Peach palm plantlet growth in different culture media in a temporary immersion system / Crescimento de mudas de pupunheira em diferentes meios de cultura em um sistema de imersão temporária

ABSTRACT: Peach palm is a domesticated palm commercially important for the production of fruits and hearts of palm. Somatic embryogenesis, an effective technique for mass propagation, was successfully established for this species. Furthermore, a temporary immersion system improved plant regeneration. However, production can be further improved by understanding the peach palm's growth dynamic and modifications of culture media. The aims of this study were to evaluate the growth of plantlets cultured in different culture media in a temporary immersion system and to correlate the results with nutrient uptake during the growth period. Somatic embryo-derived young plantlets approximately 1 cm in length were cultivated for 12 weeks in a twin flask system containing MS, Y3 or N6 salts, Morel and Wetmore vitamins and 3% sucrose, with a monthly medium refreshment. Growth was measured and mineral analysis of the plantlets was carried out after 12 weeks of culture. The Y3 and MS salts were the most appropriate for the plant growth. Number of roots was 52.52% higher and the root size was 40.42% between the N6 and MS medium and the root number in Y3 medium was 37.74% greater than in MS medium, which is important for post acclimatization survival. K and Na are important elements for peach palm. N is not required at such a high concentration as in Murashige and Skoog formulation. The Chu (N6) medium did not generate high quality plantlets, possibly due to the absence of some micronutrients, like Mo, Cu and Co.

RESUMO: A pupunheira é uma palmeira comercialmente importante para a produção de palmito. A embriogênese somática, técnica efetiva para propagação massal, foi estabelecida com sucesso para essa espécie. Além disso, um sistema de imersão temporária aumentou a regeneração de plantas. Entretanto, a produção pode ser melhorada através da compreensão da dinâmica de crescimento e modificações do meio de cultura. O estudo objetivou avaliar o crescimento de plantas em diferentes meios de cultura em um sistema de imersão temporária e correlacionar os resultados com a absorção de nutrientes durante o período de crescimento. Plantas derivadas de embriões somáticos, com aproximadamente 1 cm de comprimento, foram cultivadas por 12 semanas em um sistema tipo frasco gêmeo contendo sais do MS, Y3 ou N6, vitaminas de Morel e Wetmore e 3% de sacarose, com renovação mensal do meio de cultura. O crescimento e os teores de nutrientes nas plantas foram determinados após 12 semanas de cultivo. Os sais do MS e Y3 foram os mais apropriados para o crescimento vegetal. O número e comprimento de raízes foi 52,52% e 40,42% maior no meio MS do que no meio N6, respectivamente, e o número de raízes no meio Y3 foi 37,74% maior que no meio MS, o que é importante para a sobrevivência após a aclimatização. K e Na foram os nutrientes mais importantes para a pupunheira. O N não foi requerido em altas concentrações como verificado na formulação do meio Murashige e Skoog. O meio de Chu (N6) não gerou plantas de boa qualidade, possivelmente devido à ausência dos micronutrientes Mo, Cu e Co.

Cloning and molecular characterization of a putative habanero pepper SERK1 cDNA expressed during somatic and zygotic embryogenesis

Background: Plant gene homologs that control cell differentiation can be used as biotechnological tools to study the in vitro cell proliferation competence of tissue culture-recalcitrant species such as peppers. It has been demonstrated that SERK1 homologs enhance embryogenic competence when overexpressed in transformed tissues; therefore, cloning of a pepper SERK1 homolog was performed to further evaluate its biotechnological potential. Results: A Capsicum chinense SERK full-length cDNA (CchSERK1) was cloned and characterized at the molecular level. Its deduced amino acid sequence exhibits high identity with sequences annotated as SERK1 and predicted-SERK2 homologs in the genomes of the Capsicum annuum CM-334 and Zunla-1 varieties, respectively, and with SERK1 homologs from members of the Solanaceae family. Transcription of CchSERK1 in plant tissues, measured by quantitative RT-PCR, was higher in stems, flowers, and roots but lower in leaves and floral primordia. During seed development, CchSERK1 was transcribed in all zygotic stages, with higher expression at 14 days post anthesis. During somatic embryogenesis, CchSERK1 was transcribed at all differentiation stages, with a high increment in the heart stage and lower levels at the torpedo/cotyledonal stages. Conclusion: DNA sequence alignments and gene expression patterns suggest that CchSERK1 is the C. chinense SERK1 homolog. Significant levels of CchSERK1 transcripts were found in tissues with cell differentiation activities such as vascular axes and during the development of zygotic and somatic embryos. These results suggest that CchSERK1 might have regulatory functions in cell differentiation and could be used as a biotechnological tool to study the recalcitrance of peppers to proliferate in vitro.

Genes involved in the deformations of the shoot apical meristem in somatic embryos of Capsicum chinense Jacq.

Somatic embryos (SE) of habanero pepper (Capsicum chinense Jacq.) represent persistent deformations in the shoot apical meristem (SAM), which inhibits their capacity to form organs and subsequently plants. In dicotyledonous plants, SAM is formed in the apex, between cotyledons and it plays a central role in postembryonic shoot organ formation. Based on the previous knowledge on the role of some families of gene in the formation, organization and maintenance of the SAM, the expression patterns of WUS, WOX2, NAM, STM, PIN1 and PIN7 genes were analysed, which would allow us to elucidate the possible implication of these genes in SAM deformations in the SE of C. chinense. The results show that the expression patterns of STM and PIN1 in the SE were completely opposite to the respective expression pattern obtained in zygotic embryos (ZE). Moreover, NAM and PIN7 showed an over accumulation of transcripts in SE, compared with ZE. This is the first time in the genus Capsicum that alterations in the expression pattern of key genes of the SE development are reported, as well as its possible implication in the persistent deformations of the SAM.

Plant Regeneration via Somatic Embryogenesis in Solanum nigrum L. (Black nightshade) (Solanaceae)

Aim: To study the effect of various plant growth regulators (PGRs) for induction of somatic embryogenesis and plantlet formation from cotyledon and leaflet explants in S. nigrum (night shade) an important medicinal plant used in treatment of digestive problems and skin infections. Place and Duration of Study: Department of Biotechnology, Kakatiya university, Warangal. Telangana, India, 3 years. Methodology: Cotyledon (0.8 cm2) and leaflet explants (0.8-1.0 cm2) from 3 week and 4 week old were cultured on MS medium supplemented with 30 g/L sucrose along with different concentrations of 0.5 mg/L BAP+NAA (0.5 – 6.0 mg/L) . Results: Maximum percentage of somatic embryogenesis was observed in cotyledon(89%) and leaf (98%) explants on MS medium augmented with 0.5mg/L BAP in combination with 2.0 mg/L NAA whereas the highest number of somatic embryos per explant (86 ± 0.19) was formed in leaflet explant. Conclusion: Somatic embryogenesis was induced from both cotyledon and leaf explants. Since it is threatened and medicinally important species S. nigrum, the present protocol can be used for its conservation and genetic transformation experiments.

Embryogenic potential of the callus of gabirobeira, Campomanesia adamantium(Cambess) O. Berg

Campomanesia adamantiumis a native plant species of Brazilian Cerrado with diverse economic potential and great medicinal importance. Its sexual propagation is impaired by the recalcitrance of its seeds, which prevents effective and profitable propagation. With the purpose of establishing commercial crops and minimizing the extractive use of vegetal resources, the aim of the present study was to induce embryogenic calli in nodal segments of gabirobeira, and to determine and characterize their embryogenic phase through the establishment of a growth curve based on cellular characteristics. Calli were induced using nodal segments inoculated in WPM culture medium without the addition of hormones (control) and with different concentrations of 2,4-D, IAA, IBA, NAA or picloram. Cytochemical and SEM analyses revealed cellular characteristics of the formation of meristematic centers that indicated 4.14 μM of picloram to be the best treatment for induction of embryogenic calli, and demonstrating their embryogenic potential. The treatment was used to establish a callus growth curve, from which it was inferred that calli should be transferred to new culture media on the 28thday to maintain cell viability.

Somatic embryos from Phalaenopsis classic Spotted Pink (Orchidaceae) protocorms / Embriões somáticos provenientes de protocormos de Phalaenopsis classic Spotted Pink (Orquidaceae)

ABSTRACT: The aim of the present study was to induce the formation of somatic embryos in protocorms from Phalaenopsis Classic Spotted Pink hybrids at two physiological maturation stages, namely: 80 and 120 days after seed inoculation (DASI). Protocorms were inoculated in ½ MS medium supplemented with 0.1 mg L-1 ANA and 3 mg L-1 TDZ. Protocorms were inoculated 120 days after sowing were more developed at the 15th cultivation day due to the formation of pro-embryogenic structures. It was possible seeing the formation of globular- and torpedo-stage somatic embryos at the 30th day of cultivation in somatic embryogenesis (SE) induction medium. The protocorms inoculated at the 80th DASI did not formed somatic embryos; they oxidized 20 days after cultivation in SE-induction medium. The formation of somatic embryos happened directly on the explant, thus characterizing a direct somatic embryogenesis. The embryos converted into plants when the somatic embryos were transferred to the nutrient medium containing no growth regulator. Therefore, it was concluded that the somatic embryos induction from protocorms 120 days after sowing was positive, since the embryos were able to become plants and presented vegetative organs with morphological traits similar to those of the matrix plant.

RESUMO: O presente estudo buscou induzir a formação de embriões somáticos em protocormos do híbrido Phalaenopsis Classic Spotted Pink em dois estádios de maturação fisiológica aos 80 e 120 dias após a inoculação das sementes (DAIS). Os protocormos foram inoculados em meio ½ MS suplementado com 0,1 mg L-1 de ANA e 3 mg L-1 de TDZ. Aos 15 dias de cultivo os protocormos, inoculados aos 120 dias após a semeadura, apresentavam estádio mais desenvolvido, apresentando formação de estruturas pró-embriogênicas. Aos 30 dias de cultivo em meio de indução de ES observou-se a formação de embriões somáticos na fase globular e torpedo. Os protocormos inoculados com 80 DAIS não evoluíram para a formação de embriões somáticos, ocorrendo a oxidação destes aos 20 dias após o cultivo em meio de indução de ES. A formação dos embriões somáticos ocorreu diretamente no explante, caracterizando uma embriogênese somática direta. Quando os embriões somáticos foram transferidos para o meio nutritivo sem regulador de crescimento, houve a conversão em plantas. Diante disso, conclui-se que a indução de embriões somáticos a partir de protocormos com 120 dias após a semeadura, foi positiva, em que os embriões obtidos apresentaram competência em converter-se em plantas, apresentando os órgãos vegetativos com características morfológicas satisfatórias.

Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition

Background: Somatic embryogenesis receptor-like kinase 1 (SERK1) is a cell membrane receptor active in different plant tissues and involved in cell differentiation activities including somatic embryogenesis. The identification of promoter elements responsible for SERK1 expression during the onset of somatic embryogenesis can be useful to understand the molecular regulation of the cell-to embryo transition, and these promoter elements represent biotechnological tools in plant organ tissue culture. Results: A −1,620 bp DNA sequence located upstream of the Coffea canephora SERK1 gene homologue (CcSERK1) was isolated, and then, different segments containing key response elements (REs) for somatic embryogenesis onset and development were fused to the uidA (encoding a ß-glucuronidase, GUS) reporter gene to evaluate its expression in transgenic leaf explants. DNA segments of −1,620 and −1048 bp in length directed uidA expression with patterns in leaf explants similar to those occurring during somatic embryogenesis. When a −792-bp fragment was used, uidA expression disappeared only in leaf explants and pro-embryogenic mass but persisted in developing embryos. No uidA expression was detected in any embryogenic stage when a −618-bp fragment was used. Conclusion: DNA deletions showed that a −1048-bp sequence located upstream of the CcSERK1 gene is sufficient to direct gene expression during the onset and the development of C. canephora somatic embryogenesis. The DNA segment located between −1048 and −792 bp (containing BBM and WUS REs) is needed for gene expression before embryogenesis onset but not during embryo development. The promoter segment between −792 and −618 bp (including GATA, ARR1AT, and ANT REs) regulates gene expression in developing embryos.

Embriogénesis no zigótica en Passiflora maliformis Nonzygotic / Embryogenesis in Passiflora maliformis

La importancia económica, ecológica y medicinal de las pasifloras ha fomentado el desarrollo de investigaciones multidisciplinarias en estas plantas. En cultivo in vitro, la embriogénesis no zigótica representa una alternativa para regeneración de plantas; no obstante, esta técnica ha presentado dificultades en la reproducibilidad de los protocolos, así como formación de ciertos embriones y plántulas anormales en algunas especies. En este estudio se evaluó la capacidad morfogénica de embriones zigóticos maduros (EZM) de Passiflora maliformis L para desarrollar embriones no zigóticos (ENZ), y posterior regeneración de plántulas. Se ensayó una etapa de inducción en la que se probaron 12 tratamientos en medio MS+VitB5 suplementado con 2,4-D, KIN y BA adicionados solos o combinados y, otra, de expresión en MS, MS con Carbón activado (CA) o MS con reguladores de crecimiento (RC) en concentraciones reducidas a 1/10 parte de las adicionadas en la etapa inducción. Altos porcentajes, 70%, de regenerantes fueron obtenidos en el medio suplementado con 1 mgL-1 BA y transferidos a medio de expresión MS, así como en el medio con 1 mgL-1 BA + 3 mgL-1 2,4-D, y transferidos a medio de expresión con CA, 60%. El 70% de las plántulas regeneradas se desarrollaron exitosamente en invernadero. Este trabajo es el primero que aborda la expresión del potencial embriogénico de EZM de P. maliformis induciendo exitosamente respuestas embriogénicas no zigóticas a través de rutas directas e indirectas; los resultados obtenidos son aplicables al conocimiento de la embriogénesis no zigótica en otras pasifloras, con fines de mejoramiento y uso comercial.

The economic, ecological and medical significance of passion fruits has encouraged the development of multidisciplinary research in this group. In vitro culture, non-zygotic embryogenesis represents an alternative for plant regeneration; however, this technique has presented difficulties in the reproducibility of protocols as well as the development of certain embryos and abnormal plantlets in some species. We assessed in Passiflora maliformis L the morphogenic capacity in the mature zygotic embryos (MZE) to develop nonzygotic embryos (NZE), and subsequent plantlets regeneration. We tested an induction stage with 12 treatments in MS + VitB5 medium supplemented with 2,4-D, KIN and BA added alone or combined and, another with a MS, MS with Activated Charcoal (AC) or MS with growth regulators (GR) in 1/10 concentrations reduced to 1/10 of those added in the induction stage. We obtained high percentages, 70%, of regenerants in the medium supplemented with 1 mgL-1 BA and transferred to the MS expression medium, as well as in the medium with 1 mgL-1 BA + 3 mgL-1 2,4-D, and transferred to the expression medium with AC, 60%. 70% of the regenerated seedlings were successfully grown in the greenhouse. This is the first research that addresses the expression of the MZE embryogenic potential in P. maliformis, inducing a successfully embryogenic non-zygotic responses through direct and indirect routes; the obtained results are relevant to the knowledge of non-zygotic embryogenesis in other passion fruits, for breeding and commercial uses.

Plantlet Regeneration from Unfertilized Ovule of Mandarin (Citrus reticulata)

The genus Citrus contains numerous fresh and processed fruit cultivars that are economically important worldwide, many genotypes are amenable to somatic embryogenesis, which became a key regeneration pathway in many experimental approaches of cultivar improvement. in this objective We aime at studying the effects of various culture media on the induction and the development of citrus somatic embryos.Callus cultures were initiated from the infertlized ovules of six varieties of mandarin (Anana, Lee, Murcott, Ortanique, Temple, and Wilking) within 3 media: MT (Murashig and Tuker, 1969) without hormones, MT + 1 mg/l BAP, MT + 1 mg/l Kinetin, the experiments show a highly significant effect (P < 0.001) of the culture media and genotype. No reactivity was observed on the MT environment in the absence of growth regulator, while the culture media MT in addition to 1 mg/l BAP gave the best results of induction of embryogenic callus induction. The induction of somatic embryogenesis was obtained on MT media without hormones. For the plantlets regeneration the favorable media was MT without hormones or added to ANA and active coal.

Date palm micropropagation: Advances and applications / Micropropagação de tamareira: Avanços e aplicações

ABSTRACT Date palm (Phoenix dactylifera L.) is a fruit tree resilient to adverse climatic conditions predominating in hot arid regions of the Middle East and North Africa. The date fruit contains numerous chemical components that possess high nutritional and medicinal values. Traditional propagation by offshoots is inefficient to satisfy current demands for date palm trees. Alternatively, micropropagation provides an efficient means for large-scale propagation of date palm cultivars. Both somatic embryogenesis and organogenesis, either directly or indirectly though the callus phase, have been demonstrated in date palm in vitro regeneration. Culture initiation commonly utilizes shoot-tip explants isolated from young offshoots. Recently, the immature inflorescences of adult trees were utilized as an alternative nondestructive source of explants. In addition to the nature of the explant used, successful plant regeneration depends on the cultivar, composition of the culture medium and physical status. Challenges of date palm micropropagation include long in vitro cycle, latent contamination, browning, somaclonal variation as well as ex vitro acclimatization and transplanting. A remarkable amount of research investigating these factors has led to optimized protocols for the micropropagation of numerous commercially important cultivars. This has encouraged the development of several international commercial tissue culture laboratories. Molecular characterization provides an assurance of genetic conformity of regenerated plantlets, a key feature for commercial production. This article describes date palm micropropagation protocols and also discusses recent achievements with respect to somaclonal variation, molecular markers, cryopreservation and future prospects.

RESUMO A tamareira (Phoenix dactylifera L.) é uma arvore frutífera adaptada à condições climáticas adversas predominantemente em regiões áridas do Oriente Médio e Norte Africano. As tâmaras possuem vários componentes químicos com alto valor medicinal e nutricional. A propagação tradicional por estacas não é suficiente para satisfazer a demanda por mudas e assim, a micropropagação apresenta-se como uma alternativa eficiente para a produção de mudas em larga escala. Embriogênese somática e organogênese, tanto direta quanto indireta via calos, tem sido usada para obter a regeneração in vitro de tamareira. O inicio do cultivo in vitro normalmente utiliza meristemas excisados de brotações jovens. Recentemente, inflorescências imaturas de árvores adultas são usadas como fonte alternativa de explantes não destrutiva. Além da origem do explante, o sucesso da regenerção depende do cultivar, da composição do meio de cultura e de condições físicas. Desafios na micropropagação de tamareira incluem um longo ciclo in vitro, contaminação, escurecimento do tecido, variação somaclonal além do enraizamento e aclimatização ex vitro. Diversos estudos investigando esses fatores tem conduzido à otimização de protocolos de micropropagação de inúmeros cultivares comerciais proporcionando o estabelecimento de vários laboratórios de cultura de tecidos de plantas. A caracterização molecular permite uma segura conformidade genética do material regenerado, considerado uma característica chave na produção comercial. Essa revisão descreve protocolos de micropropagação de tamareira e aborda as mais recentes conquistas relacionadas à variação somaclonal, marcadores moleculatres, criopreservação e perspectivas futuras.

Regeneración in vitro de plantas de piña ( Ananas comosus ) ecotipo amazónico Gobernadora / In vitro regeneration of Amazonian pineapple ( Ananas comosus ) plants ecotype Gobernadora

Resumen En Venezuela existen cultivares y ecotipos de piña (A. comosus) de importancia local, entre ellos los amazónicos, cultivados principalmente por los aborígenes Piaroa. Ellos siembran los propágulos lo cual restringe la disponibilidad de material para el cultivo a gran escala. Se abordó la limitación recurriendo al cultivo de tejidos vegetales para la propagación in vitro de plantas de piña, ecotipo amazónico Gobernadora, mediante embriogénesis somática (ES) y organogénesis adventicia (OA). El material vegetal empleado correspondió a secciones basales e intermedias de hojas. Sólo las secciones de base foliar (SBF) fueron morfogénicamente inducidas. El mayor número de vitroplantas (1,58 plantas/explante) se obtuvo del callo embriogénico inducido en medio MS con Picloram 10 mg.L-1 + Tidiazuron 2 mg.L-1, transferido a MS sin hormonas. En el proceso organogénico, se obtuvo el mayor número de plantas/explante (5) por vía directa en MS con ácido naftalenoacético 5 mg.L-1 + bencilaminopurina 0,25 mg.L-1, transferido a MS. Siendo este último el mejor sistema de cultivo in vitro por su productividad y por ser una ruta que minimiza la variación somaclonal.

Abstract There are a number of pineapple (Ananas comosus) cultivars and ecotypes of local commercial importance in Venezuela, among them the Amazonian ones, cultivated mainly by the aboriginal Piaroa, are of relevance. They sow the propagules, which restricts the availability of material for large-scale cultivation. This limitation was approached by plant tissue culture for in vitro propagation of Amazonian pineapple plants, Gobernadora ecotype, through somatic embryogenesis (ES) and adventitious organogenesis (OA). Basal and intermediate sections of leaves were tested. Only the leaf base sections (FBS) were morphogenically induced. The highest number of vitroplants (1.58 plants / explant) was obtained from the embryogenic callus induced in MS medium with Picloram 10 mg.L-1 + Thidiazuron 2 mg.L-1, transferred to MS medium without hormones. In the organogenic process, the highest number of plants / explants (5) was obtained directly in MS with naphthaleneacetic acid 5 mg.L-1 + benzylaminopurine 0.25 mg.L-1, transferred to MS. The latter being the best in vitro culture system due to its productivity and for being a method that minimizes somaclonal variation.

ESTABLECIMIENTO DE UN SISTEMA DE SELECCIÓN IN VITRO DE VARIEDADES VENEZOLANAS DE ARROZ (Oryza sativa L.) RESISTENTES AL HONGO Pyricularia grisea / Establishment of an in vitro Selection of Venezuelan Cultivars of Rice (Oryza sativa L.) Resistant to the Fungus Pyricularia grisea

RESUMEN. El arroz, luego del trigo, es el cereal más importante del mundo, sin embargo, es susceptible al ataque de numerosos patógenos, siendo Pyricularia grisea, el más dañino. Este trabajo estableció un sistema de selección in vitro de variedades venezolanas a P. grisea, optimizando el sistema de regeneración por embriogénesis somática (inducción, regeneración y estrés por desecación), sometiendo el callo embriogénico (E) a presión de selección del filtrado crudo "FC" a través de cambios a la misma concentración "MC" o por incrementos progresivos en la concentración "IPC", obteniendo plantas tolerantes al fitopatógeno. El máximo porcentaje de inducción de callo embriogénico oscilo entre 30-65 %, en las cuatro variedades (Araure-4 y Venezuela 21: 1 mg.L-1 + 2 mg.L-1 K; Cimarrón: 3 mg.L-1 + 2 mg.L-1 K; Centauro: 1 mg.L-1 + 2 mg.L-1 BAP), mientras que la regeneración estuvo entre 44 y 52 % con 0.5 mg.L-1 + 2 mg.L-1 BAP a 48 h de desecación para Centauro y 24 h para las otras tres variedades. La frecuencia regenerativa de los callos E disminuyo a medida que se incrementó la concentración del FC, independientemente del método de presión selectiva. El promedio de plantas diferenciada por variedad, dependió del método de presión usado, siendo el sistema IPC (25 % para Centauro y 50 % para las otras tres variedades) el que mostro los resultados más favorables, evidenciándose que para las condiciones de los sistemas selectivos de FC evaluados, la resistencia expresada a nivel de planta in vivo no corresponde a la encontrada in vitro.

ABSTRACT. Rice after wheat is the most important cereal in the world, however, it is susceptible to attack by many pathogens, which Pyricularia grisea being the most harmful. In the current study we established an in-vitro selection system of P. grisea on Venezuelan rice varieties. A somatic embryogenesis regeneration system was optimized (induction, regeneration and desiccation) to expose embryogenic callus (E) to crude filtrate "CF" selection pressure through changes at the same concentration "SC" or progressive concentration increments " PIC "of P. grisea, and thus obtain plants tolerant to the pathogen. The maximum percentage of embryogenic callus induction ranged between 30-65 % in the four varieties (Araure-4 and Venezuela- 21: 1 mg.L-1 + 2 mg.L-1 K; Cimarron: 3 mg.L-1 mg.L-1 + 2 K; Centauro: 1 mg.L-1 + 2 mg.L-1 BAP) while regeneration was between 44 and 52 % with 0.5 mg.L-1 + 2 mg.L- 1 BAP 48 h of desiccation for Centauro and 24 h for the other three varieties. The regeneration frequency of embryogenic callus decreased as the concentration of FC increased, regardless of the method of selective pressure. The average differentiated plants per variety, depended on the pressure method used, with the PIC system being the most favorable (25 % for Centauro and 50 % for the other three varieties). The result demonstrated, to resistance expressed for plants in vivo does not correspond to the in vitro conditions

EVALUACIÓN DE UN SISTEMA DE REGENERACIÓN POR EMBRIOGÉNESIS SOMÁTICA DE NEEM (Azadirachta indica) / Evaluation of a Somatic Embryogenesis Regenetation System for Neem (Azadirachta indica)

Azadirachta indica, también conocida como neem, es una especie arbórea leñosa perteneciente a la familia Meliaceae, de gran importancia en diversas disciplinas científicas, tales como la forestal y la médico-farmacéutica. Se estableció un método para la propagación in vitro de esta planta, evaluándose como explantes, secciones foliares de vitro-plantas, cotiledones y esquejes. Se emplearon medios semisólidos con combinaciones variables de la citocinina 6-benzylaminopurina (BAP) y las auxinas ácido 2,4-diclorofenoxiacético (2,4-D) y ácido indolacético (AIA). Se observó la formación de callo regenerativo, a partir del cual se generó embriogénesis somática primaria y secundaria, mediante los reguladores de crecimiento BAP (1,0 mg.L-1) y 2,4-D (0,2 mg.L-1), mientras que la formación de callo no regenerativo fue promovida por concentraciones mayores a 0,3 mg.L-1 de 2,4-D. De los explantes evaluados, la mayor frecuencia de regeneración de plantas (~67 %) se presentó con secciones cotiledonares.

Azadirachta indica, also known as neem, is a woody tree species belonging to the Meliaceae family, of great importance in various scientific disciplines, such as forestry, medicinal and pharmacist. It was established a method for in vitro propagation of this plant, evaluating explants as leaf sections of vitro-plants, cotyledons and stem cuttings, using semi-solid medium with various combinations of cytokinin 6-benzylaminopurine (BAP) and auxins 2,4-dicholorophenoxyacetic acid (2,4-D) and indol-3-acetic acid (IAA). Regenerative callus formation was observed, from which primary and secondary somatic embryogenesis was generated by growth regulators BAP (1.0 mg.L-1) and 2,4-D (0.2 mg.L-1), whereas non-regenerative callus formation was promoted by concentrations higher than 0,3 mg.L-1 of 2,4-D. Between the used explants, the highest frequency of plant regeneration (~ 67%) presented from cotyledon sections.

POLYPHENOLS DISTRIBUTION AND RESERVE SUBSTANCES ANALYSIS IN CACAO SOMATIC EMBRYOGENESIS / Análisis de distribución de polifenoles y sustancias de reserva en embriogénesis somática de cacao

In order to understand the causes of lack of regeneration in cacao somatic embryos, two cacao varieties with different responses to regeneration potential were described based on their capacity to store different compounds. It is well known that seed reserves play a central role in the regenerative capability of somatic embryos; thus, we followed histochemical changes and reserve fluctuations of proteins, polysaccharides and polyphenols during somatic embryogenesis (SE) in the two cacao varieties. The study showed that, in somatic embryos of the regenerating variety, polyphenols were localized mainly in the periphery of the embryo (epidermal cells) and proteins were the main storage substance in the embryo expression medium, while the non-regenerating variety had a high presence of polysaccharides with random distribution of polyphenols at the end of the embryo induction step.

Dos variedades de cacao con diferentes respuestas a la regeneración fueron descritas en función de su capacidad para almacenar diferentes compuestos, con el fin de aproximarse al entendimiento de las causas de la falta de regeneración en embriones somáticos de cacao. Es bien sabido que las reservas de semillas desempeñan un papel central en la capacidad de regeneración de embriones somáticos; por tanto, se realizó un seguimiento de cambios histoquímicos y fluctuaciones de reserva de proteínas, polisacáridos y polifenoles durante la embriogénesis somática (SE) en dos variedades de cacao. El estudio mostró que, en los embriones somáticos de la variedad regenerante, los polifenoles se localizaron principalmente en la periferia del embrión (células de la epidermis) y las proteínas fueron el componente principal de almacenamiento en el medio de expresión de embriones, mientras que la variedad no regenerante tenía una alta presencia de polisacáridos y una distribución aleatoria de los polifenoles en el final de la etapa de inducción de embriones.

Efficient In Vitro Somatic Embryogenesis and Plant Regeneration from Mature and Immature Embryos of Wheat ( Triticum aestivum L.)

ABSTRACT An efficient regeneration system is a pre-requisite for the application of genetic transformation and functional genomics study of important plants. In this study, the effect of different factors (plant growth regulators, casein hydrolysate, aspartic acid and ascorbic acid) on in vitro embryogenesis and regeneration of Arta, Bahar and Zagros cultivars from mature and immature explants were investigated. Immature and mature embryos were dissected from disinfected seeds 20-25 days after pollination and imbedded mature seeds, respectively, and cultured on MS (Murashige and Skoog) medium supplemented with different compounds. The results showed that immature embryos expose high capacity of embryogenesis and regeneration in comparison with mature embryos. There were significant differences between cultivars in terms of the percentage of callus induction and regeneration. Plant growth regulators had significant effect on percentage of callus induction in mature explants and percentage of regeneration from both explants. In immature explants, the highest percentage of regeneration (65%) was achieved with the Arta cultivar calli derived from MS medium supplemented with 1mg/L 2,4-D, 2 mg/L Picloram and 200 mg/L casein hydrolysate, and subcultured on MS medium. Also, the highest percentage of regeneration (52.38%) from mature embryo explants was achieved in the Arta cultivar with callus induction on MS medium supplemented with 1 mg/L 2,4-D, 2 mg/L Picloram and 200 mg/L casein hydrolysate and regeneration on MS medium containing 0.05 mg/L NAA.

Recurrent Somatic Embryogenesis and Plantlet Regeneration in Psidium guajava L

A simple and efficient protocol for recurrent somatic embryogenesis and plant regeneration is one of the prerequisites for genetic improvement of guava. An efficient reproducible regeneration somatic embryogenesis protocol was developed in four genotypes of Psidium guajava L. using immature zygotic embryo as starter explant. Somatic embryogenesis induction was obtained on MS basal medium supplemented with 2.0 mgL-1 2, 4-D, 400 mgL-1 L-glutamine, 6% sucrose and 500 mgL-1 Malt extract. Following SE induction different developmental stages of somatic embryos (Globular, heart-shaped, torpedo, cotyledonary) was directly obtained and further recurrent embryogenesis also obtained upon prolonged incubation in induction media. Addition of polyethylene glycol (50 mgL-1) significantly improved the embryos maturation in MS supplemented with and 3% sucrose. The regeneration in MS medium supplemented with BAP (0.5 mgL-1), NAA (0.2 mgL-1), casein hydrolysate (100 mgL-1) and 3% sucrose. High plant regeneration frequency and intensity of somatic embryos (58.5%) obtained. Plant maturation on MS medium supplemented with BAP 2.0 mgL-1 with 2% sucrose. The rooted plants was successfully acclimatize in the greenhouse with a survival rate of 85%. This somatic embryogenesis protocol developed would be helpful in establishment of genetically modified guava aimed at seedlessness, increased shelf life and wilt disease.

POTENCIAL DE REGENERACIÓN DE Psychotria ipecacuanha (Rubiaceae) A PARTIR DE CAPAS DELGADAS DE CÉLULAS / Regeneration Potential of Psychotria ipecacuanha (Rubiaceae)from Thin Cell Layers

Conociendo las propiedades medicinales de la especie vegetal Psychotria ipecacuanha (Brot.) Stokes, su crítico estado de conservación, así como las dificultades que presenta para la propagación efectiva, el presente estudio tuvo como objetivo evaluar su potencial de propagación por los sistemas de regeneración in vitro, organogénesis y embriogénesis somática. Para este propósito, capas delgadas de células (CDCs) de tallos y de hojas, así como segmentos foliares fueron sometidos a diferentes tratamientos con reguladores de crecimiento y condiciones de luz. Además se estableció el efecto de diferentes longitudes de onda vía diodos emisores de luz (LEDs), sobre la regeneración en estos explantes y nudos provenientes de plantas in vitro. Los resultados obtenidos mostraron que los segmentos de hoja y las CDCs de tallo sembrados en el medio de cultivo MS suplementado con las combinaciones de los reguladores de crecimiento IBA + BAP e IBA + TDZ formaron embriones somáticos y brotes. Los cortes histológicos realizados corroboraron estos dos tipos de origen. Se encontró que bajo la condición lumínica 16/8, se alcanzaron los mejores resultados de inducción de brotes y embriones. En cuanto al efecto de las diferentes longitudes de onda de luz, se encontró que las correspondientes al rojo, verde y blanca, favorecieron el crecimiento y desarrollo de brotes y la inducción de embriones somáticos. El desarrollo de los brotes a partir de los nudos no presentó diferencias estadísticas entre los tratamientos con LEDs, por lo que se recomienda el uso de la luz blanca continua y con fotoperiodo durante el proceso de multiplicación y desarrollo de éstos.

Knowing the medicinal properties of the plant specie Psychotria ipecacuanha (Brot.) Stokes, its critical condition and the difficulties for its effective propagation, the present study aimed to assess the potential of propagation of P. ipecacuanha by in vitro systems of regeneration, organogenesis and somatic embryogenesis. For this purpose, thin cell layers (TCL) of stems and leaves, as well as leaf segments were subjected to different treatments of plant growth regulators and light conditions. Furthermore, the effect of different wavelengths via light emitting diodes (LED's), was established for the regeneration in these explants and nodal explants from in vitro plants. The results showed that leaf segments and stem TCL, cultured in MS medium supplemented with the combinations of growth regulators IBA + BAP and IBA + TDZ, formed somatic embryos and shoots. The histological sections supported the two types of source. It was found that the best results in shoot induction and embryos were achieved under the light condition 16/8-h light/ dark. Regarding the effect of different wavelengths, it was found that those corresponding to red, green, and white supported the growth and shoot development as well as somatic embryos induction. The shoots development from the nodal explants did not show statistical differences between LEDs treatments, so the use of a continuous white light and photoperiod is recommended during their multiplication and development.

ESTABLECIMIENTO DEL SISTEMA DE REGENERACIÓN POR EMBRIOGÉNESIS SOMÁTICA DE Azadirachta indica A. Juss. / Establishment of Regeneration System for Somatic Embryogenesis of Azadirachta Indica A. Juss.

Azadirachta indica, es una planta con múltiples aplicaciones tanto forestal como farmacológica. Por ende, el establecimiento del sistema de cultivo in vitro por embriogénesis somática ofrece diversas y variadas ventajas, tales como obtener plantas altamente productivas en metabolitos. En este estudio, se utilizaron secciones foliares y cotiledonares, inducidas en medios MS (1962) suplementados con: BAP sólo y combinado con ANA / 2,4-D, TDZ sólo y con ABA. La regeneración fue con MS sólo o con K + AIA y BAP + AIA. Como resultado se estableció un sistema eficiente con secciones de cotiledones, observándose organogénesis a bajas concentraciones de BAP, mientras a altos niveles de BAP (2,5 mg.L-1), así como con TDZ + ABA (0,02 + 1 mg.L-1) respectivamente favorecieron la embriogénesis somática primaria y secundaria en un 96 % y 71 % respectivamente. La regeneración fue 71 % con MS, mientras que el enraizamiento fue de 86,67 % con MS½, obteniéndose plantas completas a corto plazo.

Azadirachta indica, is a plant with multiple forest and pharmacological application. Therefore, the establishment of in vitro culture system for somatic embryogenesis offers several distinct advantages such as obtaining highly productive plant metabolites. In this study, were used sections cotyledon and leaf, induced on MS medium (1962) supplemented with: BAP alone and combined with NAA / 2,4-D, TDZ alone and ABA. Regeneration was with MS alone or with K + BAP + IAA and IAA. As a result was established an efficient system with cotyledon sections, being observed organogenesis at low concentrations of BAP, while high levels of BAP (2.5 mg.L-1) with 96 % and TDZ + ABA (0.02 + 1 mg.L-1) with 71 %, favoring the primary and secondary somatic embryogenesis. Regeneration was 71 % with MS; rooting was 86.67 % with MS½, presenting whole plants obtained short term.

FORMACIÓN DE EMBRIONES SOMÁTICOS A PARTIR DE SEMILLAS INMADURAS EN Sorghum bicolor VARIEDAD CIAP 132-R / Formation of Somatic Embryos from Immature Seeds of Sorghum bicolor Sorghum Variety CIAP 132-R

Existen varios protocolos de regeneración de plantas vía embriogénesis somática de Sorghum bicolor (L.) Moench, sin embargo los porcentajes de formación de callos con estructuras embriogénicas y regeneración de plantas son bajos. Es por ello que esta investigación tuvo como objetivo generar embriones somáticos en sorgo rojo variedad CIAP 132-R. Se ensayaron diferentes concentraciones de 2,4-D para la formación de callos, así como tres concentraciones de ácido ascórbico para eliminar la exudación de compuestos fenólicos por el explante. También para la formación de los embriones somáticos a partir de los callos se evaluaron diferentes concentraciones de 2,4-D y 6-BAP. El mayor porcentaje de formación de callos (57,5 %) se alcanzó con 18,1 µM de 2,4-D. Con la adición al medio de cultivo de 50,0 mg.l-1 de ácido ascórbico fue posible eliminar los compuestos fenólicos en el explante y en el medio de cultivo, además permitió incrementar el porcentaje de formación de callos con estructuras embriogénicas hasta un 95 %. El número mayor de embriones somáticos por callo se alcanzó en el medio de cultivo con concentraciones de 4,52 µM de 2,4- D, combinada con 2,22 µM de 6-BAP. Por primera vez, se logró la formación eficiente de embriones somáticos a partir de los callos obtenidos de semillas inmaduras germinadas como explante inicial en la variedad CIAP 132-R.

Several protocols of plant regeneration via somatic embryogenesis from Sorghum bicolor (L.) Moench have been development, however the percentage of calluses with embryogenic structures and plant regeneration are low. Therefore this study aimed to generate somatic embryos in red sorghum variety CIAP 132-R. Different concentrations of 2,4-D for callus formation, and three concentrations of ascorbic acid to remove phenolics exudation were assayed by explant. For the formation of embryos different concentrations of 2,4-D and 6-BAP were evaluated. The highest percentage of callus formation (57.5 %) was achieved with 18.1 µM 2,4-D. With the addition to the culture medium of 50.0 mg.l-1 of ascorbic acid was possible to eliminate the phenolic compounds in the explant and in the culture medium; also it allows increasing the percentage of calluses with embryogenic structures up to 95 %. The highest number of somatic embryos per callus was achieved with a reduction in the culture medium of 2,4-D to 4.52 µM in combination with 2.22 µM 6-BAP. For the first time, the efficiency of somatic embryo formation was obtained from the freshly germinated sprouts of immature seeds as initial explant CIAP 132-R.

In Vitro Propagation of Chamomilla Recutita From Capitulum Inflorescence: A Medicinal Plant With Multiple Therapeutic Applications

A protocol has been developed for induction of somatic embryogenesis from whole inflorescence explants of Chamomilla recutita L. (chamomile). Chamomile is a well-known medicinal plant from the Asteraceae family often referred to as the “star among medicinal species.” Nowadays, it is a highly favoured medicinal plant in folk and traditional medicine. Its multitherapeutic, cosmetic and nutritional values have been established through the years of traditional and scientific use and research. Chamomile has an established domestic (Indian) and international market, which is increasing day by day. Among the various major constituents, α-bisabolol and chamazulene have been reported to be more useful than others. Chamazulene occurs in the capitula of the flowers in minute quantities and has been demonstrated to exert antiinflammatory activity in-vivo. Moreover, chamomile is a seasonal 4-5 months winter crop in India but is extensively required in various medicinal applications. Therefore, to increase the overall yield of this plant, its in-vitro propagation is needed. In the present study, somatic embryos were developed from capitulum explants after 2-4 weeks of culture on MS medium supplemented with 26.8 µM NAA and 11.5 µM Kin. The somatic embryos were further subcultured in-vitro, where new plantlets regenerated from embryos. It is concluded that in-vitro propagation is possible in case of chamomile and can be used to increase the overall yield of chamazulene present in the capitula of flowers as well as augment the overall yield of this important plant, which is conventionally propagated by seeds.