In vitro induction of polyploidy in japanese quince (Chaenomeles japonica)

In vitro conditions for Japanese quince polyploidisation were investigated. Microshoots and isolated cotyledons were treated with colchicine and oryzalin. Morphogenesis was more dependent on the concentration of colchicine or oryzalin than on the duration of exposure, genotype differences were observed. Low oryzalin concentrations had no impact on morphogenesis. Plants with changed chromosome numbers were obtained at 0.25–38 mM colchicine and 10–50 μM oryzalin concentrations. It was determined that stomata length is a suitable parameter for identifying putative Japanese quince tetraploids. Stomata of tetraploid shoots of the same clone were approximately 1/3 longer than in the diploids. It was shown that through polyploidisation gigas effect was not obtained in fruit size but tetraploids have reduced seed set and an increased proportion of fruit flesh.     

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.)

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.     

In vitro flower induction in roses

Summary Vegetatively propagated plantlets of six rose cultivars were induced to flower in vitro on media containing full-strength Murashige and Skoog (MS) inorganic salts, Gamborg's B5 organic elements with 400 mg l⁻¹ myo-inositol, and different phytohormone combinations of 6-benzyladenine (BA) with α-naphthaleneacetic acid (NAA); thidiazuron (TDZ) with NAA; and zeatin (ZT) with NAA. The most efficient flower bud induction (49.1% and 44.1%) was obtained on media supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.1 mg l⁻¹ (0.54 μM) NAA or 0.5 mg l⁻¹ (2.28 μM) ZT and 0.1 mg l⁻¹ (0.54 μM) NAA for cultivar Orange Parade. Scanning electron microscopy (SEM) showed that in vitro flower bud induction occurred mostly between 15 and 30 d in induction medium through the normal flower development processes. With TDZ and ZT as the best choice for flower induction in all six cultivars tested, different rose cultivars varied in their responses to phytohormone treatments. Our study also revealed that the total time from original culture and subculture time before flower induction were two very important factors for in vitro flower induction. Plantlets 156–561 d from original culture and subcultured for 45 d were the best for flower induction. These authors contributed equally to this work.     

Chromosome doubling in sterile Syringa vulgaris × S. pinnatifolia hybrids by in vitro culture of nodal explants

The aim was to produce tetraploid forms of hybrids of Syringa vulgaris × S. pinnatifolia to restore fertility and enable further breeding to be undertaken. Excised nodal sections of three selections were pre-cultured for 5 days then treated with a range of colchicine concentrations for 1, 2 or 3 days, after which they were washed three times in sterile distilled water and cultured on shoot proliferation medium. Frequent movement to fresh medium was beneficial to survival. Three successive experiments established the range of concentrations of colchicine, 0.05 mM to 0.25 mM, at which tetraploids were likely to be produced. Thus a protocol for the production of tetraploids was established. Cytometric analysis showed that tetraploid forms of two selections were produced. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro plant regeneration from seed explants of wild groundnut species (Genus Arachis, Section Extranervosae)

In vitro regeneration of wild groundnut species from Section Extranervosae (Arachis villosulicarpa, A. macedoi, A. retusa, A. burchellii, A. pietrarellii, A. prostrata, A. aff. prostrata and a new species) was examined for the purpose of germplasm renewal and conservation. Seeds of different ages, stored at the seed bank of CENARGEN/EMBRAPA were either inoculated on culture medium or used as a source of embryo axis and cotyledon explants. Whole seeds failed to germinate on MS either without growth regulators (MS0) or supplemented with 10 M TDZ. Embryo axes cultured on MS0 produced only single plants. In the presence of 8.8 M BAP these explants showed multi-shoot formation. Cotyledons cultured on MS supplemented with 110 M BAP developed adventitious shoots through direct organogenesis. Plant regeneration was obtained from A. villosulicarpa, A. macedoi, A. retusa, A. burchellii and A. pietrarellii both from embryo axes and cotyledons. Explants from A. prostrata and A. aff. prostrata did not produce regenerants. Rooting of shoots was induced in the presence of 5.4 M NAA. Primary plants derived from these explants were further multiplied by culturing nodal segments on MS medium plus 2.7 M NAA.     

The anti-biofilm potential of pomegranate (Punica granatum L.) extract against human bacterial and fungal pathogens

Infectious diseases caused by bacteria and fungi are the major cause of morbidity and mortality across the globe. Multi-drug resistance in these pathogens augments the complexity and severity of the diseases. Various studies have shown the role of biofilms in multi-drug resistance, where the pathogen resides inside a protective coat made of extracellular polymeric substances. Since biofilms directly influence the virulence and pathogenicity of a pathogen, it is optimal to employ a strategy that effectively inhibits the formation of biofilm. Pomegranate is a common food and is also used traditionally to treat various ailments. This study assessed the anti-biofilm activity of a methanolic extract of pomegranate against bacterial and fungal pathogens. Methanolic extract of pomegranate was shown to inhibit the formation of biofilms by Staphylococcus aureus, methicillin resistant S. aureus, Escherichia coli, and Candida albicans. Apart from inhibiting the formation of biofilm, pomegranate extract disrupted pre-formed biofilms and inhibited germ tube formation, a virulence trait, in C. albicans. Characterization of the methanolic extract of pomegranate revealed the presence of ellagic acid (2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione) as the major component. Ellagic acid is a bioactive tannin known for its antioxidant, anticancer, and anti-inflammatory properties. Further studies revealed the ability of ellagic acid to inhibit the growth of all species in suspension at higher concentrations (>75?μg?ml^?1) and biofilm formation at lower concentrations (<40?μg?ml^?1) which warrants further investigation of the potential of ellagic acid or peel powders of pomegranate for the treatment of human ailments.     

In vitro propagation of Typhonium flagelliforme (Lodd) blume

Summary An in vitro culture system was developed for Typhonium flagelliforme using buds from the rhizomes. The mineral salts of four media were tested. These were Murashige and Skoog (MS), Nitsch and Nitsch (NN), Gamborg B5 (GB5) and White (W) of which MS medium was found to be the best medium for in vitro culture of T. flagelliforme. The addition of as low as 0.1 mg l⁻¹ (0.54 μM) α-naphthalene acetic acid (NAA) with the presence or absence of N⁶-benzyladenine (BA) in the MS medium caused abnormal shoot formation. The best medium for maximizing shoot number combined with normal complete plantlets from each bud was MS medium supplemented with 0.3 mg l⁻¹ (1.33 μM) BA and 0.5 mg l⁻¹ (2.46 μM) indole-3-butyric acid (IBA). The best acclimatization process was to transfer the normal plantlets, with all the leaves removed, into sand plus coconut husks substrate (1∶1) and placed in intermittent water mists house or shaded plant house with 50% light exclusion. Ninety two percent of the plantlets survived using this acclimatization method.     

In vitro translation of maize ADH: Evidence for the anaerobic induction of mRNA

Total cellular RNA from anaerobically stressed maize seedling roots was used to stimulate in vitro translation of authentic maize alcohol dehydrogenase (ADH) in a rabbit reticulocyte lysate system. Total products from such reactions were displayed on NEPHGE-SDS two-dimensional gels and the Adhl-specific translation products were identified by using RNA from sib seedlings segregating for Adhl charge and size variants. The application of a rapid RNA isolation procedure allowed the efficient isolation of biologically active RNAs from small amounts of seedling material. Maize ADHs translated in vitro are identical in size to in vivo ADH. Further, no ADH was detected in the products of an in vitro translation reaction stimulated by total RNA from aerobically grown seedlings. This suggests that induction of ADH protein by anaerobic stress is accomplished by production of Adh mRNA rather than activation of sequestered mRNA. The mRNAs for maize ADH1 and ADH2 are among a small class of mRNAs induced during anaerobiosis.Research was supported by NSF Grant PCM 76-11009. M.D.B. is supported by National Institute of Health Grant PHS 5 T32 GM07227-04. R.J.F. is a Predoctoral Trainee in Genetics supported by National Institute of Health Training Grants 82 and 7757 from the National Institute of General Medical Sciences.     

Floral induction and development in Phalaenopsis in vitro

Adventitious shoots formed from nodal sections of floral-stalks of Phalaenopsis Pink Leopard Petra that had been propagated on Hyponex medium with 6-benzyladenine (BA). After subculturing adventitious shoots on Vacin-Went medium with BA, more than 70% of the shoots formed floral buds, and 10% of the floral buds flowered in 9-month-old shoots. Floral buds were not formed on the adventitious shoots cultured on Vacin-Went medium lacking BA. A high concentration of total nitrogen inhibited the development of floral buds, and low-temperature treatments failed to induce the formation of floral buds in vitro.     

In vitro culture of Phyllanthus caroliniensis (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus caroliniensis (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication (21–23 shoots per explant) was achieved on MS or AR media supplemented with either 5.0 μM BA, 1.25–5.0 μM kinetin or 2.5–5.0 μM 2iP. Rooting was achieved with 80–100% of the microshoots on MS medium without growth regulators, although 1.25 μM NAA and 1.25–5.0 μM IAA promoted significant increases in the number of roots per explant. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed on in vitro grown plantlets and after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated in the vertical position on MS medium supplemented with 5.0 μM 2,4-D. Root cultures were successfully established on MS medium containing 1.1 μM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro induction of polyploidy in yacon (<Emphasis Type="Italic">Smallanthus sonchifolius</Emphasis>)

In vitro chromosome doubling was induced in octoploid (2n = 58) yacon using oryzalin and colchicine as mitotic spindle inhibitors. Nodal segments of in vitro cultured plants, 5–15 mm long, were exposed to 20, 25, or 30 μM oryzalin and 1, 3, or 5 mM colchicine for 24 or 48 h. The resulting ploidy level was determined by chromosome counting and flow cytometry. Out of 240 nodal segments, 3.33% hexadecaploid (2n = 116) plants were regenerated after the application of oryzalin. The greatest proportions of hexadecaploid plants (1.6%) were obtained after 48 h of 25 μM oryzalin treatment. With the colchicine treatment, only 0.42% hexadecaploid plants were detected and their survival rate was significantly lower in comparison with the oryzalin treatment. In hexadecaploid yacon, significantly higher levels of saccharides were detected (FOS 13.9 g/100 g FM, fructose 4.6 g/100 g FM and glucose 2.1 g/100 g FM) compared to the octoploid control (FOS 5.3 g/100 g FM, fructose 2.9 g/100 g FM and glucose 1.0 g/100 g FM). These results indicate that in vitro treatment of nodal segments with oryzalin solution could be an effective procedure for chromosome doubling and the polyploidy breeding can help to increase the FOS content in the tuberous roots.     

In vitro Micrografting of Mature Pistachio (Pistacia vera var. Siirt)

The success of various in vitro micrografting methods of shoot tips of pistachio (Pistacia vera L. var. Siirt) have been examined. Excised zygotic embryos that germinated in vitro were used as rootstocks. Current year shoot tips from mature trees of pistachio micrografted onto in vitro juvenile rootstocks, resulted in the restoration of shoot-bud proliferation. Variables tested include a size of microscion, grafting method, effects of culture medium and effects of time of the year at which shoot tips were used. The results indicate that the easiest and most successful method for grafting was slit micrografting. High levels of micrograft take were achieved with 2–4 mm (56.75%) and 4–6 mm (79.25%) long scions obtained from the regenerated shoot tips. The survival rate of the shoot tips was directly related to time of the year. The best growth of microscion was obtained with the in vitro forced shoot tips rather than with shoot tips excised from tree. Slow growth and lack of axillary shoot development on the micrografts was noticeable when the micrografts were cultured on hormone-free and germination medium. In vitro micrografted plantlets were successfully weaned and no problems were encountered with the establishment of micrografted plants in vivo.     

Anthocyanins from Pomegranate (Punica granatum L.) and Their Role in Antioxidant Capacities in Vitro

As phytochemicals, anthocyanins are not only responsible for the diverse colors in nature, but are associated with broad-spectrum health-promoting effects for human beings. Pomegranate is abundant in anthocyanins which possess high antioxidant capacities. However, the pomegranate anthocyanins profile and their contributions to antioxidant capacities are not fully depicted. The purpose of this article is to review anthocyanins from pomegranate as important antioxidants. Total anthocyanin content (TAC) and six major components vary greatly with intrinsic and extrinsic factors. In pomegranate, anthocyanins mainly acted as primary antioxidants, while their action as secondary antioxidants were not conclusive. The antioxidant potentials of anthocyanins were significantly affected by factors especially chemical structure and detection assays in vitro. The current knowledge may provide insights into potential applications for pomegranate anthocyanins based on their antioxidant activities.     

Evaluation of pomegranate (Punica granatum L. var. Gabsi) peel extract for control of root-knot nematode Meloidogyne javanica on tomato

The present study was carried out to assess the nematicidal potential of Punica granatum L. against the root-knot nematode Meloidogyne javanica responsible for yield losses in tomato. Varied concentrations of methanolic, ethanolic and aqueous extracts from pomegranate peels were investigated for activity against eggs and juveniles of M. javanica in in vitro assays. All extracts used significantly inhibited egg hatch by over than 75%, but viability of second-stage juveniles (J2) was not significantly inhibited by ethanolic extract. Aqueous extract was assessed at the concentration of 10, 25 and 50% against M. javanica on tomato in greenhouse trials; pomegranate peels powder was also tested at the rate of 3, 6 and 9 g as a soil amendment. Both extracts significantly reduced nematode infestations; aqueous extract enhanced plant growth but powder amendment exhibited a phytotoxicity compared to the untreated plants. The reduction in number of galls, egg masses and nematode reproduction rate was recorded.     

Phenological stages of the pomegranate tree (Punka granatum L.)

As in other temperate species, the pomegranate tree shows different phenological characteristics through its vegetative cycle in response to changing temperature. This phenology has been studied as a basis for comparisons of the successive stages of growth and reproduction in different geographical areas and conditions. The variety Mollar de Elche was studied, since it is grown on more than 95% of the total area devoted to this crop in Spain. Thirteen phenological-stages were identified during the annual cycle, starting at bud in dormancy and finishing a year later with leaf fall. These stages are denoted using the BBCH General Scale which uses numerical codes to represent particular stages. The code uses a two-digit number to describe the different phenological states and makes it possible to distinguish between principal and secondary stages of development. The duration of stages was measured in days and as accumulated degree-day.     

In vitro culture of macrobrachium eggs

Causes for the death of the eggs in the prawn Macrobrachium nobilii are: i) shedding of eggs by ovigerous female, and ii) infection by epibionts: a Saprolegnial fungus, bacteria (gram negative) and protozoans (Vorticellids and Paramecium). A cause for the death of freshly hatched larvae of some decapods is the reduction in reserve yolk energy in the larvae hatched in the last few batches. To circumvent these disadvantages, an artificial incubator was designed, in which 70% of the 3-day old eggs can successfully be incubated and hatched simultaneously. The isolted eggs are irrigated with filtered and aerated water over a diaphragm in the incubator; the water flushed from below through the diaphragm in the artificial incubator, sways and keeps the eggs continuously in a suspended motion, simulating the irrigation technique of the mother.Presented in the Second International Symposium on Invertebrate Reproduction held in Davis, California during August, 1979     

Genetic diversity in inter‐simple sequence repeat profiles across natural populations of Indian pomegranate (Punica granatum L.)

Pomegranate (Punica granatum L.), in the monogeneric family Punicaceae, is found in Iran, Afghanistan, India and Mediterranean countries. Iran is considered to be its primary centre of origin. In India, pomegranate occurs naturally only in the Western Himalayan regions of Jammu and Kashmir, Himachal Pradesh and Uttarakhand States. However, there is no information about genetic variation in wild pomegranate at population level. In this paper, we describe genetic diversity across natural populations of Indian pomegranate based on inter‐simple sequence repeat (ISSR) markers. Forty‐nine accessions representing eight populations from two regions were analysed using ISSR. Seventeen ISSR primers resulted in 268 polymorphic bands, with 87.01% polymorphism throughout the accessions. Pair‐wise population genetic distances ranged from 0.05 to 0.45, with a mean of 0.25 between populations. amova and Nei’s genetic diversity analyses revealed higher genetic variation within populations than among populations. A higher genetic differentiation (G_ST) was observed between the spatially distant populations, indicating a low level of genetic exchange (Nm) among these populations. However, clustering of populations was not in accordance with their geographical affiliations in the tree. The results indicate that the ISSR method is sufficiently informative and powerful to assess genetic variability in pomegranate, and that patterns of genetic variability observed among populations of wild pomegranate from the Western Himalaya differ. Estimation of genetic variation reported here provides a significant insight for in situ conservation and exploitation of genetic resources for this economically important species as potential breeding material.     

In vitro Shoot Regeneration from Leaves of Sweet Cherry (Prunus avium) 'Lapins' and 'Sweetheart'

Shoot regeneration was achieved from leaves of in vitro cultures of Prunus avium L. cv. 'Lapins' and 'Sweetheart' using woody plant medium (WPM) supplemented with 1-naphthalene-acetic acid (NAA) and thidiazuron (TDZ) or benzyladenine (BA). Percent regeneration was influenced by plant growth regulators and by explant type, orientation and wounding. Optimal regeneration was observed with whole-leaf explants wounded by transverse cuts along the midrib and incubated abaxial surfaces uppermost, on media supplemented with 2.27 or 4.54 µM TDZ plus 0.27 µM NAA. The percent regeneration of the two cultivars was not significantly different. Optimum conditions for regeneration resulted in 71.4% of 'Lapins' and 54% of 'Sweetheart' explants producing one or more shoots per explant.     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.