Application of tissue culture technology of medicinal plants in sustainable development of Chinese medicinal resources / 中国中药杂志

Chinese medicinal resources are the cornerstone of the sustainable development of traditional Chinese medicine industry. However, due to the fecundity of species, over-exploitation, and limitations of artificial cultivation, some medicinal plants are depleted and even endangered. Tissue culture, a breakthrough technology in the breeding of traditional Chinese medicinal materials, is not limited by time and space, and can allow the production on an annual basis, which plays an important role in the protection of Chinese medicinal resources. The present study reviewed the applications of tissue culture of medicinal plants in the field of Chinese medicinal resources, including rapid propagation of medicinal plant seedlings, breeding of novel high-yield and high-quality cultivars, construction of a genetic transformation system, and production of secondary metabolites. Meanwhile, the current challenges and suggestions for the future development of this field were also proposed.

Phytotoxicity of plant extracts of Vismia japurensis cultivated in vivo and in vitro / Fitotoxicidade de extratos de plantas de Vismia japurensis cultivadas in vivo e in vitro

Plants that produce secondary metabolites with allelopathic activity or phytotoxicity can be biotechnologically important, serving as sources of allelochemicals, and thus contributing to the agroindustrial sector. Vismia japurensis (Hypericaceae) is an Amazonian species that grows in clumps called vismiais, from which most other plants are absent. Accordingly, the objective of this study was to identify possible phytotoxicity effects of hexane and methanol extracts of Vismia japurensis leaves and branches in vivo and from seedlings grown in vitro on Lactuca sativa. In addition, fresh and dry leaves were assayed by the sandwich method in order to determine their ability to release allelochemicals. The hexanic extract from in vitro seedlings reduced germination by 10%, while the methanol extract produced a 16% reduction in germination speed. Root growth of Lactuca sativa was inhibited by 64.7% when subjected to hexane leaf extract, by 39.3% under the influence of hexane branch extract, and by 96.09% for in vitro seedling hexanic extract. When analysed by thin layer chromatography and 1H nuclear magnetic resonance, extracts showed evidence of terpenes, anthraquinones and flavonoids, with greater intensity of signals in the aromatic region of in vitro seedling hexanic extract. Clearly, Vismia japurensis has a high biotechnological potential in terms of the production of substances of low polarity with capacity to interfere in plant development.

Plantas que produzem metabólitos secundários com atividade alelopática ou fitotóxica podem ser biotecnologicamente importantes, servindo como fontes de aleloquímicos e, assim, contribuindo para o setor agroindustrial. Vismia japurensis (Hypericaceae) é uma espécie amazônica que cresce em grupos, formando vismiais. Assim, o objetivo deste estudo foi identificar possíveis efeitos fitotóxicos de extratos hexânicos e metanólicos de folhas e ramos de Vismia japurensis in vivo e de plântulas cultivadas in vitro sobre Lactuca sativa. Além disso, folhas frescas e secas foram analisadas pelo método sanduíche, a fim de determinar sua capacidade de liberação de aleloquímicos. O extrato hexânico de plântulas in vitro reduziu a germinação em 10% e o extrato metanólico promoveu uma redução de 16% na velocidade de germinação. O crescimento radicular de Lactuca sativa foi inibido em 64,7% quando submetido ao extrato hexânico das folhas, em 39,3% sob influência do extrato hexânico dos galhos e em 96,09% para o extrato de hexânico das plântulas in vitro. Quando analisados por cromatografia em camada delgada e ressonância magnética nuclear de 1H, os extratos mostraram evidências de terpenos, antraquinonas e flavonoides, com maior intensidade de sinais na região aromática do extrato hexânico das plântulas in vitro. Assim, Vismia japurensis possui elevado potencial biotecnológico em termos de produção de substâncias de baixa polaridade com capacidade de interferência no desenvolvimento de plantas.

Phytotoxicity of plant extracts of Vismia japurensis cultivated in vivo and in vitro / Fitotoxicidade de extratos de plantas de Vismia japurensis cultivadas in vivo e in vitro

Plants that produce secondary metabolites with allelopathic activity or phytotoxicity can be biotechnologically important, serving as sources of allelochemicals, and thus contributing to the agroindustrial sector. Vismia japurensis (Hypericaceae) is an Amazonian species that grows in clumps called vismiais, from which most other plants are absent. Accordingly, the objective of this study was to identify possible phytotoxicity effects of hexane and methanol extracts of Vismia japurensis leaves and branches in vivo and from seedlings grown in vitro on Lactuca sativa. In addition, fresh and dry leaves were assayed by the sandwich method in order to determine their ability to release allelochemicals. The hexanic extract from in vitro seedlings reduced germination by 10%, while the methanol extract produced a 16% reduction in germination speed. Root growth of Lactuca sativa was inhibited by 64.7% when subjected to hexane leaf extract, by 39.3% under the influence of hexane branch extract, and by 96.09% for in vitro seedling hexanic extract. When analysed by thin layer chromatography and 1H nuclear magnetic resonance, extracts showed evidence of terpenes, anthraquinones and flavonoids, with greater intensity of signals in the aromatic region of in vitro seedling hexanic extract. Clearly, Vismia japurensis has a high biotechnological potential in terms of the production of substances of low polarity with capacity to interfere in plant development.

Plantas que produzem metabólitos secundários com atividade alelopática ou fitotóxica podem ser biotecnologicamente importantes, servindo como fontes de aleloquímicos e, assim, contribuindo para o setor agroindustrial. Vismia japurensis (Hypericaceae) é uma espécie amazônica que cresce em grupos, formando vismiais. Assim, o objetivo deste estudo foi identificar possíveis efeitos fitotóxicos de extratos hexânicos e metanólicos de folhas e ramos de Vismia japurensis in vivo e de plântulas cultivadas in vitro sobre Lactuca sativa. Além disso, folhas frescas e secas foram analisadas pelo método sanduíche, a fim de determinar sua capacidade de liberação de aleloquímicos. O extrato hexânico de plântulas in vitro reduziu a germinação em 10% e o extrato metanólico promoveu uma redução de 16% na velocidade de germinação. O crescimento radicular de Lactuca sativa foi inibido em 64,7% quando submetido ao extrato hexânico das folhas, em 39,3% sob influência do extrato hexânico dos galhos e em 96,09% para o extrato de hexânico das plântulas in vitro. Quando analisados por cromatografia em camada delgada e ressonância magnética nuclear de 1H, os extratos mostraram evidências de terpenos, antraquinonas e flavonoides, com maior intensidade de sinais na região aromática do extrato hexânico das plântulas in vitro. Assim, Vismia japurensis possui elevado potencial biotecnológico em termos de produção de substâncias de baixa polaridade com capacidade de interferência no desenvolvimento de plantas.

Phytotoxicity of plant extracts of Vismia japurensis cultivated in vivo and in vitro

Abstract Plants that produce secondary metabolites with allelopathic activity or phytotoxicity can be biotechnologically important, serving as sources of allelochemicals, and thus contributing to the agroindustrial sector. Vismia japurensis (Hypericaceae) is an Amazonian species that grows in clumps called vismiais, from which most other plants are absent. Accordingly, the objective of this study was to identify possible phytotoxicity effects of hexane and methanol extracts of Vismia japurensis leaves and branches in vivo and from seedlings grown in vitro on Lactuca sativa. In addition, fresh and dry leaves were assayed by the sandwich method in order to determine their ability to release allelochemicals. The hexanic extract from in vitro seedlings reduced germination by 10%, while the methanol extract produced a 16% reduction in germination speed. Root growth of Lactuca sativa was inhibited by 64.7% when subjected to hexane leaf extract, by 39.3% under the influence of hexane branch extract, and by 96.09% for in vitro seedling hexanic extract. When analysed by thin layer chromatography and 1H nuclear magnetic resonance, extracts showed evidence of terpenes, anthraquinones and flavonoids, with greater intensity of signals in the aromatic region of in vitro seedling hexanic extract. Clearly, Vismia japurensis has a high biotechnological potential in terms of the production of substances of low polarity with capacity to interfere in plant development.

Resumo Plantas que produzem metabólitos secundários com atividade alelopática ou fitotóxica podem ser biotecnologicamente importantes, servindo como fontes de aleloquímicos e, assim, contribuindo para o setor agroindustrial. Vismia japurensis (Hypericaceae) é uma espécie amazônica que cresce em grupos, formando vismiais. Assim, o objetivo deste estudo foi identificar possíveis efeitos fitotóxicos de extratos hexânicos e metanólicos de folhas e ramos de Vismia japurensis in vivo e de plântulas cultivadas in vitro sobre Lactuca sativa. Além disso, folhas frescas e secas foram analisadas pelo método sanduíche, a fim de determinar sua capacidade de liberação de aleloquímicos. O extrato hexânico de plântulas in vitro reduziu a germinação em 10% e o extrato metanólico promoveu uma redução de 16% na velocidade de germinação. O crescimento radicular de Lactuca sativa foi inibido em 64,7% quando submetido ao extrato hexânico das folhas, em 39,3% sob influência do extrato hexânico dos galhos e em 96,09% para o extrato de hexânico das plântulas in vitro. Quando analisados por cromatografia em camada delgada e ressonância magnética nuclear de 1H, os extratos mostraram evidências de terpenos, antraquinonas e flavonoides, com maior intensidade de sinais na região aromática do extrato hexânico das plântulas in vitro. Assim, Vismia japurensis possui elevado potencial biotecnológico em termos de produção de substâncias de baixa polaridade com capacidade de interferência no desenvolvimento de plantas.

Effect of light and sucrose on photoautotrophic and photomixotrophic micropropagation of Physalis angulata / Efeito da luz e sacarose na micropropagação fotoautotrófica e fotomixotrófica de Physalis angulata

The biotechnological interest in genus Physalis has increased in the last decades, however, there are still few micropropagation studies of this genus. The objective of this study was to evaluate P. angulata photoautotrophic and photomixotrophic micropropagation with gas exchange under seven light spectra and five concentrations of sucrose. Lighting were yellow, blue, white, red, green, red + blue LEDs and natural light filtered by mesh. Sucrose concentrations were 0, 7.5, 15, 22.5 and 30 g.L-1. Phytotechnical, anatomical features and photopigment contents were evaluated through stem and root segment length, leaf number, leaf area, chlorophyll a and b contents, carotenoids, adaxial epidermis, palisadic and spongy parenchyma and abaxial epidermis. The data were compared by Scott-Knott's mean test and principal components analysis using the R software. Comparing the variables within lighting types, it was observed that only the screen treatment, screen-filtered natural illumination, obtained assessment in all variables. Comparing the levels of sucrose, it was observed that the treatment 15 g.L-1 sucrose obtained the highest number of averages with maximum evaluation. It was concluded that the natural light filtered by screen with 50% of shading allowed the photoautotrophic micropropagation of P. angulata. Better development results were observed in photomixotrophic micropropagation with 15 g.L-1 of sucrose.

O interesse biotecnológico em Physalis aumentou nas últimas décadas, porém, ainda existem poucos trabalhos de micropropagação desse gênero. Objetivou-se avaliar sua micropropagação fotoautotrófica e fotomixotrófica com troca gasosa sob sete tipos de iluminação e cinco concentrações de sacarose. Foram utilizados LEDs amarelo, azul, branco, vermelho, verde, vermelho + azul e luz natural filtrada por malha. As concentrações de sacarose foram 0, 7,5, 15, 22,5 e 30 g.L-1. Características fitotécnicas, anatômicas e teor de fotopigmentos foram avaliados através de comprimento de segmento de caule e raíz, número de folhas, área foliar, teores de clorofilas a e b, carotenoides, epiderme adaxial, parênquimas paliçádico e esponjoso e epiderme abaxial. Os dados foram comparados por teste de média Scott-Knott e análise de componentes principais utilizando-se o software R. Comparando-se as variáveis dentro de tipos de iluminação, observou-se que apenas o tratamento screen, iluminação natural filtrada por malha, obteve avaliação máxima em todas as variáveis. Comparando-se os níveis de sucrose, observou-se que o tratamento 15 g.L-1 sacarose obteve o maior número de médias com avaliação máxima. Concluiu-se que a luz natural filtrada por tela com 50% de sombreamento permitiu a micropropagação fotoautotrófica de P. angulata. Observou-se melhores resultados de desenvolvimento na micropropagação fotomixotrófica com 15 g.L-1 de sacarose.

A comparison of callus induction in 4 Garcinia species

Background: This research is intended to determine suitable types and concentrations of plant growth regulators (PGRs) to induce callus on stem and leaf sections of 4 species of the genus Garcinia, namely, Garcinia mangostana, Garcinia schomburgkiana, Garcinia cowa, and Garcinia celebica. The base medium was MS medium containing 30 g l -1 sucrose, 0.5 g l-1 polyvinylpyrrolidone (PVP), and 7 g l-1 agar, and for the different treatments, PGRs were added to the medium as follows: thidiazuron (TDZ) at concentrations of 0, 0.1, 0.5, 1, and 2 mg l-1; 6-(3- hydroxybenzylamino) purine (meta-topolin) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; 4-amino-3,5,6- trichloro-2-pyridinecarboxylic acid (picloram) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; and 2,4- dichlorophenoxyacetic acid (2,4-D) at concentrations of 0, 0.5, 1, 2, and 4 mg l-1. The occurrence of callus was observed after 4 weeks. Results: A maximum of 100% and 93% of G. mangostana leaf explants formed callus in the 0.5 mg l-1 and 1 mg l-1 TDZ treatments, respectively, while 100% of G. schomburgkiana stem explants formed callus in the 1 mg l-1 TDZ treatment and 89% of G. schomburgkiana leaf explants formed callus in the 0.5 mg l-1 picloram treatment. The highest callus induction rate for G. cowa was 62% in the 1 mg l-1 TDZ treatment and for G. celebica was 56% in the 0.5 mg l-1•mT-1 treatment. Conclusions: For all 4 species, the greatest amount of large nodular callus was observed in the TDZ treatments. White, friable callus was observed on most of the 2,4-D and picloram treatment groups. Most meta-topolin treatments resulted in minimal callus formation.

Direct in vitro regeneration of castor bean plants (Ricinus communis) using epicotyls / Regeneração direta in-vitro de plantas de mamona ( Ricinus communis ) usando epicótilos

A regeneration protocol for castor bean plant (Ricinus communis) was successfully developed using epicotyl sections obtained from in vitro seedlings. The maximum number of induced shoots (4.3 shoots/explant) and highest shoots frequency (75,56%) was obtained in WPM medium supplemented with TDZ (1 mg/L) and zeatin (0.5 mg/L), whereas the minimum number (0.8 shoots/explant) and lowest shoots frequency (37,78%) was obtained in medium containing TDZ (1 mg/L) and BAP (0.5 mg/L). The highest percentage of rooting (93.3%) was obtained in a medium containing IBA (1 mg/L). These plants were transplanted in a mesh house and achieved a high adaptability to acclimatization, reaching 77% survival. On the other hand, the maximum elongation (height) during this stage was 7.9 cm in plants supplemented with WPM nutrients, whereas it was only 4.38 cm in control plants

Foi desenvolvido com sucesso um protocolo de regeneração para a planta de Mamona (Ricinus communis) utilizando seções de epicótilos, obtidas a partir de mudas in vitro. O número máximo de brotações induzidas (4.3 brotos/explante), assim como a maior frequência de brotações (75,56%), foi obtido em meio WPM suplementado com TDZ (1 mg/L) e zeatina (0,5 mg/L). Enquanto que o número mínimo (0,8 brotos/explante), como a menor freqüência de rebentos (37,78%), foi obtido em meio contendo TDZ (1 mg/L) e BAP (0,5 mg/L). Adicionalmente, a maior percentagem de enraizamento (93,3%) foi obtida em um meio contendo IBA (1 mg/L). Depois da regeneração, as plantas foram transplantadas em casa de vegetação e conseguiram uma alta adaptabilidade e aclimatização, atingindo 77% de sobrevivência. Por outro lado, oalongamento máximo (altura) durante este estágio foi de 7,9 cm em plantas suplementadas com nutrientes de WPM, enquanto as plantas de controle presentaram apenas 4,38 cm

De novo in vitro shoot morphogenesis from shoot tip-induced callus cultures of Gymnema sylvestre (Retz.) R. Br. ex Sm

BACKGROUND: Gymnema sylvestre is a medicinal woody perennial vine known for its sweetening properties and antidiabetic therapeutic uses in the modern and traditional medicines. Its over-exploitation for the therapeutic uses and to meet the demand of pharmaceutical industry in raw materials supply for the production of anti-diabetic drugs has led to considerable decline in its natural population. RESULTS: An efficient system of shoot bud sprouting from nodal segment explants and indirect plant regeneration from apical meristem-induced callus cultures of G. sylvestre have been developed on Murashige and Skoog (MS) medium amended with concentrations of cytokinins. Of the three growth regulators tested, N6-benzylaminopurine (BAP) was the most efficient and 2.0 mg L-1 gave the best shoot formation efficiency. This was followed by thidiazuron (TDZ) and kinetin (Kin) but, most of the TDZ-induced micro shoots showed stunted growth. Multiple shoot formation was observed on medium amended with BAP or TDZ at higher concentrations. The produced micro shoots were rooted on half strength MS medium amended with auxins and rooted plantlets acclimatized with 87% survival of the regenerates. CONCLUSIONS: The developed regeneration system can be exploited for genetic transformation studies, particularly when aimed at producing its high yielding cell lines for the anti-diabetic phytochemicals. It also offers opportunities for exploring the expression of totipotency in the anti-diabetic perennial vine.

Multiplicación in vitro y embriogénesis somática de Perezia pinnatifida (Asteraceae) planta medicinal andina

Se presentan los procedimientos para la propagación in vitro de Perezia pinnatifida (Humb. & Bonpl.) Wedd., conocida como "valeriana". Se utilizaron las metodologías de multiplicación de brotes y embriogénesis somática indirecta. El medio de cultivo basal para todas las etapas fue Murashige y Skoog a mitad de sales, suplementado con sacarosa 2.0%, phytagel 0.3% y pH 5.67; y fue usado con o sin fitohormonas en los diferentes tratamientos. Los suplementos hormonales fueron: para la multiplicación de brotes BAP 1.0 mg.L-1 + ANA 0.01 mg.L-1 ó BAP 1.0 mg.L-1; para la inducción de callos embriogénicos ANA ó 2,4-D (1.0 mg.L-1 y 2.0 mg.L-1); y para la germinación de embriones BAP (0.5 y 1.0 mg.L-1) o BAP 0.5 mg.L-1 + ANA 0.05 mg.L-1. El mayor número de brotes se obtuvo en el medio suplementado con BAP 1.0 mg.L-1. En la embriogénesis somática, ANA a 1.0 mg.L-1 indujo mayor área de callos embriogénicos, y BAP a 0.5 mg.L-1 permitió mayor germinación de los embriones somáticos

This work inform on in vitro propagation of the "valeriana" Perezia pinnatifida (Humb. & Bonpl.) Wedd. Shoot multiplication and indirect somatic embryogenesis methodologies were performed. The basal culture medium for all stages was Murashige and Skoog, middle of salts supplemented with 2.0% sucrose, 0.3% phytagel and pH 5.67; the treatments were prepared with or without phytohormones. The hormonal supplements for the shoot multiplication were: BAP 1.0 mg.L-1 + ANA 0.01 mg.L-1, and BAP 1.0 mg.L-1; for embryogenic callus induction were: ANA or 2,4-D (1.0 mg.L-1 and 2.0 mg.L-1); and for the embryo germination were: BAP (0.5 and 1.0 mg.L-1) or BAP 0.5 mg.L-1 + ANA 0.05 mg.L-1. BAP 1.0 mg.L-1 produced the higher number buds. For somatic embryogenesis, ANA 1.0 mg.L-1 induced a greater area of embryogenic callus, and BAP 0.5 mg.L-1 allowed major germination of the somatic embryos

The effects of different strength of MS media in solid and liquid media on in vitro growth of Typhonium flagelliforme

Objective To determine the effects of different strength of Murashige and Skoog (MS) media (full, ½ and ¼) in solid and liquid media on in vitro growth of Typhonium flagelliforme (T. flagelliforme), whereby an optimum media composition can be provided for mass propagation of T. flagelliforme. Methods Rhizome bud of T. flagelliforme was obtained from the axenic in vitro established T. flagelliforme plantlets in Plant Tissue Culture Laboratory, Universiti Teknologi MARA, Shah Alam. Rhizome bud was used as explant and cultured onto shoot proliferation medium under different strength of MS media (full, ½, ¼) in solid and liquid culture media. Results After 6 weeks of culture, the number of shoot, number of leaf, number of root, height of shoot, fresh weight, dry weight and chlorophyll content of T. flagelliforme were analyzed. A comparison was made between liquid and solid culture media. The results revealed that the liquid culture media were more effective for all the growth parameters (shoot height, shoot number, leaf number, root number, fresh weight, dry weight, chlorophyll a and chlorophyll b content) compared to solid culture media. Apart from that, this study revealed the positive relationship between strength of MS media and type of culture media (solid and liquid media) to the growth of T. flagelliforme. Growth of T. flagelliforme was improved when MS strength was increased in liquid media. In contrast, growth of T. flagelliforme was improved when MS strength was decreased in solid media. Conclusions Through this study, an optimum media composition for mass propagation of T. flagelliforme had been established by observing effects of MS media strength and type of culture media (solid and liquid media) on the growth of T. flagelliforme.

In Vitro Mid-Term Conservation of Acorus calamus L. via Cold Storage of Encapsulated Microrhizome

ABSTRACT In vitro rhizome production, encapsulation and cold storage of Acorus calamus were attempted for its propagation and ‘true-to-type’ conservation. Shoot cultures were initiated using underground rhizome buds, on 6-benzyladenine (BA) containing Murashige and Skoog (MS) medium. Maximum microrhizome production was observed in presence of 33.3 µM BA, on modified MS medium containing 6% sucrose, 100 mg/L citric acid and 1 g/L polyvinyl pyrrolidone-40. Synthetic seeds were produced from regenerated microtubers by encapsulation in calcium alginate beads. These synthetic seeds were stored in complete darkness at 100C temperature for different durations for mid-term conservation. After cold storage, synthetic seeds were re-cultured in vitro, 100% survival was recorded after the storage of 1, 3 or 6 months; and 80% survival was observed after the storage of 12 months. The microrhizomes were produced roots in 4.9 µM indole-3-butyric acid containing half strength MS medium. All the regenerated plantlets were successfully transferred to field after acclimatization. It is the first report on successful one year in vitro cold storage of A. calamus.

In vitro morphogenic response from zygotic embryos of Genipa americana / Resposta morfogênica in vitro de embriões zigóticos de Genipa americana

ABSTRACT: The aim of this study was to evaluate the in vitro morphogenic potential of genipap (Genipa americana L.) zygotic embryos. Seeds obtained from ripe fruits had their zygotic embryos excised and inoculated in MS medium with 4.44µM of 6-benzylaminopurine (BAP) and supplemented with 0.0; 1.07; 2.14 and 3.21µM of naphthalene acetic acid (NAA). The potential of explants regeneration and the shoot length and number of leaves in plantlets were evaluated. The in vitro regeneration of genipap is possible from the conversion of zygotic embryos in a MS medium with 4.44µM BAP supplemented with 3.21µM NAA.

RESUMO: O objetivo do trabalho foi avaliar o potencial morfogênico in vitro de embriões zigóticos de jenipapeiro (Genipa americana L.). Sementes obtidas de frutos tiveram seus embriões zigóticos excisados e inoculados em meio MS com 4,44µM de 6-benzilaminopurina (BAP) suplementado com 0,0; 1,07; 2,14 e 3,21µM de ácido naftaleno acético (ANA). O potencial de regeneração dos explantes e o comprimento da parte aérea e o número de folhas nas plântulas formadas foi avaliado. Observou-se que é possível a regeneração in vitro de jenipapeiro a partir da conversão de embriões zigóticos em meio MS com 4,44µM de BAP, suplementado com 3,21µM de ANA.

The effects of different strength of MS media in solid and liquid media on in vitro growth of Typhonium flagelliforme

Objective: To determine the effects of different strength of Murashige and Skoog (MS) media (full, 1/2 and 1/4) in solid and liquid media on in vitro growth of Typhonium flagelliforme (T. flagelliforme), whereby an optimum media composition can be provided for mass propagation of T. flagelliforme. Methods: Rhizome bud of T. flagelliforme was obtained from the axenic in vitro established T. flagelliforme plantlets in Plant Tissue Culture Laboratory, Universiti Teknologi MARA, Shah Alam. Rhizome bud was used as explant and cultured onto shoot proliferation medium under different strength of MS media (full, 1/2, 1/4) in solid and liquid culture media. Results: After 6 weeks of culture, the number of shoot, number of leaf, number of root, height of shoot, fresh weight, dry weight and chlorophyll content of T. flagelliforme were analyzed. A comparison was made between liquid and solid culture media. The results revealed that the liquid culture media were more effective for all the growth parameters (shoot height, shoot number, leaf number, root number, fresh weight, dry weight, chlo-rophyll a and chlorophyll b content) compared to solid culture media. Apart from that, this study revealed the positive relationship between strength of MS media and type of culture media (solid and liquid media) to the growth of T. flagelliforme. Growth of T. flagelliforme was improved when MS strength was increased in liquid media. In contrast, growth of T. flagelliforme was improved when MS strength was decreased in solid media. Conclusions: Through this study, an optimum media composition for mass propagation of T. flagelliforme had been established by observing effects of MS media strength and type of culture media (solid and liquid media) on the growth of T. flagelliforme.

Production of Active Compounds in Medicinal Plants: From Plant Tissue Culture to Biosynthesis / 中草药·英文版

Over past decades plant tissue culture has emerged as an alternative of whole plant cultivation in the production of valuable secondary metabolites. Adventitious roots culture of Panax ginseng and Echinacea purpure has reached the scale of 1-10 kL. Some molecular biological techniques, such as transgenic technology and genetic stability are increasingly used in the studies on plant tissue cultures. The studies on elicitors have deepened into the induction mechanism, including signal molecules, functional genes, and so on. More and more biological elicitors, such as A. niger and yeast are used to increase the active compounds in plant tissue cultures. We also discussed the application of synthetic biology in the studies on biosynthesis of artemisinin, paclitaxel, and tanshinon. The studies on active ingredients biosynthesis of medicinal plants provide unprecedented possibilities to achieve mass production of active ingredients. Plant tissue cultures can not only produce active ingredients but also as experimental materials for biosynthesis. In order to improve the contents of active compounds in medicinal plants, following aspects could be carried out gene interference or gene silencing, gene overexpression, combination with chemical synthesis, application of elicitors, and site-directed mutagenesis of the key enzymes.

Application of plant tissue culture in field of Chinese medicine resources / 中国中药杂志

Plant tissue culture technology has been widely used in the field of traditional Chinese medicine(TCM) resources with its unique advantages, playing an important role in the protection of TCM resources. In this review, some applications of plant tissue culture were summarized, including production of active compounds by using plant tissue culture, genetic diversity analysis, Dao-di herbs, elicitor application, biosynthesis and transgenic plants. Through the above researches will promote the further development of plant tissue culture technology, making it play a greater role in the field of TCM resources.

Infección de callo embriogénico friable de yuca con Xanthomonas axonopodis pv. manihotis (Xam) / Cassava friable embryogenic calli infection with Xanthomonas axonopodis pv. manihotis (Xam)

Las nuevas tecnologías para la edición de genomas, como los TALEN y el sistema CRISPR/Cas9, representan una gran oportunidad para mejorar características deseables en diferentes organismos. Los TALEN son el resultado del acoplamiento de nucleasas a los TALE (Transcription Activator-Like Effectors), los cuales son efectores naturales de gran importancia en la patogénesis de las especies de Xanthomonas. Xanthomonas axonopodis pv. manihotis (Xam) es el agente causal del añublo bacteriano de la yuca, quien durante el proceso patogénico es capaz de translocar sus efectores a la célula vegetal mediante el sistema de secreción tipo tres (SSTT). Actualmente no hay protocolos estándar para la edición de genomas en yuca. En este estudio se exploró la posibilidad de translocar efectores sobre callo embriogénico friable (CEF) a través de la inoculación con Xam, con el fin de determinar el potencial de este patógeno como sistema de entrega de TALEN. El CEF de dos variedades de yuca susceptibles (COL2215 y cv. 60444) se cocultivaron con la cepa Xam668 a diferentes tiempos. Posteriormente, se evaluó la expresión de marcadores correspondientes a los genes blanco conocidos para los TALE presentes en esta cepa bacteriana. Aunque no se logró demostrar la translocación de los mismos en el tejido embriogénico, sí se lograron establecer condiciones adecuadas de cocultivo con Xam y el efecto que la infección bacteriana tiene sobre la regeneración de embriones a partir de este tejido.

New technologies for genome edition, such as TALENs and CRISPR/Cas9 system, are a great opportunity to improve desirable features in different organisms. TALENs are the result from coupling nucleases and TALEs (Transcription Activator-Like Effectors), which are natural effectors with an important role in pathogenicity for the Xanthomonas species. Xanthomonas axonopodis pv. manihotis (Xam) is the cassava bacterial blight causal agent, and this pathogen is able to translocate its effectors to the plant cell during pathogenesis by using the type-three secretion system (TTSS). Currently, there are no standard protocols for genome edition in cassava. In this study, we explored the possibility to translocate effectors to friable embryogenic calli (FEC) through Xam inoculation, in order to establish the potential of this pathogen as a TALEN delivery system. Friable embryogenic calli derived from two different susceptible varieties (COL2215 and cv. 60444) were co-cultured with the strain Xam668 using different culture times. Subsequently, we evaluated the expression of makers corresponding to reported target genes for TALEs present in this bacterial strain. Although we were not able to demonstrate effector translocation, we established the conditions for co-culturing cassava calli and Xam and determined the effects derived from bacterial infection on embryo regeneration from FECs.

Introduction trial of medicine mulberry (Morus nigra) in Chongqing / 中国中药杂志

Medicine mulberry (Morus nigra) mainly distributed in southern areas of Xinjiang Uighur Autonomous Region and introduced by grafting, is a unique Morus species, whose plant number is little. As a traditional herbal medicine, medicine mulberry with high levels of secondary metabolites has important values of scientific research and utilization. In order to solve the introduction problems for medicine mulberry, we have established its rapid propagation system through tissue culture since 2011. The shoots of medicine mulberry through tissue culture were transplanted into the field to carry out an introduction experiment. Here, we firstly reported that the growth status and pest and disease occurrence of medicine mulberry in the field of Chongqing and found that the medicine mulberry through tissue culture had well-developed root system, it showed better growth than medicine mulberry by grafting technique, and Pseudodendrothrips moil was a major pest of medicine mulberry. The introduction technique for medicine mulberry established successfully in this study could lay the foundation for large-scale cultivation and high efficiency utilization of medicine mulberry.

Anti-Melanogenic Potentials of Nanoparticles from Calli of Resveratrol-Enriched Rice against UVB-Induced Hyperpigmentation in Guinea Pig Skin

We already reported that genetically engineered resveratrol-enriched rice (RR) showed to down-regulate skin melanogenesis. To be developed to increase the bioactivity of RR using calli from plants, RR was adopted for mass production using plant tissue culture technologies. In addition, high-pressure homogenization (HPH) was used to increase the biocompatibility and penetration of the calli from RR into the skin. We aimed to develop anti-melanogenic agents incorporating calli of RR (cRR) and nanoparticles by high-pressure homogenization, examining the synergistic effects on the inhibition of UVB-induced hyperpigmentation. Depigmentation was observed following topical application of micro-cRR, nano-calli of normal rice (cNR), and nano-cRR to ultraviolet B (UVB)-stimulated hyperpigmented guinea pig dorsal skin. Colorimetric analysis, tyrosinase immunostaining, and Fontana-Masson staining for UVB-promoted melanin were performed. Nano-cRR inhibited changes in the melanin color index caused by UVB-promoted hyperpigmentation, and demonstrated stronger anti-melanogenic potential than micro-cRR. In epidermal skin, nano-cRR repressed UVB-promoted melanin granules, thereby suppressing hyperpigmentation. The UVB-enhanced, highly expressed tyrosinase in the basal layer of the epidermis was inhibited by nano-cRR more prominently than by micro-cRR and nano-cNR. The anti-melanogenic potency of nano-cRR also depended on pH and particle size. Nano-cRR shows promising potential to regulate skin pigmentation following UVB exposure.

Effects of donor plant age and explants on in vitro culture of Cedrela montanaMoritz ex Turcz

Para evaluar el potencial organogénico de Cedrela montanaMoritz ex Turcz, se colectaron explantes de árboles maduros (10-20 años) y juveniles (7-18 meses). Los primeros incluyeron yemas,hojas y nudos de brotes juveniles (ubicados hacia la parte basal deltronco) y rejuvenecidos (obtenidos a partir de estacas). Los segundoshojas, peciolos, nudos, entrenudos y nudos de brotes elongados invitro. Los nudos de árboles juveniles presentaron el mayor potencialorganogénico, ya que el 45,8% de los explantes presentaron elongaciónde yemas axilares y el 56,2% enraizamiento en medio sin reguladoresde crecimiento. El 51% de los brotes elongados formaron brotesadventicios con 0.5 μM NAA y 0.5 μM BA, el 30% con 0.5 μM NAAy 1 μM BA, y el 30% con 1 μM BA; y el 20% raíces con 0,5 μM NAA.La formación de raíces se vio estimulada con la adición de carbónactivado (5 gL-1) en el medio. El 80% de las plántulas regeneradas apartir de nudos y el 72,5% de las provenientes de brotes generadosin vitro se aclimataron exitosamente. Por el contrario, explantesde árboles maduros presentaron baja respuesta organogénica.Elongación de yemas axilares fue registrada solamente en 10.7% delos nudos de brotes juveniles y en 6.7% de aquellos provenientes debrotes rejuvenecidos. En conclusión, la edad de la planta donadoray el tipo de explante influyen sobre el potencial organogénico de C.montana. Este estudio contribuyó al conocimiento de la respuesta deesta especie bajo condiciones in vitro...

To evaluate the organogenic potential ofCedrela montana Moritz ex Turcz, explants from mature(10-20 year-old) and juvenile (7-18 month-old) treeswere collected. The first grouping included buds, leaves,and nodes derived from juvenile basal offshoots andrejuvenated shoots from cuttings. The second, includedleaves, petioles, nodes, internodes and nodes of in vitroelongated shoots. The highest organogenic potential wasobserved in nodes from juvenile trees: 45.8% of explantspresented axillary bud elongation, while 56.2% presentedrooting in a growth regulator free culture medium. Fiftyonepercent of elongated shoots produced adventitiousshoots with 0.5 μM NAA and 0.5 μM BA; 30% with0.5 μM NAA and 1 μM BA; and 30% with 1 μM BA.Twenty percent presented roots with 0.5 μM NAA. Rootformation was stimulated in a medium supplementedwith activated charcoal (5 gL-1). The acclimatizationof eighty percent of plantlets regenerated from nodes,and of 72.5% in vitro generated shoots was successful.On the contrary, mature trees material presented loworganogenic response. Axillary bud elongation wasrecorded just in 10.7% of explants from juvenile shootsand in 6.7% of explants from rejuvenated shoots. Inconclusion the age of donor plant and type of explantaffect the organogenic potential of C. montana. Thisstudy contributes to the understanding of this species’response under in vitro conditions...

Para avaliar o potencial organogênico da Cedrela montanaMoritz ex Turcz, explantes derivados de árvores adultas (10-20 anos)e jovens (7-18 meses) foram coletados. O primeiro incluiu brotos,folhas, e nós derivados de brotações jovens (localizado na direçãoda parte basal do tronco) e rejuvenescida (obtido a partir de estacas).O segundo incluía folhas, pecíolos, nós, entrenós e nós de brotosalongados in vitro. Maior potencial organogênico foi observado emnós de árvores jovens, em que o alongamento de brotos foi obtidoem 45,8% dos explantes e o enraizamento atingiu 56,2% em meiosem reguladores de crescimento. Brotos adventícios foram induzidasem 51% dos rebentos gerados in vitro com 0.5 μM NAA e 0.5 μM BA;30% de indução ocorreu com 0.5 μM NAA e 1 μM BA; 30% com 1μM BA. Raízes adventícias foram induzidas em 20% dos rebentoscom 0,5 μM NAA. Formação de raízes foi estimulada com carvão ativado(5 gL-1) no meio. 80% das plântulas regeneradas a partir de nós e 72,5%das plântulas a partir de brotações obtidas in vitro foram aclimatizadascom sucesso. Em contraste, explantes derivados de árvores adultasapresentou resposta organogênica baixo. Alongamento de brotos degemas axilares foi registrado somente em 10,7% dos nós de brotaçõesjovens e 6,7% das brotações ejuvenescidas. Em conclusão a idade daplanta doadora e o tipo de explante afeta o potencial de organogênesein vitro da C. montana. Este estudo contribuiu para o conhecimento daresposta desta espécie sob condições in vitro...

A method for seedling recovery in Jatropha curcas after cryogenic exposure of the seeds

Actually, the germplasm of Jatropha spp. is conserved as whole plants in field collections. Under this storage method, the genetic resources are exposed to disease, pest and natural hazards such as human error, drought and weather damage. Besides, field genebanks are costly to maintain and with important requirements of trained personnel. Thus, the development of efficient techniques to ensure its safe conservation and regeneration is therefore of paramount importance. In this work we describe a method for Jatropha curcas seeds cryoexposure and seedling recovery after thawed. In a first experiment, an efficient protocol for in vitro plant recovery was carried out using zygotic embryo or seeds with or without coat. In a second experiment, desiccated seeds with or without coat were exposed to liquid nitrogen and evaluated after cryoexposure. Germination percentages were variable among treatments, and seeds demonstrated tolerance to liquid nitrogen exposure under certain conditions. Seeds of J. curcas presented up to 99.6% germination after seed coat removal. Seeds with coat cultured in vitro did not germinate, and were 60% contaminated. The germination of the zygotic embryos was significantly higher in the ½ MS medium (93.1%) than in WPM medium (76.2%), but from zygotic embryo, abnormal seedlings reached up to 99%. Seeds with coat exposed to liquid nitrogen showed 60% germination in culture after coat removal with good plant growth, and seeds cryopreserved without coat presented 82% germination, but seedlings showed a reduced vigor and a significant increase in abnormal plants. Seeds cultured in vitro with coat did not germinate, independently of cryoexposure or not. This study reports the first successful in vitro seedling recovery methodology for Jatropha curcas seeds, after a cryopreservation treatment, and is recommended as an efficient procedure for in vitro plant recovery, when seeds are conserved in germplasm banks by low or cryotemperatures.

Actualmente, el germoplasma de las especies de Jatropha ssp. se conserva como plantas enteras en las colecciones de campo. Bajo este método de almacenamiento, los recursos genéticos están expuestos a enfermedades, plagas y desastres naturales tales como el error humano, la sequía y las inclemencias del tiempo. Además, los bancos de germoplasma de campo son costosos de mantener y requieren bastante personal capacitado. Por lo tanto, el desarrollo de técnicas eficientes para asegurar su conservación segura así como su regeneración, es de suma importancia. En este trabajo se describe un método de recuperación para semillas y plántulas crioexpuestas de Jatropha curcas después de descongeladas. En un primer experimento, se llevó a cabo un protocolo eficiente para la recuperación de plantas in vitro mediante el uso de embriones cigóticos o semillas con o sin testa. En un segundo experimento, las semillas disecadas, con o sin testa fueron expuestas a nitrógeno líquido y se evaluaron después de la crioexposición. Los porcentajes de germinación fueron variables entre los tratamientos, y las semillas demostraron tolerancia a la exposición del nitrógeno líquido bajo ciertas condiciones. Las semillas de J. curcas presentaron hasta un 99.6% de germinación después de la eliminación de la testa. Las semillas con la testa cultivadas in vitro no germinaron, y el 60% se contaminaron. La germinación de los embriones cigóticos fue significativamente alta en el medio ½ MS (93.1%) en comparación con el medio WPM (76.2%), pero desde los embriones zigóticos, las plántulas anormales alcanzaron más del 99%. Semillas con la testa inmersa en nitrógeno líquido mostraron un 60% de germinacion en cultivos despúes de la remoción de la testa con un buen crecimiento de la planta, y las semillas criopreservadas sin testa presentaron un 82% de germinación, pero las plántulas mostraron un reducido vigor y un incremento significativo de plantas anormales. Semillas con testa cultivadas in vitro no germinaron, independientemente de la criopreservación o no. Este estudio reporta el primer éxito in vitro de una metodología de recuperación de plántulas para semillas de Jatropha curcas, después de un tratamiento de criopreservación, que se recomienda como un procedimiento eficaz para la recuperación de plantas in vitro, cuando las semillas se conservan en bancos de germoplasma a bajas o crio-temperaturas.