<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

Rapid germination and development of <Emphasis Type="Italic">Taxus baccata</Emphasis> L. by <Emphasis Type="Italic">in vitro</Emphasis> embryo culture and hydroponic growth of seedlings

A highly promising procedure to obtain seedlings of Taxus baccata L. has been developed, which involves a combination of in vitro embryo culture and growth under hydroponic conditions. Embryos isolated from freshly collected seeds were 100% sterile, even though the seeds were not treated with acid or soaked in water prior to culturing. The embryo germination level of non-leached seeds was slightly lower (85%) than those leached in running water for 7 d (100%). The leached embryos germinated with extended roots while the non-leached embryos had abnormal shapes. The embryos cultured on media supplemented with an absorbent (PVP or activated charcoal) had extended roots and shoots and were a larger size without any browning, as compared to those grown without the supplement; activated charcoal gave better results. There were no significant differences in germination rates of T. baccata embryos between the media with differing strengths of macronutrients; however, for further development of the shoot, it was necessary to sub-culture the seedlings in MS in the light. To obtain seedlings with longer roots, they had to be maintained in one-half strength MS in darkness. Approximately 90% of the plants survived when grown hydroponically for 2 mo. The surviving plants showed well-extended roots and were a good starting material for genomic, proteomic, and conservational studies as well as Taxol permeabilization investigations.     

Cultivating <Emphasis Type="Italic">in vitro</Emphasis> coconut palms (<Emphasis Type="Italic">Cocos nucifera</Emphasis>) under glasshouse conditions with natural light,improves <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis nursery survival and growth

Plantlets of coconut were cultured in vitro under three different ambient conditions including a standard culture room, a culture room inside a glasshouse with natural light but controlled temperature, and a standard glasshouse with natural light and natural fluctuations of temperature. Plantlets from the 3 treatments were compared in terms of growth, plant survival as well as net photosynthesis and efficiency of PSII (Fv/Fm ratio) both at the end of the in vitro stage and at 3 stages of ex vitro acclimatization. At the end of the in vitro stage, plantlets cultured in vitro under glasshouse conditions showed the best performance showing the highest photosynthesis rate, dry weight and number of leaves. Plantlets from the standard culture room showed the lowest photosynthesis and growth rate. After 6 months of ex vitro acclimatization, plantlets originally grown in vitro under glasshouse conditions maintained better field survival and growth rates in terms of fresh weight, dry weight and leaf number than plantlets originally grown in vitro in the standard culture room. Although more studies are required to define the reason for this effect, it is clear that the conditions of standard culture rooms are not the best for in vitro cultivation of coconut and perhaps other tropical species.     

Do secondary sexual dimorphism and female intolerance to drought influence the sex ratio and extinction risk of <Emphasis Type="Italic">Taxus baccata</Emphasis>?

Sex ratio and sexual dimorphism were studied in the dioecious tree Taxus baccata. We examined five populations of T. baccata in Poland and Ukraine to identify the differences between male and female individuals. The sex of all individuals, height and diameter, needle length and area, specific leaf area (SLA), the number of stomata rows, stomatal density, and content of carbon and nitrogen were measured to identify the differences between male and female individuals. The relationship between sex ratio and climatic conditions, age and population size were analysed using data collected from the field and the literature. Female trees were shorter than males, but needles of females were longer and had larger area. Although there were no differences among sexes in SLA, nitrogen and carbon concentration, we found a positive correlation between nitrogen concentration and SLA among females. The sex ratio changed with tree height within populations, and taller height classes were biased in favour of males. Regardless of population age, the percentage of females within populations was positively correlated with precipitation. Probably high reproductive effort caused female trees to lose in competition with males, and this loss may also be enhanced by lower drought tolerance in females and could contribute to risk of extinction for T. baccata. The continental geographic range of T. baccata may be restricted by limited occurrence of females, which demand higher water resources than males.     

Identification of two phenotypes of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under <Emphasis Type="Italic">in vitro</Emphasis> salt stress conditions

This study describes two phenotypes of Arabidopsis thaliana (ecotype Columbia) developed in vitro under salt stress (75 mM NaCl). The phenotypes 01 and 02 appeared visibly distinguishable by rosette morphology and competence to produce flowers. Phenotype 01, sensible to salt stress, accumulated high quantities of Na⁺, showed a slight reduction in dry mass, and high protein and chlorophyll contents. Moreover, its anatomy exhibited some xeromorphic traits. Phenotype 02, clearly salt tolerant, showed a morphology similar to control plants, displaying typical phyllotactic rosette and flowering stalk production. Accumulation of Na⁺, protein and chlorophyll contents were close to control plants. Reversion experiments on NaCl free MS medium, showed a partially recovered phenotype 01. A threshold salt stress concentration that permits the simultaneous development of two phenotypes, was found.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Long-range chromosomal engineering is more efficient <Emphasis Type="Italic">in vitro</Emphasis> than <Emphasis Type="Italic">in vitro</Emphasis>

Cre/LoxP mediated chromosomal engineering in embryonic stem (ES) cells has a variety of applications, including the creation of model systems for studying aneuploidy. Targeted meiotic recombination (TAMERE) was proposed as a high efficiency in vivo alternative to effect Cre-mediated recombination, in which Cre recombinase under control of the Synaptonemal Complex 1 promoter is expressed during male meiosis in transgenic mice. TAMERE has been successfully used with LoxP sites up to 100 kb apart. We tested TAMERE for a chromosome engineering application in which LoxP sequences were integrated into sites 3.9 Mb apart on the same (cis) or opposite (trans) copies of mouse Chromosome 16 (MMU16). TAMERE was ineffective in generating either a deletion or a translocation in vivo. The TAMERE method may be of limited use for large genomic rearrangements. The desired translocation was achieved with an in vitro method that can be used in any ES cell line. Mice produced from the reciprocal duplication/deletion of MMU16 in a region homologous to human chromosome 21 provide models that are useful in studies of Down syndrome.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Pisum sativum in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivin, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm⁻³ kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T₀ putative transformants and their seed progenies (T₁ to T₃ generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T₀ – T₃ plants were morphologically normal and fertile.This research was supported by the National Agency for Agricultural Research (grants No. QE 0046 and QF 3072) and Ministry of Education of the Czech Republic (grant No. ME 433).     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> strains: <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

<Emphasis Type="Italic">In vitro</Emphasis> germplasm conservation of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis> L. under slow growth conditions

Germplasm conservation of Podophyllum peltatum L. was attempted by using synthetic seed technology and media supplemented with osmotic agents. Excised buds from in vitro cultures were encapsulated in calcium alginate beads and cultured on different substrates then stored at 5, 10, and 25°C for up to 8 mo. Survival and vigor in re-growth were the parameters used to evaluate the germplasm storage conditions. Vigor in re-growth was measured by number of buds induced after storage, which was achieved on a substrate containing water solidified with 1% w/v agar under 10°C. In vitro storage of shoot cultures was also evaluated by supplementing osmotic agents, mannitol, or sorbitol to the media. Such treatment had a negative impact on post-storage re-growth (at 25°C), even though the inclusion of 2% w/v sorbitol and mannitol each to the media increased plantlet survival during 10°C storage treatment. A deleterious effect was noticed among cultures in re-growth when higher concentrations of these supplements were added to the media. Genetic stability was assessed following 8 mo of storage using a PCR-based multilocus DNA fingerprinting technique, amplified fragment-length polymorphism. No differences in the DNA fragment patterns were observed using eight primer combinations in stored clones. However, a polymorphic band was noticed in the accession that served as explant source, suggesting that the mutation has occurred prior to this study perhaps during the 9 years of in vitro cultivation.     

An efficient <Emphasis Type="Italic">in vitro</Emphasis> method for mass propagation of <Emphasis Type="Italic">Tylophora indica</Emphasis>

A protocol of high frequency shoot organogenesis and plant establishment from stem derived callus has been developed for Tylophora indica (Burm. f.) Merrill. - an endangered medicinal plant. Callus was developed on Murashige and Skoog (MS) medium supplemented with 10 M 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (80 %) of shoot multiplication was achieved on MS medium containing 5.0 M kinetin. The developed shoots rooted best on half-strength MS medium supplemented with 0.5 M indole-3-butyric acid (IBA). The in vitro raised plantlets with well developed shoot and roots were acclimatized successfully and grown in greenhouse.     

Attenuation of fibrosis <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> with <Emphasis Type="Italic">SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.     

<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> assays for osteoclast apoptosis

Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.     

An <Emphasis Type="Italic">in vitro</Emphasis> method to evaluate grapevine cultivars for <Emphasis Type="Italic">Erysiphe necator</Emphasis> susceptibility

Powdery mildew, caused by the obligate biotrophic ascomycete Erysiphe necator, is one of the most destructive grapevine diseases worldwide. Cultivars of Vitis vinifera L, for wine and table grape production, are all susceptible to E. necator, whose attacks result in severe epidemics under the warm and dry conditions of the Mediterranean basin. The aim of the present study was to compare the susceptibility of different grapevine cultivars to E. necator by an in vitro assay for assessing the potentiality of this method in breeding programs for resistance to the pathogen. Leaves of 12 grapevine cultivars were spot-inoculated in vitro with about 10 conidia from five different isolates of E. necator, using colony growth and conidiation 3 wk post-inoculation as indicators of susceptibility to the disease. A remarkable difference was observed between highly susceptible cultivars like ‘Baresana’, ‘Malvasia’, ‘Bianca’, and ‘Italia’, and the less susceptible ‘Alphonse Lavallée’ and ‘Ohanez’, in accordance with their behavior in the field. No statistically significant differences were found in the virulence of E. necator isolates.