Use of Electron Microscopy for the Detection of Contaminant Endophytic Bacteria In Vitro.

The success of in vitro cultivation, particularly for micropropagation purposes, depends on the efficient control of contaminants. In this context, the sterilization of plant material constitutes a fundamental step in initiating cultures. Microbial contaminants can be found either on the surface (epiphyte) or inside plant explants (endophyte). However, the latter is generally challenging to detect and may not always be eradicated through surface sterilization alone. Endophyte contaminants, such as bacteria, can persist within plant material over several cultivation cycles, potentially interfering with or inhibiting in vitro establishment, growth, or recovery of cryopreserved materials. Therefore, microscopy techniques, such as electron microscopy, can yield valuable insights into bacterial endophytes' localization, tissue colonization patterns, and functions in in vitro plant culture. This information is essential for adopting effective strategies for eliminating, preventing, or harmonious coexistence with contaminants.

Quantification of DNA Methylation by ELISA in Epigenetic Studies in Plant Tissue Culture: A Theoretical-Practical Guide.

This chapter describes a step-by-step protocol for rapid serological quantification of global DNA methylation by enzyme-linked immunosorbent assay (ELISA) in plant tissue culture specimens. As a case study model, we used the coconut palm (Cocos nucifera), from which plumules were subjected to somatic embryogenesis followed by embryogenic calli multiplication. DNA methylation is one of the most common epigenetic markers in the regulation of gene expression. DNA methylation is generally associated with non-expressed genes, that is, gene silencing under certain conditions, and the degree of DNA methylation can be used as a marker of various physiological processes, both in plants and in animal cells. Methylation consists of adding a methyl radical to carbon 5 of the DNA cytosine base. Herein, the global DNA methylation was quantified by ELISA with antibodies against methylated cytosines using a commercial kit (Zymo-Research™). The method allowed the detection of methylation in total DNA extracts from coconut palm embryogenic calli (arising from somatic embryogenesis) cultivated in liquid or solid media by using antibodies against methylated cytosines and enzymatic development with a colorimetric substrate. Control samples of commercially provided Escherichia coli bacterial DNA with previously known methylation percentages were included in the ELISA test to construct an experimental methylation standard curve. The logarithmic regression of this E. coli standard curve allowed methylation quantification in coconut palm samples. The present ELISA methodology, applied to coconut palm tissue culture specimens, is promising for use in other plant species and botanical families. This chapter is presented in a suitable format for use as a step-by-step laboratory procedure manual, with theoretical introduction information, which makes it easy to apply the protocol in samples of any biological nature to evaluate DNA global methylation associated with any physiological process.

Differential expression of genes potentially related to the callogenesis and in situ hybridization of SERK gene in macaw palm (Acrocomia aculeata Jacq.) Lodd. ex Mart.

For the purpose of understanding the molecular processes triggered during callus formation in macaw palm, the expression of seven genes potentially involved in this process, identified in previous studies and from the literature, was investigated by RT-qPCR. In addition, in situ hybridization of the SERK gene was performed. Leaf tissues from adult plants from two macaw palm accession were inoculated in a medium combined with Picloram at a concentration of 450 µM to induce callus. The expression analysis was performed from leaf samples from two accessions of different origins (Municipalities of Tiros, MG, and Buriti Vermelho, DF, Brazil), which are characterized as non-responsive (NR) and responsive (R), respectively. The material was collected before callus induction (0 DAI, initial day) and 120 days after callus induction (120 DAI). Genes related to development (SERK, OASA, EF1, ANN1) and stress (LEA, CAT2, and MDAR5) were evaluated. The results obtained showed that all the genes involved with the development had their expressions downregulated at 0 DAI when the accession R was compared with the accession NR. On the other hand, it was possible to observe that these genes were upregulated at 120 DAI. The LEA stress gene showed a tendency to increase expression in the NR accession, while the R accession showed decreased expression and the CAT2 and MDAR5 genes showed upregulation in both accessions. In situ hybridization showed SERK transcripts in the vascular bundles, indicating the expression of SERK in this region, in addition to its expression in calluses. The results obtained in this study support our hypothesis that the regulation of genes involved in the control of oxidative stress and development is crucial for the formation of calluses in macaw palm.

Cellular responses of oil palm genotypes during somatic embryogenesis involve participation of procambial cells, DNA demethylation, and auxin accumulation.

KEY MESSAGE: Cell markers of somatic embryogenesis initiation from leaf tissues in oil palm involve the participation of procambial cells, DNA demethylation, and auxin accumulation. Low callogenesis and genotype-dependent response have been mentioned in the development of somatic embryogenesis protocols of Elaeis oleifera × E. guineensis elite hybrids, which requires more detailed investigations of the process. Thus, the initial cellular responses of immature leaves of adult genotypes of this hybrid were investigated for the first time, emphasizing histological, epigenetic, and endogenous auxin changes. Leaf segments from two genotypes, one responsive to somatic embryogenesis (B351733) and another non-responsive (B352933), were inoculated in Murashige and Skoog medium with 450 µM of 4-amino-3, 5, 6-trichloropicolinic acid. For anatomical analysis, samples of both genotypes were collected at 0, 20, 90, and 105 days of cultivation. Samples of both genotypes were also taken at different cultivation periods to analyze DNA methylation status (% 5-mC-5 methylcytosine) via ELISA test. Immunolocalization assays were performed with anti-indole-3-acetic acid and anti-5-methyl-deoxycytosine antibodies from samples of hybrid B351733. We distinguished two groups of cells reactive to the induction of embryogenic callogenesis, parenchymatous sheath cells, and procambial cells; however, only the latter are directly involved with the formation of calluses. The data obtained indicate that the formation of calluses in hybrid B351733 is related to DNA hypomethylation, while the non-responsiveness of leaf explants in hybrid B352932 is related to DNA hypermethylation. The in situ immunolocalization enabled the identification of initial markers of the callogenic process, such as IAA accumulation and hypomethylation. Identifying these events brings the possibility of establishing strategies for efficient manipulation of somatic embryogenesis protocols in palm trees.

Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry.

BACKGROUND: Piper hispidinervum is a species native from the Amazon region with great economic potential, given its scientifically proven insecticidal properties. In this study, an efficient protocol of plant regeneration via indirect somatic embryogenesis has been established for the first time. In a first experiment, for the induction of calluses, foliar explants of non-discriminated accesses of P. hispidinervum were inoculated in MS medium supplemented with α-naphtalenacetic acid (NAA) and 6-benzylaminopurine (BAP), in different combinations. For a second experiment, foliar explants from five different accesses of P. hispidinervum (PH17, PH21, PH28, PH37, and PH39) were analyzed regarding the formation of calluses when cultivated in MS medium with 5 mg L-1 NAA + 2.5 mg L-1 BAP. To obtain somatic embryos-like structures, calluses were cultivated in MS medium with 10 mg L-1 NAA + 2.5 mg L-1 of BAP. The somatic embryos-like structures obtained were inoculated in MS medium devoid of growth regulators and the plantlets were subjected to acclimatization. Calluses and somatic embryos-like structures were subjected to anatomical analysis and genetic stability of regenerated plants was analyzed by flow cytometry. RESULTS: The treatments 2.5 mg L-1 BAP and 5 mg L-1 NAA + 2.5 mg L-1 BAP, after 60 days of cultivation, provided each 32% of primary callus, not being verified the formation of calluses in medium devoid of BAP. It was found that accesses differed among them with respect to the formation of primary calluses, with emphasis on accesses PH28, PH37, and PH39, with mean percentage of 95.3%. Regarding the percentage of embryogenic calluses and formation of somatic embryos-like structures, there were no statistical differences between accesses, with mean values of 90.6% and 77.3%, respectively. The somatic embryos-like structures of P. hispidinervum have conspicuous morphoanatomical similarities with the zygotic embryo, and flow cytometry analysis showed no significant variation in nuclear DNA size among plants regenerated in vitro and plants coming from seed germination, which indicates ploidy level stability. CONCLUSION: This protocol is the first cited in the literature that demonstrates an efficient micropropagation process by somatic embryogenesis of P. hispidinervum. It can be used either to enable large-scale vegetative production or to subsidize germplasm conservation or genetic engineering of P. hispidinervum.

Morphostructural and histochemical dynamics of Euterpe precatoria (Arecaceae) germination.

The fruits of Euterpe precatoria, popularly known as açaí palm, have been commercially exploited for use in food and beverages because of their medicinal and energetic properties. However, despite the growing demand, little is known about the seeds germination, until now, its main form of propagation. In this context, we have characterized the structure of the zygotic embryo and described temporally the germination process of E. precatoria with emphasis on the morpho-anatomical and histochemical aspects. For this end, we have collected and analyzed zygotic embryo and seedlings samples before sowing and at different periods after sowing-2, 4, 6, 8, 10, 15, 20, 30, 40, 50, and 60 days. The embryo has an oblique embryonic axis and mainly protein reserves. Seed germination is classified as cryptocotyledonar, hypogeal, and adjacent ligular and we observed seedlings at 20 days after sowing (33.3%), although anatomical evidence of the beginning of the germination process have been observed at 15 days. The day-20 was histologically marked by the expansion of the ligule, beginning of second eophyll differentiation, and complete root protrusion. This stage was characterized by the total consumption of protein reserves and increased starch grains. The occurrence of 100% of root and ligula emission was verified at day-60, which characterizes a slow and heterogeneous process. The morphological marker of the E. precatoria germination is the exit of the proximal region (cotyledonary petiole) of the embryo from within the seed by the lifting of the operculum and the species has some peculiarities, such as the presence of high concentrations of phenolic compounds and idioblasts before and throughout the germination process, and starch grains located on the embryonic axis. We can verify that the consumption of protein reserves of the embryo is related to the energy supply necessary for root protrusion.

MALDI TOF MS-profiling: Applications for bacterial and plant sample differentiation and biological variability assessment.

In this study, we evaluated the potential use of MALDI-TOF MS Profiling for the differentiation of biological samples submitted to different treatments. We compared the bacterium Xanthomonas campestris pv. campestris (Xcc), grown in culture medium and in vivo (recovered from the plant). Plant samples were also analyzed and included explants at different somatic embryogenesis (SE) stages, as well as leaves from Brassica oleracea and Arabidopsis thaliana inoculated with Xcc, at different time points. The results showed that bacteria and highly divergent plant samples, such as those from embryogenic stages, can be unequivocally differentiated and the clustering was in accordance with proteomic analysis performed by 2-DE. These results show an important application of MALDI-TOF MS Profiling to select and prioritize samples to be analyzed prior to more complex approaches including transcriptomics and proteomics. We also show that in plant-pathogen interactions, when more subtle differences are obtained, the main contribution of MALDI-TOF MS Profiling is in the assessment of experimental variability. This is relevant since reproducibility is a challenging issue when dealing with complex experimental conditions such as plant-pathogen interactions. We propose the use of MALDI-TOF MS Profiling to aid researchers in minimizing experimental variability unrelated to the condition being analyzed. SIGNIFICANCE: MALDI-Profiling offers an inexpensive, rapid and reliable approach for investigating the protein profile to assess sample differentiation and experimental variability in microorganisms and plants and can be highly useful to analyze samples prior to more complex and expensive techniques such as proteomics and transcriptomics.

Stress and cell cycle regulation during somatic embryogenesis plays a key role in oil palm callus development.

Oil palm is an oleaginous plant of relevant economic importance since its fruits are rich in vegetable oil. These plants have a single apical meristem and the main method for vegetative propagation is somatic embryogenesis. The aim of this study was to identify differentially abundant proteins from oil palm genotypes contrasting in the capacity of embryogenic competence acquisition, using shotgun proteomics. Oil palm leaves were subjected to callus induction and the material was collected in biological triplicates at 14 and 90 days of callus induction. LC-MS/MS analysis was performed and revealed a total of 4695 proteins. Responsive and non-responsive genotypes were compared at 14 and 90 days of callus induction and 221 differentially abundant proteins were obtained. The data analysis revealed several proteins mainly related to energy metabolism, stress response and regulation of cell cycle, further analyzed by qRT-PCR, which seem important for embryogenic development. We suggest some of these proteins as key factors for the success of callus formation in oil palm including antioxidant and cell division proteins as well as proteins involved in the ubiquitination pathway. These proteins may also be potential biomarkers for the acquisition of embryogenic competence. SIGNIFICANCE: Antioxidant and cell division proteins as well as proteins involved in the ubiquitination pathway are key factors for the success of callus formation in oil palm. The proteins identified in this study may be potential biomarkers for embryogenic competence acquisition.

Biochemical events during somatic embryogenesis in Coffea arabica L.

Several biochemical components associated with different stages of somatic embryogenesis in coffee (Coffea arabica L.) are investigated using foliar explants. Soluble sugar, starch, free amino acids and total proteins were extracted and quantified at different stages of somatic embryogenesis, such as foliar segments (initial explants), primary calluses, embryogenic calluses, globular embryos, torpedoes, cotyledonary embryos and mature fruit zygotic embryos. Total soluble sugar levels increased sixfold at the initial stages of somatic embryogenesis induction. During this period, total soluble sugar in the cultures contained approximately 99.3% glucose and fructose. At 67.4 µg/mg MS, no significant changes were observed in total sugar content during the embryo's somatic maturation and regeneration. During this stage, total soluble sugar was composed of 60% sucrose. After primary callus formation, starch contents increased gradually until the culture's conclusion. Total free amino acids, particularly arginine, lysine, methionine, asparagine, glutamine and histidine, revealed a higher synthesis until the formation of the primary callus, after which they remain statistically constant up to the end of the process. During the induction of calluses, a gradual increase of total proteins occurred, which, in the differentiating and maturing of somatic embryos, did not differ statistically till the formation of a cotyledonary embryo, when rates decreased 21.8%.

Genotype-dependent changes of gene expression during somatic embryogenesis in oil palm hybrids (Elaeis oleifera x E. guineensis).

To understand the molecular processes triggered during the different steps of somatic embryogenesis (SE) in oil palm, the expression of 19 genes associated to SE identified in proteomic and transcriptomic studies was investigated by qRT-PCR. To evaluate the differential expression of these genes, two interspecific hybrid genotypes (Elaeis oleifera x Elaeis guineensis) contrasting for the acquisition of embryogenic competence were used. Aclorophyllated leaves of both hybrids, one responsive (B351733) and the other non-responsive (B352933) to SE were submitted to callus induction and collected at different time points: 0 (before induction), 14, 30, 90 and 150 days of callus induction (doi). The results obtained showed that all evaluated genes were downregulated at 14 doi in the responsive genotype when compared to the non-responsive. It was also possible to observe that most of the genes changed their expression behavior at 30 doi and were upregulated thereafter until 150 doi, with the exception of the pathogenesis-related PRB1-3-like (PRB1-3) gene, which did not show differential expression at 30 doi and was downregulated at 90 and 150 doi when compared to the non-responsive hybrid. These results indicate that 30 doi is a turning point in gene expression, probably associated to embryogenic competence acquisition. We also show that the expression behavior of the responsive genotype is more stable than that of the non-responsive when the different induction time points are compared to 0 doi (before induction). Moreover, the results obtained in this study corroborate our hypothesis that the regulation of genes involved in the control of oxidative stress and energy metabolism are crucial for the acquisition of embryogenic competence in oil palm.

Assessment of mint (Mentha spp) species for large-scale production of plantlets by micropropagation / Avaliação de espécies de menta (Mentha spp) para a produção de mudas em larga escala por micropropagação

Species of the genus Mentha produce essential oils which are widely used in pharmaceutical and cosmetic industries. Current study evaluates the potential for in vitro propagation and estimates mass production of plantlets of Mentha species. Nine species (M. piperita, M. suaveolens, M. canadensis, M. longiflora, M. aquatica, M. arvensis, Mentha x gracilis, M. gracilis and M. spicata) were propagated with five successive 30-day subcultures in MS medium supplemented with NAA (0.05 µM) and BAP (4.4 µM). Shoots were rooted in MS with IBA, IAA or NAA (0.0; 0.25; 0.5; 2.5 or 5.0 µM). The rooted plantlets were finally acclimatized in a greenhouse. Studied species increased in multiplication rates between 4.2 and 9.0-fold per month. M. piperita, M. longiflora, M. arvensis, M. x gracilis and M. gracilis showed the greatest potential for plantlet production since the estimated production varied between 6,000 and 27,000 plantlets after five 30-days subcultures. The addition of auxin to the medium did not influence root induction. However, IAA at a concentration of 5 µM provided the best results for root length and fresh weight, with averages 11.1 cm and 0.16 g, respectively. Survival of plantlets reached 100% during the greenhouse acclimatization process.

Espécies do gênero Mentha produzem óleos essenciais largamente usados na indústria farmacêutica e de cosméticos. O estudo avaliou o potencial de propagação in vitro e estimou a produção de mudas de espécies de menta. Nove espécies (M. piperita, M. suaveolens, M. canadensis, M. longiflora, M. aquatica, M. arvensis, Mentha x gracilis, M. gracilis e M. spicata) foram propagadas por até cinco sucessivos subcultivos de 30 dias em meio de MS adicionado de ANA (0,05 µM) e BAP (4,4 µM). Os brotos foram enraizados em meio de MS com AIB, AIA ou ANA (0,0; 0,25; 0,5; 2,5 ou 5,0 µM). Finalmente, as mudas foram aclimatizadas em casa de vegetação. As espécies estudadas apresentaram aumentos nas taxas de multiplicação, variando entre 4,2 e 9,0 vezes por mês. M. piperita, M. longiflora, M. arvensis, M. x gracilis e M. gracilis mostraram os melhores potenciais para propagação, uma vez que a produção variou entre 6.000 e 27.000 mudas após cinco subcultivos de 30 dias. A adição de auxina no meio não influenciou a indução de raízes. Entretanto, o AIA na concentração de 5 µM promoveu os melhores resultados quanto ao comprimento e massa fresca das raízes, com médias de 11,1 cm e 0,16 g, respectivamente. No processo de aclimatização houve 100% de sobrevivência das mudas.

Proteomic identification of differentially expressed proteins during the acquisition of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.).

In the present study we have identified and characterized the proteins expressed during different developmental stages of Elaeis guineensis calli obtained from zygotic embryos. We were interested in the possible proteomic changes that would occur during the acquisition of somatic embryogenesis and therefore samples were collected from zygotic embryos (E1), swollen explants 14days (E2) in induction medium, primary callus (E3), and pro-embryogenic callus (E4). The samples were grinded in liquid nitrogen, followed by total protein extraction using phenol and extraction buffer. Proteins were analyzed by two-dimensional electrophoresis (2-DE) and the differentially expressed protein spots were analyzed by MALDI-TOF mass spectrometry (MS and MS/MS). Interestingly, we have identified proteins, which can be used as potential candidates for future studies aiming at the development of biomarkers for embryogenesis acquisition and for the different stages leading to pro-embryogenic callus formation such as type IIIa membrane protein cp-wap13, fructokinase and PR proteins. The results obtained shed some light on the biochemical events involved in the process of somatic embryogenesis of E. guineensis obtained from zygotic embryos. The use of stage-specific protein markers can help monitor cell differentiation and contribute to improve the protocols for successfully cloning the species. BIOLOGICAL SIGNIFICANCE: Understanding the fate and dynamics of cells and tissues during callus formation is essential to understand totipotency and the mechanisms involved during acquisition of somatic embryogenesis (SE). In this study we have investigated the early stages of somatic embryogenesis induction in oil palm and have identified potential markers as well as proteins potentially involved in embryogenic competence acquisition. The use of these proteins can help improve tissue culture protocols in order to increase regeneration rates. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Production of pre-basic potato seed by polyvinyl chloride PVC: articulate gutters hydroponic system

The development of more efficient and productive systems for pre-basic seed potato production would improve the quality of the propagative material used by the potato growers, directly affecting the crop yields. A two-year experiment was carried out to evaluate the potato pre-basic seed production by two types of hydroponic systems (fibrocement tiles and articulated PVC gutters), two cultivars (`Baronesa` and `Eliza`) and two types of propagative material (plants coming from in vitro culture and minitubers). The PVC gutters system was highly efficient. When using minitubers, this system reached multiplication rates up to 74 tubers per plant. Minitubers were more productive than in vitro plants, independent of cultivar and hydroponic system utilized.

Um experimento realizado por dois anos consecutivos avaliou a produção de sementes pré-básicas de batata por meio de sistemas de cultivo hidropônico. O trabalho testou a combinação de dois sistemas de cultivo (telha de fibrocimento e calhas de PVC articuladas), duas cultivares (Baronesa e Eliza) e dois tipos de material propagativo (plântulas oriundas do cultivo in vitro e minitubérculos). O sistema de calhas de PVC foi altamente eficiente. Quando foi utilizado minitubérculos, este sistema alcançou taxas de multiplicação de até 74 tubérculos por planta. De modo geral, o uso de minitubérculos como material propagativo apresentou os melhores resultados de produtividade quando comparada ao material in vitro, independentemente da cultivar e sistemas hidropônicos utilizados.

Modificações na anatomia foliar de bananeiras durante o processo de micropropagação / Modifications on leaf anatomy of banana during the micropropagation process. / Modificaciones en la anatomía foliar de bananas durante el proceso de micropropagación.

O estudo e compreensão das alterações decorrentes do processo de micropropagação constituem caminhos interessantes para o aprimoramento desta tecnologia. O objetivo deste trabalho foi avaliar modificações anatômicas em folhas de bananeira (Musa spp.) durante o processo de adaptação das plantas micropropagadas às condições ex vitro. Brotações axilares assépticas da cv. Preciosa (AAAB) foram enraizadas em meio MS, adicionado de ANA (1mg·l-1) e ágar (6g·l-1) por 24 dias, e aclimatizadas por 120 dias. Os tratamentos consistiram de folhas em diferentes estádios de desenvolvimento: T1 - folhas de plantas ao final da fase de enraizamento in vitro, T2 - folhas persistentes de plantas aos 30 dias de aclimatização, T3 - novas folhas de plantas aos 30 dias de aclimatização (folhas de transição), T4 - folhas de transição de plantas aclimatizadas por 60 dias, T5 - novas folhas de plantas aclimatizadas por 60 dias, e T6 - novas folhas de plantas aclimatizadas por 120 dias. Maior grau de diferenciação e, portanto melhor adaptação ocorre em folhas provenientes de primórdios foliares diferenciados em condição ex vitro. A fase de aclimatização é imprescindível para o maior espessamento e diferenciação dos parênquimas clorofilianos e para corrigir as modificações desenvolvidas nas plantas in vitro. O estudo sobre a anatomia foliar possibilita uma melhor compreensão das alterações que ocorrem em plantas de bananeira micropropagadas.

The study and understanding of alterations taking place during the micropropagation process can provide valuable information about this technology. The objective of this work was to evaluate the anatomical modifications in leaves of micropropagated banana (Musa spp.) plants during their adaptation to ex vitro conditions. Aseptic axillary shoots of ‘Preciosa’ cultivar (AAAB) were rooted for 24 days in MS medium containing NAA (1mg·l-1) and agar (6g·l-1), and acclimatized for 120 days. The treatments consisted of leaves at different stages of development: T1 - leaves from plants at the end of in vitro rooting phase, T2 - persistent leaves from plants after 30 days of acclimatization, T3 - new leaves from plants after 30 days of acclimatization (transition leaves). T4 - transition leaves from plants after 60 days, T5 - new leaves from plants after 60 days of acclimatization, and T6 - new leaves from plants after 120 days of acclimatization. A higher degree of differentiation and, thereby, better adaptation took place in leaves from leaf primordial differentiated in ex vitro conditions. The acclimatization phase is crucial for a greater thickness and differentiation of spongy and palisade parenchyma, and to correct the modifications of plants developed in vitro. The study of leaf anatomy provides a better understanding of alterations occurring in micropropagated banana plants.

El estudio y comprensión de las alteraciones ocurridas durante el proceso de micropropagación pueden proveer información valiosa acerca de esta tecnología. El objetivo de este trabajo fue evaluar las modificaciones anatómicas que tienen lugar en las hojas de plantas de banano (Musa spp.) micropropagadas durante el proceso de adaptación de las mismas a las condiciones ex vitro. Brotes axilares asépticos de cv. Preciosa (AAAB) fueron enraizados por 24 días en medio MS, adicionado con ANA (1mg·l-1) y agar (6g·l-1); y aclimatados por 120 días. Los tratamientos consistían de hojas en diferentes estadios de desarrollo: T1 - hojas de plantas en el final de la fase de enraizamiento in vitro, T2 - hojas persistentes de plantas con 30 días de aclimatación, T3 - hojas nuevas de plantas con 30 días de aclimatación (hojas de transición), T4 - hojas de transición de plantas aclimatadas por 60 días, T5 - hojas nuevas de plantas aclimatadas por 60 días, y T 6 - hojas nuevas de plantas aclimatadas por 120 días. Mayor grado de diferenciación y, por tanto, mejor adaptación ocurrió en hojas provenientes de primordios foliares diferenciados en condiciones ex vitro. La fase de aclimatación es fundamental para un mayor espesor y diferenciamiento de los parénquimas clorofilianos y para corregir las modificaciones desarrolladas en las plantas in vitro. El estudio de la anatomía foliar posibilita una mejor comprensión de las alteraciones que ocurren en bananeras micropropagadas.