Plant regeneration from callus cultures of Vitex trifolia (Lamiales: Lamiaceae): a potential medicinal plant.

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25 +/-2 degrees C under a light intensity of 61 micromol/m2s from cool white fluorescent lamps and a 16 h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10 microM BAP in combination with 0.54 microMNAA. Addition of 135.74-271.50 microM adenine sulphate (Ads) and 0.72-1.44 microM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10 microM BAP in combination with 0.54 microM NAA, 271.50 microM Ads and 1.44 microM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23 microM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Plant regeneration from callus cultures of Vitex trifolia (Lamiales: Lamiaceae): a potential medicinal plant / Regeneración de plantas a partir de cultivos de callos de Vitex trifolia (Lamiales: Lamiaceae): una planta medicinal potencial

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supple mented Murashige & Skoog medium, and incubated at 25±2ºC under a light intensity of 61µmol/m2s from cool white fluorescent lamps and a 16h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10µM BAP in combination with 0.54µMNAA. Addition of 135.74-271.50µM adenine sulphate (Ads) and 0.72-1.44µM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10µM BAP in combination with 0.54µM NAA, 271.50µM Ads and 1.44µM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23µM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Vitex trifolia es una especie arbustiva de uso popular como planta medicinal, sus hojas, raíces y flores se han reportado para la cura de diferentes aflicciones. El aumento de la explotación de estas plantas ha puesto en peligro su conservación y ha justificado el uso de herramientas biotecnológicas para su propagación. El objetivo de esta investigación fue presentar un protocolo eficiente para la regeneración de estas plantas a través de la organogénesis, y analizar la homogeneidad genética de las líneas clonales establecidas por ADN polimórfico amplificado aleatoriamente (RAPD) mediante la repetición de marcadores de inter secuencia simple (ISSR). La regeneración de plántulas se logró en cultivos de callos derivados de explantes de tallo, hoja y pecíolo de V. trifolia en un medio diferenciado Murashige & Skoog, que se incubaron a 25±2ºC bajo una intensidad de luz de 61μmol/m2s con lámparas fluorescentes blancas y un fotoperíodo de 16h. La tasa de regeneración de brotes se correlacionó positivamente con la concentración de las hormonas en el medio nutritivo. Los brotes se regeneraron más rápidamente a partir de explantes de tallo y pecíolos en comparación con explantes de hoja. La mayor tasa de regeneración de brotes se obtuvo en los explantes de tallo utilizando 11.10μM BAP en combinación con 0.54μM NAA, 271.50μM Ads y 1.44μM GA3. Este protocolo puede ayudar a la propagación masiva y conservación de esta importante planta medicinal de gran potencial terapéutico.

Factors influencing rapid clonal propagation of Chlorophytum arundinaceum (Liliales: Liliaceae), an endangered medicinal plant.

Chlorophytum arundinaceum is an important medicinal plant and its tuberous roots are used for various health ailment treatments. It has become an endangered species in the Eastern Ghats, and a rare medicinal herb in India, due to its excessive collection from its natural habitat and its destructive harvesting techniques, coupled with poor seed germination and low vegetative multiplication ratio. In order to contribute to its production systems, an efficient protocol was developed for in vitro clonal propagation through shoot bud culture. For this, multiple shoots were induced from shoot bud explants on Murashige and Skoog's medium supplemented with 2.5-3.0 mg/L BAP, 0.01-0.1 mg/LNAA and 3% (w/v) sucrose. Inclusion of Adenine Sulphate (25mg/L) in the culture medium improved the frequency of multiple shoot production and recovered the chlorotic symptoms of the leaves. Media having pH 5.9 and 4% sucrose showed significant improvement on shoot bud multiplication and growth. In vitro flowering was observed when the subcultures were carried out for over four months in the same multiplication media. Rooting was readily achieved upon transferring the shoots on to half- strength MS medium supplemented with 0.1 mg/L IBA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house, successfully established, and flowered in the field. This method could effectively be applied for the conservation and clonal propagation to meet the demand of planting materials.

Factors influencing rapid clonal propagation of Chlorophytum arundinaceum (Liliales: Liliaceae), an endangered medicinal plant

Chlorophytum arundinaceum is an important medicinal plant and its tuberous roots are used for various health ailment treatments. It has become an endangered species in the Eastern Ghats, and a rare medicinal herb in India, due to its excessive collection from its natural habitat and its destructive harvesting techniques, coupled with poor seed germination and low vegetative multiplication ratio. In order to contribute to its production systems, an efficient protocol was developed for in vitro clonal propagation through shoot bud culture. For this, multiple shoots were induced from shoot bud explants on Murashige and Skoog’s medium supplemented with 2.5-3.0mg/L BAP, 0.01-0.1mg/L NAA and 3% (w/v) sucrose. Inclusion of Adenine Sulphate (25mg/L) in the culture medium improved the frequency of multiple shoot production and recovered the chlorotic symptoms of the leaves. Media having pH 5.9 and 4% sucrose showed significant improvement on shoot bud multiplication and growth. In vitro flowering was observed when the subcultures were carried out for over four months in the same multiplication media. Rooting was readily achieved upon transferring the shoots on to half- strength MS medium supplemented with 0.1mg/L IBA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house, successfully established, and flowered in the field. This method could effectively be applied for the conservation and clonal propagation to meet the demand of planting materials. Rev. Biol. Trop. 59 (1): 435-445. Epub 2011 March 01.

Chlorophytum arundinaceum es una planta medicinal importante y sus raíces se utilizan en diversos tratamientos contra enfermedades. Se ha convertido en una especie en peligro de extinción en el Ghats Oriental y una hierba medicinal rara en la India, debido a la recolecta excesiva en su hábitat natural y la manera destructiva de cosecharla, asociado con una mala germinación y pobre multiplicación vegetativa. Para contribuir con sus sistemas de producción, se desarrolló un protocolo eficiente para la propagación clonal in vitro a través del cultivo de brotes. Para ello, los retoños múltiples fueron inducidos a partir de sus brotes en un medio Murashige y Skoog enriquecido con 2.5-3.0mg/L de BAP, 0.01-0.1mg/L de NAA y el 3% (w/v) sucrosa. La inclusión de sulfato de adenina (25mg/L) en el medio de cultivo mejoró la frecuencia de producción de brotes múltiples y se recuperaron los síntomas de clorosis de las hojas. Los medios con un pH de 5.9 y 4% de sucrosa mostraron una mejoría significativa en la multiplicación y crecimiento de las yemas. En la floración in vitro se observó cuando los subcultivos se llevaron a cabo durante más de cuatro meses para los mismos medios de multiplicación. El enraizamiento se logró fácilmente al transferir los brotes a un medio MS de intensidad media enriquecido con 0.1 mg/l de IBA y 2% (w/v) de sucrosa. Las plántulas micropropagadas maduraron en el invernadero, se establecieron exitosamente y florearon en el campo. Este método se podría aplicar para la conservación y propagación clonal con el fin de satisfacer la demanda de material de siembra.