In vitro regeneration and micro-propagation of enset from Southwestern Ethiopia

Three clones of enset (Ensete ventricosum Welw. Cheesman) from southwestern Ethiopia (Keffa-Shaka zone) were investigated for their potential for micropropagation and regeneration in tissue culture. Corm and leaf tissue of greenhouse-grown plants as well as in vitro germinated zygotic embryos were used as starting material for micro-propagation and regeneration studies. Embryos were excised from disinfected seeds and cultured in vitro. Multiple shoots from both corm- and embryo-explants were obtained using regeneration medium supplemented with 10 μM or 20 μM BAP. Rooting of shoots was achieved using medium with 5 μM IBA, 1 μM BAP and 1 g l⁻¹ activated charcoal. Plantlets obtained by this process were transferred to soil under greenhouse conditions. Optimal conditions, which were determined for clonal propagation of three different genotypes of enset, allowing both in vitro micropropagation and regeneration, are described. This protocol makes for conservation of enset clones, rapid propagation of selected disease-free germplasm and more efficient breeding procedures. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro micropropagation and long-term conservation of the endangered moss Splachnum ampullaceum

Protonema explants of Splachnum ampullaceum Hedw. were grown in vitro on 10 different mineral media with different sources and contents of nitrogen, in each case with or without added sucrose (30 g dm⁻³) and/or B5 vitamins. The cultures were maintained at day/night temperatures 24 ± 4/20 ± 2 °C and a 16-h photoperiod (irradiance of 25 μmol m⁻² s⁻¹). Sucrose had little or no effect on protonema diameter and bud number in nitrate-only media or in high-ammonium media but markedly reduced bud number in low-ammonium media. Sucrose markedly reduced one-year explant survival rate in the low-ammonium media. The presence of B5 vitamins in such media markedly improved one-year survival, suggesting that the best medium for long-term culture of Splachnum ampullaceum is a medium containing ammonium at relatively low concentration as ammonium phosphate or sulphate (e.g. Gamborg's B5 medium), with added B5 vitamins but without added sucrose.     

In vitro production of microtubers for conservation of potato germplasm: Effect of genotype,abscisic acid,and sucrose

Summary With the objective of using microtubers for conservation of potato germplasm, the main effects of genotype, abscisic acid (ABA), and sucrose level, and of their interactions on biomass production, microtuberization, microtuber dormancy, and dry matter content, were studied. ABA decreased both microtuber production and microtuber dormancy, whereas higher concentrations (60–80 gl⁻¹) of sucrose promoted biomass production, microtuber production as well as microtuber dry matter content. Microtubers stored under diffused light had longer dormancy than those kept continuously in the dark. Interactions among various factors conditioned the main effects for some characters. In vitro performance of the genotypes studied was related to their known performance under in vivo conditions for most of the characters. Microtubers produced on media devoid of ABA and containing high sucrose concentrations and N⁶-benzyladenine (44.38 μM) could be stored for 12 mo. under diffused light at 6±1°C.     

Identification of bacteria and yeasts from <Emphasis Type="Italic">in vitro</Emphasis> and surface-sterilized field samples of <Emphasis Type="Italic">Ensete ventricosum</Emphasis> by rDNA analysis

Bacterial and fungal contaminants of enset (Ensete ventricosum) cultures and microbes associated with surface-sterilized field material were identified by 16S/26S rDNA sequencing. Ten bacterial species were identified in 16 isolates from in vitro cultures and seven in 10 isolates from field clones. Three yeast species and one filamentous fungus were recorded as in vitro contaminants, whereas five yeast species were isolated from the field material. The bacterium, Pseudomonas reactans (6 isolates), and the yeast, Torulaspora delbrueckii (8 isolates), were the most frequent in vitro contaminants. Most of the bacterial species isolated from in vitro enset were Gram-positive and hitherto unrecorded as in vitro contaminants. The difficulty in controlling the in vitro contaminants is due to their apparent endogenous nature and their resistance to antimicrobial drugs.     

Biotechnology and the conservation of forest genetic resources: in vitro strategies and cryopreservation

In vitro techniques have a clear role within ex situ conservation strategies for trees and crop genetic resources, particularly where it is important to conserve specific genotypes or where normal propagules such as recalcitrant seed may not be suitable for long-term storage. These involve the use of conventional micropropagation, restricted growth techniques and cryopreservation. Although these techniques have been used primarily with herbaceous species, increasing attention is being given to woody species. Cryopreservation techniques for both woody and herbaceous species and new approaches which do not require freeze-induced cell dehydration, referred to as the encapsulation-dehydration and the vitrification techniques are described. Illustrative data are presented for the cryopreservation of willow using the encapsulation-dehydration technique.     

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.     

In vitro storage of Eucalyptus grandis germplasm under minimal growth conditions

Slow growth-storage, for up to 10 months, has been achieved for Eucalyptus grandis shoot cultures by either the addition of 10 mg l⁻¹ abscisic acid to the growth medium or by the halving of nutrient supply (half MS) and removal of exogenous plant growth regulators. Reduction of light intensity or the addition of mannitol to the media were less effective in reducing growth rate. Isolated in vitro axillary buds encapsulated in calcium alginate and stored under low temperature and low light intensities survived for up to 3 months without loss in viability. Storage of such encapsulated fresh axillary buds at higher temperature resulted in a loss in viability. These methods have immediate applications to forestry breeding and clonal programs. This revised version was published online in June 2006 with corrections to the Cover Date.     

Minimal growth in vitro conservation of coffee (Coffea spp.)

We have studied the influence of low concentrations of 6-benzyladenine on growth limitation, in order to preserve coffee germplasm through a microcutting collection. Concentrations of 0 M, 1.3 M and 4.4 M were compared in four species: Coffea congensis, C. canephora, C. liberica and C. racemosa. After six months, microcutting behaviour varied between the different treatments, and a species effect was observed. The slow growing species (C. liberica and C. congensis) needed 1.3 M; the others coffee species (C. canephora and C. racemosa) exhibited moderate caulogenesis on 6-benzyladenine-free medium. Zero and low concentrations did not affect survival rates. In conclusion 1.3 M seems most appropriate for conserving all four species.Abbreviation BA 6-benzyladenine     

Long-termin vitro storage ofColocasia esculenta under minimal growth conditions

The storage of a clone ofColocasia esculenta at 28/24°C over a 12-h photoperiod in the absence of mannitol, was not feasible with transfer intervals of more than eight months. Mannitol had a positive effect on survival at this temperature, but caused abnormalities at high concentrations. At 9°C in total darkness,C. esculenta could be stored for more than eight years with transfer intervals of approximately three years. After this period, more than 90% of the cultures showed living shoots, but not all shoots per culture showed regrowth. Cultures which were transferred three times during the experimental period, had a significantly (=0.05) lower number of regrowing shoots than cultures which were transferred twice. This might be due to the fact that the former cultures were transferred at a less favourable developmental stage. The addition of mannitol to the storage medium did not improve survival and regrowth, nor did a more gradual lowering of the temperature to 9°C.     

Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds

In southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple food crop, supporting the diet of a quarter of the Ethiopian population. Due to difficulty in germinating seeds and the long vegetative period, breeding enset is extremely difficult. Adventitious buds and somatic embryos have been induced from callus derived from corm tissues and cultured on Murashige and Skoog's (MS) basal medium supplemented with benzylaminopurine (BAP) or 6 --dimethylallylamino purine 2iP. Elongation of somatic embryos was achieved on the same medium and rooting was induced on half-strength MS basal medium supplemented with IBA. No phenotypic variation was observed among more than 200 potted regenerants. The possible implications for mutation breeding in this crop are discussed.Abbreviations IAA Indole-acetic acid - IBA Indole-butyric acid - BAP Benzylaminopurine - 2iP 6 --dimethylallylamino purine     

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 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 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.     

Growing of potato microplants in the presence of alginate-silverthiosulfate capsules reduces ethylene-induced culture abnormalities during minimal growth conservation in vitro

Alginate capsules containing anionic complex silverthiosulfate (STS) [Ag(S₂O₃)₂ ^3-] were placed in the culture tubes over minimal growth media for studying whether STS could be used at higher concentrations to sustain ethylene-inhibiting effect during conservation of microplants in six potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of STS (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 mM) were incorporated into the alginate capsules, and 12 alginate-STS capsules were tested in semisolid (7 g l^–1 agar) minimal growth medium containing 20 g l^–1 mannitol and 40 g l^–1 sucrose. This indirect supplementation of STS through alginate capsules rendered reduced total availability of STS in the minimal growth medium as compared to when it was directly supplemented in the medium at a given concentration. Growing of microplants in the presence of alginate-STS capsules improved the microplant growth and reduced the culture abnormalities over a period of 16 months under minimal growth conditions. Most significant improvement in microplant growth was in terms of green leaf production and leaf senescence. Vitrification, flaccidity and other growth abnormalities, viz., leaf loss, abnormal stem swelling and necrosis were not observed when the microplants were conserved in the presence of alginate-STS capsules. To foster optimum microplant growth and reduce culture abnormalities, potato microplants could favourably be maintained in the presence of 0.5–1.0 mM alginate-STS capsules during minimal growth conservation. Higher concentrations of alginate-STS capsules (>1.0 mM) were in general detrimental to potato microplant growth and survival during prolonged storage in vitro. Release kinetics of STS from the alginate-STS capsules, its distribution in the medium and accumulation of silver in potato microplants were studied using ^110mAg. The release rate of STS from the capsules was found to be directly proportional to the concentrations of alginate-STS capsules. A distinct concentration gradient of ^110mAg in the medium with increasing depth from top to bottom, and its accumulation in the potato microplants may be attributed to the improved anti-ethylene action of STS at higher concentrations through alginate capsules.     

Efficient micropropagation of <Emphasis Type="Italic">Ensete ventricosum</Emphasis> applying meristem wounding: a three-step protocol

A highly efficient three-step protocol for in vitro propagation of Ensete ventricosum (enset) was developed that consisted of initiation, bud proliferation, and shoot elongation and rooting stages. At the initiation stage, it was crucial to use shoot tips (5–8 mm) with subtending corm tissues as explants to obtain growth. The addition of 0.5–1% (w/v) activated charcoal to the medium was essential to prevent phenol exudation which otherwise leads to the loss of cultures. During the bud proliferation stage, modified MS macronutrients and micronutrients together with a combination of cytokinins (1.6 M naphthaleneacetic acid, 4.4 M 6-benzylaminopurine, 23.2 M kinetin, 22.6 M N⁶ 2-isopentyladenine) was used. This novel composition of macronutrients was based on the analysis of leaf nutrient content of glasshouse-grown enset sprouts. Multiple bud formation on the enlarged corm tissue was induced only when the meristem region was wounded before transfer to the bud proliferation medium. Up to 75 healthy shoots per explant were produced, whereas unwounded explants produced, only one to two shoots per explant. A third stage with a low concentration of cytokinin enabled shoot elongation as well as root development. The plantlets were acclimatized with 100% success and they showed no apparent phenotypical deviation.Abbreviations BAP 6-Benzylaminopurine - IBA Indole-3-butyric acid - DW Dry weight - EV Ensete ventricosum medium - 2-iP N⁶ 2-Isopentyl adenine - NAA -Naphthaleneacetic acid     

Micropropagation of Telopea speciosissima R. Br. (Proteaceae). 2: Rhizogenesis and acclimatisation to ex vitro conditions

Conditions affecting rhizogenesis in vitro and ex vitro and subsequent acclimatisation of Telopea speciosissima (waratah) were investigated. Clonal selections were successfully rooted in vitro in agar, on filter paper bridges or using crushed quartz-sand, the last substrate resulting in superior growth of roots. The in vitro substrates were impregnated with half-strength MS, 7.5 gl^-1 sucrose and various concentrations of IBA. For the quartz-sand, an IBA concentration of 50 M was optimal, 70% of microcuttings were rooted. No plantlets rooted in vitro were acclimatised to ex vitro conditions (using mist, fog or humidity tent regimes). Microcuttings (25–45 mm in length) were rooted ex vitro in a fog humidity regime (droplet size <10 m) using an IBA powder dip (3 g IBA kg^-1). Neither a mist nor a humidity-tent regime was suitable for rooting of waratah microshoots ex vitro. A peat and perlite mixture was superior to crushed quartz-sand or potting mix for the rooting of microshoots; this appeared to be related to the air-filled porosity (>20%) of the mixture, measured after the medium was saturated and then drained for 24h. Plantlets must be left under the high humidity regime until shoot growth resumes (four to eight weeks) otherwise plant mortality increase significantly. In vitro-produced leaves abscised between eight and 12 weeks after transfer to ex vitro conditions, indicating that these structures did not acclimatise ex vitro.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indole-3-butyric acid - LSD least significant difference - MS Murashige and Skoog medium     

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     

Induction of stem elongation on in vitro chicory root explants under unfavourable photoperiodic conditions by gibberellin A3

Flowering stems are formed under long-day conditions on root explants of chicory (Cichorium intybus L.) cultured in vitro while under short days, only vegetative growth is observed. Under short-day conditions (12 h), stem elongation is induced by treating newly formed shoot apices with GA₃ (1 l, 10^–3M). No flower buds were formed on the GA₃-induced stems. Long days seem to be indispensable for the induction of flower buds on the elongated stem.     

In vitro study of gas effects on alveolar macrophages

Summary To evaluate the biological effects of gas pollutants on alveolar macrophages several in vitro systems ave been developed. We described here an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. We studied the long term (24 h) and short term (30 min) effects of NO₂ on alveolar macrophages. Our results demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either cell injury or cell activation associated with the release of various bioactive mediators (superoxide anion, neutrophil chemotactic activity). Cell culture in aerobiosis opens new ways for the research on the biological effects of gas pollutants.Abbreviations AM alveolar macrophages - CL Chemiluminescence     

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.