Gregory Franklin

Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress.

Xanthone production in Hypericum perforatum (HP) suspension cultures in response to elicitation by Agrobacterium tumefaciens co-cultivation has been studied. RNA blot analyses of HP cells co-cultivated with A. tumefaciens have shown a rapid up-regulation of genes encoding important enzymes of the general phenylpropanoid pathway (PAL, phenylalanine ammonia lyase and 4CL, 4-coumarate:CoA ligase) and xanthone biosynthesis (BPS, benzophenone synthase). Analyses of HPLC chromatograms of methanolic extracts of control and elicited cells (HP cells that were co-cultivated for 24h with A. tumefaciens) have revealed a 12-fold increase in total xanthone concentration and also the emergence of many xanthones after elicitation. Methanolic extract of elicited cells exhibited significantly higher antioxidant and antimicrobial competence than the equivalent extract of control HP cells indicating that these properties have been significantly increased in HP cells after elicitation. Four major de novo synthesized xanthones have been identified as 1,3,6,7-tetrahydroxy-8-prenyl xanthone, 1,3,6,7-tetrahydroxy-2-prenyl xanthone, 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone and paxanthone. Antioxidant and antimicrobial characterization of these de novo xanthones have revealed that xanthones play dual function in plant cells during biotic stress: (1) as antioxidants to protect the cells from oxidative damage and (2) as phytoalexins to impair the pathogen growth.

Factors affecting in vitro flowering and fruiting of green pea (Pisum sativum L.)

Multiple shoots were efficiently regenerated from cotyledonary node and shoot tip explants of Pisum sativum within 15 days on MS medium containing B5 vitamins and supplelmented with 2.0 mgl-1 6-benzylaminopurine. The elongated shoots produced on the same medium were excised and transferred to MS medium containing half strength ammonium nitrate (8.25 gml-1) and supplemented with auxins (indole-3-butyric acid or naphthalene acetic acid) either alone or in combinations with gibberellic acid. Rooting and flowering were observed on the 7th and 15th day after their transfer to rooting medium. The flowers self-fertilised in vitro and produced mature pods within 25 days of rooting. These seeds were germinable both in vitro and in vivo. In vitro seeds sown in pots under field conditions developed into flowering plants, and subsequently produced pods with viable seeds.

A study on the effect of genotypes, plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus

We have developed an efficient protocol for callus induction and plant regeneration in three elite soybean cultivars (Williams 82, Loda and Newton). The technique is most novel in that the shoot buds developed from the nodal callus. Callus induction and subsequent shoot bud differentiation were achieved from the proximal end of cotyledonary explants on modified Murashige and Skoog (MS) media containing 2.26 μM 2,4-dichlorophenoxy-acetic acid (2,4-D) and 8.8 μM benzyladenine (BAP), respectively. Varying the carbon source optimized the regeneration system further. Among the various carbon sources tested, sorbitol was found to be the best for callus induction and maltose for plant regeneration.

REGENERATION OF EGGPLANT (SOLANUM MELONGENA L.) FROM ROOT EXPLANTS

SummaryEggplant (Solanum melongena L.) was efficiently regenerated from cultured roots of 15-d-old seedlings on Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron and 13.3 μM 6-benzyladenine. Within 28d of culture initiation, induction of organogenic calluses and subsequent differentiation into shoot buds were observed. Shoot buds upon subculture to MS basal medium elongated into healthy shoots. Excised shoots (2–4 cm) were rooted on Soilrite® irrigated with water either in vitro or in vivo. Plants with well-developed root systems were established under field conditions after hardening in the glasshouse, where they developed into flowering plants and produced mature fruits with viable seeds.

Factors influencing regeneration of soybean from mature and immature cotyledons

Soybean (Glycine max L.) plantlets were e?ciently regenerated from mature and immature cotyledons of?ve different genotypes by studying various parameters affecting regeneration. Green organogenic noduleswere induced at the proximal end, which subsequently differentiated into shoot buds on modi?ed MS(Murashige T. and Skoog F. 1962. Physiol. Plant. 15: 473-497) medium. The presence of 6-benzylaminopurine(BAP) and thidiazuron (TDZ) in the medium exerted a synergistic effect, in that regenerationeffciency was higher than for either cytokin alone. The regenerated shoot buds elongated and rooted onMS medium containing 0.29 lM gibberellic acid (GA3) and 2.69 lM a-naphthaleneaceticacid (NAA),respectively. Rooted plants were established in the greenhouse with 87% success and produced viable seeds.Preliminary studies with Agrobacterium show great promise for soybean transformation based on theregeneration protocol reported here.

ORGANOGENESIS AND EMBRYOGENESIS IN SEVERAL HYPERICUM PERFORATUM GENOTYPES

SummarySt John’s wort (Hypericum perforatum) is a valuable plant used as a herbal remedy or in phytopharmaceutical drugs to treat a variety of physical ailments. Much research has been performed to study the biochemical production of secondary metabolites of in vitro cultured plants or organs. However, all of these studies have looked at the regeneration of plants from explants in only one genotype. In addition, no study has revealed the mechanism of plant regeneration in H. perforatum, i.e. organogenesis or somatic embryogenesis. We found that different genotypes Helos, Topas, Elixir, and Numi responded similarly to regeneration medium. The regeneration responses (i.e. callus, root, or shool production) of identical explants from different genotypes were similar. However, the source of explant material (leaves, hypocotyls, and roots) from the same genotype had significant effects on the response to media and plant regeneration frequency. Using scanning electron microscopy and light microscopy, the progress of organogenesis and embryogenesis under similar culture conditions was recorded. Root segments were the most responsive explants, producing the maximum number of shoots per explant of all the genotypes.

Hypericum sp.: essential oil composition and biological activities

Phytochemical composition of Hypericum genus has been investigated for many years. In the recent past, studies on the essential oils (EO) of this genus have been progressing and many of them have reported interesting biological activities. Variations in the EO composition of Hypericum species influenced by seasonal variation, geographic distribution, phenological cycle and type of the organ in which EO are produced and/or accumulated have also been reported. Although many reviews attributed to the characterization as well as biological activities of H. perforatum crude extracts have been published, no review has been published on the EO composition and biological activities of Hypericum species until recently (Crockett in Nat Prod Commun 5(9):1493–1506, 2010; Bertoli et al. in Global Sci Books 5:29–47, 2011). In this article, we summarize and update information regarding the composition and biological activities of Hypericum species EO. Based on experimental work carried out in our laboratory we also mention possible biotechnology approaches envisaging EO improvement of some species of the genus.

Factors affecting regeneration of pigeonpea (Cajanus cajan L. Millsp) from mature embryonal axes

Regeneration of viable plants was obtained via organogenesis from mature embryonal axes explants of pigeonpea. Shoots were produced from the apical region of embryonal axes after 20 days of dark incubation on modified Murashige and Skoog (1962) medium containing 8.86 μM benzylaminopurine (BAP) and 1.07 μM α-naphthalene acetic acid (NAA). When the explants were cultured under light-dark (16--8 hrs) conditions, shoots were initiated only after 65 days of culture initiation. The explants lost their ability to regenerate plantlets when they were cultured in continuous light. Regenerated shoots elongated either in the same medium or in MS basal medium. About 40% of the elongated shoots sequentially produced simple, bifoliate and finally trifoliate leaves instead of producing trifoliate leaves directly. The elongated shoots were rooted efficiently upon transferring them to half strength MS medium supplemented with 2.41 μM indole-3-butyric acid (IBA). Rooted plantlets were successfully transferred to soil after hardening.

Chlorogenic acid participates in the regulation of shoot, root and root hair development in Hypericum perforatum

Chlorogenic acid (CGA), a product of the phenylpropanoid pathway, is one of the most widespread soluble phenolic compounds in the plant kingdom. Although CGA is known to have important roles in plant function, its relevance in plant de novo organogenesis is not yet understood. With a series of experiments, here we show that CGA has a potential role in shoot, root and root hair development. In the first phase of our investigation, we developed an efficient and novel thin cell layer (TCL) regeneration protocol for Hypericum perforatum which could bridge all the in vitro morphogenetic stages between single cell and complete plant. Tissues at different morphogenetic states were analysed for their phenolic profile which revealed that shoot differentiation from callus tissues of H. perforatum is accompanied by the onset of CGA production. Further, the relevance of CGA in de novo organogenesis was deciphered by culturing highly organogenic root explants on media augmented with various concentrations of CGA. Results of this experiment showed that CGA concentrations lower than 10.0 mg l⁻¹ did not affect shoot organogenesis, whereas, higher concentrations significantly reduced this process in a concentration-dependent manner. In spite of the differential concentration-dependent effects of CGA on shoot regeneration, supplementation of CGA did not have any effect on the production of lateral roots and root hairs. Interestingly, CGA showed a concentration-dependent positive correlation with lateral roots and root hairs production in the presence of α-naphthaleneacetic acid (NAA). When the culture medium was augmented with 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), induction of shoots, lateral roots and root hairs from the explants was significantly affected. Addition of an optimum concentration of CGA in these cultures partially restored all these organogenic processes.

Antimicrobial activity of cream incorporated with silver nanoparticles biosynthesized from Withania somnifera

We report on the antimicrobial activity of a cream formulation of silver nanoparticles (AgNPs), biosynthesized using Withania somnifera extract. Aqueous extracts of leaves promoted efficient green synthesis of AgNPs compared to fruits and root extracts of W. somnifera. Biosynthesized AgNPs were characterized for their size and shape by physical-chemical techniques such as UV-visible spectroscopy, laser Doppler anemometry, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, X-ray diffraction, and X-ray energy dispersive spectroscopy. After confirming the antimicrobial potential of AgNPs, they were incorporated into a cream. Cream formulations of AgNPs and AgNO3 were prepared and compared for their antimicrobial activity against human pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Candida albicans) and a plant pathogen (Agrobacterium tumefaciens). Our results show that AgNP creams possess significantly higher antimicrobial activity against the tested organisms.