In vitro propagation of a medicinal plant: Tripterygium wilfordii Hook f.

In this study a reliable protocol was developed for the establishment of commercial in vitro cultures of Tripterygium wilfordii Hook f.. Juvenile shoots from one-year-old elite plants were used as the source of explants. New axillary shoots were obtained after 30 days of culture on a MS medium supplemented with BAP （2.0 mg.L^-1） and NAA （0.1 mg.L^-1）. The optimal multiplication medium was a modified MS medium supplemented with BAP （1.0 mg.L^-1） and NAA （0.1 mg.L^-1）. This yielded a multiplication rate of 2.4 for each subculture. Slightly more than 92% of shoots rooted when cultured on a modified MS medium containing IBA （0.2 mg.L^-1） and activated charcoal （0.5 mg.L^-1）. Activated charcoal promoted both a strong and a high rooting rate during the rooting phase. Plantlets were transferred to pots for a short acclimatization stage in a greenhouse where 95% of the plantlets survived. This highly reproducible procedure can be adopted for large-scale propagation of T. wilfordii.

In Vitro Propagation Potential via Somatic Embryogenesis of the Two Maturing Early Cultivars of European Chestnut (Castanea sativa Mill.)

Turkey ranks the third in the production of chestnuts in the world having an important place both in domestic and global markets. However, the chestnut production and the number of trees have been diminishing in recent years. Therefore, in vitro propagation of the chestnut, in addition to the classical propagation techniques, should be applied. Especially the propogation of the early maturing cultivars and production of the quality chestnuts will provide a better income to the producer. Here, somatic embryo production and regeneration from the immature cotyledons of the early maturing cultivars of the European chestnut (Castanea sativa Mill), Haciibis and Karamehmet, were studied using the somatic embryogenesis, one of the in vitro propagation techniques. To induce the somatic embryogenesis, 168 different combinations were applied to both cultivars. The somatic embryogenesis rate in Haciibis cultivar, in which the interactions were observed among the applications, was found to be 9.9% while it was 11.1% for the Karamehmet cultivar. Dessication, cold treatment, gibberellic acid (GA3) and benzyladenine (BA) + naphthaleneacetic acid (NAA) applications were performed on the regeneration of the somatic embryos, and 40% conversion to plant was obtained with desiccation together with BA + NAA supplementation to the medium.

In vitro Rapid Propagation of Ficus carica L.‘Masui Dauphine’

In this study, Ficus carica L. ‘Masui Dauphine＇ was used as the experi- mental material to investigate the effects of explant type, basal medium, hormone types and concentrations on in vitro rapid propagation of‘Masui Dauphine＇. Accord- ing to the results, the most suitable explants for in vitro rapid propagation of ‘Masui Dauphine＇ were axillary buds and the best medium was modified MS ＋ 1.0 mg/L 6-BA ＋ 0. 05 mg/L NAA ＋ 1.0 mg/L GA3 ＋ 20 mg/L sucrose ＋ 7 mg/L agar, pH 5.8. This study provided scientific basis for barge-scale cultivation of‘Masui Dauphine ＇.

In Vitro Regeneration of Style-stigma-like Structure from Stamens of Crocus sativus

Style-stigma-like structures were regenerated from stamens of Crocus sativus L. The age of the stamen explant has an obvious effect on the induction rate. Auxin NAA. has larger effect on the induction of filament style-stigma-like structure. Auxin NAA of higher concentration can lead to higher induction rate. Temperature and light have different effects on the induction of style-stigma-like structure from anther's filament of C. sativus with exogenous hormones at different levels. Ultraviolet tests show that style-stigma-like structure from anther's filament of C. sativus contains crocin, safranal and picrocrocin, contents of which are obviously more than those contained in the style-stigma-like from style. Floral reversion was observed in the induction of style-stigma-like structure from petals, ovaries and styles.

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect na＇ive cells. The presence of minus （antisense） RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques （flow cytometry and Western blot） respectively. RESULTS： The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies （anti-core, and anti-E1）. Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION： HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

Effects of chemical sterilization of the culture media,porous membranes and luminosity on in vitro culture of Eucalyptus grandis×Eucalyptus urophylla

Hybrid combinations of Eucalyptus have increased due to expansion of plantations into unconventional areas and to the search for higher quality timber.However,most of these species have difficulties surviving in vitro cultivation.Active chlorine and sealing systems are often used to reduce contamination and increase gas exchange.The aim of the present study is to evaluate the establishment,multiplication,elongation and adventitious rooting of E.grandis × E.urophylla.Two clones(C1 and C2) and four active chlorine concentrations(0.000%,0.001%,0.003%,and 0.005%) were tested in the establishment and multiplication phases.Three sealing forms(W/M,1/M and 3/M) and the same four active chlorine concentrations were applied to the elongation phase.Two luminosities(dark and light)and three sealings(W/M,1/M and 3/M) were tested during adventitious rooting.Active chlorine concentration of0.005% led to the lowest fungal contamination rate and to the highest in vitro establishment.Active chlorine concentration of 0.003% resulted in the greatest length and highest number of shoots per explant in the multiplication phase.There were no phytotoxicity problems and the quality of plants grown in an environment with active chlorine was maintained in comparison with those grown in an autoclave.The increase in gas exchange in ventilation systems had a positive impact on the in vitro growth and development of plants.

Micropropagation of yellow mangosteen:a valuable endemic tree of India

We developed a method for in vitro regenera- tion of Garcinia xanthochymus （yellow mangosteen） from matured seed segments. Multiple shoots were induced on woody plant （WP） medium supplemented with cytokinins. An average of 11 shoots per explant were regenerated from mature seed segments on WP medium containing 20 μM 6-benzylaminopurine. Histological analysis revealed that hypodermal cells of seed segments were initially involved in active division, which later developed into meriste- moids, subsequently leading to the formation of shoot buds. Shoot elongation was achieved by repeated subculturing of seed explants in shoot regeneration medium. Rooting of shoots was achieved on WP medium supplemented with indole-3-butyric acid or s-naphthalene acetic acid. Plant- lets were transplanted to pots containing soil： compost （1：1） and survival rate was 90 %.

Sterile Germination of the Hybrid Seeds of [Den. Burana Green Star × Den. Rainbow-compactum] and Rapid Propagation of F_1 Plantlets via Tissue Culture

[Objective] This study was to germinate the hybrid seeds of [Den.Burana Green Star × Den.Rainbow-compactum] under aseptic condition and to explore the parameters for rapid propagation of F1 plantlets via tissue culture.[Method]Hybridization between Den.Burana Green Star（female parent）and Den.Rainbow-compactum（male parent）was performed and the in vitro culture and proliferation of F1 hybrids were studied using eight different basic media including MS,1/2MS,1/3MS,1/4MS,B5,N6,modified Knudson and H.[Result]Improved Knudson medium appended with 1.0 mg/L 6-BA,1.0 mg/L NAA and 10% mature banana puree performed best in F1 seed germination under aseptic condition,as well as the rapid propagation of protocorm-like body.Of all the eight media tested,1/2MS is the medium most suitable for the in vitro rapid propagation of the F1 seedlings.Efficiency of eight media in the in vitro rapid propagation was in order 1/2 MS MS1/3 MS1/4 MS ≈N6 improved Knudson ≈B5H.NAA presented better rooting and growth-promoting effect in the in vitro rapid propagation of the F1 seedlings than IBA.And the optimal NAA concentration to recommend from our experiment results was 2.0 mg/L.[Conclusion]Our experimental results provided mature method and important technological information for hybrid breeding dendrobium.

Comparative effects of plant growth regulators on leaf and stem explants of Labisia pumila var. alata

Objective: Labisia pumila var. alata, commonly known as 'Kacip Fatimah' or 'Selusuh Fatimah' in Southeast Asia, is traditionally used by members of the Malay community because of its post-partum medicinal properties. Its various pharmaceutical applications cause an excessive harvesting and lead to serious shortage in natural habitat. Thus, this in vitro propagation study investigated the effects of different plant growth regulators (PGRs) on in vitro leaf and stem explants of L. pumila. Methods: The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L. Results: Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34±19.55)% and (70.40±14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00±7.07)% and (77.78±16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5±5.0) and (30.0±8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00±0.00)%) that was obtained in 1 mg/L zeatin after (11.0±2.8) d of culture. Conclusions: Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs.