Population Structure Of Kaempferia Galanga L. From Eastern India

Objective: India has been a producer of a large number of aromatic medicinal plants which serves as a valuable genetic resource for future quality improvement to meet the ever-growing demand of human essential products. Thus, an urgent need arises for germplasm conservation of these high yielding varieties to help the pharmaceutical and other industries. For this understanding, the population structure is essential in order to explore their genetic identification by fingerprinting and molecular characterization. Methods: In the present study DNA was isolated using modified Cetyl Trimethyl Ammonium Bromide (CTAB) method and Polymerase Chain Reaction (PCR) was performed according to standardized method along with its data analysis. This study was undertaken to characterize the highly medicinal Kaempferia galanga collected from 4 different populations of Odisha using the molecular markers as Random Amplified Polymorphic DNA and Inter-Simple Sequence Repeats for the first time. Results: A dendrogram constructed through Sequential Agglomerative Hierarchical and Nested (SAHN) clustering and Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis showed an average similarity of 0.993 ranging between 0.967 to 1.000. Jaccard’s similarity coefficient of combined markers segregated the genotypes into two main clusters, 1 with six samples and the others at 0.98 similarity coefficient. Conclusion: Hence, the molecular analysis could be further used for the identification of important novel gene present in Kaempferia galanga which can be utilized for future crop improvement as well as pharmacological activities.

ISSR analysis of genetic background of triploid female and diploid male strains of Siraitia grosvenorii / 中草药

To evaluate the genetic background of triploid female and diploid male strains of Siraitia grosvenorii and provide the biological reference for good varieties breeding of seedless S. grosvenorii. Inter simple sequence repeats (ISSR) marker was developed to analyze the genetic background among 28 samples of S. grosvenorii, and cluster analysis and double principal coordinate analysis were revealed by the NTSYS-pc software and GenAIEx software, respectively. Out of 100 ISSR primers selected, 13 primers were used for amplification and a total of 131 unambiguous bands were obtained, among which 99 (PPB = 75.57%) were polymorphic. The results of cluster analysis and double principal coordinate analysis showed that there was a certain rich of genetic background in triploid female and diploid male strains of S. grosvenorii. But the genetic similarity coefficients of majority were bigger and the genetic distance was closer. The complexity of the genetic background in triploid female and diploid male strains of S. grosvenorii is lower and germplasm innovation strategies should be carried out to enrich the genetic background of the parents of seedless S. grosvenorii.

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.

Establishment and optimization of ISSR reaction system for Anoectochilus formosanus / 中草药

Objective: To establish a stable, reproducible, and suitable reaction system of Anoectochilus formosanus for ISSR analysis of genetic differences according to the ISSR-PCR characters in plants of Anoectochilus Bl. Methods: The initial ISSR reaction system in plants of Anoectochilus Bl. was established and then the primers were screened. After that, PCR amplifications were carried out until the different concentrations of the factors in the ISSR reaction system were designed, and based on the principle of high stability and reproducibility, the stable and reliable ISSR reaction system was eventually established after a series of adjustment and optimization of the reaction system. Results: The optimal ISSR reaction system in plants of Anoectochilus Bl. was reported for the first time, and the volume of 25 μL contained approximately 20 ng template DNA, 1 U Taq DNA polymerase, 0.2 mmol/L deoxyribonucleotide triphosphate (dNTP), 2 mmol/L MgCl2, 200 umol/L dNTP Mixture, and 0.2 μmol/L primer within 1x reaction buffer [10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl]. The reaction mixtures were denatured at 94°C for 7 min and subjected to 45 cycles of 30 s at 94 °C, 45 s at 52 °C, 2 min at 72 °C, and a final extension step of 10 min at 72 °C and eventually stored at 4 °C. Conclusion: The ISSR-PCR systems established for studying on the plants of Anoectochilus Bl. show the characters of stable reaction system, better repeatability, clear marker site, and reliable abundant polymorphisms. It is suitable for the study on genetic diversity in plants of Anoectochilus Bl. in difference wild populations and genetic variation of its sterile seedlings after long time tissue culture.

Analysis of genetic relationship and quality assessment on plants in Flemingia Roxb. ex Ait. et Ait. f / 中草药

Objective: To analyze the genetic relationship of the plants in Flemingia Roxb. ex Ait. et Ait. f. and make preliminary quality accessment, in order to provide the theoretical basis for screening the new medicinal resource. Methods: Inter-simple sequence repeats (ISSR) markers were used to analyze the genetic relationship of 14 species in Flemingia Roxb. ex Ait. et Ait. f.. Meanwhile, spectrophotometry was used to measure total flavones. Results: Thirty primers were screened out of 60 ISSR random primers and produced 367 bands totally. Among them 363 bands (98.91%) were polymorphic, four bands were owned by all; The genetic similarity (GS) was 0.5668-0.8338, showing abundant genetic diversity; F. wallichii and F. philippinensis have the closest genetic relationship, F. lineate and F. chappar have the largest genetic distance; The highest content of total flavones is in F. mengpengensis and secondary is in F. philippinensis, total content of flavones in F. wallichii is above average. Conclusion: The plants in Flemingia Roxb. ex Ait. et Ait. f. have the abundant genetic diversity. As medicinal plants, F. wallichii has the potential for replacing F. philippinensis.

Application of ISSR-PCR to identification of different Dendrobium Sw. species / 中草药

Objective To identify nine species of Dendrobium Sw. by inter-simple sequence repeats-polymerase chain reaction (ISSR-PCR) method and to investigate the difference among the species of Dendrobium Sw. at DNA molecular level. Methods Ten primers constituted by simple sequence repeats (SSR) were tested for PCR and sepharose electrophoresis of the nine species. Results Seven of the ten primers amplified polymorphic bands. The number of polymorphic band positions of each primer ranged from 7 to 14, and the size of fragments ranged from 220 to 1 260 bp. The amplification patterns of two primers, namely UBC-807 and UBC-864, were higher in terms of polymorphic and amplified band ratio. Each of them was able to distinguish all the examined species. Conclusion ISSR-PCR provides a quick, reliable molecular marker technique for identification of different species of Dendrobium Sw.