In Vitro Calli Production Resulted in Different Profiles of Plant-Derived Medicinal Compounds in Phyllanthus amarus.

The efficient production of plant-derived medicinal compounds (PDMCs) from in vitro plants requires improvements in knowledge about control of plant or organ development and factors affecting the biosynthesis pathway of specific PDMCs under in vitro conditions, leading to a realistic large-scale tool for in vitro secondary metabolite production. Thus, this study aimed to develop an in vitro technique, through the induction and proliferation of calli, for production of plant fresh weight, and to compare the PDMC profile obtained from the plants versus in vitro calli of Phyllanthus amarus. It was successfully possible to obtain and proliferate two types of calli, one with a beige color and a friable appearance, obtained in the dark using Murashige and Skoog (MS) medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and a second type with a green color, rigid consistency, and nonfriable appearance obtained under light conditions and MS medium plus 6-benzyladenine (6-BA). In vitro micropropagated plants that gave rise to calli were also acclimatized in a greenhouse and cultivated until obtaining the mass for PDMC analysis and used as a control. While the micropropagated-derived plants concentrated the lignans niranthin, nirtetralin, and phyllanthin, the Phyllanthus amarus calli proliferated in vitro concentrated a completely different biochemical profile and synthesis of compounds, such as betulone, squalene, stigmasterol, and ß-sitosterol, in addition to others not identified by GC-MS database. These results demonstrate the possibility of applying the calli in vitro from Phyllanthus amarus for production of important PDMCs unlike those obtained in cultures of differentiated tissues from field plants.

Influencia de reguladores de crecimiento sobre el establecimiento de cultivos in vitro de Salvia hispanica L. ("chía") y sobre su contenido de ácidos grasos / Influence of growth regulators on the establishment of in vitro cultures of Salvia hispanica L. ("chia") and its content of fatty acids

Salvia hispanica L. (Labiateae), comúnmente conocida como "chía", es una especie herbácea anual cuyo cultivo está ampliamente extendido por América del Sur. Es utilizada como cultivo industrial y para la ela-boración de alimentos funcionales por su contenido en proteínas, antioxidantes, fibras y lípidos esenciales. El aceite de sus semillas contiene la mayor proporción (68 %) de omega-3 que cualquier fuente vegetal conocida. El objetivo de este trabajo fue establecer cultivos in vitro de "chía" y analizar la influencia de diferentes reguladores de crecimiento sobre la inducción de callos, sobre el crecimiento de estos y sobre su contenido de ácidos grasos. Se iniciaron cultivos de callos in vitro a partir de explantos de tallos sin nudos y de hojas de plántulas axénicas de 20 días de edad, utilizando 3 tratamientos de reguladores de crecimiento diferentes. El medio Murashige & Skoog modificado (MSRT) con el agregado de ácido 2,4-diclorofenoxia-cético (2,4-D) a una concentración de 2,25 µM y un fotoperíodo de 16 horas fueron las condiciones óptimas para la inducción de callos. Para el mantenimiento de los callos el tratamiento más adecuado resultó ser bencilaminopurina (BAP) a una concentración de 1 µM. La cinética de crecimiento se caracterizó por un período de latencia hasta el día 20 de cultivo, seguido de un período de crecimiento exponencial entre los días 20 y 54. El tratamiento con 2,4-D (2,25 µM) mostró la más alta velocidad específica de crecimiento (0,22 ± 0,01 /día), el tiempo de duplicación más bajo (31,51 ± 1,00 día) y la mayor biomasa máxima (1,46 ± 0,01 g PF). El contenido de ácidos grasos en los callos de "chía" fue 0,83 % en promedio de todos los tratamientos después de 6 meses en cultivo y no mostró variaciones significativas (0,2 % - 0,3 %) entre los tratamientos aplicados (p < 0,05). (AU)

Using foliar spectral properties to assess the effects of drought on plant water potential.

Drought frequency is predicted to increase in future environments. Leaf water potential (ΨLW) is commonly used to evaluate plant water status, but traditional measurements can be logistically difficult and require destructive sampling. We used reflectance spectroscopy to characterize variation in ΨLW of Quercus oleoides Schltdl. & Cham. under differential water availability and tested the ability to predict pre-dawn ΨLW (PDΨLW) using spectral data collected hours after pressure chamber measurements on dark-acclimated leaves. ΨLW was measured with a Scholander pressure chamber. Leaf reflectance was collected at one or both of two time points: immediately (ΨLW) and ~5 h after pressure chamber measurements (PDΨLW). Predictive models were constructed using partial least-squares regression. Model performance was evaluated using coefficient of determination (R2), root-mean-square error (RMSE), bias, and the percent RMSE of the data range (%RMSE). ΨLW and PDΨLW were well predicted using spectroscopic models and successfully estimated a wide variation in ΨLW (light- or dark-acclimated leaves) as well as PDΨLW (dark-acclimated leaves only). Mean ΨLWR2, RMSE and bias values were 0.65, 0.51 MPa and 0.09, respectively, with a %RMSE between 8% and 20%, while mean PDΨLWR2, RMSE and bias values were 0.60, 0.44 MPa and 0.01, respectively, with a %RMSE between 9% and 20%. Estimates of PDΨLW produced similar statistical outcomes when analyzing treatment effects on PDΨLW as those found using reference pressure chamber measurements. These findings highlight a promising approach to evaluate plant responses to environmental change by providing rapid measurements that can be used to estimate plant water status as well as demonstrating that spectroscopic measurements can be used as a surrogate for standard, reference measurements in a statistical framework.

Influence of light and plant size on the reproduction and growth of small palm tree species: Comparing two methods for measuring canopy openness.

PREMISE OF THE STUDY: The reproduction and growth of large palms are influenced by many factors, including light and plant size, but few studies have examined smaller species (up to 2 m tall). We examined the effect of incident light and individual size on growth rates, the probability of reproduction, and the number of inflorescences of three small palm species and compared two methods for estimating canopy openness. METHODS: We measured canopy openness above the crowns of 132 adult plants and in the centers of 72 subplots (10 × 10 m) where individuals were sampled. We also recorded individual size and the number of leaves and inflorescences produced in two years. KEY RESULTS: Reproductive individuals of Butia paraguayensis tend to occur in illuminated microhabitats. Reproduction of Acrocomia emensis was correlated with stem diameter, but not with light. Reproduction was inversely related to individual size and light in Syagrus petraea, probably because this clonal palm invests heavily in sexual reproduction during its younger stages and clonal activity in older stages and may be adapted to the low-light conditions of the woodland understory. Growth was not predicted by light or individual size. Stronger correlations were found when incident light was measured directly above the crown, as opposed to the subplot center. CONCLUSIONS: The influences of light on reproduction are dependent on plant life histories, even among related and sympatric species. Light measurements directly above individual crowns provide better understanding of the reproductive effort rather than in subplot center.

A Novel Method for the Separation of Overlapping Pollen Species for Automated Detection and Classification.

The identification of pollen in an automated way will accelerate different tasks and applications of palynology to aid in, among others, climate change studies, medical allergies calendar, and forensic science. The aim of this paper is to develop a system that automatically captures a hundred microscopic images of pollen and classifies them into the 12 different species from Lagunera Region, Mexico. Many times, the pollen is overlapping on the microscopic images, which increases the difficulty for its automated identification and classification. This paper focuses on a method to segment the overlapping pollen. First, the proposed method segments the overlapping pollen. Second, the method separates the pollen based on the mean shift process (100% segmentation) and erosion by H-minima based on the Fibonacci series. Thus, pollen is characterized by its shape, color, and texture for training and evaluating the performance of three classification techniques: random tree forest, multilayer perceptron, and Bayes net. Using the newly developed system, we obtained segmentation results of 100% and classification on top of 96.2% and 96.1% in recall and precision using multilayer perceptron in twofold cross validation.

DNA-Based Authentication of Botanicals and Plant-Derived Dietary Supplements: Where Have We Been and Where Are We Going?

Herbal medicines and botanicals have long been used as sole or additional medical aids worldwide. Currently, billions of dollars are spent on botanicals and related products, but minimal regulation exists regarding their purity, integrity, and efficacy. Cases of adulteration and contamination have led to severe illness and even death in some cases. Identifying the plant material in botanicals and phytomedicines using organoleptic means or through microscopic observation of plant parts is not trivial, and plants are often misidentified. Recently, DNA-based methods have been applied to these products because DNA is not changed by growth conditions unlike the chemical constituents of many active pharmaceutical agents. In recent years, DNA barcoding methods, which are used to identify species diversity in the Tree of Life, have been also applied to botanicals and plant-derived dietary supplements. In this review, we recount the history of DNA-based methods for identification of botanicals and discuss some of the difficulties in defining a specific bar code or codes to use. In addition, we describe how next generation sequencing technologies have enabled new techniques that can be applied to identifying these products with greater authority and resolution. Lastly, we present case histories where dietary supplements, decoctions, and other products have been shown to contain materials other than the main ingredient stipulated on the label. We conclude that there is a fundamental need for greater quality control in this industry, which if not self-imposed, that may result from legislation.

Optimized method for growing in vitro Arabidopsis thaliana pollen tubes.

Pollen tubes elongate by tip growth toward the ovule to deliver the sperm cells during fertilization. Since pollen tubes from several species can be grown in vitro maintaining their polarity, pollen tube growth is a suitable model system to study cell polarity and tip growth. A. thaliana pollen tubes germinated in vitro for 6 h can reach up to 800 µm. By studying the phenotype of mutants of T-DNA insertion lines, genes involved in pollen tube growth can be identified. Moreover, components involved in the regulation of pollen tube growth such as calcium ions and reactive oxygen species (ROS) can be analyzed.

O uso de plantas medicinais por moradores de Quixadá­Ceará / The use of medicinal plants to habitants from Quixada-Ceara

RESUMO O presente trabalho teve por objetivo observar o uso de plantas medicinais por moradores de Quixadá-Ceará. A amostragem adotada foi 5% da população urbana adulta na sede do município, o que resultou em 263 domicílios visitados, onde foi aplicado um questionário semi-estruturado. De acordo com os resultados ainda temos um número expressivo de pessoas que fazem uso de plantas medicinais na cidade de Quixadá-Ceará (57,41%), e que a maioria dos entrevistados (97%) considera que o uso indiscriminado de plantas medicinais não causa nenhum problema à saúde. A maioria dos entrevistados foram mulheres (63,57%) e o maior número de informações e conhecimentos sobre plantas medicinais concentrou-se na faixa de 51-60 anos (24,71%). Com relação ao nível de escolaridade, 34,22% dos entrevistados afirmou ter ensino médio completo. A família botânica mais representativa foi a Lamiaceae (21,05%) e a espécie mais citada foi a erva-cidreira. Dos entrevistados 93,37% consideraram utilizar o chá como medicamento e 6,63% alegaram fazer a utilização do mesmo como alimento. Quanto à frequência do uso de plantas medicinais de forma contínua, 27% alegaram fazer o uso diariamente.

ABSTRACT This study aimed to observe the use of medicinal plants by residents from Quixada-Ceara. The sample used was 5% of the adult urban population in the county seat, which resulted in 263 households visited, where we applied a semi-structured questionnaire. According to the results, there is still a significant number of people who uses medicinal plants in the city of Quixada-Ceara (57.41%), and the majority of participants (97%) consider that the indiscriminate employment of medicinal plants do not cause any health problem. Most respondents were female (63.57%) and the highest number of information and knowledge about medicinal plants were concentrated among 51-60 years old individuals (24.71%). In regard of the educational level, 34.22% of the participants claimed to have completed high school. The botanical family Lamiaceae was the most representative (21.05%) and the most cited specie was the lemongrass. 93.37% of the respondents pledged to use tea as medicine and 6.63% claimed to use it as food. Regarding the employment frequency of the medicinal plants, 27% claimed to take them daily.

Empirical trials of plant field guides.

We designed 3 image-based field guides to tropical forest plant species in Ghana, Grenada, and Cameroon and tested them with 1095 local residents and 20 botanists in the United Kingdom. We compared users' identification accuracy with different image formats, including drawings, specimen photos, living plant photos, and paintings. We compared users' accuracy with the guides to their accuracy with only their prior knowledge of the flora. We asked respondents to score each format for usability, beauty, and how much they would pay for it. Prior knowledge of plant names was generally low (<22%). With a few exceptions, identification accuracy did not differ significantly among image formats. In Cameroon, users identifying sterile Cola species achieved 46-56% accuracy across formats; identification was most accurate with living plant photos. Botanists in the United Kingdom accurately identified 82-93% of the same Cameroonian species; identification was most accurate with specimens. In Grenada, users accurately identified 74-82% of plants; drawings yielded significantly less accurate identifications than paintings and photos of living plants. In Ghana, users accurately identified 85% of plants. Digital color photos of living plants ranked high for beauty, usability, and what users would pay. Black and white drawings ranked low. Our results show the potential and limitations of the use of field guides and nonspecialists to identify plants, for example, in conservation applications. We recommend authors of plant field guides use the cheapest or easiest illustration format because image type had limited bearing on accuracy; match the type of illustration to the most likely use of the guide for slight improvements in accuracy; avoid black and white formats unless the audience is experienced at interpreting illustrations or keeping costs low is imperative; discourage false-positive identifications, which were common; and encourage users to ask an expert or use a herbarium for groups that are difficult to identify.

Effects of light and temperature on isoprene emission at different leaf developmental stages of eschweilera coriacea in central Amazon / Efeitos da luz e da temperatura sobre a emissão de isopreno em diferentes estádios de desenvolvimento foliar de eschweilera coriacea na Amazônia Central

Isoprene emission from plants accounts for about one third of annual global volatile organic compound emissions. The largest source of isoprene for the global atmosphere is the Amazon Basin. This study aimed to identify and quantify the isoprene emission and photosynthesis at different levels of light intensity and leaf temperature, in three phenological phases (young mature leaf, old mature leaf and senescent leaf) of Eschweilera coriacea (Matamatل verdadeira), the species with the widest distribution in the central Amazon. In situ photosynthesis and isoprene emission measurements showed that young mature leaf had the highest rates at all light intensities and leaf temperatures. Additionally, it was observed that isoprene emission capacity (Es) changed considerably over different leaf ages. This suggests that aging leads to a reduction of both leaf photosynthetic activity and isoprene production and emission. The algorithm of Guenther et al. (1999) provided good fits to the data when incident light was varied, however differences among E S of all leaf ages influenced on quantic yield predicted by model. When leaf temperature was varied, algorithm prediction was not satisfactory for temperature higher than ~40 °C; this could be because our data did not show isoprene temperature optimum up to 45 °C. Our results are consistent with the hypothesis of the isoprene functional role in protecting plants from high temperatures and highlight the need to include leaf phenology effects in isoprene emission models.

O isopreno emitido pelas plantas corresponde em cerca de um terço das emissões globais de compostos orgânicos voláteis anualmente. A maior fonte de emissão de isopreno para a atmosfera global é a Bacia Amazônica. Este estudo objetivou identificar e quantificar a emissão de isopreno e fotossíntese em diferentes níveis de intensidade de luz e temperatura foliar, em três fases fenológicas (folha madura recente, folha madura tardia e folha senescente) de Eschweilera coriacea (Matamatá verdadeira) - a espécie com maior distribuição na Amazônia central. In situ, as medidas de fotossíntese e emissão de isopreno da folha madura recente apresentaram as maiores taxas em todos os níveis de luz e de temperatura. Adicionalmente, a capacidade de emissão de isopreno (ES) mudou consideravelmente entre as diferentes idades foliares, sugerindo que o envelhecimento reduz a atividade fotossintética e a produção/emissão de isopreno. O algoritmo de Guenther et al. (1999) demonstrou bom ajuste para a emissão de isopreno em diferentes intensidades de luz, entretanto, diferenças na ES entre as idades foliares influenciaram no rendimento quântico estimado pelo modelo. Em relação à temperatura foliar, a estimativa do algoritmo não foi satisfatória para as temperaturas acima de ~40 °C; isto provavelmente ocorreu pelo fato dos dados não apresentarem temperatura ótima até 45 °C. Nossos resultados são consistentes com a hipótese do isopreno ter um papel funcional para proteger as plantas de altas temperaturas e apontam a necessidade de incluir os efeitos da fenologia foliar em modelos de emissão de isopreno.

Effects of light and temperature on isoprene emission at different leaf developmental stages of eschweilera coriacea in central Amazon / Efeitos da luz e da temperatura sobre a emissão de isopreno em diferentes estádios de desenvolvimento foliar de eschweilera coriacea na Amazônia Central

Isoprene emission from plants accounts for about one third of annual global volatile organic compound emissions. The largest source of isoprene for the global atmosphere is the Amazon Basin. This study aimed to identify and quantify the isoprene emission and photosynthesis at different levels of light intensity and leaf temperature, in three phenological phases (young mature leaf, old mature leaf and senescent leaf) of Eschweilera coriacea (Matamatل verdadeira), the species with the widest distribution in the central Amazon. In situ photosynthesis and isoprene emission measurements showed that young mature leaf had the highest rates at all light intensities and leaf temperatures. Additionally, it was observed that isoprene emission capacity (Es) changed considerably over different leaf ages. This suggests that aging leads to a reduction of both leaf photosynthetic activity and isoprene production and emission. The algorithm of Guenther et al. (1999) provided good fits to the data when incident light was varied, however differences among E S of all leaf ages influenced on quantic yield predicted by model. When leaf temperature was varied, algorithm prediction was not satisfactory for temperature higher than ~40 °C; this could be because our data did not show isoprene temperature optimum up to 45 °C. Our results are consistent with the hypothesis of the isoprene functional role in protecting plants from high temperatures and highlight the need to include leaf phenology effects in isoprene emission models.(AU)

O isopreno emitido pelas plantas corresponde em cerca de um terço das emissões globais de compostos orgânicos voláteis anualmente. A maior fonte de emissão de isopreno para a atmosfera global é a Bacia Amazônica. Este estudo objetivou identificar e quantificar a emissão de isopreno e fotossíntese em diferentes níveis de intensidade de luz e temperatura foliar, em três fases fenológicas (folha madura recente, folha madura tardia e folha senescente) de Eschweilera coriacea (Matamatá verdadeira) - a espécie com maior distribuição na Amazônia central. In situ, as medidas de fotossíntese e emissão de isopreno da folha madura recente apresentaram as maiores taxas em todos os níveis de luz e de temperatura. Adicionalmente, a capacidade de emissão de isopreno (ES) mudou consideravelmente entre as diferentes idades foliares, sugerindo que o envelhecimento reduz a atividade fotossintética e a produção/emissão de isopreno. O algoritmo de Guenther et al. (1999) demonstrou bom ajuste para a emissão de isopreno em diferentes intensidades de luz, entretanto, diferenças na ES entre as idades foliares influenciaram no rendimento quântico estimado pelo modelo. Em relação à temperatura foliar, a estimativa do algoritmo não foi satisfatória para as temperaturas acima de ~40 °C; isto provavelmente ocorreu pelo fato dos dados não apresentarem temperatura ótima até 45 °C. Nossos resultados são consistentes com a hipótese do isopreno ter um papel funcional para proteger as plantas de altas temperaturas e apontam a necessidade de incluir os efeitos da fenologia foliar em modelos de emissão de isopreno.(AU)

Confirmation of a purple-leaved plum graft hybrid.

Fifty-seven scions from an adult purple-leaved plum tree were grafted onto the crown of a 6-year-old Yuhuang plum tree and compared to the control of a non-grafted tree. The floral buds of the purple-leaved plum were fully removed before blossoming to avoid sexual hybridization between the two species. The seeds of the Yuhuang plum were picked in July and sown in the spring after stratification. Three, eleven and eight variants with purplish red leaves were found among the seedlings that grew from the seeds picked in 1999, 2000, and 2001, respectively. The ratio of variant occurrence ranged from 2.3 to 15.8%. Our results confirmed the observation of a graft hybrid by Luther Burbank.

Identificação botânica e química de espécies vegetais de uso popular no Rio Grande do Norte, Brasil / Botanical and chemical identification of plant species of popular use in Rio Grande do Norte, Brazil

Plantas medicinais são utilizadas mundialmente como uma das principais formas de cuidado primário de saúde. No entanto, a literatura indica que muitas espécies podem apresentar composição química variável, toxicidade ou difícil identificação. O objetivo do presente estudo foi obter critérios úteis para o controle de qualidade farmacognóstico das principais espécies vegetais de uso popular no estado do Rio Grande do Norte, utilizando metodologias de análise botânica (morfodiagnose macro e microscópica) e química (triagem fitoquímica e cromatografia em camada delgada), evitando assim adulterações ou uso inadequado dessas plantas medicinais no estado. No total, sete espécies foram analisadas Acmella oleracea, Chenopodium ambrosioides, Lippia alba, Mentha piperita, Ocimum gratissimum, Peumus boldus e Rosmarinus officinalis. Diversos marcadores botânicos e fitoquímicos foram identificados, contribuindo dessa forma para a correta identificação destas espécies de plantas medicinais importantes no estado do Rio Grande do Norte.

Medicinal plants are worldwide used as one of the main forms of primary healthcare. However, the literature indicates that many species may have variable chemical composition, toxicity, or even difficult identification. The aim of this study was to obtain useful criteria for pharmacognostic quality control of the main plant species of popular use in the state of Rio Grande do Norte, using methods of botanical (macro and microscopic morphodiagnosis) and chemical (phytochemical screening and thin-layer chromatography) analysis, thus preventing adulteration or inappropriate use of these medicinal plants in the state. In total, seven species were analyzed Acmella oleracea, Chenopodium ambrosioides, Lippia alba, Mentha piperita, Ocimum gratissimum, Peumus boldus and Rosmarinus officinalis. Several botanical and phytochemical markers were identified, thereby contributing to the correct identification of these important medicinal plant species in the state of Rio Grande do Norte.

Two measurement methods of leaf dry matter content produce similar results in a broad range of species.

BACKGROUND AND AIMS: Leaf dry matter content (LDMC) is widely used as an indicator of plant resource use in plant functional trait databases. Two main methods have been proposed to measure LDMC, which basically differ in the rehydration procedure to which leaves are subjected after harvesting. These are the 'complete rehydration' protocol of Garnier et al. (2001, Functional Ecology 15: 688-695) and the 'partial rehydration' protocol of Vendramini et al. (2002, New Phytologist 154: 147-157). METHODS: To test differences in LDMC due to the use of different methods, LDMC was measured on 51 native and cultivated species representing a wide range of plant families and growth forms from central-western Argentina, following the complete rehydration and partial rehydration protocols. KEY RESULTS AND CONCLUSIONS: The LDMC values obtained by both methods were strongly and positively correlated, clearly showing that LDMC is highly conserved between the two procedures. These trends were not altered by the exclusion of plants with non-laminar leaves. Although the complete rehydration method is the safest to measure LDMC, the partial rehydration procedure produces similar results and is faster. It therefore appears as an acceptable option for those situations in which the complete rehydration method cannot be applied. Two notes of caution are given for cases in which different datasets are compared or combined: (1) the discrepancy between the two rehydration protocols is greatest in the case of high-LDMC (succulent or tender) leaves; (2) the results suggest that, when comparing many studies across unrelated datasets, differences in the measurement protocol may be less important than differences among seasons, years and the quality of local habitats.

Micropropagation of Glandularia perakii Cov. et Schn. (Verbenaceae), a native species with ornamental potential.

Glandularia perakii is a perennial species with beautiful violet flowers that grows in the stony soil of Mendocine pedemont. A plentiful and prolonged flowering confers it an important ornamental potential. In this paper, a method of propagation of G. perakii from nodal segments is reported. Proliferating microshoot cultures were obtained by placing nodal segment on Murashige and Skoog medium (MS) supplemented with 20 g.L-1 of sucrose without growth regulators. In this medium multiplication rate after 20 days was 7.9. Rooted plants were acclimatized successfully.

Micropropagation of Glandularia perakii Cov. et Schn. (Verbenaceae), a native species with ornamental potential

Glandularia perakii is a perennial species with beautiful violet flowers that grows in the stony soil of Mendocine pedemont. A plentiful and prolonged flowering confers it an important ornamental potential. In this paper, a method of propagation of G. perakii from nodal segments is reported. Proliferating microshoot cultures were obtained by placing nodal segment on Murashige and Skoog medium (MS) supplemented with 20 g.L-1 of sucrose without growth regulators. In this medium multiplication rate after 20 days was 7.9. Rooted plants were acclimatized successfully. (AU)

Micropropagation of Glandularia perakii Cov. et Schn. (Verbenaceae), a native species with ornamental potential

Glandularia perakii is a perennial species with beautiful violet flowers that grows in the stony soil of Mendocine pedemont. A plentiful and prolonged flowering confers it an important ornamental potential. In this paper, a method of propagation of G. perakii from nodal segments is reported. Proliferating microshoot cultures were obtained by placing nodal segment on Murashige and Skoog medium (MS) supplemented with 20 g.L-1 of sucrose without growth regulators. In this medium multiplication rate after 20 days was 7.9. Rooted plants were acclimatized successfully.