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 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.     

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 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 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.     

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 vivo and in vitro investigation of salt tolerance in three anthocyaninless mutants in tomato (Lycopersicon esculentum Mill.)

Growth responses of a tomato cultivar Ailsa Craig and the ah-, aw- and bls-isogenic/near isogenic lines (IL/NIL) from it were evaluated and compared at cotyledons stage under salt treatment in vivo and in vitro experiments. No differences in hypocotyl and root growth responses were detected between the anthocyanin-containing and the anthocyaninless lines within the in vivo experiments. The anthocyaninless mutants, (except in some cases the bls mutant), exhibited higher callogenic and shoot-forming capacity on both, control and salinized media. It was concluded that for this reason it would be difficult to determine the relationship between the in vivo and in vitro responses of the lines studied and as well as to evaluate the usefulness of the in vitro method in testing these lines for salt tolerance.     

In vitro tests for screening of immuno-modulating mycobacterial strains in leprosy

There is an urgent need for the development of anin vitro assay for the initial screening of a large number of organisms from which potential candidates as vaccines can be identified. Our previous studies have demonstrated a crucial defect in the lepromatous macrophage. In this study by monitoring this defective macrophage response we have screened various mycobacteria for their ability to reverse the alterations induced byMycobacterium leprae. Among the limited Mycobacteria testedMycobacterium vaccae appears to be the most promising as an immunomodulator. Our results also indicate the need for caution in using the mouse model for this purpose     

Efficient flower induction from cultured buckwheat (Fagopyrum esculentum L.) node segments in vitro

Flower induction from shoot segments of buckwheat seedlings was examinedin vitro. Cytokinin, (especially kinetin at 0.1M), short day conditions and a high concentration ofsolidifying agent improved the flower induction from node segments invitro, in up to about 50% of node segments. The use of anaeration membrane on bottle caps and a high content of sucrose in the mediumimproved flower induction in vitro considerably. In theimproved conditions, flowers were induced from 100% cultures and 10bloomed flowers per explant were induced in vitro in 8weeks. Both long and short types of stigmas, and normal set of flowers wereobserved under the microscope. When pollen produced invitro was cultured on an artificial medium, 70% of the pollengrains germinated, indicating normal viability of in vitropollen, and indicating the potential for artificial pollination invitro. All the varieties examined flowered at a similar percentage,suggesting that the process was independent of variety and that flowers couldbeproduced in vitro. Flower induction from buckwheat plantsin vitro and possible cross breeding invitro are also discussed.     

In vitro cold storage of cork oak shoot cultures

Friable callus cultures were initiated from cotyledons and hypocotyls of Opuntia ficus-indica. Explants from cotyledons produced significantly more callus than those from hypocotyls. Optimum callus growth was observed on Murashige & Skoog medium supplemented with 0.9 μM 6-furfurylaminopurine, 2.3 μM 2,4-dichlorophenoxyacetic acid, 1.0 μM 4-amino 3,5,6-trichloropicolinic acid, 400 mg l^-1 casein hydrolysate and 3% sucrose. The same medium without agar was used for establishing cell suspensions. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro micropropagation of the macarronesian evergreen treePersea indica (L.) K. Spreng

Summary Shoot propagation ofPersea indica (L.) K. Spreng was achieved using seedling axillary buds cultured on MS (Murashige and Skoog, 1962) medium with 1 mg/l (2.8 μM) N⁶-benzyladenine (BA). Forty percent of the obtained shoots did not elongate, but showed bud proliferation, which was maximal (three axillary buds per shoot) at the end of the seventh subculture. Sixty percent of the shoots elongated, did not show bud proliferation, and formed calluses at their base. Successful rooting (84.6%) was achieved dipping the base of each elongated shoot in 3 g/l (16.11 mM) indolebutyric acid (IBA) for 1–2 s, and transferring to half strength MS medium without growth regulators. These shoots presented an acclimatization success of 100%. Results suggest that micropropagated elongated shoots ofP. indica can be adequately used in reforestation programs.     

In vitro corm production of Gladiolus dalenii and G. tristis

The ability of the summer flowering Gladiolus dalenii Van Geel and the winter flowering G. tristis L. to form corms in vitro was investigated. G. dalenii spontaneously formed corms on a shoot induction medium consisting of the basal medium of Murashige & Skoog (1962) with up to 2.0 mg l^-1 benzyladenine (BA), 3% sucrose and solidified with 2 g l^-1 Gelrite^®. The effect of different BA and sucrose concentrations as well as different temperatures on in vitro corm production of G. tristis was further investigated. The best production of shoots per explant was achieved on a medium containing 0.5 to 1.0 mg l^-1 BA, sucrose concentrations of 6 to 9% and cultured at 15°C. The best corm production was achieved at the same temperature and with the same medium with the exception that BA was omitted from the medium. To test the effect of the osmotic potential on the formation of shoots and corms, sucrose was substituted by mannitol at various concentrations. Sucrose proved to be essential for both shoot and corm production and the use of mannitol had no beneficial effect.     

In vitro culture of Ginkgo

Summary Ginkgo biloba L. is an important landscape tree, is resistant to insect, fungi and other pests, and produces a number of chemicals that have pharmaceutical properties (termed ginkgolides). Studies were initiated to establish an in vitro culture protocol for Ginkgo. Explants (intact embryos, embryos with cotyledons removed, and cotyledon tissue) were removed from disinfested seeds and cultured on Murashige and Skoog minimal organics medium with various combinations of either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and either kinetin or benzyladenine (BA). Cultures were incubated in the light and morphological development was recorded. Both embryo and cotyledon explants produced callus (cotyledon tissue produced the most callus). Ginkgolides A and B were detected in callus tissue extracts. Intact embryo cultures initiated on media with 2,4-D plus NAA for 5 wk produced shoots and roots when transferred to media with 4.5 μM 2,4-D alone for an additional 5 wk. Plants were transferred from the 2,4-D media to pots and maintained in the greenhouse.     

In vitro conservation of enset under slow-growth conditions

Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 μM BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 μM BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of banana (Musa spp.): initiation,proliferation and development of shoot-tip cultures on defined media

In vitro multiplication of banana (Musa spp.) from shoot-tip explants isolated from lateral suckers is described. Using explants with apical domes, a total of 22 banana cultivars were successfully cultured on a modified Murashige and Skoog's medium containing 6-benzylaminopurine (BA) and indolebutyric acid (IBA). Shoot-tip explants could be induced to produce multiple shoot initials in the presense or absence of apical domes, but the survival rates were higher if apical domes were retained. Cultivars varied widely in their multiplication rates in response to cytokinins, BA being consistently more effective than kinetin (Kn). Although Kn was less effective in this regard, it stimulated vigorous root growth. Rooted plantlets were successfully established in soil.