Development of plant tissue culture of Plumbago rosea Linn. for enhanced production of secondary metabolites

P rosea syn. Indica belong to the family of plumbaginaceae, is an important medicinal plant, cultivated widely in India. The roots of these plant are generally used for medicinal purposes mainly as diuretic, germicidal, vessicant, and abortifacient. It is also used for anaemia, diarrhea, leprosy and common wart. The bark of the root contains orange yellow pigment named plumbagin, a crystalline substance, belongs to the class of naphthoquinone. Its chemical structure is 5-hydroxy 2-methyl 1,4naphthoquinone. Apart from P rosea, P zeylanica, P europea, Drosera and Drosophyllum also contains plumbagin. The most exploited source of plumbagin is, of course, P. rosea roots. The roots contain O.9mg/ g D.Wt. of plumbagin in the roots. These plants grow very slowly and the roots suitable for plumbagin extraction can be obtained only after several years of growth. The productivity of the plant is also rather poor. The focus of the present study was to develop alternative strategies to obtain plumbagin. The tissue culture of P rosea for micropropagation has been studied

Method of plant tissue culture and regeneration

Plants may be regenerated from stomatal cells or protoplasts of such cells. Prior to regeneration the cells or protoplasts may be genetically transformed by the introduction of hereditary material most preferably by a DNA construct which is free of genes which specify resistance to antibiotics. The regeneration step may include callus formation on a hormone-free medium. The method is particularly suitable for sugar beet.

Exploring plant tissue culture to improve the production of phenolic compounds: A review

Plant tissue and organ culture has been extensively used from the beginning of the XX century for the study and comprehension of some primary biological mechanisms such as morphogenesis. However, with the increasing demand of the market for novel products derived from plants, in vitro culture became a reliable technique for the mass production of plant material. Moreover, the potential to use this technique for the production of some bioactive compounds, such as phenolic compounds, is immense since it allows the manipulation of the biosynthetic routes to increase the production and accumulation of specific compounds. This work intends to make a brief historical review of in vitro culture, highlighting its use for the production of bioactive compounds. Also, emphasizes the importance of phenolic compounds for the consumer as well reviews the metabolic pathways involved in its production in plant cells. Furthermore, it was carried out a comprehensive study on the work developed for the production of plant phenolic compounds in in vitro cultures, as well as on the type of elicitors used to increase of the same production; also a brief highlighting of the phenolic compounds which serve as elicitors. There are numerous reports directed to the production of phenolic extracts in in vitro plant cultures, however there is a lack in the production of individual phenolic compounds mainly due to the complexity of the biosynthetic routes and extraction procedures. Elicitation procedures are often used to increase the production of phenolics, archieving in most cases higher yields than in non-elicitated cultures. The increasing production of bioactive phenolic extracts/compounds allows for their further applicability, namely in the industry of functional foods or in pharmaceutical/medical fields.

Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

Tissue culture propagation alters plant-microbe interactions in tobacco rhizosphere

We have compared properties of roots from different lines (genotypes) of tobacco raised either in tissue culture or grown from seed. The different lines included unmodified plants and plants modified to express reduced activity of the enzyme cinnamoyl-CoA reductase, which has a pivotal role in lignin biosynthesis. The size and structure of the rhizosphere microbial community, characterized by adenosine triphosphate and phospholipid fatty acid analyses, were related to root chemistry (specifically the soluble carbohydrate concentration) and decomposition rate of the roots. The root material from unmodified plants decomposed faster following tissue culture compared with seed culture, and the faster decomposing material had significantly higher soluble carbohydrate concentrations. These observations are linked to the larger microbial biomass and greater diversity of the rhizosphere communities of tissue culture propagated plants.

Tissue culture propagation alters plant-microbe interactions in tobacco rhizosphere

We have compared properties of roots from different lines (genotypes) of tobacco raised either in tissue culture or grown from seed. The different lines included unmodified plants and plants modified to express reduced activity of the enzyme cinnamoyl-CoA reductase, which has a pivotal role in lignin biosynthesis. The size and structure of the rhizosphere microbial community, characterized by adenosine triphosphate and phospholipid fatty acid analyses, were related to root chemistry (specifically the soluble carbohydrate concentration) and decomposition rate of the roots. The root material from unmodified plants decomposed faster following tissue culture compared with seed culture, and the faster decomposing material had significantly higher soluble carbohydrate concentrations. These observations are linked to the larger microbial biomass and greater diversity of the rhizosphere communities of tissue culture propagated plants.

A dehydrorotenoid produced by callus tissue culture and wild plant roots of Boerhaavia coccinea

“Um desidro-rotenóide produzido por cultura de calos e por raízes de plantas silvestres de Boerhaavia coccinea”. Cultura de calos foram estabelecidos de folhas e galhos finos de plântula de B. coccinea produzida in vitro e analisada para isofl avonóide. A quantificação do 6,9,11-triidroxi-6a,12a-desidro-rotenóide isolado das raízes de B. coccinea P Miller, coletada em seu habitat natural, e do mesmo rotenóide produzido na cultura de células estão descritos neste artigo. A análise rotineira em CLAE mostrou que a cultura de calos produziu o mesmo isoflavonóide encontrado nas raízes da planta do campo. A quantidade do metabólito secundário produzido in vitro foi de 955.35

Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.

In vitro propagation of Eucalyptus grandis L. by tissue culture

Multiple shoots were induced from nodal segments of five year old trees of Eucalyptus grandis L. on solid medium containing Murashige and Skoog's (MS) Basal medium supplemented with additional thiamine, BAP and NAA. Rooting could be achieved from shoot culture on half strength MS salts or white's medium supplemented with low auxins like IAA, IBA and NAA.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Axenic cell culture of the seagrass Cymodocea nodosa from cotyledonary tissue

[EN] Plant Tissue Culture, also called “micropropagation”, is the propagation of plants from different tissues (or explants) in a shorter time than conventional propagation, making use of the ability that many plant cells have to regenerate a whole plant (totipotency).There are two alternative mechanisms by which an explant can regenerate an entire plant, namely organogenesis and somatic embryogenesis. Since the last decades, the number of higher terrestrial plants species from which these techniques have been successfully applied has continually increased. However, few attempts have been carried out in marine plants. Previous seagrasses authors have focused their studies on i) vegetative propagation of rhizome fragments as explants in Ruppia maritima, Halophila engelmannii, Cymodocea nodosa and Posidonia oceanica; ii) culture of meristems in Heterozostera tasmanica, C. nodosa or P. oceanica; and iii) culture of germinated seeds on aseptic conditions, in Thalassia testudinum, H. ovalis, P. coriacea, P. oceanica, and H. decipiens. All these studies determine the most adequate culture medium for each species (seawater, nutrients, vitamins, carbon sources, etc...), often supplemented with different plant growth regulators and the necessary conditions for the culture maintenance, such as light and temperature. On the other hand, several studies have previously established protocols for cell or protoplast isolation in the species Zostera marina, Z. muelleri, P. oceanica, and C. nodosa, using shoots collected from natural meadows as original vegetal source, but further cell growth was never accomplished. Due to the absence of somatic embryogenesis or organogenetic studies in seagrasses we wonder: IS THE SUCCESSFUL APPLICATION OF TISSUE CULTURE TECHNIQUES POSSIBLE IN SEAGRASSES?