Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John's wort).

In vitro-grown roots: a superior explant for prolific shoot regeneration of St. John's wort (Hypericum perforatum L. cv ‘New Stem’) in a temporary immersion bioreactor

Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures

Hypericum perforatum L. is a medicinal plant that has been extensively studied because of its bioactive properties. The objective of this study was to establish a system that could lower the cost of in vitro propagation by using liquid medium, as well as to evaluate the secondary metabolism in the systems tested. Nodal segments of H. perforatum were obtained from in vitro shoots and grown in three liquid culture systems: total immersion (TI), partial immersion (PI), and paper bridge support (PB). Semi-solid medium (3 g L−1 Phytagel™) was used as control (SS). The organogenic responses were evaluated, and phenolic compounds, hypericin, and the activity of polyphenol oxidases (PPO) and peroxidases (POX) were quantified. After 80 days of culture, induction and proliferation of adventitious shoots were similar in the PI and SS systems (65.3 and 71.3 shoots, respectively), whereas PB resulted in the fewest shoots per explant (29.5 shoots). Longer shoots were obtained under the PI conditions. Hyperhydricity was observed in the shoots from the TI system. Browning was visible in shoots from the TI and PB systems. The highest concentrations of phenolic compounds and hypericin were observed in shoots derived from PI and PB, at 80 days of culture. POX activity was higher in shoots cultured in PI at 40 days, whereas PPO was significantly more active at 80 days of culture. Likely, POX was more related to shoot growth, whereas PPO played a later role in response to the culture environment and medium stress.

Secondary metabolism in micropropagated Hypericum perforatum L. grown in non-aerated liquid medium

Tylophora indica (Burm. f.) Merrill is a threatened medicinal climber distributed in the forests of northern and peninsular India. An efficient and reproducible protocol for high-frequency callus regeneration from immature leaf explants of T. indica was developed. Organogenic callus formation from immature leaf pieces was obtained by using Murashige and Skoog (MS) medium supplemented with 7 μM 2,4-dichlorophenoxyacetic acid and 1.5 μM 6-benzyladenine. On this medium 92% explants produced callus. The optimal hormone combination for plantlet regeneration was 8 μM thidiazuron, at which shoot regeneration was obtained from 100% of the cultures, with an average of 66.7 shoots per culture. Histological studies of the regenerative callus revealed that shoot buds were originated from the outermost regions. For root formation, half-strength MS medium supplemented with 3 μM indole-3-butyric acid was used. Plants were transferred to soil, where 92% survived after 3 mo. of acclimatization.

Thidiazuron-induced high-frequency shoot organogenesis from leaf-derived callus of ia medicinal climber, Tylophora Indica (Burm. F.) merrill

Use of Hypericum perforatum L. has increased in the past few years due to the antidepressant and antiviral activities found in extracts of this plant. As a result of its potential as a pharmaceutical, a new system was developed for in vitro culture of this species. Leaf explants were inoculated onto MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 or 4.5 μM) and 6-benzyladenine (BA, 0.44 or 4.4 μM) or kinetin (0.46 or 4.6 μM). Explants were cultivated under dark or light conditions to induce callus formation. Callus initiation was observed in all media evaluated and the highest cell proliferation was obtained from explants cultivated in the presence of 4.4 μM BA and 4.5 μM 2,4-D in the dark. Shoot induction was obtained from callus induced on 4.6 μM kinetin and 0.45 μM 2,4-D 6 weeks after transferring the callus to a MS medium supplemented with 4.4 μM BA. Roots were induced from shoots on full and half-strength MS media with or without indolebutyric acid (IBA, 4.9 μM) and the highest rooting frequencies were obtained on half-strength MS medium, regardless of the presence of IBA. Regenerated plants were easily acclimated in greenhouse conditions. The procedure reported here allows the micropropagation of H. perforatum in five months of culture and the proliferation of cell masses which could be used for studies on organic compounds of pharmaceutical interest.

Callus formation and plant regeneration from Hypericum perforatum leaves

Several methods for gene transfer into plants have been developed and it has allowed many ofthe world's importam crop lants to be transformed. Transformation using Agrobacterium has several advantages over vectorless systems, although its efficiency on transferring DNA is still low for many species. Recentty, a technology called SAAT (Sonication Assisted Agrobacterium-mediated Transformation) has been described, which involves subjecting lhe plant tissue to brief periods of ultrasound m the presence of Agrobacterium. Scanning and light microscopy revealed tiiat SAAT treatment produces a large number of small and uniform wounding through the tissue allowing the Agrobacterium easy access to internai plant tissues. SAAT improved the transfonnation efficiency in several different plant tissues including immature cotyledons, leaf tissue, suspension cultores, somatic and zygotic embryos from dijferent species such as soybean, cowpea, white spruce, wheat and maize. This new method allows the increase of transfonnation rates in several species, especially those more recalcitram to Agrobacterium -mediated transfonnation.

http://www.scielo.br/pdf/cr/v30n4/a29v30n4.pdf

Eliane Romanato Santarém

Papers

Callus formation and plant regeneration from Hypericum perforatum leaves

SAAT: tranformação de plantas mediada por ultra-som e Agrobacterium