Does soaking time during disinfestation affect germination rates in Dendrobium? / O tempo de imersão durante a desinfestação influencia nas taxas de germinação de Dendrobium?

Asymbiotic germination is considered an efficient and viable technique that can increase germination rates. The effect of type and concentration of disinfestants, and the exposure time to disinfestants may differ according to the plant species. Therefore, species-specific standardization of disinfestation agent and procedure is necessary to achieve optimal germination rates. The objective of this study was to determine a disinfestation methodology to increase in vitro germination rates and the early development of seedlings of Dendrobium nobile and Dendrobium phalaenopsis, using different times for seed disinfestation and different culture media. Seeds were disinfected by soaking in a 0.8% sodium hypochlorite solution for 5 or 15 min under aseptic conditions, after which seed suspensions were either washed with water or left unwashed. Next, they were seeded in culture flasks containing four different culture media (MS, ½MS, K, and VW). The flasks were then transferred to a growth room under controlled photoperiod and temperature, where they remained under an irradiance of 20 µmol m-2 s-1. Germination rates of the species were evaluated 45 days after placement in the culture flasks. A higher germination rate was observed when the seeds were triple washed, regardless of the culture medium or soaking time. Seed soaking disinfestation for 5 min is also recommended. MS and ½MS media were the most effective culture media in promoting in vitro germination of the species under study.

A germinação assimbiótica é considerada uma técnica eficiente e viável resultando em elevados percentuais de germinação. Apesar do sucesso dessa técnica, o tipo, a concentração e o tempo de exposição do agente desinfestante diferem, necessitando padronização para cada espécie. Assim, a padronização do agente desinfestante e do procedimento são necessários para o aumento das taxas germinativas. Objetivou-se com este trabalho determinar metodologia que aumente a taxa de germinação in vitro e favoreça o desenvolvimento inicial de plântulas de Dendrobium nobile e Dendrobium phalaenopsis, utilizando diferentes métodos de desinfestação de sementes e diferentes meios de cultura. Sementes, sob condições assépticas, foram desinfestadas em solução de hipoclorito de sódio a 0,8%, por cinco ou quinze minutos. Após esses períodos, as suspensões de sementes receberam ou não a tríplice lavagem com água. Em seguida, foram semeadas em frascos de cultivo que continham quatro diferentes meios de cultura (MS, MS½, K e VW). Posteriormente, foram transferidos para sala de crescimento com fotoperíodo e temperatura controlados, onde permaneceram sob irradiância de 20 µmol m-2 s-1. Quarenta e cinco dias após a semeadura foi avaliada a porcentagem de germinação das espécies estudadas. Os resultados neste trabalho indicam que, independentemente do meio de cultura ou do tempo de desinfestação, as sementes quando submetidas à tríplice lavagem apresentaram porcentagem de germinação superior a àquelas que não receberam este procedimento. Recomenda-se a desinfestação das sementes por 5 minutos. Os meios MS e MS½ foram os mais efetivos em promover a germinação in vitro dessas espécies.

Molecular phylogeny of orchids mycorrhiza isolated from native tropical orchids in Indonesia

@#Aims: Mycorrhiza has an important role as a biocontrol agent. Its association with Phalaenopsis amabilis was molecularly identified through rDNA-ITS sequence analysis. The aims of the study were to identify molecular of orchids mycorrhiza isolate from native tropical orchids in Indonesia, conducted as one of native orchid conservation efforts in Indonesia. Methodology and results: One group of Ceratobasidium were isolated from the root of orchid plant in Yogyakarta based on morphological and microscopical analysis. The results of molecular analysis showed 600-750 bp of DNA products located on the ITS1-5.8S-ITS4 region. The sequenced products showed insertion and substitution occurances, which may result in strain diversity and possible variation. Reconstruction of phylogenetic trees using Maximum Parsimony and Bootstrap-1000 approach showed showed the Indonesian isolate is at the basal clade and already far apart from the other isolates. Conclusion, significance and impact of study: Isolate Ceratobasidium from Yogyakarta, Indonesia successfully isolated based on identification of rDNA-ITS sequences. Results of this study were expected to become the basic information in an effort of native orchid cultivation and protection against infectious diseases in Indonesia. The study was the first to report regarding Ceratobasidium isolated from native tropical orchids in Indonesia.

Effectiveness Test of Orchid Mycorrhizal Isolate (Ceratorhiza and Trichoderma) Indonesia and Its Role as a Biofertilizer

Aim: The existence of Orchid Mycorrhizal Fungi (OMF) has a role to stimulate growth and support the supply of orchid nutrition as a biofertilizer agent. This study aimed to determine the association of mycorrhizal with Phalaenopsis amabilis (L.) Blume which was carried out through the effectiveness test of two Indonesian orchid mycorrhizal isolates i.e. Ceratorhiza and Trichoderma. Study Design: This study consisted of 4 treatments. Each treatment was repeated 3 times, each repetition of 5 plantlets, so that the total plantlet used was 60. Place and Duration of Study: Laboratory of Plant Biotechnology, Department of Biology, Universitas Gadjah Mada, Indonesia, between June 2017 and April 2018. Methodology: The method of inoculating orchid mycorrhizal by placing a plantlet in a petri dish containing orchid mycorrhizal for 1, 2, 3, and 4 days. Then plantlets are grown on sterile moss growing media and acclimatized in a greenhouse. Observation of each treatment is carried out every day for the next month. Observation variables include the number of initial and final roots, the number of live and dead roots, and the number of living and dead plants. Results: The results of the orchid mycorrhizal induction test showed that the Ceratorhiza inoculation treatment showed a fluctuation in the mean increase in the number of final roots, live roots, dead roots, and dead plantlets that were higher than the Trichoderma inoculation treatment. The results also showed that the best inoculation time on Ceratorhiza and Trichoderma was day 3 and 4. The adaptation process had the effect of increasing the number of dead roots in weeks 1 and 2. The adaptation process stopped at the beginning of week 4 with the number of new roots appearing a lot. Conclusion: Orchid mycorrhizal Ceratorhiza shows the value of effectiveness test compared with Trichoderma. The results of this study are expected to be basic information in efforts to cultivate natural orchids in Indonesia.

The Effects of Different Media, Sucrose Concentrations and Natural Additives on Plantlet Growth of Phalaenopsis Hybrid 'Pink'

ABSTRACT The effects of various sucrose concentrations as carbon source and natural additives in different media on plantlet growth of Phalaenopsis hybrid 'Pink' were studied. Plantlets were cultured on two media (Murashige and Skoog [MS] and Vacin and Went [VW]) supplemented with 0, 10, 20, 30 and 40 g L-1 sucrose either with 0, 10 and 20% (v/v) coconut water (CW) or carrot juice (CJ) as natural additives. After four months of culture, the combination of sucrose and CW supplemented with both media affected plantlet growth where most of the plantlets showed slow growth and survival frequency (0-80%) with increasing concentrations of CW in all sucrose concentrations. However, plantlet growth on both media containing only 20 g L-1 sucrose without CW was optimal in terms of root number, root length, leaf number, leaf length, leaf width, fresh weight, dry weight and plant height. The combination of sucrose and CJ supplemented with MS medium resulted in overall good plantlet growth with 100% survival frequency. The combination of sucrose (20 g L-1) and CJ (10%) supplemented with MS medium increased root length, leaf length, leaf width and plant height. Plantlet growth was also optimal in the combination of 20 g L-1 sucrose and 10% CJ supplemented with VW medium. The results of this study indicate that Phalaenopsis hybrid 'Pink' cultured on the combination of sucrose (20 g L-1) and CJ (10%) supplemented with either MS or VW media can be used for plantlet growth of this species.

AHL-lactonase characteristics of Bacillus thuringiensis SGT3g and its effectiveness in inhibiting pathogenicity of Dickeya dadantii

ABSTRACT Aims: Dickeya dadantii is a pathogenic bacterium causing bacterial soft rot disease in plants. The bacterium uses a homoserine lactone signal in its quorum sensing process to express the virulence factor genes. Anti-quorum sensing is a new approach to control plant pathogenic bacteria. The aims of this study are to characterize AHL-lactonase enzyme produced by Bacillus thuringiensis SGT3g and to determine its effectiveness in inhibiting virulence of D. dadantii. Methodology and results: Activity of AHL-lactonase was determined using Chromobacterium violaceum as a bacterial biosensor. The crude extract enzymes of AHL-lactonase on both as extracellular and intracellular enzymes were analyzed their enzyme activity of protein precipitation and dialysis products. The optimum activity of AHL-lactonase was found at 30 °C and pH 5-8. Bacillus thuringiensis SGT3g was capable to reduce soft rot symptom disease caused by D. dadantii on Phalaenopsis orchid leaves after 24 h of incubation. Conclusion, significance and impact study: Bacillus thuringiensis SGT3g was capable to degrade AHL signal of C. violaceum and D. dadantii. The activity AHL-lactonase of B. thuringiensis SGT3g had a wide range of pH and temperature. The lactonase could reduce soft rot symptom disease caused by D. dadantii without any growth inhibition of D. dadantii on orchid leaves. Bacillus thuringiensis SGT3g can be used as an alternative biopesticide to control phytopathogenic bacteria due to its capability to suppress bacterial pathogenic virulence.

Evaluación de diferentes medios de cultivo in vitro en el desarrollo de híbridos de Phalaenopsis (Orchidaceae) / Evaluation of different in vitro culture media in the development of Phalaenopsis hybrid (Orchidaceae)

Los híbridos de Phalaenopsis tienen una gran importancia económica a nivel mundial, como flor cortada y planta ornamental, debido a sus flores vistosas y a la capacidad de adaptación a diferentes condiciones ambientales. Las técnicas de cultivo in vitro resultan indispensables para mejorar la eficacia germinativa, el crecimiento y desarrollo de orquídeas con fines comerciales e investigativos. En esta investigación se determinó el medio de cultivo más apropiado para la germinación in vitro de un híbrido de Phalaenopsis. Inicialmente se evaluó la viabilidad de las semillas utilizando la prueba de tetrazolio (TZ). Las semillas se desinfectaron y se cultivaron aplicando el método de la jeringuilla. El porcentaje de viabilidad en promedio fue de 92,2 % (P≤ 0,05: Tukey HSD), con un porcentaje de germinación entre todos los medios de 95,1 % (P≤ 0,05: Tukey HSD). El medio de cultivo más eficiente para la germinación de híbridos de Phalaenopsis a las 18 semanas de cultivo fue el Murashige & Skoog (MS) suplementado con agua de coco, y jugo de piña con diferencias estadísticamente significativas (P≤ 0,05: Tukey HSD), con respecto a los demás medios de cultivo, contribuyendo de esta manera al uso de componentes orgánicos con el fin de mejorar la germinación y desarrollo de Phalaenopsis.

The Phalaenopsis hybrids have a significant economic importance throughout the world, as ornamental flower or plant. It is because of its attractive flowers and its adaptation capacity into different environments. The different culture media in vitro are vital to improve the efficacy of germination, growing and development of the Orchids for commercial and research purposes. In this research, the most appropriated medium for in vitro propagation of Phalaenopsis hybrid was determined. At first, the seeds viability was evaluated by using tetrazolium test (TZ). The seeds were disinfected and cultivated by means of the syringe method. The viability percentage average was 92.2 % (P≤ 0.05: Tukey HSD), with a percentage of germination of 95.1 % (P≤ 0.05: Tukey HSD) in all the environments. The most efficient culture Medium for Phalaenopsis hybrid phenological development, at 16 weeks, was Murashige & Skoog (MS). Coconut water and pineapple juice were used as supplement showing statistically significant differences (P≤ 0,05: Tukey HSD), in comparison with the other culture media, contributing this way to the usage of organic components, which will be employed to improve the germination and development of the Phalaenopsis.

Development of pollen mediated activation tagging system for Phalaenopsis and Doritaenopsis

In the present study, a novel plant transformation system for Doritaenopsis and Phalaenopsis has been developed. The pollen-mediated activation tagging system was established by artificial pollination. The pollens, co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring an activation tagging vector (pTAG-8), were used for pollination. In order to optimize the transformation efficiency, several factors (concentration of A. tumefaciens, concentration of acetosyringone during co-cultivation and the duration of co-cultivation) known to influence Agrobacterium-mediated DNA transfer were examined. A concentration of 0.5-1 x 10(8) CFU/ml for A. tumefaciens, 0.1 mM acetosyringone, and 6 hrs of co-culture period were found to be the optimal condition for high transformation efficiency. Integration of T-DNA into the genome of putative transgenic plants was confirmed by PCR and DNA blot analyses. Single copy of the transgene was observed in all transgenic plants analyzed. Most of the transgenic plants had a morphologically normal phenotype and the overall capsule formation efficiency was similar to control plant. Our results showed a new approach of genetic transformation in orchids and this method can be employed for genetic improvement of the orchids.

Adubação de orquídeas em substratos com fibra de coco / Fertilization of orchids in substrates with coconut fiber

Objetivou-se, nesta pesquisa, avaliar a eficiência de substratos e doses de adubação sobre o crescimento e teores foliares de nutrientes de híbridos de orquídeas do gênero Phalaenopsis. O estudo constou de dois experimentos conduzidos em substrato com fibra de coco industrializada (Sub 1) e não industrializada (Sub 2), em mistura com casca de Pinus e brita zero (1:1:1 v/v/v), em delineamento de blocos casualizados. O primeiro experimento, em esquema de parcelas subdivididas no tempo (06 e 12 meses), com cinco repetições, avaliou o híbrido RJ 343, nos dois substratos, sob quatro doses de adubo mineral (0; 0,9; 1,2; 1,5 g L-1) e dois tratamentos adicionais [aplicação foliar de Aminon® (0,5 ml L-1) na dose de 1,2 g L-1, no Sub 1 e Sub 2]. O segundo avaliou o híbrido RJ 84-2, nos dois substratos, com as mesmas doses de adubo mineral e aplicação foliar de Aminon® (0 e 0,5 ml L-1), em esquema de parcelas subsubsubdivididas no tempo (6 e 12 meses), com três repetições. Avaliaram-se número de folhas, área foliar, área superficial de raízes, massa seca de folhas, raízes e total, e teores foliares de N, P e K. O Aminon® não teve efeito sobre o crescimento dos híbridos. O aumento da adubação promoveu aumento de crescimento, exceto de raízes, sendo o maior crescimento observado sob a dose de 1,5 g L-1 de adubo mineral. O Sub 2 promoveu maior crescimento das plantas do híbrido RJ 343, enquanto o Sub 1 promoveu maior crescimento do híbrido RJ 84-2.

The research objective was to evaluate the efficiency of substrates and fertilization levels on the growth and leaf nutrient contents of orchid hybrids of the genus Phalaenopsis. The study consisted of two experiments in carried out with industrialized coconut fiber (Sub 1) and non-industrialized coconut fiber (Sub 2), in mixture with Pinus bark and grade zero gravel (1:1:1 v/v/v) in randomized blocks. The first experiment in a split-plot scheme in time (6 and 12 months) with five replications, evaluated the hybrid RJ 343, in both substrates, under four levels of mineral fertilizer (0; 0.9; 1.2; 1.5 g L-1) and two additional treatments [leaf application of Aminon® (0.5 ml L-1) at 1.2 g L-1, in Sub1 and Sub 2]. The second one evaluated the hybrid RJ 84-2, in both substrates, with the same mineral fertilizer levels and leaf application of Aminon® (0 e 0.5 ml L-1), in a split-split-split plot scheme in time (6 and 12 months) with three replications. The number of leaves, leaf area, superficial root area, leaf, root and total dry weight, and N, P and K leaf contents were evaluated. Aminon® had no effect on the growth of the hybrids. Increase in fertilization led to increased growth, except for roots, with greater growth being observed at 1.5 g L-1 of mineral fertilizer. The Sub 2 caused greater growth of plants of the hybrid RJ 343, whereas the Sub 1 led to greater growth of the hybrid RJ 84-2.

Propagación in vitro de Phalaenopsis (Orchidaceae) a partir de protocormos, mediante el sistema de inmersión temporal RITA / In vitro propagation of Phalaenopsis (Orchidaceae) from protocorms, using the temporary inmersion system RITA

Protocormos in vitro de Phalaenopsis de tres meses de edad se transfirieron a contenedores RITA® con el fin depropagarlos masivamente. Los factores evaluados fueron la concentración de sacarosa en el medio y la frecuencia de inmersión. Se dispusieron cinco pares de contenedores RITA® con medio de cultivo líquido a concentraciones de sacarosa de 0, 15, 30, 45 y 60 g/L. El medio utilizado fue el MS a la mitad de la concentración de las sales, suplementado con vitaminas y tidiazuron (5 mg/L). El experimento se realizó en dos etapas, cada una con duración de dos meses. La primera etapa con una frecuencia de inmersión de cuatro horas y la segunda con una frecuencia de inmersión de ocho horas, ambas con un tiempo de inmersión de un minuto. Los resultados mostraron que la mejor respuesta proliferativa, con 8,2 protocormos adventicios por protocormo por mes, se obtuvo en el medio con 15 g/L de sacarosa y un tiempo de inmersión de un minutocada cuatro horas.

In order to massively propagate Phalaenopsis orchids, three months old in vitro protocorms were transferred to RITA® vessels. The evaluation factors were the sucrose concentration in the culture medium and the frequency immersion. There was a set of five pairs of RITA® vessels with liquid culture medium containing sucrose at 0, 15, 30, 45, 60 g/L. A half-strength MS medium plus vitamins, supplemented with vitamins and thidiazuron (5 mg/L) was used. The experiment had two stages, each lasting two months. Each stage had a one minute immersion. The first stage had a four hour immersion frequency and the second one had an eight hour immersion frequency. Results showed that the best proliferating response was reached in a 15 g/L of sucrose medium with one minute of immersion time every four hours, resulting in 8,2 adventitious protocorms per protocorm per month.