Topophysis and Growth Regulators Effects on Buds and Zygotic Embryos Regeneration of African Bush Mango Tree Cultured In vitro

Background: African bush mango tree is an important fruit plant with high nutritional, medicinal, and commercial values. However, its seedling system remained a deep understanding. This study aimed to evaluate the effect of topophysis and growth regulators on the reactivity of different types of buds and zygotic embryos of wild mango. Methodology: Ripe fruits from two local varieties (Wossro and Sissro) were pulped. The nuts were extracted and dried for one week under greenhouse. Zygotic embryos were excised from nuts and disinfected with the bleach solution (NaClO 10% with 8% active chloride) for 10 min following by three successive rinsing using sterilized water. The second set of nuts was cultivated for under greenhouse in the polybags containing the sand. One month later, buds from different positions (apical, axillary and cotyledonary) were excised and disinfected with NaClO 10% for 10 min follow by the immersion in a mercuric chloride 0.1% added with two drops of Tween 20 especially for axillary and cotyledonary buds for 5 min while 0.01% of mercuric chloride was used for apical buds. The explants were cultured on 糓S and 絎PM media supplemented with BAP, KIN at different concentrations (0.2 mg/L and 3.5 mg/L respectively) and NAA (0.05 mg/L). Results: The best sprouting rate was obtained with the variety Wossro which showed a high bud break rate (26.47%) against (9.88%) for the variety Sissro. The topophysis significantly influenced the response of the buds in tissue culture. 24.48% of axillary buds were sprouted on � MS medium + 3.5 mg/L BAP+ 0.05mg/L NAA. The in vitro germination of embryos was significantly (p? 0.05) influenced by the genotype of the variety. 50.76% of buds were germinated for Wossro while 18.32% were germinated for Sissro. � MS + 0.2 mg/L KIN has significant influenced the plants growth and development. Implication: The findings will help to improve the regeneration rate and plantlets production of African bush mango tree.

Genetic transformation of the Brazilian BR 451 maize variety by the Agrobacterium tumefaciens method / Transformação genética da variedade brasileira de milho BR 451 pelo método da Agrobacterium tumefaciens

ABSTRACT: The asexually gene introduction by genetic engineering has brought enormous possibilities to innovate plant breeding. However, principally because of the low in vitro response, genetic transformation has been restricted to only certain genotypes of agronomically significant species. With the objective of establishing a protocol for genetically transforming the Brazilian BR 451 maize variety through Agrobacterium tumefaciens, it was studied the capacity of plant regeneration in vitro from embryogenic calli cultivated in three regeneration media, each having different growth regulators. It was also evaluated the temperature stress effect on the transformation of the immature embryos with A. tumefaciens EHA 101 containing the plasmid pTF102 with uidA and bar genes. The BR 451 variety embryos and those of the Hi-II hybrid (control) were exposed to three treatments applied as they were being infected with the agrobacteria (a) infection at 25°C; (b) infection at 40°C; (c) pretreatment at 40°C for 5 seconds followed by infection at 25°C. Transformation was determined by uidA gene expression and through the callus resistant to the herbicide Bialaphos® formation. Embryos infected at 40°C showed a higher degree of genetic transformation in the Hi-II, although the same was not noted in BR 451. When growth regulators were added to the culture medium the number of regenerated BR 451 plants showed no increase.

RESUMO: A introdução de genes de forma assexual por meio da engenharia genética tem ampliado as possibilidades do melhoramento genético vegetal. No entanto, devido principalmente a baixa resposta in vitro, a transformação genética tem se limitado a poucos genótipos das espécies de interesse agronômico. Visando estabelecer protocolo de transformação genética da variedade de milho BR 451 via Agrobacterium tumefaciens, foi estudada a capacidade de regeneração de plantas in vitro a partir de calos embriogênicos cultivados em três meios de regeneração contendo diferentes reguladores de crescimento. Também foi avaliado o efeito do estresse de temperatura na transformação de embriões imaturos com a A. tumefaciens EHA 101portadora do plasmídeo pTF102 que contém os genes uidA e bar. Para tal, três tratamentos foram aplicados aos embriões da variedade BR 451 e do híbrido Hi-II (controle) durante a infecção com a agrobactéria: (a) infecção em 25oC; (b) infecção a 40oC; (c) pré-tratamento de 40oC por cinco segundos seguido por infecção em 25oC. A transformação foi avaliada mediante a expressão do gene uidA e a formação de calos resistentes ao herbicida Bialaphos®. A infecção de embriões a 40oC aumentou a transformação genética em Hi-II, mas não em BR 451. A adição de reguladores de crescimento no meio de regeneração não incrementou o número de plantas regeneradas.

A rapid and efficient protocol for in vitro multiplication of genetically uniform Stevia rebaudiana (Bertoni).

Stevia rebaudiana (Bertoni), commonly called candy leaf or sweet leaf, endemic to South America, is an important medicinal plant. As a source of low calorie natural sweetener ‘stevoside’, it is used in obesity, diabetes, treatment of heartburn and tooth decay, and also serves as a food supplement. Large scale commercial propagation of S. rebaudiana demands a suitable protocol. Here, we propose an improved protocol for in vitro multiplication of S. rebaudiana from nodal explants. In this protocol, the effect of laboratory grade urea on multiple shoot induction from nodal explants was studied. The nodal explants were initially cultured on Murashige and Skoog (MS) basal media for 2 weeks which facilitated the axillary bud break. Further, culturing of these explants on MS medium fortified with 6 benzyl aminopurine (BAP) (2 mg/L) and Naphthalene acetic acid (NAA) (1 mg/L) with and without urea (5 mg/L) for a period of 40 days revealed maximum shoot production of 44.56 from a single nodal explant in media supplemented with urea as compared to 22.44 without urea. The differences in the number of shoots produced were significant and these shoots readily rooted in MS media with NAA (4 mg/L). Primary and secondary hardening was successful in these plants. There were no visible morphological abnormalities observed in the micropropagated plantlets. Genetic analysis from random samples also revealed that these plants are genetically uniform. The advantage of the present protocol is that the complete process of multiple shoot induction, rooting and hardening could be completed within a period of 6 months as compared to the existing protocols.

Investigating the In vitro Regeneration Potentiality of Three High Yielding Indica Rice (Oryza sativa L.) Varieties.

Aims: An efficient and reproducible In vitro regeneration protocol is vital for varietal improvement research. The current research was conducted to optimize the callus induction, shoot and root regeneration of three indica rice varieties. Place, Duration and Design of Study: The experiment was conducted in the tissue culture and biotechnology laboratory of the department of Genetics and Plant Breeding of Bangladesh Agricultural University using completely randomized experimental design. Methodology: Dehusked mature seeds of three indica rice varieties namely, BRRI dhan28, BRRI dhan29 and BINA dhan6 were cultured In vitro in MS medium supplemented with different concentrations and combinations of phytohormones. Results: The callus induction ranged from 14 - 84% which showed a general increasing trend with the increase in the concentration of 2,4-Dichlorophenoxyacetic acid (2,4-D) starting from 1.0 mg L-1 till 2.5 mg L-1. A further increase in the concentration of 2,4-D to 2.5 mg L-1, however, decreased the percentage of callus induction in all three varieties. MS medium supplemented with 2.5 mg L-1 2,4-D and 0.5 mg L-1 6-Benzylaminopurine (BAP) was better than any other composition for callus induction. For size of callus and nature of callus, however, MS medium supplemented with 2.0 mg L-1 2,4-D and 0.5 mg L-1 BAP was found to perfume best. The highest percentage of callus induction was observed in the variety BRRI dhan29 (84%) followed by BRRI dhan28 (74%). Almost all the varieties produced yellowish and compact calli except BINA dhan6 which produced creamy and friable calli. The desiccation treatment has shown to increase size but decrease the compactness of the callus. The differences are, however, not statistically significant. MS medium supplemented with 0.6 mg L-1 1-Naphthaleneacetic acid (NAA) and 6 mg L-1 Kinetin (Kn) showed highest shoot regeneration in BRRI dhan29 (85%) followed by BRRI dhan28 (60%). Higher frequency of root formation was observed in all three varieties using Indole-3-butyric acid (IBA). The survival rate of the plantlet in acclimatization chamber (96%) and in field condition (93.33%) was higher for BRRI dhan29. BINA dhan6 has shown the least regeneration potentiality for all the aforementioned traits. Conclusion: Of the three varieties, BRRI dhan29 and BRRI dhan28 has shown higher regeneration potentiality. This optimized protocol will thus be useful in genetic improvement of these varieties using biotechnological manipulations.

Avances en la propagación vía embriogénesis somática de Psychotria ipecacuanha (Brot. ) Stokes, planta medicinal en peligro crítico / Advances in vitro propagation via somatic embryogenesis of Psychotria ipecacuanha (Brot. ) Stokes, medicinal plant critically endangered

Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae Juss), es una especie vegetal con reconocidas propiedades medicinales. Esta especie se encuentra en peligro crítico de extinción, debido a la sobreexplotación de las poblaciones naturales. Conociendo además las dificultades para su propagación por medio de semillas (debido a la baja tasa de germinación y elevada muerte prematura de las plántulas) y por vía vegetativa (lento crecimiento), el presente trabajo tuvo como objetivo evaluar el potencial de propagación vía embriogénesis somática directa. Segmentos de hojas jóvenes de plantas mantenidas en casa malla fueron desinfectados y sembrados en el medio de cultivo MS (Murashige y Skoog) suplementado con diferentes concentraciones y combinaciones de reguladores de crecimiento. Las combinaciones IBA y BAP a 1 y 2 mg/L y 2 y 1 mg/L, respectivamente; mostraron ser efectivas en la formación de embriones somáticos en esta especie. La procedencia de la planta donadora parece tener influencia en la sensibilidad del tejido foliar a la respuesta. Este es el primer reporte de embriogénesis somática directa para esta especie y el primer reporte de cultivo in vitro de poblaciones colombianas.

Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae Juss), is a specie with known medicinal properties. This species is critically endangered due to overexploitation of natural populations.Besides knowing the difficulties in propagation by seed (due to the low rate of germination and high seedling premature death) and by vegetative (slow growth), the present study evaluated the potential for propagation by using direct somatic embryogenesis.Young leaves segments from plants cultivated in a greenhouse were disinfected and planted in MS medium (Murashige y Skoog), supplemented with different concentrations of growth regulators. IBA and BAP combinations at 1 and 2 mg / L and 2 and 1 mg / L, respectively, shown to be effective in the formation of somatic embryos in this species. The origin of the donor plant seems to influence foliar tissue sensitivity to the answer. This is the first report of direct somatic embryogenesis for this species and the first report of in vitro culture of Colombian populations.

In vitro regeneration of Coelogyne nervosa A.Rich. and Eria pseudoclavicaulis Blatt., threatened orchids of Western Ghats, India.

The seeds of C. nervosa and E. pseudoclavicaulis were germinated asymbiotically on Knudson C (KC) and Schenk and Hildebrandt basal medium (SH). Growth regulators such as 2,4-Dichlorophenoxyacetic acid (2,4-D) individually and in combinations with benzyladenine (BA) and kinetin were used for callus induction from the protocorm like bodies. Coelogyne nervosa showed maximum (90%) callus induction in Knudson C medium supplemented with 2,4-D (2.26 µM) and Eria pseudoclavicaulis showed 60% callus induction in Schenk and Hildebrandt medium supplemented with 2,4-D (2.26 µM). Calli developed a route of production of protocorm-like bodies and eventually developed into plantlets on transfer to growth regulator free half strength basal medium. The well rooted plants were hardened successfully in the potting mixture containing coconut husk, charcoal, and brick pieces in the ratio 2:1:1.

Cultivo in vitro de Brosimum gaudichaudii Tréc. (Moraceae) / In vitro cultivation of Brosimum gaudichaudii Tréc. (Moraceae)

O objetivo desta pesquisa foi avaliar o potencial organogenético de entrenós, discos foliares, e raízes de Brosimum gaudichaudii utilizando 12 diferentes combinações dos fitorreguladores 6-benzilaminopurina e ácido naftaleno acético, em meio MS (diluído à metade), sólido (6,5 g de ágar), e com 20 g.L-1 de sacarose. Independentemente das combinações hormonais testadas verificou-se a formação de calos friáveis (2 a 20 mm de diâmetro) em 90% dos entrenós usados como explantes. No entanto, os tratamentos testados não foram capazes induzir calos ou gemas em raízes e em discos foliares. O estudo anatômico revelou a formação de meristemóides nas regiões mais externa e mais interna dos calos. Os resultados obtidos poderão servir de base para novos testes de indução de calos na espécie.

The objective of this research was to evaluate the organogenic potential of internodes, leaf discs and roots of Brosimum gaudichaudii using 12 different combinations of the plant growth regulators 6-benzylaminopurine and naphthalene acetic acid in MS medium (half strength), solid medium (6.5 g agar) and sucrose medium (20 g.L-1). Regardless the hormonal combination tested, we observed the formation of friable calluses (2 - 20 mm wide) in 90% of the internode explants. However, the treatments were not able to induce callus or buds on roots and leaf discs. The anatomical analysis revealed meristemoid formation in the outer and inner regions of the calluses. The results may serve as the basis for further testing of callus induction in this species.

Efficient Regeneration of Eucalyptus urophylla x Eucalyptus grandis from Stem Segment

The aim of the present study was to establish an efficient regeneration system for the hybrid E. urophylla x E. grandis by means of organogenesis. Stem segments from seedlings were used as explants and cultured in a modified Murashige and Skoog medium (MS), supplemented with 13.2 µM N-phenyl-N'-[6-(2-chlorobenzothiazol)-yl] urea (PBU) and 0.285 µM indole-3-acetic acid (IAA). PBU was a useful growth regulator. After cultivating for 5 d, 96% explants formed callus. After 30 d, the calli obtained were transferred to MS medium containing different combinations of 6-benzyladenine (BA) and naphthalene acetic acid (NAA). Compared with other growth regulator combinations, PBU stimulated more vigorous calli and restrained browning. In addition, a large percentage (91.3%) of the calli induced by PBU showed adventitious buds formation. Shoot elongation was then stimulated on half-strength MS mineral salts medium supplemented with 6.6 µM PBU and 0.285 µM IAA for 20 d. For rooting, the elongated shoots were cultivated on root induction medium containing 2.46 µM indole-3-butyric acid (IBA). Plantlets were then successfully transplanted to a greenhouse. This procedure represented an efficient way of E. urophylla x E. grandis plant regeneration.

In vitro Regeneration of Plantlets from Leaf and Nodal explants of Aristolochia indica L.- An Important Threatened Medicinal Plant

Objective: An efficient reproducible protocol has been developed for in vitro regeneration of plantlets from leaf and nodal explants of Aristolochia indica L. Methods: Wild grown plants Aristolochia indica L. were collected and grown in the departmental garden. Leaf and nodal segments (0.5-1.0 cm) from young healthy plants were first washed thoroughly under running tap water for 15 - 20 minutes and then treated with liquid detergent [5% (v/v) Tween-20] for 5-10 minutes. Later these explants were washed with double-distilled water for 5 minutes. Subsequently, explants were immersed in 70% (v/v) ethanol for 2 - 3 minutes and washed with sterile glass double distilled water for 2-3 times. Eventually, the explants were treated with an aqueous solution of 0.1% (w/v) HgCl2 for 1 - 2 minutes and rinsed for two-to-three times in sterile ddH2O to remove all traces of HgCl2. The sterilized explants were inoculated aseptically onto solid basal Murashige and Skoog’s medium with different concentrations and combinations of BAP and NAA for in vitro regeneration of plants. Results: Both leaf and nodal explants cultured on MS medium supplemented with 0.8 mg/L BAP developed into mass of callus. These calli were subcultured for the induction of shoots and roots. Shoots were induced from both calli on MS medium supplemented with 0.8 mg/L BAP+0.5 mg/L NAA. Roots were induced from in vitro shoots on MS medium supplemented with 0.8 mg/L NAA for 4 weeks. Nodal explants were more regenerative with 95 % response compared to leaf explants with 85%. Finally, these in vitro regenerated plantlets were hardened, acclimatised and successfully transferred to the field. Conclusions: The present protocol for in vitro regeneration of Aristolochia indica L. can be used to make this plant available throughout the year for traditional healers, pharmaceutical usages, germplasm conservation, commercial cultivation, and also for the production of secondary metabolites.

Efeito de diferentes reguladores de crescimento na regeneração in vitro de pau-rosa (Aniba rosaeodora Ducke) / Effect of differents growth regulators in vitro propagation of Aniba rosaeodora Ducke

O objetivo deste trabalho foi estabelecer um protocolo para a regeneração in vitro de pau-rosa (Aniba rosaeodora Ducke), utilizando brotações apicais e segmentos nodais inoculados em meio de cultura com distintas concentrações de diferentes reguladores de crescimento. Explantes esterilizados com soluções de benomyl (4,0 g.L-1) por 24 horas e hipoclorito de sódio a 20% + tween 20 por 20 minutos, foram submetidos a um experimento de indução de broto, raiz e calo em meio MS1 acrescido de 30g.L-1 de sacarose e 9g.L-1 de agar, suplementado com BAP (0,0 e 4,0 mg.L-1), ANA, AIA e 2,4-D (0,0; 3,0 e 6,0 mg.L-1), e suas respectivas combinações. O delineamento utilizado foi o inteiramente casualizado em esquema fatorial 7 X 2, com 14 tratamentos e 15 repetições cada, onde foram analisados o número médio de brotos, raízes e calo. Após 90 dias, os resultados mostraram que a presença de auxinas é fundamental para a formação dos parâmetros induzidos nos explantes de pau-rosa. O meio de cultura contendo 4,0 mg.L-1 de BAP + 6,0 mg.L-1 de AIA apresentou a melhor média para a brotação com 2,13 brotos/explante. Para o enraizamento o meio contendo 3,0 mg.L-1 de ANA foi o mais eficiente, apresentando uma média de 2,53 raízes/explante. Em relação à indução de calo, todos os tratamentos apresentaram calogênese, porém o meio suplementado com 4,0 mg.L-1 de BAP + 6,0 mg.L-1 de 2,4-D, apresentou a melhor média, 1,67 calos/explante.

The objective of this work was to establish a protocol for in vitro regeneration of rosewood (Aniba rosaeodora Ducke), using apical and nodal segments inoculated in culture medium with various concentrations of growth regulators. Explants disinfected with solutions of benomyl (4,0 g.L-1) for 24 hours and sodium hipoclorite in 20% + tween 20 for 20 minutes were submitted in an experiment of shoot, root and callus induction in MS medium, with 30 g.L-1 of sucrose and 9,0 g.L-1 of agar added with the growth regulators: BAP (0,0 and 4,0 mg.L-1), ANA, AIA and 2,4-D (0,0; 3,0 and 6,0 mg.L-1), and their respective combinations. The design was complete randomized in arranged factorial 7 X 2, with 14 treatments and 15 replications, where about the explants were analysed the number of shoot, root and callus . After 90 days, the results showed that the presence of auxins is fundamental to induce buds, roots and callus . The medium was contained 4,0 mg.L-1 BAP + 6,0 mg.L-1 AIA showed the best average for shooting with 2,13 buds/explants. In the rooting, the medium was contained 3,0 mg.L-1 of ANA was the more efficient, showed 2,53 roots/explant. However, all the treatments obtained formation of callus, but the medium with 4,0 mg.L-1 BAP + 6,0 mg.L-1 2,4-D showed the best result,1.67 callus/explant.

Adventitious shoots from nodule cluster cultures of Vriesea reitzii: an endemic and endangered bromeliad from atlantic forest / Brotos adventícios a partir de culturas nodulares de Vriesea reitzii: uma bromélia endêmica e em extinção da Mata Atlântica

The Atlantic Forest is a biome with megadiversity and a number of bromeliads take part of it. This is the case of Vriesea reitzii, an endemic bromeliad threatened with extinction. Tissue culture techniques are valuable tools for the mass propagation of bromeliads, thus reducing pressure in the natural habitat. The aim of the present work was to establish an in vitro protocol based on the induction and proliferation of nodule cluster cultures of this species. Plantlets maintained in MS liquid culture medium plus NAA (2µM) and BAP (4µM) had the basal regions of leaves excised and then inoculated in gelled with agar (7g L-1) MS culture medium plus with Dicamba (2.5; 5; 10; 20 e 30µM) and Kin (2µM) or free of plant growth regulators. Nodule cluster cultures arose from the basal region of explants. The subculture to MS liquid medium plus GA3 (10µM) and in MS liquid medium free of plant growth regulators resulted in a high proliferation rate. The mean regenerative rate was 39 plantlets/0.03g of nodule culture. Plantlets were acclimatized in a mix substrate of 1:1 (v:v) of carbonized rice coat and Turfa Fertil® mineral supplement.

A floresta tropical atlântica é um bioma de alta diversidade, rico em espécies endêmicas de bromélias, entre elas Vriesea reitzii, que se encontra ameaçada de extinção. Técnicas de cultura de tecidos possibilitam a propagação massal de bromélias, reduzindo a pressão de coleta na natureza. O presente trabalho teve como objetivo desenvolver um protocolo regenerativo baseado na indução e no desenvolvimento de culturas nodulares desta espécie. Plantas multiplicadas em meio MS líquido com ANA (2µM) e BAP (4µM) tiveram suas folhas excisadas e inoculadas em meio MS geleificado com ágar (7g L-1) e suplementado com Dicamba (2,5; 5; 10; 20 e 30µM) e Kin (2µM) ou isento de fitorreguladores. A indução de culturas nodulares foi observada na região basal dos explantes. Estas culturas foram subcultivadas em meio de cultura MS líquido suplementado com GA3 (10µM) e subseqüentemente para meio MS líquido isento de fitorreguladores, resultando em altas taxas de proliferação de culturas nodulares que originaram brotos adventícios e microbrotos. A taxa média de regeneração foi de 39 brotos/0,03g de culturas nodulares. As mudas foram aclimatizadas com sucesso em substrato composto por fertilizante organomineral Turfa Fértil® e casca de arroz carbonizada na proporção de 1:1 (v:v).

Controle genético da regeneração in vitro em progênies de Eucalyptus grandis / Genetic control of in vitro regeneration of Eucalyptus grandis

Com o objetivo de avaliar o controle genético da regeneração direta in vitro a partir de plântulas de Eucalyptus grandis, foram utilizadas sementes de 10 progênies de polinização aberta da população base, origem Atherton, localizada em Anhembi, Estado de São Paulo. Vinte dias de cultivo após a germinação, 196 segmentos distais dos hipocótilos por progênie foram inoculados in vitro num Delineamento em Blocos Completos Aleatorizado Generalizado, com duas unidades experimentais por bloco e sete repetições por bloco, usando a interação blocos por progênie como estimadora do erro experimental. Após 14 semanas de cultivo, foram feitas avaliações da regeneração. Houve diferenças significativas de regeneração entre as progênies (P<0,0001) com extremos de regeneração de 11 por cento a 60 por cento. A herdabilidade no sentido restrito entre as médias das unidades experimentais do caráter foi alta (h2m=0,94), indicando que houve um forte controle genético na regeneração in vitro dentro da população. Houve também alta variabilidade dentro da amostra estudada, assim como um forte efeito do progenitor materno sobre a regeneração.

The genetic control of in vitro direct regeneration was tested on seedlings of ten open-pollinated progenies from the base population of Atherton origin of Eucalyptus grandis at University of São Paulo (Brazil). Seeds were germinated in vitro, after twenty days, distal hypocotyls segments from 196 seedlings per progeny were inoculated in culture media at Generalized Complete Randomized Block Design, with two experimental units per block and seven repetitions, using the interaction blocks by progenies as an estimate of the experimental error. At week 14 from the inoculation bud induction was evaluated. Regeneration among progenies were significantly different (P<0.0001). Regeneration varied from 11 to 60 percent. The narrow-sense heritability between means of experimental units for in vitro regeneration was height. (h2m=0.94), indicating a strong genetic control of the trait within the population and also a high maternal effect. High variability within the study sample was found.

Regenerácion in vitro de tres variedades de crisantemo (Dendrathema grandiflora) a través de organogénesis y embriogénesis somática / In vitro regeneration of three chrysanthemum (Dendrathema grandiflora) varieties via organogenesis and somatic embryogenesis

El crisantemo (Dendrathema grandiflora) presenta alta demanda en los mercados de flor cortada, tanto colombianos como internacionales. La producción de esta especie se ve seriamente afectada por enfermedades fúngicas como la roya Blanca (Puccinia horiana), lo que ocasiona que se empleen grandes cantidades de fungicidas aumentando los costos de producción a nivel económico y ecológico. La evaluación de sistemas de regeneración in vitro de crisantemo a partir de discos de hoja constituyó un primer paso hacia el empleo de la transformación genética, como apoyo a las técnicas de mejoramiento convencional para la obtención de plantas resistentes al hongo. Se establecieron discos de hoja de D. grandiflora var. Escapade, var. White albatross y var. Yellow albatross sobre medio MS en presencia ANA (0 - 4.83 mM) y BAP (0 - 13.32 mM) solos y en combinación. Así mismo, se establecieron discos foliares de las tres variedades en estudio sobre el medio MumB en presencia 2,4-D (0 - 4.52 mM) durante 7, 14 y 21 días, tiempo en el cual los explantes fueron transferidos a medio a medio Mum B sin 2-4D. Los brotes regenerados, fueron individualizados, enraizados y endurecidos. Los resultados obtenidos indican que el medio MS suplementado con: ANA 4.83 mM + BAP 4.44 mM ó ANA 4.83 mM y BAP 13.32 mM permite la regeneración de plantas vía organogénesis para las tres variedades y que es posible obtener embriones somáticos de las tres variedades, sobre medio Mum B en presencia de 2,4-D 2.26 mM, con periodos de incubación de 14 días para White Albatross y 21 días para Yellow Albatros y Escapade. El medio Mum B sin 2,4-D, permite el desarrollo de los brotes, a partir de embriones somáticos en los tres casos. El 85 por ciento de los brotes obtenidos presentaron enraizamiento espontáneo, lo que facilitó el endurecimiento y transferencia exitosa a suelo.

Chrysanthemum (Dendrathema grandiflora) has a high demand in the Colombian and international cut flower markets. Since commercial production of this ornamental species is strongly affected by fungal diseases such as chrysanthemum white rust (Puccinia horiana), high doses of fungicides are being used posing increased environmental and commercial costs. Assessment of in vitro regeneration systems from leaf discs was a first step in developing a plant genetic transformation protocol to obtain fungi-resistant plants. Leaf discs of White Albatross, Yellow Albatross, and Escapade varieties were established in vitro on MS medium supplemented with NAA (0 - 4.83 μM) and BAP (0 - 13.32 μM) alone and in combination. Leaf discs were also cultured on MumB medium containing 2,4-D (0 - 4.52 μM) for 7, 14, and 21 days prior to their transferral to a 2,4-D free MumB medium. Regenerated shoots were individualized, rooted, and hardened. Results show that MS with 4.83 μM NAA + 4.44 μM BAP and 4.83 μM NAA + 13.32 μM BAP induce organogenesis, and MumB with 2.26 μM 2,4-D induces somatic embryogenesis on all three varieties, with exposition periods to 2,4-D of 14 days for White Albatross and 21 days for Yellow Albatross and Escapade. Shoot development from somatic embryos was observed in the three varieties when cultured on a 2,4-D free MumB medium. Spontaneous rooting was recorded in 85% of the shoots thus facilitating hardening and successful transfer to soil.