Morpho-anatomy of in vitro germination and cryopreservation of the orchid Cattleya crispa (Orchidaceae Morfoanatomía de la germinación in vitro y criopreservación de la orquídea Cattleya crispa (Orchidaceae) / Morfoanatomía de la germinación in vitro y criopreservación de la orquídea Cattleya crispa (Orchidaceae)

Introduction: Cattleya crispa is an ornamental epiphytic orchid with geographic distribution restricted to the Brazilian Atlantic Forest. Due to predatory extractivism and human-induced habitat loss, this species appears on the Red List of Brazilian Flora. Objective: To characterize morpho-anatomical aspects regarding germination and post-seminal development from C. crispa seeds; as well as studying the effect of cryopreservation on these seeds. Methods: We used light microscopy and electron microscopy to describe the microstructure of a 100 ripe seeds. We evaluated seed viability, seed germination, survival rate and protocorm weight in cryopreserved and non-cryopreserved material, with four replicas per treatment using 20 mg of plant material. Results: The seeds are fusiform, whitish yellow with a length from 700 to 900 µm and a water content of 5 %. Germination began seven days after sowing, the formation of the globular protocorm at 30 days and the formation of the seedling occurred 150 days. The persistent seed coat can compress the protocorm and cause it to collapse. The cryopreserved seeds presented 87.15 % viability, 78.32 % germination, 8.48 % survival and protocorms with 104.27 mg five months after sowing. Data wasn't different to non-cryopreserved seeds. Conclusions: The cryocapability of the seeds shows that cryopreservation can be used for long-term conservation. The results of this work contribute to the overall biology of C. crispa and to the propagation and storage of genetic material for conservation purposes.

Introducción: Cattleya crispa es una orquídea epífita ornamental con distribución geográfica restringida a la Mata Atlántica brasileña. Debido al extractivismo depredador y a la pérdida de hábitat inducida por el hombre, esta especie aparece en la Lista Roja de la Flora Brasileña. Objetivo: Caracterizar aspectos morfoanatómicos de la germinación y desarrollo inicial de semillas de C. crispa; así como estudiar el efecto de la criopreservación de estas semillas. Métodos: Utilizamos microscopía óptica, microscopía electrónica de barrido y microscopía electrónica de transmisión para describir la microestructura en 100 semillas maduras. Evaluamos la viabilidad de la semilla, la germinación de la semilla, la tasa de supervivencia y el peso de los protocormos en el material criopreservado y no criopreservado, con cuatro réplicas por tratamiento de 20 mg de material vegetal. Resultados: Las semillas son fusiformes, amarillo blanquecinas, con una longitud de 700 a 900 µm y un contenido de agua del 5 %. La germinación comenzó siete días después de la siembra, la formación del protocormo globular a los 30 días y la formación de la plántula a los 150 días. La cubierta de semilla persistente puede comprimir el protocormo y provocar su colapso. Las semillas criopreservadas presentaron 87.15 % de viabilidad, 78.32 % de germinación, 8.48 % de supervivencia y protocormos con 104.27 mg a los cinco meses de la siembra. Los datos no fueron diferentes a las semillas no criopreservadas. Conclusiones: La capacidad criogénica de las semillas muestra que la crioconservación puede utilizarse para la conservación a largo plazo. Los resultados de este trabajo contribuyen a la biología general de C. crispa y a la propagación y almacenamiento de material genético con fines de conservación.

Phytoremediation of Cu-contaminated vineyard soils in Brazil: A compendium of Brazilian pot studies.

Vineyard soils can be contaminated by copper (Cu) due to successive applications of fungicides and organic fertilizers. Soil remediation can be addressed by altering soil properties or selecting efficient Cu-extracting cover crops tolerant to Cu toxicity. Our objectives were to synthesize the Cu-extracting efficiency by plant species tested in Brazil, classify them according to Cu resistance to toxicity, and assess the effect of soil properties on attenuating Cu toxicity. We retrieved results from 41 species and cultivars, totaling 565 observations. Freshly added Cu varied between 50 and 600 mg Cu kg-1 of soil across studies. The partition of Cu removal between the above- and below-ground portions was scaled as a logistic variable to facilitate data synthesis. The data were analyzed using the Adaboost machine learning model. Model accuracy (predicted vs. actual values) reached R2  = 0.862 after relating species, cultivar, Cu addition, clay, SOM, pH, soil test P, and Cu as features to predict the logistic target variable. Tissue Cu concentration varied between 7 and 105 mg Cu kg-1 in the shoot and between 73 and 1340 mg Cu kg-1 in the roots. Among soil properties, organic matter and soil test Cu most influenced the accuracy of the model. Phaseolus vulgaris, Brassica juncea, Ricinus communis, Hordeum vulgare, Sorghum vulgare, Cajanus cajan, Solanum lycopersicum, and Crotolaria spectabilis were the most efficient Cu-extracting cover crops, as shown by positive values of the logistic variable (shoot removal > root removal). Those Cu-tolerant plants showed differential capacity to extract Cu in the long run.

Seeds cryopreservation of Vriesea reitzii Leme & A.F. Costa endemic bromeliad from Atlantic Rainforest / Criopreservação de sementes de Vriesea reitzii Leme & A.F. Costa bromélia endêmica da Mata Atlântica

Vriesea reitzii is an endemic bromeliad from the Atlantic Rainforest. The objective of this research was to evaluate the cryopreservation using the method of direct immersion of its seeds, collected from capsules at 120, 135, and 150 days after anthesis (DAA). The water content was determined before cryopreservation, while the germination percentage, germination speed index (GSI), and total soluble carbohydrates were quantified after cryopreservation. The highest percentage of moisture (17.6%) was observed in 120 DAA, while the highest percentage of germination (89.6%) and GSI (17.0) were observed in 150 DAA. Optical and transmission electron microscopy analyses were performed, and no cell damage or changes at the morpho-histological and ultrastructural levels were observed after the cryopreservation process. From these results, V. reitzii seeds can be classified as orthodox seeds and the cryopreservation (+LN) is an efficient tool for an ex situ conservation of this species.

Vriesea reitzii é uma bromélia endêmica da Mata Atlântica. O objetivo deste trabalho foi avaliar a criopreservação de sementes utilizando o método de imersão direta, coletadas de cápsulas aos 120, 135 e 150 dias após a antese (DAA). O teor de água foi determinado antes da criopreservação. A porcentagem de germinação, o índice de velocidade de germinação (GSI) e os carboidratos solúveis totais foram quantificados após a criopreservação. A maior porcentagem de umidade (17,6%) foi observada aos 120 DAA, enquanto a maior porcentagem de germinação (89,6%) e GSI (17,0) foram observadas aos 150 DAA. Em análises de microscopia ótica e eletrônica de transmissão não foram observados danos celulares ou alterações a nível morfohistológico e ultraestrutural após o processo de criopreservação. A partir desses resultados, as sementes de V. reitzii podem ser classificadas como ortodoxas e a criopreservação (+LN) é uma ferramenta eficiente para a conservação ex situ desta espécie.

Identification and characterization of SSR markers of Guadua chacoensis (Rojas) Londoño & P.M. Peterson and transferability to other bamboo species.

The aim of this study was to develop simple sequence repeat (SSR) markers for genetic studies on G. chacoensis, as well as to evaluate their transferability to other bamboo species. Genomic DNA was isolated from G. chacoensis and its partial sequencing was used to find SSR loci. The obtained sequencing data were de novo assembled using the software CLC Genomics Workbench® 8.0v. The SSR loci primers were identified and designed with the software SSRLocator. The selected markers were validated using 56 plants sampled in seven populations from southern Brazil. The markers with potential polymorphism were selected and fluorescently labeled for characterization by capillary electrophoresis. In total, 92 SSR loci were found in G. chacoensis contigs. Suitable primers were designed for 70 SSR loci, and the remaining 22 SSR loci did not have sequences for primer development. Out of 35 selected SSR markers, after PCR optimization, 10 with high polymorphism potential were characterized. These loci can be used in genetic analyses of G. chacoensis and all of them were successfully transferred to other bamboo species. Non-polymorphic loci require further tests with additional plants, from different populations, to identify possibilities of their use.

Male sterility in Bambusa tuldoides Munro.

Despite their great economic importance, relatively little is known about bamboo sexual reproduction because they usually spread through rhizomes and have long intervals between flowering periods. Bambusa tuldoides is no exception; the intervals between flowering periods are about 23 years and often do not result in successful caryopsis production. The aim of the present work was to characterize Bambusa tuldoides sexual reproduction at three stages of flower development and investigate possible male sterility. Pollen was cultured onto several types of culture medium in order to encourage germination, but not a single of the thousands of observed pollen germinated under any condition. Anthers and microspores were analyzed by scanning electron microscopy, transmission electron microscopy, and optical microscopy techniques. Anther dehiscence appeared to be normal when compared to other species. In contrast, microspores began to develop abnormally starting as early as the first flower development stage: retraction of the cytoplasm and rupture of the nuclear and mitochondria membrane. As the interior machinery of the microspores degenerated, starch accumulated within numerous amyloplasts during stages two to four of flower development. The sporoderms of these microspores were similarly incomplete: though they possessed an exine, they lacked an intine. The results here obtained suggest that the non-viability of these abnormal pollen grains prevents the development of Bambusa tuldoides caryopses.

Determination of endogenous IAA and carbohydrates during the induction and development of protocorm-like bodies of Cattleya tigrina A. Richard / Determinação de AIA e carboidratos endógenos durante a indução e desenvolvimento deestruturas semelhantes a protocormos de Cattleya tigrina A. Richard

Tissue culture techniques have been employed for orchid mass propagation by means of themorphogenetic route of protocorm like-bodies (PLBs). This study aimed to analyze and compare Indoleaceticacid (IAA) and sugar endogenous levels in protocorm-like bodies (PLBs) induction and developmentin Cattleya tigrina, in order to better understand this process and to optimize micropropagation procedureprotocols. Leaves grown on MS (Murashige and Skoog) culture medium, suplemented with 9 μM TDZfor PLBs induction and development were collected after 0, 2, 7, 14, 30, 60 and 100 days of cultivation, forfurther analysis. Increase of IAA and reduction of sugar levels are strongly related to morphogeneticresponse, that is, PLBs formation over the preexisting ones and leaf primordia formation. Sucrose, fructoseand glucose presence in this study is related to cell signaling. Thus, hormonal signals and carbohydratesalter metabolism, triggering PLBs initiation and development in C. tigrina.

Técnicas de cultura de tecidos têm sido empregadas para a propagação em massa de orquídeaspor meio da via morfogenética de estruturas semelhantes a protocormos (ESPs). O estudo teve comoobjetivo analisar e comparar os níveis endógenos de ácido indol-3-acético (AIA) e de açúcares na indução eno desenvolvimento de estruturas semelhantes a protocormos (ESPs) em Cattleya tigrina, visando à melhorcompreensão deste processo e à otimização de protocolos de micropropagação. Folhas cultivadas em meiode cultura MS (Murashige & Skoog), suplementadas com 9 μM de TDZ para indução e desenvolvimentode ESPs foram coletadas após 0, 2, 7, 14, 30, 60 e 100 dias de cultivo, para posterior análise. O aumento dosníveis de AIA e a redução dos níveis de açúcar estão fortemente relacionados à resposta morfogenética, ouseja, à formação de ESPs sobre os pré-existentes e à formação de primórdios foliares. A presença desacarose, frutose e glicose neste estudo está relacionada à sinalização celular. Assim, sinais hormonais ecarboidratos alteram o metabolismo, desencadeando a iniciação e o desenvolvimento de ESPs em C. tigrina.

Dynamics in global DNA methylation and endogenous polyamine levels during protocorm-like bodies induction of Cattleya tigrina A. Richard / Dinâmica da metilação do DNA global e níveis endógenos de poliaminas durante a indução de estruturas semelhantes a protocormos de Cattleya tigrina A. Richard

Cattleya tigrina is endemic to the Atlantic forest biome and classified as vulnerable in the Red Book of Brazilian Flora. In vitro techniques comprise valuable tools for the conservation of endangered plant species. The aim of the present study was to evaluate the morphological features, global DNA methylation levels and free polyamines during protocorm- like bodies (PLBs) induction of C. tigrina. Along with that, an efficient protocol for in vitro propagation of this species is proposed. The first evidences of PLBs induction in C. tigrina occurred at seven days in culture, starting from the basal portion of the leaf abaxial surface. A hypomethylation marked the beginning of cell differentiation, followed by an increased global DNA methylation at 35 days in culture, coinciding with a subtle change in the structures morphogenetic development. During PLBs induction, putrescine exhibited higher levels as compared to spermidine and spermine, and apparently presents a major role during the PLBs induction in C. tigrina. Due to the apparent secondary PLBs formation, this protocol can represent a highly efficient method for in vitro propagation of this species.

Cattleya tigrina é uma espécie endêmica do bioma Mata Atlântica e classificada como vulnerável no Livro Vermelho da Flora Brasileira. As técnicas in vitro compreendem ferramentas valiosas a serem empregadas na conservação de espécies de plantas ameaçadas. O objetivo do presente estudo foi avaliar as características morfológicas, os níveis globais de metilação do DNA e as poliaminas livres durante a indução de estruturas semelhantes a protocormos (ESPs). Paralelamente, um protocolo eficiente para a propagação in vitro desta espécie é apresentado. As primeiras evidências de indução de ESPs em C. tigrina foram observadas aos sete dias de cultivo, a partir da porção basal da superfície abaxial da folha. Uma hipometilação foi observada concomitante ao início da diferenciação celular, e um aumento da metilação global do DNA foi encontrada aos 35 dias de cultivo, coincidindo com uma sutil mudança no desenvolvimento morfogenético das estruturas. Durante a indução de ESPs, a putrescina exibiu níveis aumentados em comparação a espermidina e espermina e, aparentemente, apresenta um papel importante durante a indução dessas estruturas em C. tigrina. Devido à aparente formação secundária de ESPs, este protocolo pode representar um método altamente eficiente para a propagação in vitro desta espécie.

Histodifferentiation and ultrastructure of nodular cultures from seeds of Vriesea friburgensis Mez var. paludosa (L.B. Smith) L.B. Smith and leaf explants of Vriesea reitzii Leme & A. Costa (Bromeliaceae).

Micropropagation via induction, multiplication and development of nodular cultures (NCs) is an efficient regeneration system for Bromeliaceae, a family of endangered monocot plants with ornamental value. Therefore, the present work aimed to induce NCs from seeds and leaf explants of Vriesea in order to characterize the morphological and histochemical aspects of induction and formation of these cultures. Seeds of Vriesea friburgensis var. paludosa were sterilized and inoculated into liquid culture media supplemented with different concentrations and combinations of growth regulators. Leaf explants of Vriesea reitzii were inoculated into medium supplemented with 4 µM α-naphthalene acetic acid (NAA) and 2 µM 6-benzylaminopurine (BAP). The addition of NAA (4 µM) in the culture medium used for seeds led to an induction rate of 72% in NCs. First, the embryo began to germinate, and afterwards, nodular structures started to form. While NCs formed from seeds is associated with root and shoot meristems, the formation of NCs from leaf explants involves the intercalary meristem. Meristematic cells generate an appropriate response in the induction medium, producing NCs by the proliferation of small cells with meristematic characteristics and large vacuolated cells. These results provide a better understanding of morphogenetic responses in bromeliads and, hence, the opportunity to develop optimized micropropagation protocols. Abbreviations: BAP, 6-benzylaminopurine; 2-iP, N6 (2-isopentyl) adenine; CBB, Coomassie Brilliant Blue; CLSM, confocal laser scanning microscopy; MSB, MS basal medium; NAA, α-Naphthalene acetic acid; NCs, nodular cultures; PAS, Periodic Acid-Schiff; SEM, scanning electron microscopy; TDZ, N-phenyl-N'-1,2,3-thidiazol-5-ylurea; TB-O, Toluidine Blue O; TEM, Transmission electron microscopy.

In vitro propagation of Vriesea reitzii, a native epiphyte bromeliad from the Atlantic rainforest / Propagação in vitro de Vriesea reitzii, uma bromélia epífita nativa da Mata Atlântica

The induction of nodular culture (NC) and the subsequent development of microshoots of V. reitzii are considered an in vitro propagation model-system with high regenerative performance. Current research analyzed the determinant factors of the in vitro morphogenesis control of bromeliads. Seeds excised from mature capsules were grown on medium MS basic (MSB), liquid or gelled, supplemented or not with α- naphthaleneacetic acid (NAA), 6-benzilaminopurine (BAP) or thidiazuron (TDZ). The regeneration and elongation of microshoots were evaluated from NC sub-cultivated on MSB medium on liquid culture medium supplemented with different concentrations of indolyl-3-acetic acid (IAA) and gibberellic acid (GA3). Plant growth regulators (PGR) supplemented into the medium MSB inhibited the germination of the seeds and induced NC in the second week of growth. The induced NC on MSB medium with NAA (4 µM) and sub-cultivated on MSB medium with NAA (2 µM) plus N6(2-isopentenyl) adenine (2-iP) (2 µM) showed granular texture and high rate of proliferation. NC sub-culture in MSB medium with IAA (4 µM) provided a higher average number of microshoots (1,478 shoots g-1 of NC). Shoots over 3.0 cm resulted in more than 95% ex vitro survival.

A indução de culturas nodulares (CNs) e subsequente desenvolvimento de microbrotos de V. reitzii configuraram-se em um sistema de alta performance regenerativa in vitro. No presente trabalho foram estudados os fatores determinantes do controle da morfogênese in vitro das CNs. Sementes excisadas de cápsulas maduras foram cultivadas em meio básico MS (MSB) líquido ou geleificado e suplementado ou não com ácido naftalenoacético (ANA), 6-benzilaminopurina (BAP) ou thidiazuron (TDZ). A partir das CNs subcultivadas em meio MSB, foi avaliada a regeneração e o alongamento de microbrotos em meio de cultura líquido suplementado com diferentes concentrações de ácido indolil-3-acético (AIA) combinados com ácido giberélico (AG3). A suplementação de fitorreguladores ao meio MSB inibiram a germinação das sementes e promoveram a indução de CNs na segunda semana de cultivo. As CNs induzidas em meio MSB suplementado com ANA (4 µM) e subcultivadas em meio MSB suplementado com ANA (2 µM) mais N6(2-isopentenil) adenina (2-iP) (2 µM) apresentaram textura granular e alta taxa de proliferação. O cultivo destas CNs em meio MSB suplementado com AIA (4 µM) resultou no maior número médio de microbrotos (1.478 brotos g -1 de CN). Brotos maiores de 3,0 cm resultaram em mais de 95% de sobrevivência em ambiente ex vitro.

Physiological characteristics of the Atlantic Forest native bromeliads: Nidularium campo-alegrense Leme and Aechmea ornata Baker / Características fisiológicas de bromélias nativas da Mata Atlântica: Nidularium campo-alegrense Leme e Aechmea ornata Baker

Despite the ecological importance of bromeliads, the basic knowledge about the physiological aspects in some species is poorly understood. The aim of this study was to physiologically characterize the species Nidularium campo-alegrense Leme and Aechmea ornata Baker to contribute to a better understanding of their metabolic processes. From mature leaves of bromeliads N. campo-alegrense and A. ornata held at the Agricultural Sciences Center of the Federal University of Santa Catarina, it was quantified photosynthetic pigments, carbohydrates and starch, and determined stomata and trichome density. The chlorophyll and carotenoids content did not differ significantly between species. However, the total carbohydrates content of A. ornata (3.2 mg g- 1 FM) was significantly higher than N. campo-alegrense, but with similar starch content (1.7 mg g -1 FM). Both species present hypostomatic leaves, with more trichomes in the species A. ornata. This information provides the groundwork for future studies on the biochemical mechanisms related to their photosynthetic process, contributing in establishing strategies for the conservation and propagation of these species.

Apesar da importância ecológica das bromélias, o conhecimento básico sobre os aspectos fisiológicos em algumas espécies é pouco compreendido. O objetivo do presente trabalho foi caracterizar fisiologicamente as espécies Nidularium campo-alegrense Leme e Aechmea ornata Baker, para contribuir ao melhor entendimento dos seus processos metabólicos. Pelas folhas maduras, das bromélias N. campo-alegrense e A. ornata mantidas no Centro de Ciências Agrárias (CCA) da Universidade Federal de Santa Catarina (UFSC), foram quantificados os pigmentos fotossintéticos, carboidratos e amido e foi determinada a densidade estomática e tricomática. Os teores de clorofila e carotenoides não apresentaram diferença significativa entre as espécies. No entanto, na espécie A. ornata, o teor de carboidratos totais (3,2 mg g-1 MF) foi significativamente maior e o teor de amido (1,7 mg g-1 MF) semelhante quando comparado com N. campo-alegrense. As duas espécies apresentam folhas hipoestomáticas e o número de tricomas foi maior na espécie A. ornata . Estas informações fornecem as bases para futuros estudos sobre os mecanismos bioquímicos relacionados com seu processo fotossintético para contribuir no estabelecimento de estratégias de conservação ou propagação destas espécies.

Micropropagation systems of Feijoa (Acca sellowiana (O. Berg) Burret).

Acca sellowiana (O. Berg) Burret sin. Feijoa sellowiana (Myrtaceae) is a semiwoody fruit species native to South Brazil, Uruguay, and Argentina; edible fruits are tasty. The naturally occurring populations in Santa Catarina State show high variability in fruit size, color, and other features. A breeding program launched in 1990 resulted in the release of four Brazilian commercial varieties. The conventional clonal propagation methods of this species, such as cutting and grafting, have shown low efficiency. Therefore, tissue culture techniques were developed for mass propagation. This chapter describes several protocols based on organogenesis and somatic embryogenesis. Additional techniques including synthetic seed technology and temporary immersion system are also described.

5-Azacytidine combined with 2,4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels.

UNLABELLED: DNA methylation is an epigenetic regulatory mechanism of gene expression which can be associated with developmental phases and in vitro morphogenetic competence in plants. The present work evaluated the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2,4-D) on Acca sellowiana somatic embryogenesis (SE) and global DNA methylation levels by high-performance liquid chromatography mass spectrometry (HPLC/MS/MS). 2,4-D-free treatments revealed no somatic embryo formation in both accessions tested. Treatments supplemented with 2,4-D pulse plus AzaC in the culture medium resulted in increased embryo formation. In AzaC-free treatment, HPLC/MS/MS analysis showed a gradual increase in methylation levels in cultures of both accessions tested during SE induction. Treatment with AzaC and 2,4-D-free resulted in a marked decrease in methylation for both accessions, ranging from 37.6 to 20.8 %. In treatment with 2,4-D and AzaC combined, the 85 accession showed increasing global methylation levels. Otherwise, the 101X458 accession, in the same treatment, showed a decrease between 10 and 20 days, followed by an increase after 30 days (39.5, 36.2 and 41.6 %). These results indicate that 2,4-D pulse combined with AzaC improves SE induction. However, the conversion phase showed that although positively influencing SE induction, AzaC had a dysregulatory effect on the stage of autotrophic plant formation, resulting in significantly lower conversion rates. The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and global DNA methylation dynamics are related to morphogenetic response. KEY MESSAGE: 5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.

Dark-induced hormone changes coincide with the resumption of light-inhibited shoot growth in Catasetum fimbriatum (Orchidaceae).

Catasetum fimbriatum plants cultivated in the absence of light exhibit continuous shoot growth leading to the formation of nodes and internodes. On the other hand, when these plants are incubated in the presence of light, shoot longitudinal growth is inhibited and pseudobulbs develop just below the shoot apical meristem. These facts provide evidence of a possible influence of light on mitotic cell division in the shoot apex as well as on pseudobulb initiation. The effects of light and dark on the interruption and/or maintenance of shoot apex mitotic activity and the subsequent formation of pseudobulbs in the sub-meristematic regions were investigated by means of histological and hormonal studies. The interruption of shoot apex development occurred around the 150th d of light incubation and seems to have resulted from the establishment of a strong storage sink in the region of the future pseudobulb, in detriment to the continuous activity of the shoot apical meristem. The reduced total cytokinins/IAA ratio in the apex, mainly due to high levels of IAA, could be a key factor in the interruption of cell divisions. Transfer to the dark brings about the resumption of shoot apex development of plants through the re-entrance of cells in the cell cycle which coincides with a significant increase in the total cytokinins/IAA ratio.

Thidiazuron influences the endogenous levels of cytokinins and IAA during the flowering of isolated shoots of Dendrobium.

This study reports the effects of thidiazuron (TDZ) on the endogenous levels of indoleacetic acid (IAA), zeatin, zeatin riboside ([9R]Z), isopentenyladenine and isopentenyladenosine ([9R]iP) as well as structural changes in the apical meristem of Dendrobium Second Love shoots during flower induction and initial development in vitro. The results revealed that the presence of 1.8microM TDZ had a profound effect on the endogenous cytokinins (CKs) and IAA levels of the explants, when compared to those grown on a TDZ-free medium. A significant increase in CKs (especially [9R]iP and [9R]Z) and IAA in the first samples (taken at day 5) grown on TDZ-enriched medium was associated with flower induction, while a second increase in the level of these hormones after 25d of culture was related to flower development. The histological changes detected in the shoot apical meristem of explants grown in the presence of 1.8microM TDZ during 30d of culture are also described. Based on these findings, it is suggested that both auxin and CKs seem to be involved with the floral transition of Dendrobium Second Love in vitro. However, a possible direct effect of TDZ on flower formation is not discarded.