Embryogenic Cell Suspension Research Articles

Genome-wide analysis of transcription factors during somatic embryogenesis in banana (Musa spp.) cv. Grand Naine.

Transcription factors BABY BOOM (BBM), WUSCHEL (WUS), BSD, LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), VIVIPAROUS1 (VP1), CUP SHAPED COTYLEDONS (CUC), BOLITA (BOL), and AGAMOUS LIKE (AGL) play a crucial role in somatic embryogenesis. In this study, we identified eighteen genes of these nine transcription factors families from the banana genome database. All genes were analyzed for their structural features, subcellular, and chromosomal localization. Protein sequence analysis indicated the presence of characteristic conserved domains in these transcription factors. Phylogenetic analysis revealed close evolutionary relationship among most transcription factors of various monocots. The expression patterns of eighteen genes in embryogenic callus containing somatic embryos (precisely isolated by Laser Capture Microdissection), non-embryogenic callus, and cell suspension cultures of banana cultivar Grand Naine were analyzed. The application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the callus induction medium enhanced the expression of MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the embryogenic callus. It suggested 2, 4-D acts as an inducer for the expression of these genes. The higher expression of MaBBM2 and MaWUS2 in embryogenic cell suspension (ECS) as compared to non-embryogenic cells suspension (NECS), suggested that these genes may play a crucial role in banana somatic embryogenesis. MaVP1 showed higher expression in both ECS and NECS, whereas MaLEC2 expression was significantly higher in NECS. It suggests that MaLEC2 has a role in the development of non-embryogenic cells. We postulate that MaBBM2 and MaWUS2 can be served as promising molecular markers for the embryogencity in banana.

Indirect Somatic Embryogenesis of Date Palm Using Juvenile Leaf Explants and Low 2,4-D Concentration.

This chapter describes an efficient protocol for large-scale micropropagation of date palm. Somatic embryo-derived plants are regenerated from highly proliferating suspension cultures. Friable embryogenic callus is initiated from juvenile leaves using slightly modified Murashige and Skoog (MS) medium supplemented with 0.1mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures consisting of proembryonic masses are established from highly competent callus for somatic embryogenesis using half-strength MS medium enriched with 0.1mg/L 2,4-D and 300mg/L activated charcoal. The productivity of cultures increased 20-fold when embryogenic cell suspensions were used instead of standard protocols on solidified media. The overall production of somatic embryos mostly exceeds 10,000units per liter per month. Partial desiccation of mature somatic embryos, corresponding to a decrease in water content from 90 down to 75%, significantly improved germination rates.

Plant Regeneration from Somatic Embryogenic Suspension Cultures of Date Palm.

Somatic embryogenesis is one of the most important technologies for plant regeneration of elite date palm cultivars. Recently, considerable progress has been made in the development and optimization of this technique from embryogenic cell suspension cultures. This chapter describes a procedure for the rapid development of a large number of somatic embryos from embryogenic cell suspension cultures. An efficient plant regeneration protocol via somatic embryogenesis from cell suspension cultures starting with shoot-tip explants to plantlet acclimatization also is fully described. Low concentrations of 6-benzylaminopurine (BAP) to 0.3mg/L and high rate of subcultures each 7 days lead to improve the establishment and multiplication of somatic embryos in suspension cultures by limiting oxidative browning, associated with high total phenols and peroxidase activities. The detailed morphological observations have revealed the cells destined to become somatic embryos. Activated charcoal (AC) at 0.15g/L has a positive effect on growth rate of somatic embryos by reducing tissue and medium browning, phenolics, and peroxidase activity.

Validation of reference genes for qPCR analysis of Coffea arabica L. somatic embryogenesis-related tissues

Quantitative real-time PCR (qPCR) is a sensitive method used to investigate relevant changes in gene expression during somatic embryogenesis. Understanding its regulatory network might be helpful to the process of induction of embryos and facilitate the development of efficient plant regeneration procedures. In this study, a set of 12 genes was selected and their stability was assessed in different tissues of somatic embryogenesis-related cultures of Coffea arabica. Analyses of gene expression stability were performed using the RefFinder tool that integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms. Among the all candidate reference genes studied, APRT/EF1a, UBQ/ACT, ACT/24S, RPL39/24S, PP2A/RPL39, PP2A/AP47, emerged as the most stable for normalization of qPCR analyses of embryogenic cell suspensions, non-embryogenic calli, embryogenic calli, combined embryogenic and non-embryogenic calli, somatic embryos and plantlet, respectively. A combination of two genes, 24S and PP2A, was identified as most suitable reference genes across all samples for the C. arabica tissues studied. The commonly employed reference gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found to be inappropriate as a reference gene for embryogenic tissues of C. arabica. In addition, Baby boom (BBM) gene expression was investigated to confirm the validity of the selected reference genes, the transcript levels of gene were overestimated when unsuitable reference genes were used for normalization. The results shown herein will permit a more precise and reliable normalization of qPCR in experiments involving somatic embryogenesis of C. arabica.

Efficient regeneration and transformation of Spathiphyllum cannifolium

An efficient and stable transformation system was developed based on embryogenic cell suspension culture by Agrobacterium tumefaciens-mediated method in Spathiphyllum cannifolium. Embryogenic calli induced from leaves on MS medium with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L thidiazuron were incubated in liquid medium with identical growth regulators for suspension cultivation. The plantlet regeneration rate of embryogenic suspension cell aggregates (ESCAs) incubated on MS medium achieved 100 %. The ESCAs were transformed using A. tumefaciens strain EHA105 harboring a vector pCAMBIA1301 containing the β-glucuronidase (gusA) reporter gene and the hygromycin phosphotransferase II (hpt II) selection marker gene. The optimized transformation conditions were as follows: infiltration of ESCAs with Agrobacterium for 10 min under 60 kPa vacuum pressure, followed by co-cultivation on medium containing 200 μM acetosyringone for 3 days. In total, 858 plants were regenerated from 1200 infected ESCAs. Analyses of randomly selected regenerated plants by GUS assays, PCR and dot blot showed that 91.30 % were transgenic. Southern blot analyses revealed that the copy number of T-DNAs integrated into the genome ranged from one to three. This newly developed transformation system provides a basis for the future development of genetic engineering techniques to improve flower color and enhance the disease resistance of peace lily.

English

Fusarium oxysporum f. sp. cubense (Foc) is one of the major threats to dessert banana (Musa spp.) production. In Uganda, ‘Sukali Ndiizi’ is one of the most popular dessert banana cultivars and it is highly susceptible to Fusarium wilt. Development of resistant cultivars through transgenic approaches has shown to offer one of the most effective control options for most diseases. The transgenic approaches for providing plant disease resistance have mainly been through either enzymatic destruction of pathogen structures, neutralization of pathogen and its products or production of metabolites that eventually kill the pathogen. However in recent years, methods that prevent cell death of host plant after infection especially for necrotrophic pathogens like F. oxysporum have registered success in providing resistance in several crops. We investigated whether the transgenic expression of a programmed cell death inhibition gene in Sukali Ndiizi could be used to confer Fusarium resistance to Foc race 1. Embryogenic cell suspensions of cv. ‘Sukali Ndiizi, were stably transformed with a synthetic, plant-codon optimise mCed-9 gene. Twenty-eight independently transformed plant lines were regenerated. The lines were inoculated with Foc race 1 and observed for 13 weeks in small-plant glasshouse. Three transgenic lines showed significantly lower internal and external disease symptoms than the wild-type susceptible ‘Sukali Ndiizi’ banana plants used as controls. This is the first report from Africa on the generation of Fusarium wilt tolerant transgenic ‘Sukali Ndiizi’, a very popular but rapidly diminishing African dessert banana. Key words: Fusarium wilt, banana, Sukali Ndiizi, Fusarium oxysporum f. sp. Cubense race 1, programmed cell death, disease resistance.

The proteome profile of embryogenic cell suspensions of Coffea arabica L.

Somatic embryogenesis, is a process by which new viable embryos are produced from somatic tissues. Somatic embryogenesis is not only a useful biotechnological tool for the massive clonal propagation and genetic engineering but it also allows to obtain fundamental knowledge about the molecular changes that take place during embryogenesis. We present the proteome profile of two embryogenic cell suspensions. We identified 1052 non-redundant proteins. We present their known GO annotations and show two protein networks sharing the GO annotations related to stress and embryogenic capacity via the free program Cytoscape. To our knowledge these results give the first high-throughput proteome description of embryogenic cell suspensions and provide new information about somatic embryos for the whole plant community. The published proteome is a first step toward understanding somatic embryogenesis in coffee and toward a better annotation of proteins in an important non-model crop. All data are available via ProteomeXchange with identifier PXD002963.

Initiation of coconut cell suspension culture from shoot meristem derived embryogenic calli: A preliminary study

<p>An attempt was made to establish highly competent embryogenic cell suspension culture in coconut, a species recalcitrant to in vitro culture. Embryogenic calli were initiated from shoot meristem explants of coconut. Y3 medium supplemented with 2.4-D (4.5 μM) and glutamine (34.2 μM) was found to be the best medium to initiate cell suspension. Growth evaluation was done by packed cell volume (PCV) and it was found that maximum growth volume of 9.9% was reached at 200 days of culture initiation. About 52% of viable cells were detected through fluorescent microscopy. Cell aggregation was noticed in Y3 medium supplemented with glutamine (34.2 μM), malt extract (100mg/l), biotin (40.9 μM) and kinetin (9.3 μM), but further progress could not be achieved. It was also observed that embryogenic calli were not of a friable type, but were associated with densely aggregated cells. Because of its hard nature, we were unsuccessful to obtain high quality cell suspension.</p>

Are Genetic and Epigenetic Instabilities of Plant Embryogenic Cells a Fatality? The Experience of Coffee Somatic Embryogenesis

In plants, undifferentiated or totally differentiated cells can be easily in vitro cultured to generate undifferentiated embryogenic cells that can regenerate complete plants. This is the most spectacular expression of totipotency. Embryogenic cells represent a key material in plant biotechnologies as they are used in many processes aiming at asexual reproduction by somatic embryogenesis (SE), genetic transformation, protoplast regeneration and cryopreservation. Nevertheless, tissue culture systems that involve the acquisition of competence for totipotency and extensive cell division remain risky with respect to genome and epigenome instabilities. Particularly, the use of embryogenic cell suspensions has frequently been associated with an increased likelihood of genetic instability and somaclonal variation (SV) in the regenerated plants. SV is a major concern in all in vitro vegetative plant propagation systems because it leads to the loss of genetic fidelity. Since 20 years, embryogenic cell suspension based SE techniques have been applied to coffee for the large-scale dissemination of exceptional Arabica hybrids. Here, we present our production and research experience showing that SE is efficient and reliable for true-to-type propagation. Over 99% of coffee trees regenerated fully conform to the mother plant, both morphologically - they grow, flower and produce normally. Hence strong genetic and epigenetic changes in proliferating embryogenic cells are not a fatality. The results also demonstrated the importance of embryogenic culture age on SV and hence the non-random nature of this phenomenon. The genetic and epigenetic alterations are particularly limited during SE. The main change in most of phenotypic variants was aneuploidy showing that mitotic aberrations play a major role in SV in coffee. These results provide a proof of concept for the use of embryogenic cell suspensions with other plant species: a revolution in the world of plant micropropagation on an industrial scale.

Overexpression of Arabidopsis CBF1 Gene in Transgenic Furenzhi banana (Musa spp. AA group) Improves Resistance to Low Temperature

Cold is an environmental factor that limits the growing season of banana and adversely affects fruit quality and productivity. C-repeat/dehydration responsive element binding factor 1 (CBF1) play important roles in the responses of plants to low-temperature and cold. To test the function and potential use of cold-induced factor CBF1 in improving the chill tolerance of banana, AtCBF1 driven by a cauliflower mosaic virus 35S promoter was introduced into banana plants by Agrobacterium -mediated transformation of embryogenic cell suspensions (ECSs) of banana cultivar Furenzhi (AA). The presence of AtCBF1 transgenes in regenerated plants was confirmed by polymerase chain reaction (PCR) and Southern blots. s-glucoronidase (GUS) histochemical assays reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analysis demonstrated that foreign genes were stably expressed in regenerated plants. Transgenic banana showed growth retardation, thicker leaves and higher chlorophyll content than non-transformed plants. While relative electrolyte leakage (REL) and malondialdehyde (MDA) content were significantly lower in transgenic banana plants than in non-transformed plants under low temperature stress. An obviously higher cold tolerance was observed among the transgenic plants in the cold detection. These results suggest that over-expression of AtCBF 1 in transgenic banana plants plays an important role in improving tolerance to low-temperature.

Somatic Embryogenesis: Still a Relevant Technique in Citrus Improvement.

The genus Citrus contains numerous fresh and processed fruit cultivars that are economically important worldwide. New cultivars are needed to battle industry threatening diseases and to create new marketing opportunities. Citrus improvement by conventional methods alone has many limitations that can be overcome by applications of emerging biotechnologies, generally requiring cell to plant regeneration. Many citrus genotypes are amenable to somatic embryogenesis, which became a key regeneration pathway in many experimental approaches to cultivar improvement. This chapter provides a brief history of plant somatic embryogenesis with focus on citrus, followed by a discussion of proven applications in biotechnology-facilitated citrus improvement techniques, such as somatic hybridization, somatic cybridization, genetic transformation, and the exploitation of somaclonal variation. Finally, two important new protocols that feature plant regeneration via somatic embryogenesis are provided: protoplast transformation and Agrobacterium-mediated transformation of embryogenic cell suspension cultures.

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.

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana.

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties “Cavendish Williams” and “Gros Michel” were developed using multiple buds, whereas ECS of “Sukali Ndiizi” was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000–50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20–70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa.

English

The plasmids pULGU1 and pEmuGN were introduced by biolistics in embryogenic cell suspensions of pearl millet, Pennisetum glaucum . The plasmid pULGU1 contained the mutant acetolactate synthase (ALS) gene of Arabidopsis thaliana , responsible for resistance to the chlorsulfuron herbicide. The plasmid pEmuGN carried the reporter β-glucuronidase (GUS) gene of Escherichia coli . Two months after bombardment of the cells, transformed calli were selected on Murashige and Skoog medium containing 30 nM chlorsulfruron, a completely concentration inhibitory for non-transformed cells. A concentration of 200 nM chlorsufuron did not affect the growth of these selected resistant calli. Genomic DNA of resistant cells presented an electrophoretic pattern revealing the integration of the mutant ALS gene into the genome of Pennisetum glaucum . Expression of the GUS reporter gene was revealed by histochemical assay of the blue coloration of the calli in the presence of the substrate Xgluc (5-bromo-4-chloro-3-indolyl-β-D-Glucuronic acid). The presence of GUS gene was furthermore confirmed, by southern blot hybridization of non-radioactive probes, labelled with digoxigenin, on genomic DNA extracted from selected chlorsulfuron-resistant calli. GUS gene activity observed in all selected calli was high during the first 6 months after bombardment and then decreased. Pearl millet plants were regenerated from cell lines derived from chlorsulfuron resistant calli. Southern blot hybridization of non-radioactive probes with genomic DNA extracted from these regenerated plants showed the presence of the GUS and ALS transgenes, confirming the stable transformation of these plants. Key words : Pennisetum glaucum , stable transformation, chlorsulfuron (ALS), β-glucuronidase (GUS), CaMV35S and Emu promoters.

Somatic embryogenesis, cell suspension, and genetic stability of banana cultivars

The induction of somatic embryogenesis in banana is extremely difficult because of endogenous problems of the species and genotype dependency. Establishing a suitable protocol for somatic embryogenesis is necessary for applying biotechnological approaches to assist the genetic improvement, by facilitating the access to individual cells or groups of cells for use in genetic transformation, and the induction of polyploidy and mutagenesis. Embryogenic cultures were induced from immature male flowers of two important cultivars of banana, ‘Grand Naine’ (AAA) and ‘Tropical’ (AAAB), using immature male flowers. Cell suspensions were established, and regenerated plants were evaluated for their genetic stability using 11 simple sequence repeat markers. For induction of embryogenesis, different doses of autoclaved glutamine in the induction medium were used, and somatic embryos were converted into plants in medium supplemented with benzylaminopurine and naphthalene-1-acetic acid. ‘Grand Naine’ formed somatic embryos in glutamine-free media, while ‘Tropical’ somatic embryos grew in the presence of autoclaved glutamine. During regeneration, ‘Grand Naine’ showed better results for formation of embryos in culture medium without growth regulators, but these embryos were not converted into plants when kept in the same medium, while ‘Tropical’ produced a large number of plants regenerated when kept in the same medium. The simple sequence repeat markers used did not detect any genetic variation. These results suggest that the establishment of embryogenic cell suspension cultures of banana may be effective for production of genetically stable plants on a large scale as well as being a biotechnological tool to support banana genetic improvement.

Embriogénesis somática en el cultivar de plátano ‘FHIA – 25’ (AAB) a partir de ápices meristemáticos

<p>El cultivar de plátano ‘FHIA – 25’ (AAB), posee excelente rendimiento y alta resistencia a “Sigatoka negra”, pero con la limitante del bajo contenido de azúcar en su fruto, lo cual hace que sea necesario disponer de un método de regeneración de plantas a nivel celular como la embriogénesis somática, que se complemente a técnicas biotecnológicas de transformación genética para mejorar la calidad del fruto. El presente trabajo se realizó con el objetivo de establecer una metodología de regeneración vía embriogénesis somática a partir del explante inicial ápices de brotes axilares establecidos directamente en medio de cultivo líquido. Se obtuvieron suspensiones celulares embriogénicas homogéneas a partir del explante antes mencionado. Se lograron las mayores tasas de multiplicación a la densidad celular de 3,0%. La incubación de los embriones somáticos durante 30 días en el medio de cultivo de maduración permitió incrementar la germinación de los mismos. Durante la fase de aclimatización las plantas provenientes de los embriones somáticos, así como las plantas regeneradas por organogénesis, mostraron un alto porcentaje de supervivencia (98 y 97 %, respectivamente), sin la presencia de variación somaclonal.</p>

Acidogenic growth model of embryogenic cell suspension culture and qualitative mass production of somatic embryos from triploid bananas

A young male flower-derived embryogenic suspension cell population of AAA ‘Pei Chiao’, ‘Dwarf Cavendish’, and AAB ‘Raja’ was used for developing an acidogenic growth model . We hypothesized that a close relationship exists between the self-regulated pH medium and the corresponding changes in the growth phases. Studies have reported that a pH below 4.6 may prevent the embryogenic cells from undergoing polar growth. Controlling pH up to a level 4.6 within 2 days during the changes of pre-embryogenic cells (PECs) and proembryogenic masses into embryogenic determined cells (EDCs) uniformly resulted in unequal cell division. The hydrogen ion buffer 2-N-morpholino-ethanesulfonic acid at 10 g L−1 was added to MA2 and MA3 media, showing the medium pH of MA3 up to 5.0, thus maintaining a relatively stable pH in AAA ‘Pei-Chiao’ and AAB ‘Raja’ cells that autoregulate acidification, significantly increasing the number of somatic embryos. When the proliferation and globularization phases were acidified to pH 3.5 ± 0.2, cells were released to free single cells of PECs and EDCs after 21 days. This study provides possible explanation that PECs deposit callose on their cell walls as a possible protector from strong acidic condition. Regulation at pH 5.0 ± 0.2 resulted the production efficiency achieved was 0.9 million somatic embryos per 1 mL of the settled cell volume.

Retraction Note to: Plant regeneration in Chlorophytum borivilianum Sant. et Fernand. from embryogenic callus and cell suspension culture and assessment of genetic fidelity of plants derived through somatic embryogenesis.

[This retracts the article DOI: 10.1007/s12298-012-0113-y.].

Improved Agrobacterium-mediated transformation of Carica papaya cultivar ‘Kapoho’ from embryogenic cell suspension cultures

The adoption of Agrobacterium-mediated transformation to agronomically important Carica papaya cultivars and genotypes, such as “Kapoho’, has been challenging. To address this problem, an alternative transformation protocol was developed for papaya using embryogenic suspension-derived cultures. The ability of the tissues (cultivar ‘Kapoho’) to regenerate plantlets from these cultures was demonstrated for both transgenic lines and untransformed controls. Suspension-derived cultures at the young globular calli stage contained embryos highly receptive to Agrobacterium infection and did not require the problematic wounding treatments inherent in other protocols. The optimum parameters selected to obtain transgenic calli were as follows: 1-d co-cultivation with Agrobacterium, the cell density used for transformation was A600 of 0.005, and elimination of Agrobacterium post-transformation was done using 250 mg/L carbenicillin and cefotaxime. Putative transgenic calli were confirmed positive for the presence of the eGFP transgene, Cp45 promoter sequence, and hygromycin resistance gene (hptII) using PCR. The presence of eGFP messenger RNA (mRNA) and protein were detected using reverse transcription PCR (RT-PCR) and Western blot analysis, respectively. Visualization of qualitative eGFP fluorescence in roots, stems, and leaves further confirmed the expression of Cp45:eGFP fusion in the transformed papaya plantlets. This technique serves as an alternative and efficient method to generate transgenic plants in a simple laboratory setup that facilitates Agrobacterium-mediated transformation of previously difficult papaya cultivars and genotypes.

TDZ and 4-CPPU Induce Embryogenic Response on Scalps of Recalcitrant East African Highland Banana

Breeding of banana conventionally is very difficult due to high levels of sterility arising from aneuploidy. Transformation of East African highland banana (EA-AAA banana) has been limited by difficulties in induction of embryogenic callus and/or embryogenic cell suspension (ECS), a requirement for efficient genetic transformation. In this study N-phenyl-N’-1,2,3-thidiazol-5-ylurea (TDZ) and N-(2-chloro-4-pyridyl)-N’-phenylurea (4-CPPU) that are highly active in regulating morphogenesis in tissue culture of many plant species were tested for enhancing embryogenic callus induction in recalcitrant EA-AAA banana through scalps. In vitro derived shoot-tips of banana cultivars Mbwazirume, Mpologoma, Nakabululu, Nakinyika and Nfuuka were subcultured on various combinations of TDZ and 4-CPPU till scalps were formed. In all the cultivars, ideal scalps for callus induction were achieved in over 50% of the shoot-tip cultures within four subculture cycles in the medium containing equal proportions of 9–13 µM of TDZ and 4-CPPU, and 26 µM of each. Embryogenic callus was formed on 2.5–20% of the scalp cultures depending on cultivar and, TDZ and 4-CPPU combination. Cultivars Nakinyika, Nakabululu, Mbwazirume and Nfuuka developed regenerable embryogenic callus. Of the plantlets regenerated 5 out of 12 and 4 out of 5 plantlets of cultivars Nakinyika and Mbwazirume, respectively, developed normal roots and were weaned onto soil. These results suggest that TDZ and 4-CPPU increases embryogenic response from scalps of EA-AAA banana which can enhance their genetic transformation.