In Vitro Propagation of Three Strawberry Varieties and Field Evaluation

A study was done to produce a rapid in vitro propagation of three strawberry genotypes and tested in the field under Bangladeshi circumstances. Festival, RABI-3, and Neho strawberry genotypes’ runner tips were cultivated in vitro to induce root induction and multiple shoot proliferation. MS (Murashige and Skoog) media that were basally containing three different concentrations at 1.0, 1.5, and 2.0 of BA (6-benzyl adenine), KIN (6-furfuryl amino purine), or GA3 (gibberellic acid) at 0.5 mg/L increasing tips of the runner was attained. The culture grew on the medium provided with 1.5 mg/L 6-benzyl adenine and 0.5 mg/L 6-furfuryl amino acid to increase shoot at the best level. Micro-cuttings were rooted on MS media at half strength combined with 0.5 mg/L - 1.5 mg/L IBA (indole butyric acid) or IAA (indole acetic acid). IBA attained 4 - 9 roots and 91% - 96% rooting at 1.0 mg/L. The resulting plantlets grew into hardy plants and took root in the earth. The genotype festival had the highest response rate, followed by RABI-3 and Neho.

Effect of natural and synthetic growth stimulators on <i>in vitro</i>rooting and acclimatization of common ash (<i>Fraxinus excelsior</i>L.) microplants

Application of growth stimulators can be especially effective on plantlets in vitro of tree species which are usually worse rooted and adapted in comparison with annual plants. In our work we evaluate effects of natural (dihydroquercetin, Zircon) and synthetic growth stimulators (Melafen, Fumar, Epin-Extra) on rooting and acclimatization of common ash (Fraxinus excelsior L.) microplants. The 0.05% -?0.2% Zircon and 10-5%?Melafen enhanced in vitro rooting by 29% -?37% and 31%, respectively. Melafen also stimulated root formation faster compared to control plants. The dihydroquercetin concentration of 0.01% increased rooting by 24% and root number per shoot by 1.8 times. In vitro plants rooted on media supplemented with Melafen, Fumar and Zircon demonstrated enhanced ability to adapt to non-sterile conditions and accelerated growth. Two months after planting to the greenhouse, plants rooted on 0.01% dihydroquercetin were 45% taller than the control. Weekly spraying of plantlets with 0.02% Epin-Extra containing 24-epibrassinolid stimulated growth of uniform plants with large leaves. The obtained results support the use of growth stimulators for application in clonal micropropagation of common ash both for large-scale production of planting stock and for conservation of rare and valuable genotypes.

In vitro Rooting of Podophyllum Hexandrum and Transplanting Technique

Podophyllum hexandrum Royle is an important medicinal plant that produces podophyllotoxin with anti-cancer properties. In China, it is used as a traditional Chinese medicine. Recent years, unplanned exploiting and collection have led to the disappearance of this species in China and India. Effective methods such as tissue culture should be adopted to conserve it to abtain a large scale. It was an crucial process for the final success of tissue culture that seedlings with roots and true leaves in flasks were transferred to soil in field. A protocol has been development for in vitro rooting and hardening and en vitro transplant of Podophyllum hexandrum Royle. Roots were efficiently established on WPM media supplemented with IAA 1.5 mg·L-1 and NAA 0.5 mg·L-1. Before transferred to soil containing turfysoil and perlite (2:1), rooted plants were exposed to air for 5d for adaptation. Two periods were good for transplanting this plantlets.

Simple Protocol for the Micropropagation of Teak(Tectona grandis Linn.)in Semi-Solid and Liquid Media in RITA^(█) Bioreactors and ex Vitro Rooting

In Latin America the forestry of exotic species such as teak has been increasing in recent decades, due to their advantages in wood quality, rapid growth;and the relative ease of producing clones and their multiplication with respect to native species. Therefore, there is great interest in developing larger-scale propagation strategies that reduce costs and intensive manual labor. Culture in liquid media with temporary immersion and the semi-automation of the system has raised expectations for large-scale micropropagation. We report a protocol for teak, which reuses the primary explants in several culture cycles in semi-solid medium to produce nodal explants for the multiplication phase in temporary immersion bioreactors (RITA&reg;). The control of factors such as cytokinin concentration, explants density, immersion frequencies and culture duration was analyzed. The number of shoots increased with 0.5 mg&middot;l-1 of BA (6-Benzyladenine), alone or in combination with 0.5 mg&middot;l-1 of Kinetin, with 2 daily immersions of 1 minute each;however, these shoots showed a high degree of hyperhydricity. When 0.05 mg&middot;l-1 of BA was used with 1 immersion of 1 minute every 2 days, the hyperhydricity decreased. Although the number of shoots was lower, they showed good length to be used during multiplication and rooting ex vitro. Our results suggest that teak micropropagation can be simplified in two phases in vitro, the establishment and multiplication;followed by rooting ex vitro and acclimatization. This would imply a reduction in production costs, since most of the multiplication would take place in RITA&reg;containers.

Effects of Lanthanum and Europium on Rooting of Plantlet Eriobotrya Japonica Lindl. in vitro

The effect of La 3+ and Eu 3+ on the rooting of Eriobotrya japonica Lindl. plantlet in vitro was studied with adding La 3+ and Eu 3+ to the rooted medium. The rooting rate, the number of root and the length of root were studied after transplanting 20 d. The activity of peroxidase, nitrate reductase and fresh weight of roots were determined after transplanting 44 d. The results show that the optimum concentration range of La 3+ (1.0～3.0 μmol·L -1), Eu 3+ (2.0～3.0 μmol·L -1) in the rooted medium can increase the rooting rate and the fresh weight of roots, and promote the length of root and raise the activities of peroxidase and nitrate reductase significantly. La 3+ has more effect in improving the rooting rate, root length and the activities of peroxidase and less effect in promoting root fresh weight and the activities of nitrate reductase than Eu 3+.

Evaluation of in-vitro antibacterial activity and anti-inflammatory activity for different extracts of Rauvolfia tetraphylla L.root bark

Objective:To assess the in-vitro antihacterial activity and anti-inflammatory activity of orally administered different extracts(Hydro-alcoholic,methanolic,ethyl acetate and hexane)of Rauvolfia tetraphylla(R.tetraphylla)root bark in Carrageetiaii induced acute inflammation in rats.Methods:In-vitro antibacterial activity was evaluated for extracts against four Gram positive and four Gram negative bacteria by using cylinder plate assay.Hydro-alcoholic extract(70%v/v ethanol)at 200,400 and 800 mg/kg doses and methanolic,ethyl acetate and hexane extracts at doses 100,200 and 400 mg/kg were tested for anti-inflammatory activity in Carrageenan induced rat paw oedema model and paw thickness was measured every one hour up to 6 hrs.Results:All extracts of R.tetraphylla root bark showed good zone of inhibition against tested bacterial strains.In Carrageenan induced inflammation model,hydro-alcoholic and methanolic extract of R.tetraphylla root bark at three different doses produced significant(P<0.00l)reduction when compared to vehicle treated control group and hexane,ethyl acetate extracts.Conclusions:In the present study extracts of R.tetraphylla root bark shows good in-vitro antibacterial activity and in-vivo anti-inflammatory activity in rats.

Studies on the Isolation, Identification and In Vitro Growth Rates of the Three Pathogenic Fungi from Panax notoginseng Cultivated in Wenshan Eparchy

Objective] The aim of this study was to simultaneously isolate and identify the main pathogenic fungi of the root rot, black spot and round spot from the Panax notoginseng plants cultivated in Wenshan Eparchy of Yunnan Province of China. [Method] The pathogenic fungi were isolated and purified by using potato dextrose agar （PDA） medium. The morphological identification was accomplished first according to the colony forms of the fungi when cultivated in vitro, then accord-ing to the symptom characteristics and colony forms of the re-isolated fungi in the reverse inoculation experiments. The molecular identification was performed accord-ing to the amplification and alignment of the internal transcribed space （ITS） se-quences of the fungi. The increases of the diameters and thickness of the colonies of the fungi cultivated in vitro were employed to indicate the growth rates of the fungi. [Results] The consistency of the colony forms and symptom characteristics and the 96%-99% similarities revealed in the ITS sequence alignments al proved that the main pathogenic fungi of the root rot, black spot and round spot of the P. notoginseng plants raised in Wenshan were Cylindrocarpon didymium, Alternaria panax and Mycocentrospora acerina, respectively. When cultivated in vitro in the same temperature, humidity and il umination, the increases of the colony diameters and thickness of C. didymium were the highest, fol owed by those of A. panax, then those of M. acerina. During different cultivation periods, the differences of the colony diameters and thickness of the three fungi al reached extremely significant level. However, at the same cultivation time, the differences of the diameters and thickness among the three fungi only reached significant level. [Conclusion] The main pathogenic fungi which result in the root rot, black spot and round spot of the P. notoginseng in Wenshan are C. didymium, A. panax and M. acerina, respec-tively. When these three diseases break out at the same time, the root rot wil spread fastest, fol owed orderly by the black spot and the round spot.

Rooting and acclimatization of in vitro plantlets of mtl-D gene modified Robinia pseudoacacia 'Idaho'

Robinia pseudoacacia ‘Idaho＇ （Robinia x ambigua ‘Idahoensis＇, R. pseudoacacia x R. viscosa） modified by a mtLD gene went through five lines and had characteristics of drought tolerance. Three stages of their micropropagation had been studied by pre- vious investigators. The other two stages, in vitro shoot rooting and plantlet acclimatization, still remained unsolved in the laboratory. For this paper, we studied the later two stages based on the previous achievements. Results showed that the highest rooting rate of Idaho locust was 98.4% when the in vitro shoots, over 2.5 cm in height and 0.08 cm in diameter, were placed on a half strength MS basal medium with 0.4 mg.U1 IBA and 0.1 mg＇U1 NAA as supplements and were solidified with 0.5% agar; the highest survival rate was 98.3% when the rooted plantlets were potted in vermiculite. All the stages for micropropagation of the Idaho locust, modified by the mtl-D gene, were assembled completely. The tissue culture plants grow well in the field.

In Vitro Probiotic Effect of Pseudostellaria heterophylla Fibrous Roots on Feed Bacillus subtilis and Preliminary Study on Its Solid Fermentation

Traditional Chinese medicine Pseudostellaria heterophylla fibrous roots have been widely studied and applied in non-antibiotic clinical breeding of livestock and poultry. In vitro probiotic effect of P. heterophylla fibrous roots extract on feed probiotics Bacillus subtilis BD-K010 was studied, and the feasibility of solid fermentation of P. heterophylla fibrous roots was preliminarily evaluated. For in vitro probiotic effect, the increased concentration of P. heterophylla fibrous roots extract dependently increased the total biomass of B. subtilis BD-K010;1.0% P. heterophylla fibrous roots extract received the best effect, and the final p H of 1.0% experimental group was closer to neutral. Meantime, B. subtilis BD-K010 with optimum concentration of P. heterophylla fibrous roots showed significantly higher in vitro antibacterial effect than the control group(P<0.01),and the antibacterial effects on Escherichia coli and Staphylococcus aureus were improved by 51.99% and 63.16%, but it was ineffective to Salmonella. For solid fermentation, the profile of substrate complex and appendage flocculent structure on substrate surface at the end of fermenta-tion in experimental groups added with B. subtilis BD-K010 and cellulase plus BD-K010 were more complex;the live bacteria number, polysaccharide content and saponin content at fermentation end-point in two experimental groups were extremely higher than those in the control group(P<0.01).P. heterophylla fibrous roots extract had good in vitro probiotic effect on B. subtilis BD-K010 and promoted its antibacterial effect, and it is feasible to use probiotics for solid fermentation of P. heterophylla fibrous roots and to improve effective components. It is of great significance to further de-velop and utilize P. heterophylla fibrous roots resources in modern animal husbandry.

Acclimatization of <i>in Vitro</i>Propagated Pineapple (<i>Ananas comosuss</i>(L.), var. Smooth cayenne) Plantlets to <i>ex Vitro</i>Condition in Ethiopia

Pineapple (Ananas comosuss, var. Smooth cayenne), which is a popular tropical fruit, is propagated vegetatively. Conventional propagation alone does not provide clean and adequate planting material demanded in Ethiopia. Recently, in vitro multiplication has become a promising technique for large-scale production. However, the acclimatization to the external environment procedure impedes the efficiency, which needs carefully optimized acclimatization techniques. We report optimized acclimatization procedures following first- and second-stage hardening methods for in vitro pineapple plantlets. Primarily, Jiffy-7 peat pellet allowed growing plants vigorously and provided above 8% survival rate over soil mix. Nevertheless, in Ethiopia, soil mix is cheaper and locally accessible. The primarily acclimatized plantlets are needed to be hardened further for better establishment and survival in the field. Black polybag and polysleeve pots filled with soil mix were evaluated in the greenhouse. A significant difference was obtained between pots for number of roots and substrate weight. Polybags had higher root number than polysleeves and saved about 27% of substrates per plant, which is a reduction of 25% of total transportation cost. Hence, the soil mix and polybags were found to be preferable over substrates and pots, for subsequent in vitro pineapple acclimatization.

Direct Regeneration of Plants Derived from in vitro Cultured Shoot Tips and Leaves of Poplar （Populus×euramericana ＇Neva＇）

The purpose of the present study was to establish a regeneration procedure for Populus × euramericana ＇Neva＇ by using in vitro shoots tips and leaves. For sterilization, 0.1% （w/v） mercuric chloride （HgCl2） solution for 8 to 10 min was the optimal treatment for this poplar cultivation. The effects of benzyladenine （BA） and α-naphthaleneacetic acid （NAA） added to Murashige and Skoog （MS） medium were tested on organogenesis. The highest regeneration rate and numbers of shoots/explant from shoot tips （96.7%, 9.8） and leaves （90.0%, 8.7） were obtained on the half-strength MS medium supplemented with 0.5 mg/L BA and 0.1 mg/L NAA. The optimal medium for in vitro rooting of shoots was on a half-strength MS medium containing 1 mg/L indolebutyric acid （IBA） with the highest rooting frequency （93.3%） and numbers of roots/explant （8.2）. For acclimatization, in vitro rooted plantlets were transferred to plastic cups containing vermiculite and peat （1： 1）. After acclimatization, transplanted plantlets grew well in a shade house. Therefore, we believe that this efficient plant regeneration protocol especially by leaf explants is very important for in vitro clonal propagation of Populus×euramericana ＇Neva＇.

Effects of Lanthanum on Root Growth and Senescence of GF_(43) (Prunus domestica) Plantlet in Vitro

The effect and the action mechanism of lanthanum on GF_(43) plantlet in vitro were studied. The results of experiments show that root growth rate and dry weight of GF_(43) by LaCl_3 treatments increase. The activities of antioxidant enzymes in root system such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) apparently enhance. Moreover ·O^-_2 and malond ialdehyde (MDA) contents and cell membrane permeability of GF_(43) are decreased by LaCl_3. The relatively stable membrane structure of cell could also be maintained and the root ageing of GF_(43) plantlet in vitro delays.

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.

The Effects of Auxins and Cytokinin on Growth and Development of (<i>Musa</i>sp.) Var. “Yangambi” Explants in Tissue Culture

The aim of this study was to investigate the effects of concentration of different growth regulators (auxins and cytokinins) on growth and development of banana shoot tips cultured in vitro. Explants were taken from young suckers of field grown plants of var. “Yangambi”. The shoot tips were cultured on MS media supplemented with different concentrations of BAP (0, 2, 4, 6 and 8 mg/l) with or without IAA at concentration of 0.34 mg/l. At the rooting phase, the media was supplemented with different concentrations of IBA (0.1, 0.5, 1.0, 1.5 and 2.0 mg/l) with or without BAP at concentration of 0.2 mg/l. The results indicated that 6.0 mg/l BAP significantly increased the number of shoots formed and the interaction of 6 mg/l BAP with 0.35 mg/l IAA significantly increased the fresh weight. For rooting, 2.0 mg/l IBA was more efficient in number and length of roots produced than all other treatments.

Effects of Neodymium on Nitrogen Metabolism of Plantlet Loquat in Vitro

The effect of NdCl_3 on the rooting and nitrogen metabolism of loquat in vitro was studied when NdCl_3 was added to the rooted medium. The results show that 0.4 μmol·L^(-1) NdCl_3 in the rooted medium can obviously increase the rooting rate, length of root and fresh weight of roots, and enhance the activities of nitrate reductase, glutamine synthetase, glutamate dehydrogenase in the root system and in the leaves. The transformation of NO_3^- to NH_4^+ in root system and leaves are promoted and the nitrogen metabolism is accelerated with 0.4 μmol·L^(-1) NdCl_3 treatment.

Effect of Lanthanum on Deferring Root Ageing of Peach Plantlet in Vitro

Poor adventitious root formation is a major obstacle in micropropagation and in conventional propagation. The effects of LaCl_3 on root growth and deferring the root ageing of peach plantlet in vitro were investigated. The results show that the optimum concentration of LaCl_3 (2.5 μmol·L -1) in the rooted medium can significantly increase the rooting rate, the root length and the fresh weight, promote the activities of superoxide dismutase (SOD); catalase (CAT) and peroxidase (POD) and decrease the production rate of O_2 ·-, the malondialdehyde (MDA) contents and the plasma membrane permeability. There are important theory meaning and practical value in applying LaCl_3 in the rooting medium to raise the rate of rooting and transplant for stone fruit tree.

<i>In Vitro</i>Propagation of Mature Carob Trees (<i>Ceratonia siliqua</i>L.) from the Axillary Buds

The influence of tree age and the effect of growth regulators on the micropropagation of the carob (Ceratonia siliqua L.) from the axillary buds of mature trees have been described. Significant differences (P < 0.005) in results are obtained in the stages of initiation, multiplication, and rooting according to their response to the various concentrations of different growth regulators examined, namely BA, IBA, AG3. The use of 0.5 mg/l BA and 0.2 mg/l IBA was the most favorable for shoots neoformation. The leafy shoots are propagated in MS medium with BA at a concentration of 1.5 mg/l. The addition of gibberellic acid at 0.2 mg/l in the culture medium allows a good elongation and development of the shoots of the carob. The effect of the age of the plant material used has shown that explants taken from mature carob trees have a low capacity for bud sprouting and shoot proliferation compared to those taken from juvenile trees. Rooting has been successful when the plant material used is taken from young trees on an MS medium containing 2 mg/IBA, with an average number of 3 to 4, roots 1 to 2 cm long, then for the adult material, no rooting was observed. Based on these tests, it appears that micropropagation of the carob from the axillary buds is feasible, but additional work must be done to root this recalcitrant material.

High Light Intensity Increases the CAM Expression in “MD-2” Micro-Propagated Pineapple Plants at the End of the Acclimatization Stage

This work describes the evaluation of morpho-physiological and biochemical changes in “MD-2” micro-propagated pineapple plants (Ananas comosus (L.) Merr.) grown after 30 days under low light intensity (LL, greenhouse light conditions at 250 μmol·m-2·s-1) or high light intensity (HL, field light conditions at 800 μmol·m-2·s-1). Gas exchange, leaf pH, protein content and superoxide dismutase activity (SOD) (EC 1.15.1.1) were measured every 3 h during one day. Chlorophylls content and succulence index (SI) were determined at 9 h. Results showed significant differences in CO2 exchange rates, with a maximum occurring at 6 h (3.00 and 8.25 μmol CO2 m-2·s-1 for leaves under LL and HL conditions respectively). Plants under HL conditions had higher CO2 uptake and lower pH values between 0 h and 6 h respective to LL plants. The maximum pH value was attained 3 h before in HL plants. Leaf SI was increased and chlorophyll content decreased by HL conditions. SOD activity was higher in plants under HL conditions, near doubling those of LL plants at 18 h (2.8 versus 1.5 U·mg-1 Protein respectively). Both groups showed a typical CAM phenotype, but it was stronger in HL conditions, which may confer these plants with a better acclimation to transfer to the field.

Hepatitis C virus: Morphogenesis, infection and therapy

Hepatitis C virus(HCV) is a major cause of liver diseases including liver cirrhosis and hepatocellular carcinoma. Approximately 3% of the world population is infected with HCV. Thus, HCV infection is considered a public healthy challenge. It is worth mentioning, that the HCV prevalence is dependent on the countries with infection rates around 20% in high endemic countries. The review summarizes recent data on HCV molecular biology, the physiopathology of infection(immune-mediated liver damage, liver fibrosis and lipid metabolism), virus diagnostic and treatment. In addition, currently available in vitro, ex vivo and animal models to study the virus life cycle, virus pathogenesis and therapy are described. Understanding of both host and viral factors may in the future lead to creation of new approaches in generation of an efficient therapeutic vaccine.

Cloning and activity analysis of in vitro expression of plant NAD-IDH genes

The plant NAD-dependent isocitrate dehydro-genase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidop-sis thaliana ecotype Columbia gl1 and three lines (Shaan 2A, Shaan 2B and Ken C1) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted into the multiple cloning sites of pMID1 vector for the expression of recombinant protein. All the recombinants were successfully expressed in the in vitro expression system. When the transcripts of subunits 0, 1 and 2 were added to-gether to the in vitro system with 36 靏/mL of protein disul-fide isomerase, the expressed products had NAD-IDH enzy-matic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designated subunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either subunit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of subunit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.