusionsRNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-655) contains supplementary material, which is available to authorized users.

Enhanced plumbagin accumulation in embryogenic cell suspension cultures of Plumbago rosea L. following elicitation

This study focused on enhancing the production of plumbagin, an anticancer compound, in embryogenic cell suspension cultures of Plumbago rosea. Elicitation techniques have been reported to enhance plumbagin production. Cell suspension cultures raised from embryogenic calli induced from in vitro leaf explants were exposed to different concentrations of jasmonic acid, yeast extract and different auxin combinations. Influence of these on cell growth, biomass and plumbagin production was studied. To our knowledge this is the first report on elicitation of embryogenic cell suspension cultures of P. rosea for enhanced plumbagin production. Elicitor treated suspension cultures exhibited decreased culture viability and increased plumbagin synthesis. A maximum of 5.59-fold enhancement of plumbagin production was observed in cultures added with 1 mg L−1 naphthalene acetic acid after 6 days of incubation. Viability of cultures decreased with increased concentration of elicitors and prolonged incubation period. Application of elicitors in cell suspension cultures induces defense related responses which lead to increased secondary metabolite production for making the cells adapt to the situation. If the stressed condition persists or is in intolerable level this will eventually lead to programmed cell death and loss of culture viability.

Agrobacterium-mediated transformation of embryogenic cell suspension cultures and plant regeneration in Lilium tenuifolium oriental × trumpet ‘Robina’

A method has been developed for embryogenic cell suspension cultures, plant regeneration and transformation of the important ornamental lily genotype (Lilium tenuifolium oriental × trumpet ‘Robina’). Bulb scales, filaments, ovaries and stem axis tissues were used as explants for callus induction in Murashige and Skoog (MS) medium with additions of growth regulators: picloram on its own, or in combination with 1-naphthaleneacetic acid (NAA), and thidiazuron (TDZ). The results show that the optimum medium for callus induction in bulb scale and filament tissue is MS + picloram 1.0 mg L−1, and for the ovary, it is MS + picloram 1.5 mg L−1. The stem axis had the highest rate (89.2 %) of callus induction with MS + NAA 2.2 mg L−1 + TDZ 0.1 mg L−1. The suspension cultures were established with the combination of NAA and TDZ with 2–5 mm cell clusters. These took a long time compared with suspension cultures established by picloram with 1–3 mm cell clusters. In three suspension cultures induced by picloram, the best callus from the point of view of proliferation and regeneration was derived from filaments. For plant regeneration, the growth rate of suspension cultures from the stem axis was higher than from the other three suspension culture induced by picloram. Vector pCAMBIA1301 with the β-glucuronidase (GUS) gene as reporter was transformed by Agrobacterium mediation into suspension cultures initiated from filament and stem axis material. After co-cultivation, the numbers of blue spots in material from the two sources were 26.8 ± 4.3 and 24.0 ± 4.7, respectively (difference not significant). Hygromycin-resistant callus was successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were also confirmed by the GUS histochemical assay, polymerase chain reaction.

Developing a cell suspension system for Musa-AAA-EA cv. ‘Nakyetengu’: a critical step for genetic improvement of Matooke East African Highland bananas

Embryogenic cell suspensions of triploid East African Highland bananas (Musa AAA-EA) were initiated and generated using cooking cultivar ‘Nakyetengu’ belonging to the Nakabululu clone set. Immature male flowers produced embryogenic calli consisting of embryos and friable tissue after 4 mo culture on a modified MA1 callus induction medium. Friable calli were initiated and maintained in liquid MA2 medium. A cell growth rate of 1.5–2.0 sedimented cell volume (SCV) per month was observed. Embryo development was observed at 2.18 × 103 embryos per mL SCV. Germination of these embryos was observed at 2.8% and 6.2% for two cell suspension lines. Plant regeneration efficiency was 60–100%, all producing normal plants with a shoot and roots at weaning. In the field, somatic cell-derived plants were all normal morphology and comparable to control plants during vegetative and reproductive stages. This study is a breakthrough for recalcitrant East African Highland banana and offers a system that can provide essential raw materials for associated germplasm improvement through genetic engineering approaches.

Improved sanguinarine production via biotic and abiotic elicitations and precursor feeding in cell suspensions of latex-less variety of Papaver somniferum with their gene expression studies and upscaling in bioreactor

Elicitors play an important role in challenging the plant defense system through plant-environment interaction and thus altering the secondary metabolite production. Culture filtrates of four endophytic fungi, namely, Chaetomium globosum, Aspergillus niveoglaucus, Paecilomyces lilacinus, and Trichoderma harzianum were tested on embryogenic cell suspensions of latex-less Papaver somniferum in dose-dependent kinetics. Besides this, abiotic elicitors salicylic acid, hydrogen peroxide, and carbon dioxide were also applied for improved sanguinarine production. Maximum biomass accumulation (growth index (GI) = 293.50 ± 14.82) and sanguinarine production (0.090 ± 0.008 % dry wt.) were registered by addition of 3.3 % v/v T. harzanium culture filtrate. Interestingly, it was further enhanced (GI = 323.40 ± 25.30; 0.105 ± 0.008 % dry wt.) when T. harzanium culture filtrate was employed along with 50 μM shikimate. This was also supported by real-time (RT) (qPCR), where 8-9-fold increase in cheilanthifoline synthase (CFS), stylopine synthase (STS), tetrahydroprotoberberine cis-N-methyltransferase (TNMT), and protopine 6-hydroxylase (P6H) transcripts was observed. Among abiotic elicitors, while hydrogen peroxide and carbon dioxide registered low level of sanguinarine accumulation, maximum sanguinarine content was detected by 250 μM salicylic acid (0.058 ± 0.003 % dry wt.; GI = 172.75 ± 13.40). RT (qPCR) also confirms the downregulation of sanguinarine pathway on CO2 supplementation. Various parameters ranging from agitation speed (70 rpm), impeller type (marine), media volume (2 l), inoculum weight (100 g), and culture duration (9 days) were optimized during upscaling in 5-l stirred tank bioreactor to obtain maximum sanguinarine production (GI = 434.00; 0.119 ± 0.070 % dry wt.). Addition of 3.3 % v/v T. harzanium culture filtrate and 50-μM shikimate was done on the 6th day of bioreactor run.

Isolation of endophytic bacteria from embryogenic suspension culture of banana and assessment of their plant growth promoting properties

Endophytic microorganisms have been reported from various plants. In the current study, somatic embryogenic cultures of banana (Musa accuminata AAA cv. Grand Naine) were found to have association with endophytic bacteria which were present initially in a covert state. The presence of bacteria was detected only in suspension cultures derived from the somatic embryogenic cultures. The bacteria isolated from embryogenic cell suspension culture were identified as Ralstonia sp. and Bacillus sp. The Ralstonia sp. interestingly showed the presence of various plant growth promoting properties including indole acetic acid and siderophore production. Also the strain was found to have the potential to solubilize phosphate and produce ammonia. Growth enhancement effect of Ralstonia sp. on Vigna radiata seedlings showed promising results and the growth parameters were found to be statistically significant when compared to control. Identification and confirmation of the plant growth promoting properties of Ralstonia sp. makes the study significant with promising applications.

Enhancement of regeneration efficiency in banana (Musa acuminata cv. Berangan) by using proline and glutamine

Regeneration of Musa acuminata cv. Berangan (AAA) from embryogenic cell suspension was enhanced by using proline and glutamine. Sixteen embryo development media consisting of different concentrations of proline and glutamine were tested. Glutamine was preferred as the source of organic nitrogen supplement compared to proline. The embryos developed in liquid media were shown to produce 10.6-fold higher number of plantlets in lesser duration (1.5 to 2.5 months) as compared to solid media. Optimum somatic embryo development was observed in liquid media supplemented with 400 mg/l glutamine (M4GL) with an average regeneration of 33,844 ± 402 plantlets per ml of settled cell volume (SCV). Although both amino acids have proven to promote the embryo development, concentrations of proline between 200 to 400 mg/l in liquid media caused abnormal embryo differentiation.

Host‐induced post‐transcriptional hairpin RNA‐mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

Primary results of 2-dimensional electrophoresis for protein studies of Gentiana kurroo Royle somatic embryos derived from long-term embryogenic cell suspensions

Two-dimensional gel electrophoresis was used to compare protein expression profiles between various stages of <em>Gentiana kurroo</em> Royle somatic embryos. Seven distinct stages (I–VII) were pointed out and measured from long-term embryogenic cell suspension. Isoelectric focusing was done in the pH intervals 3–10, and the second dimension was carried out with 13% SDS-polyacrylamide gel electrophoresis. Dependent on the stage from about 400 (stage IV) to more than 700 (stage II) protein spots were totally detected. The molecular weight of abundant proteins range from 12 to 70 kDa, however, majority of proteins were located between 20–49 kDa spots on the gels. The highest difference in the number of spots appeared in the case of globular embryo (stage I) and elongated cotyledonary stage (stage VII) with differences being about 130 spots. The relevance of embryogenic cell suspension choice for proteomic analysis as well as expediency of the increasing number of particular embryo stages is discussed.

Somatic embryogenesis in Lolium multiflorum suspension culture

The embryogenic cell suspension was obtained from immature embryos of <em>Lolium multiflorum</em> through a callus culture. Somatic embryogenesis was induced by addition of 2,4-D, dicamba and picloram in 0,5 mg/l concentrations in MS liquid nutrient medium. It was shown that somatic embryos arised from single cells. In globular embryoids, the meristematic cells are characterized by the presence of phytoferritin inclusions in the leucoplasts.

EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine

Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration. Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.

Transgenic banana cv. Rasthali (AAB, Silk gp) harboring Ace-AMP1 gene imparts enhanced resistance to Fusarium oxysporum f.sp. cubense race 1

Banana cultivar Rasthali (AAB, Silk gp) is one of the favored varieties known for its flavor and keeping quality in southern India. The cultivar is affected by Fusarium wilt which has reached serious proportions, almost wiping it out. Highly regenerative embryogenic cell suspensions of cultivar Rasthali were developed using male floral meristems. Agrobacterium strain LB4404 harboring binary vector pCAMBIA2301 with a gene encoding antimicrobial peptide (Ace-AMP1) cloned from onion seeds driven by CaMV35S promoter and nos terminator was used in transformation. Embryogenic cells were transformed with the gene and 148 putative transformants were developed. Molecular characterization of transformants demonstrated that 20 plants were PCR positive and the transgenes had been stably integrated into the banana genome in ten lines and expression of the gene was confirmed by ELISA and Western blot in them. Transformants were root challenged with Fusarium oxysporum f.sp. cubense race1 (Foc) in earthen pots and observed over a period of six months. External symptoms appeared after 4–5 months and by 6 months plants showed only 0–20% of PDI and 38–48% VDI as compared to untransformed control plants which showed severe disease symptoms (100%) within three months and died. Significantly higher AMP protein content in transgenic lines was negatively correlated and higher root colonization was positively correlated with disease symptoms. Six transgenic lines were selected for field evaluation.

Using flow cytometry and cytological analyses to assess the genetic stability of somatic embryo-derived plantlets from embryogenic Musa acuminata Colla (AA) ssp. malaccensis cell suspension cultures

Flow cytometry and chromosome counts were used to analyze the genetic stability of plants regenerated via Musa acuminata Colla (AA) ssp. malaccensis embryogenic cell suspension (ECS) cultures. These cultures were initiated from immature zygotic embryos (IZE) on Murashige and Skoog medium using nine different plant growth regulator (PGR) treatments. Highest percentage of embryogenic calli (EC) formation occurred on media with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D, 97 %), and 8.2 μM of picloram (Pi, 80 %) followed by 2.2 μM 2,4-D (75 %). Embryonic development was synchronized in liquid medium by filtration, and somatic embryo development was achieved with ECS aliquots overlaid on PGR-free medium. The EC medium composition and elapsed time of both short-term (~5 months old) and long-term (~2 years old) ECS cultures influenced plant regeneration, resulting in 65–99 % embryo germination and 50 to 100 % plant conversion. The mean 2C DNA content (1.23 ± 0.002 pg) and chromosome number (2n = 2x = 22) of M. acuminata ssp. malaccensis IZEs, seedlings and sucker plantlets were similar to the reported values. No significant differences were detected among IZEs before culturing, and none were found among the IZEs and leaves of control plants and the plants regenerated from short-term ECS lines when initiated with 2,4-D or Pi vis-a-vis the accession from which they originated. However, plants regenerated from the long-term ECS-L3 culture remained diploid, had the highest DNA content (2C = 1.283 ± 0.01 pg) and were clearly separate from the other regenerated and control plants.

Characterization of a Putative Serk-Like Ortholog in Embryogenic Cell Suspension Cultures of Coffea arabica L.

The acquisition of embryogenic cell suspension cultures (ECS) has been the objective of studies on in vitro induction of somatic embryogenesis with biotechnological tools, due to the high efficiency of ECS as plant material for genetic transformation and large-scale production and cryopreservation of germplasm. The objective of this work was to identify and analyze one of the main gene families involved in somatic embryogenesis, somatic embryogenesis receptor-like kinase (SERK) in coffee (Coffea arabica L.). Coffee SERKs were identified by searching an EST (expression sequences tag) database generated by the Brazilian Coffee Genome Project starting from candidate sequences obtained from the NCBI database (National Center for Biotechnology Information) . In silico analysis and quantitative PCR results imply that the identified EST-contig C166 might directly be involved in somatic embryogenesis. The results suggest that C166 is the possible ortholog of SERK in C. arabica (CaSERK) and indicate that C166 might be a valuable bio-marker for ECS, and in that context can increase the methodological efficiency for ECS formation in C. arabica. Functional analysis of CaSERK with mutants of a more manageable species will lead to a better understanding of the molecular regulation as well as the specific functions of genes involved in somatic embryogenesis in coffee.