Embryogenic Suspension Cultures Research Articles

Cassava for thefuture: embryogenic liquid cultures suitable for new biotech techniques.

Cassava, a crop of importance for subsistence farming in Africa, Asia, and Latin America, has the potential to benefit from global economic integration as a versatile industrial resource. Enhancing cassava productivity is not just a matter of agricultural competitiveness but a crucial step toward ensuring many communities' food security and livelihoods. Given its high performance in marginal environments, where climate change poses threats, ensuring food security and livelihoods relies on rapidly adapting cassava. This study aimed to develop a protocol that swiftly transitions cassava embryogenic short-period liquid suspension cultures, facilitating the regeneration of genetically stable in vitro plants. The resulting protocol, with its potential to be a foundational component in future technologies employing various genome editing or genetic modification techniques, holds promise for the advancement of cassava biotechnology.

An efficient embryogenic cell suspension culture system through secondary somatic embryogenesis and regeneration of true-to-type plants in banana cv. Sabri (silk subgroup AAB)

An efficient embryogenic cell suspension culture system through secondary somatic embryogenesis and regeneration of true-to-type plants in banana cv. Sabri (silk subgroup AAB)

Vinblastine synthesis under the influence of CaCl2 elicitation in embryogenic cell suspension culture of Catharanthus roseus

Vinblastine synthesis under the influence of CaCl2 elicitation in embryogenic cell suspension culture of Catharanthus roseus

Polish Contribution to Global Research on Somatic Embryogenesis

AbstractSomatic embryogenesis (SE) is a complex process that begins with regaining totipotency in some somatic cells, proceeds through embryo development and maturation, and ends with the formation of a whole plant. Since the first publications on SE in 1958, this regeneration process has been applied to the in vitro propagation of many plant species and has led to the development of some specific model systems. SE has been used to expand our understanding of the cytomorphological, physiological, biochemical, and genetic processes that govern the earliest developmental events in the life of plants. This paper summarizes the achievements of Polish research groups working on SE systems established for several plants (gentians, the tree fernCyathea delgadiiSternb., and conifers) and three model species (Arabidopsis thaliana,Medicagosativa, andM. truncatula). SE systems have used a broad spectrum of experimental approaches involving genomic tools (transcriptomics, proteomics, and chromatin analyses), physiological methods which focus on phytohormones, and cytological techniques. Studies on the experimental models ofA. thalianaandMedicagospp. have resulted in the identification of new genetic and epigenetic elements of the complex regulatory network controlling embryogenic induction in plant somatic cells. The protocol developed for ferns has provided a unique and simple system for cytological analysis of early SE events that occur in a single cell of initial explants. Gentian embryogenic suspension cultures have successfully been used in broad biotechnological applications, including plant transformation, protoplast isolation, culture, and fusion. Systems described for coniferous species effectively produced many vigorous somatic seedlings and cost-efficient storage of genotypes during clonal field-testing. The research undertaken by Polish scientists has resulted in developing experimental systems that have enabled significant advances in SE knowledge.

Development of a New Genetic Transformation System for White and Green Ash Using Embryogenic Cultures

All North American ash (Fraxinus spp.) species are threatened by the emerald ash borer (EAB; Agrilus planipennis), an exotic beetle which has already destroyed millions of ash trees in the U.S. and Canada. Although both chemical insecticides and biological control can be effective, and host resistance appears possible, the speed of the invasion has defied traditional management approaches. One potential, innovative approach to managing this destructive insect is to develop a host tree-induced gene silencing strategy using RNA interference (RNAi) constructs targeting EAB-specific genes. An important requirement for applying RNAi technology is a reliable transformation/regeneration system for the host tree species. We developed an Agrobacterium-mediated gene transfer system for white ash (F. americana) and green ash (F. pennsylvanica) using the embryogenic cultures of these species as target material. Embryogenic suspension cultures of multiple genotypes of both species were plated and inoculated with A. tumefaciens carrying the pFHI-GUSi expression vector, which carries the nptII selectable marker and intron-GUS reporter genes, followed by selection on a semi-solid medium containing geneticin. Putative transgenic events showed expression of the GUS gene at all tested developmental stages from callus to plantlets, and transgene presence in the leaves of regenerated plants was confirmed using PCR. The overall average transformation efficiency achieved was 14.5 transgenic events per gram of tissue. Transgenic somatic seedlings of two white ash and three green ash genotypes were produced and acclimated to greenhouse conditions.

Banana somatic embryogenesis and biotechnological application

As one of the most important economic crop for both staple food and fruit widely cultivated in tropics and subtropics, banana (<italic>Musa</italic> spp.) is susceptible to a plethora of abiotic and biotic stresses. Breeding cultivars resistant to abiotic and biotic stressors without adverse effects on yield and fruit quality are the objectives of banana improvement programs. However, conventional breeding approaches are time-consuming and severely hampered by inherent banana problems (polyploidy and sterility). Therefore, genetic transformation is becoming increasingly popular and can provide rapid solutions. Numerous efforts have been made to develop superior banana cultivars with better resistance to abiotic and biotic stresses and optimum yields using genetic modification strategies. Somatic embryogenesis (SE) through embryogenic cell suspension (ECS) cultures is an ideal recipient system for genetic transformation in banana. The purpose of this paper is to review the current status of banana somatic embryo research, clarify the way of banana somatic embryo induction and culture, and summarize the main influencing factors in the process of somatic embryogenesis. At the same time, their applications in breeding technologies such as cryopreservation, protoplast culture, genetic transformation and gene editing were also summarized, in order to provide reference for the research and practical application of banana somatic embryogenesis in the future.

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

Secretome-Derived Cultured Cell System: Overview Towards Extracellular Protein Characterization and Biotechnological Applications

Secretome released by plant cells into the extracellular space, play crucial roles during development, embryonic potential acquisition, nutrient backing and stress acclimation. The dynamic nature of the extracellular proteome presents the challenge of identifying an array of extracellular proteins involved in the regulation of somatic embryogenesis in embryogenic suspension cultures. Extracellular proteins produced by cell cultures are perceived here as a central node of overlapping regulator factor network of totipotent somatic embryo developmental process. This paper reviews in a morphogenetic aspect the biological processes associated with extracellular protein-derived plant cultured cells and explores their prospective biotechnological applications in laboratories and biofactories retated to cell signaling and metabolism, developmental process, and biotic / abiotic stress tolerance. The role of extracellular proteins in acquisition and maintenance of embryonic potential and their relevance are especially emphasized.

Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.)

BackgroundSwitchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration.ResultsRegenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety ‘Performer’ tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines.ConclusionThe new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6–7 months from culture establishment.

Robust CRISPR/Cas9 mediated genome editing tool for banana and plantain (Musa spp.)

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables precision editing of the genome of many plant species. Developing robust gene editing tools in banana and plantain would pave the way for the improvement of these crops. Here, we developed efficient CRISPR/Cas9 genome editing protocol for banana and plantain using multiple gRNAs targeting phytoene desaturase (PDS) gene. CRISPR/Cas9 construct containing two gRNAs was delivered into embryogenic cell suspension cultures of banana cultivar ‘Sukali Ndiizi’ and plantain cultivar ‘Gonja Manjaya’ by Agrobacterium-mediated transformation. The regenerated genome-edited events of ‘Sukali Ndiizi’ and ‘Gonja Manjaya’ showed albino and variegated phenotypes indicating mutations at the targeted sites disrupting the function of PDS gene. The majority of events (52/77 for ‘Sukali Ndiizi’ and 16/17 for ‘Gonja Manjaya’) were albino. Sequencing of the target sites confirmed presence of indels in all the 18 events sequenced demonstrating mutation efficiency of 100 % in both cultivars. The majority of events (6/8) of ‘Gonja Manjaya’ showed indels at both the target sites of PDS gene, however only 2/10 events of ‘Sukali Ndiizi’ showed indels at both sites, with one event (S24) having a knockout of large fragment (723 bp) indicating that both gRNAs were effective. Several of the albino events of both ‘Sukali Ndiizi’ and ‘Gonja Manjaya’ showed homozygous mutations. Further sequencing of four potential off-target sites in five events showed no mutations indicating CRISPR/Cas9 based editing in banana and plantain is targeted and precise with a very low probability of off-target sites. This study could provide a methodological framework for single or multiple knockouts in banana and plantain.

Somatic Embryogenesis in the Family Gentianaceae and Its Biotechnological Application.

The family Gentianaceae consists of 1736 species, which play an important role in human being existence due to their pharmacological and horticultural values. Many species accumulate bitter iridoid substances used medicinally and in flavorings, while others are cultivated because of beauty of their flowers showing a wide range of colors and patterns. Out of 99 genera belonging to the gentian family, process of somatic embryogenesis (SE) was reported for 5. The first reports, aimed at micropropagation of ornamental cultivars and production of secondary metabolites, concerned Centaurium erythraea Rafn., Eustoma russellianum Grieseb. and Exacum affine Balf. Somatic embryos were induced on different explants cultured in the liquid Murashige and Skoog medium supplemented with auxins and cytokinins. In the 1990s of the last century, significant progress in the exploration of the phenomenon of SE and its biotechnological application was made for the genus Gentiana. The process was induced on various explants and studied at the structural and ultrastructural levels. Regenerated plants were screened for genetic stability using flow cytometry, chromosome counting, and molecular markers. Besides typical indirect SE, the use of leaf fragments enabled to obtain single-cell origin of somatic embryos. On the other hand, proliferation of embryogenic callus in liquid medium resulted in the establishment of long-term embryogenic cell suspension cultures, paving the way not only to study the formation of somatic embryos and the development of regenerants but also to preserve the morphogenic potential of cell aggregates by cryopreservation. Cell suspensions re-established after storage in liquid nitrogen maintained their embryogenic character and allowed to obtain somatic embryo-derived regenerants that were true-to-type at both genetic and epigenetic levels. Another application of SE was related to genetic manipulation purposes. Efficient protocols of plant regeneration from callus-, cell suspension-, or leaf mesophyll-derived protoplasts allowed engaging procedures of somatic hybridization or protoplast electroporation for gentian genome modifications. Also, high embryogenic potential existing in the numerous gentian species enabled successful Agrobacterium-mediated transformation of G. cruciata L. and G. dahurica Fisch.

AaMps1 protein inhibition regulates the protein profile, nitric oxide, carbohydrate and polyamine contents in embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

The Monopolar Spindle 1 (Mps1) protein is a dual-specificity kinase that plays a critical role in the progression of the cell cycle. Studies on biochemical changes promoted by Mps1 protein inhibition are important for the development of somatic embryogenesis in plants; however, such work has not been previously performed for Araucaria angustifolia. We analyzed the effects of Mps1 protein inhibition on differential protein regulation and the effects of such differential regulation on the carbohydrate, nitric oxide (NO) and polyamine (PA) contents in embryogenic suspension cultures of this species. A proteomic analysis by the shotgun method was performed using mass spectrometry. The PA and carbohydrate contents were determined by high-performance liquid chromatography, while NO was analyzed by fluorescence microscopy. A total of 1518 proteins were identified, and 157 and 162 proteins were down- and up-regulated, respectively, in embryogenic suspension cultures incubated with 10 µM Mps1 inhibitor. Inhibition of the AaMps1 protein affected the abundance of proteins related to cell cycle division by reducing the accumulation of both cell division control protein 48 and NAP1-related protein 2. In addition, the abundance of the sucrose synthase 3 protein was down-regulated, which reduced sucrose contents. Moreover, a significant reduction in endogenous NO and free putrescine contents was observed in the embryogenic suspension cultures treated with the Mps1 inhibitor. These results show that AaMps1 protein inhibition affects the metabolism of proteins, carbohydrates, NO and PAs, which ultimately interferes in the development of somatic embryogenesis in A. angustifolia.

Biolistic Transformation of Cotton Embryogenic Cell Suspension Cultures.

Genetic transformation of cotton (Gossypium hirsutum L.) is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependent. However, once embryogenic cell cultures are available, they can be effectively used for transformation by Agrobacterium or biolistic bombardment methods. Here I describe a detailed procedure to transform cotton embryogenic cell suspension cultures by biolistic bombardment. A commercially available, helium-driven biolistic device (Bio-Rad PDS1000/He) was used to bombard gold particles coated with plasmid DNA (for visual identification of transformed cells and/or selection) into embryogenic cells. Stable transformation at a high frequency (up to 4% of the transiently expressing cells) is possible. Regeneration of fertile transgenic plants from embryogenic cells takes only about 2months. Another advantage of the embryogenic cell suspension cultures is that they are amenable for cryopreservation and long-term storage. It is highly preferable to transform commercial varieties of choice than obsolete varieties to avoid genetic drug due to backcrossing.

High-frequency plant regeneration from embryogenic cell suspension cultures of Gynura procumbens

The efficient plant regeneration system from embryogenic cell suspension cultures of Gynura procumbens (Lour.) Merr. is described. Leaf, stem and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) in various concentrations (0, 0.1, 0.3, 1.0 and 3.0 mg l−1). Leaf, stem and petiole explants formed pale-yellow nodular callus and off-white calluses at a frequency of 100% when cultured on MS medium supplemented with more than 1 mg l−1 of 2,4-D after 4 weeks incubation. However, only 20% of pale-yellow nodular callus derived from petiole explants developed into white embryonic structures. Upon transfer to MS basal medium without growth regulators, these white embryonic structures differentiated into somatic embryos. Embryogenic cell suspension cultures were initiated from petiole-derived pale-yellow nodular calluses. More than 73.2% of regenerated plantlets via somatic embryogenesis produced roots on MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid and 1 mg l−1 indole-3-butyric acid (IBA), respectively. Rooted plantlets were successfully transplanted to soil mixture of sterile vermiculite and potting soil (1:1) and grown to maturity in a growth chamber, achieving a survival rate of > 95%. The plant regeneration system from embryogenic cell suspension cultures of G. procumbens established in this study could be applied as an alternative for mass proliferation as well as molecular breeding for quality improvement of G. procumbens.

Growth, morphology and maturation ability of Pinus pinea embryogenic suspension cultures

Stone pine (Pinus pinea L.) is a Mediterranean forest species used for the commercial harvesting of its edible seeds, known as pine nuts. Plant regeneration by somatic embryogenesis on semi-solid medium has been achieved in stone pine, but suspension culture systems for the large-scale production of somatic embryos need to be developed. Suspension cultures were established from several embryogenic lines. Growth parameters as settled cell volume (SCV) and dry weight (DW) in suspension cultures were influenced by genotype, initial inoculum density, orbiting speed and type of carbohydrate in the culture medium. A higher linear growth (DW) up to week four was obtained using 20 mg mL−1 inoculum. The orbiting speed affected the SCV as well as the morphology of embryogenic cultures. Growth parameters decreased when orbiting speed was increased from 50 rpm to 150 rpm. Cultures at 50 rpm most commonly grew as clusters of embryonal cell masses, whereas at 150 rpm there were large numbers of single cells. Cultures grew slowly in medium with maltose instead of sucrose, but growth was not affected when plant growth regulators (PGRs) concentration was decreased. A sustainable suspension culture could be maintained by transferring 250 mg of filtered cell mass to 25 mL of liquid medium at 3-week intervals. Culture scaling-up in liquid medium resulted in a 23-fold DW increase in 6 weeks, compared with 12 fold increase on semi-solid medium. Cotyledonary embryos were obtained on semi-solid maturation medium from lines proliferated in suspension cultures. Increased somatic embryo production was obtained when proliferation was performed at 100 rpm orbiting speed in liquid medium with low PGRs concentration and sucrose instead of maltose.

Responses of Arabica coffee (Coffea arabica L. var. Catuaí) cell suspensions to chemically induced mutagenesis and salinity stress under in vitro culture conditions

Crop improvement of Coffea arabica L. (coffee) via mutagenesis could accelerate breeding programs; thus, the present study aimed to develop an in vitro protocol using the chemical mutagens sodium azide (NaN3) and ethyl methanesulfonate (EMS) on embryogenic cell suspensions of Arabica coffee variety Catuai and, subsequently, to evaluate the responses of the resulting mutagenized tissues to salinity stress. Embryogenic suspension cultures were incubated with 0.0, 2.5, 5.0, or 10.0 mM NaN3 or 0.0, 185.2, 370.5, or 741.0 mM EMS. As the concentration of NaN3 or EMS increased, the survival of embryogenic suspension cultures decreased compared to controls. The median lethal dose (LD50) for NaN3 was 5 mM for 15 min and for EMS it was 185.2 mM for 120 min. Embryogenic suspension cultures treated with NaN3 or EMS were cultured on selective medium supplemented with 0, 50, 100, 150, 250, or 300 mM NaCl showed that 50 mM NaCl could be used as selection pressure. Plantlet growth and total amino acid content were affected by NaCl stress; some mutants had longer shoots and higher amino acid content than controls. Random amplified polymorphic DNA (RAPD) analysis was performed to determine whether the NaN3 or EMS treatments could induce genetic variability and resulted in identifiable polymorphic markers. A total of 18 10-mer primers were used to amplify genomic DNA of putative mutant and non-mutant arabica coffee embryogenic cultures and produced 50 scorable bands, of which 22% were polymorphic.

Studies on growth dynamics of embryogenic cell suspension cultures of commercially important Indica rice cultivars ASD16 and Pusa basmati.

Significant efforts are being directed towards the improvement of rice using genetic manipulations. A good and reproducible system for recovering fertile Indica rice plants is imperative. The aim of this study was to study the influence of initial cell density on growth dynamics of suspension cultures. The cultures were initiated from 3-week-old embryogenic calli derived from mature seeds of Indica rice cultivars ASD16 and Pusa basmati. Growth kinetics of the rice cell suspensions were measured and the obtained data reveal that viable cells at 3% PCV using 20ml liquid (N6D) medium and sub-culturing at 7-day intervals resulted in rapid increase in fresh and dry weightsand the embryogenic competency of the cells were found to be high. The growth kinetics analysis revealed that ASD16 showed better efficiency for high frequency and viable somatic embryo formation as compared to Pusa basmati. The technique was found to be suitable for developing somatic embryos for both cultivars ASD16 and Pusa basmati, which can be used for many important applications including micropropagation and secondary metabolites production.

Somatic embryogenesis of a seedless sweet orange (Citrus sinensis (L.) Osbeck)

Somatic embryogenesis is an important in vitro technique used to obtain Citrus sinensis (L.) Osbeck (sweet orange) plantlets for conservation, sanitation, propagation, and breeding. The induction of somatic embryogenesis from adult tissues of sweet orange could be an alternative to in vitro organogenesis from epicotyl segments, especially in seedless cultivars, where the latter is not feasible. The aim of this study was to obtain plantlets from ovary-derived somatic embryos of sweet orange cv. ‘Washington Navel’, an important seedless cultivar for citrus fresh fruit production. The explants used were pistils from flower buds, pre-anthesis, from 20-y-old plants cultivated in the field. Forty plantlets from 47 somatic embryos were obtained, in vitro-grafted, and acclimatized in greenhouse conditions. Ploidy evaluation through flow cytometric analysis, as well as the results of target region amplification polymorphism (TRAP) molecular markers confirmed the somatic origin of embryos as genetically similar to donor plants. This technique could be used for obtaining embryogenic cell suspension cultures or regenerated plants from mature tissues other than seed-derived tissues, especially for seedless genotypes.

CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali genome.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1-5) or deletion (1-4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.

Establishment and biochemical characterization of tamarillo (Solanum betaceum Cav.) embryogenic cell suspension cultures

Somatic embryogenesis (SE) is an important biotechnological tool with great potential for large-scale cloning. In Solanum betaceum Cav. (tamarillo), embryogenic (EC) and non-embryogenic (non-EC) cells can be obtained from the same explant on auxin-containing medium, making this system ideal for the evaluation of biochemical changes occurring during embryogenic induction. Liquid cultures offer additional possibilities for the analysis of factors controlling SE induction, and the main objectives here were the establishment of cell suspensions and the characterization of the extracellular protein profiles in EC and non-EC cultures. Growth kinetics of liquid cultures, starting with different amounts of EC or non-EC callus or with different weight per volume ratios, were analyzed. Embryogenic suspension cultures were efficiently established starting with 40 mg of cells in 20 mL of liquid medium. Mass spectrometry and fluorometric techniques were employed to identify extracellular proteins, their hydrolytic activity, and the main classes of proteases secreted into the media of EC or non-EC cultures. Extracellular protein profiles revealed quantitative and qualitative differences between EC and non-EC suspension cultures, mainly for several hydrolytic enzymes, such as glucanases and xylanases. Proteolytic activity analysis found serine proteases, aspartic proteases, and metalloproteases in EC cultures, whereas serine proteases were dominant in non-EC lines. For the first time, a protocol for the growth of tamarillo EC and non-EC suspensions was achieved. Moreover, the comparison of protein profiles between EC and non-EC lines showed pronounced differences in the proteolytic and glycolytic enzymes secreted.