Characterization and in vitro selection for iron efficiency inPyrus andCydonia

Summary Responses to low Fe were characterized in tissue cultures ofPyrus amygdaliformis andCydonia oblonga (quince), two species used as rootstocks for pear. Cultured shoots and plantlets ofP. amygdaliformis had a higher chlorophyll concentration and Fe²⁺/total Fe ratio than those ofC. oblonga when grown under low Fe conditions. This tolerance to low Fe was correlated with high Fe³⁺-reducing ability and medium acidification. The adaptive responses were manifested in roots of plantlets, shoot bases, root cultures, and cell suspension cultures. Shoots were regenerated from leaves of quince and subjected to Fe-deficient conditions. Two somaclonal variants (IE-1 and IE-2) were recovered; each displayed higher ability to reduce Fe³⁺ and acidify the medium. These variants may be useful as rootstocks for regions with calcareous soils, which limit Fe availability.     

Micropropagation of juvenile and adult Quercus suber L.

This paper describes research on the application of tissue culture techniques to the micropropagation of cork oak (Quercus suber L.), a forest species of ecological and industrial importance in the Mediterranean area. Apical buds and nodal stem segments were employed as initial explants. Their origins were young seedlings, stump sprouts and sprouts formed on cuttings collected from old trees.The action of the mineral medium and growth regulators was studied in the multiplication stage. Media with low concentrations of ions, such as Sommer's or Heller's, are more suitable for growth and proliferation of explants than other media richer in salts. It was also observed that cytokinin (BA) must be present for the culture development. Adding low concentrations of auxin (NAA) to the medium improves the multiplication rate, especially in vegetative material of adult origin.The auxin type is the most important factor in the promotion of rhizogenesis. The method of application determines the quality of the root system. Treatment with low concentrations of IBA added to the rooting medium gives the best results.High sucrose concentration also improves rooting. Diluting the mineral rooting medium is slightly favourable, although there is no significant difference between it and the standard mineral concentration.Abbreviations D Durzan's - GD Gresshoff & Doy's - H Heller's - L Lepoivre's - MS Murashige & Skoog's - SH Schenk & Hildebrandt's - S Sommer's medium     

Micropropagation of Campanula isophylla Moretti

A method for micropropagation of Campanula isophylla Moretti is described. The method is based on division of the basal parts of shoot clusters into sections, each with four 3 mm stem stubs. Shoots from the shoot clusters are easy to root and give plants without apparent phenotypic aberrations. It is thus possible to propagate the stock and produce rooted plantlets in the same process. Basal sections of shoot clusters formed more shoots than shoot tips or single nodes. The medium used for propagation was MS with 4.4 M benzyladenine (BA). Addition of naphthaleneacetic acid or raising the concentration of BA did not improve the results significantly. As primary explants 2 mm stem segments with an axillary or apical bud were used; smaller explants often failed to grow. For rooting the concentration of macronutrients was reduced to one-half, and BA was omitted. The cultures received an irradiance of 20 mol m^-2 s^-1 fluorescent light; dry weight of shoots decreased if the irradiance was reduced. The method was used for propagation of 113 genotypes; shoot numbers and days to first root differed significantly among genotypes.     

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy,immunofluorescence, and rhodamine-phalloidin

Summary The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.     

Micropropagation of parsley through axillary shoot proliferation

A micropropagation protocol of parsley,Petroselinum crispum (Mill.) Nyman (curled type) has been developed. Surface-sterilized axillary buds cultured on Murashige and Skoog medium supplemented with benzyladenine, kinetin, or thidiazuron developed axillary shoots (rosettes). Kinetin resulted in only a low proliferation rate. The concentrations of thidiazuron or benzyladenine that were optimal for shoot proliferation, resulted in shoots with a low capability to root. During the rooting treatment, these shoots showed wilting signs. Rooting was increased significantly by using a two-week inductive stage with 2.5 M naphthaleneacetic acid directly followed by acclimatization. Two proliferation media (5 M benzyladenine and 0.5 M naphthaleneacetic acid or 5 M kinetin and 2.5 M naphthaleneacetic acid) resulted in moderate proliferation but produced shoots that were easy-to-root. These media have been tested by repeated axillary proliferation on the same medium. The medium with 5 M benzyladenine and 0.5 M naphthaleneacetic acid was optimal.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige & Skoog - NAA -Naphthaleneacetic acid     

Micropropagation of the endangered Aloe polyphylla

A rapid propagation protocol was establish for thehighly endangered Aloe polyphylla (Schönland ex Pillans). Seed was germinated in vitro on Murashige & Skoog 1962 medium [6] with or without sucrose. Plantlets were cultured onmedium containing benzyladenine only, or a combinationof BA and NAA. After initial problems with browning,the explants rapidly formed axillary and adventitiousbuds. Maximal shoot formation was obtained on MSmedium containing 1.0 mg l^–1 BA. Some shootsrooted spontaneously on MS medium, but the rootingpercentage was improved with a 0.5 mg l^–1 IBAsupplement. Rooted plantlets were acclimatized togreenhouse conditions. The success of this projectindicates that micropropagation can be a useful toolin the conservation of this endangered species of thegenus Aloe.     

Micropropagation of Actinidia kolomikta

Nodal segments of female A. kolomikta shoots were cultured on Murashige and Skoog modified medium with different growth regulator concentrations. The highest multiplication rate (9.5) was achieved on a medium with 10 M benzyladenine and 0.1 M indolebutyric acid. Over 90% of shoots rooted in vivo after pulse stimulation with indolebutyric acid.Abbreviations BA benzyladenine - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid     

Micropropagation of camphor tree (Cinnamomum camphora)

Multiple shoots were induced from shoot tips and nodal segments of a 12-year-old tree of Cinnamomum camphora on Woody Plant Medium (WPM) supplemented with BA and kinetin. The nodal segments from the in vitro developed plantlets could be induced again to produce a large number of harvestable shoots. Harvested shoots were rooted in vitro in WPM supplemented with activated charcoal (AC) and IBA. The plantlets were transferred to thermocol cups after which they were replanted into polybags and then to field. These plants survived with over 90% success under field conditions and exhibited vigorous growth. This system could be utilized for large-scale multiplication of C. camphora by tissue culture.     

Micropropagation ofPinus sylvestris

Plantlet regeneration from axillary buds, induced on seedling apices ofPinus sylvestris, is described. The influence of medium containing 1, 5, 10 or 50 M BA on the initiation of buds on 3, 6 and 9-week-old seedlings was investigated. With higher BA concentrations the number of apices that formed buds and the total number of buds increased, wheres their length decreased. Soaking the explants for 2 h in 111 M BA and for 5 h in 44 M BA gave better results, best expressed in longer buds. After 30 days, axillary buds were separated and transferred to a medium with 2% sucrose. Separated buds exposed to far-red light during the first week elongated better in comparison with the control. 64% of shoots rooted after a 24 h period of treatment with 53.8 M NAA incorporated in 0.6% water agar and transferred to 1/16-strength Murashige & Skoog medium as modified by Cheng supplemented with 1% sucrose.     

Micropropagation ofPrunus mume

Shoot proliferation from axillary buds ofPrunus mume Sieb. et Zucc. was obtained on Woody Plant Medium (WPM) supplemented with 1 to 5 M benzyladenine, 3% sorbitol and solidified with 0.5 to 0.7% agar. Effects of different carbon sources on shoot proliferation were examined. Glucose provided better shoot proliferation than sucrose, sorbitol and fructose. In the presence of sucrose, leaf chlorosis occurred and shoots gradually declined. Best rooting percentage was obtained on WPM supplemented with 1 M naphthaleneacetic acid. Rooted plantlets were acclimatized under intermittent mist. However, survival rate was relatively low (20 to 30%).     

Micropropagation of Camptotheca acuminata decaisne from axillary buds, shoot tips, and seed embryos in a tissue culture system

Summary Micropropagation of the anti-cancer plant Camptotheca acuminata Decaisne from axillary buds and seed embryos was investigated. Axillary buds from greenhouse seedlings required a period of culture in media free of N⁶-benzyladenine (BA) before multiple shoot induction began. Direct induction of multiple shoots on BA-containing medium resulted in high mortality of the axillary buds. Multiple shoot induction from the greenhouse axillary buds was best achieved on B5 with 4.4 μM BA+0.5μM α-naphthaleneacetic acid, forming an average of three 2-mm tall shoots per bud in 8 wk. Elongation of these multiple shoots was successful at a lower BA level (0.22 μM) on B5 medium. Both in vitro and ex vitro rooting of the microcuttings was feasible with indole-3-butyric acid in the culture media, but ex vitro rooting led to high plantlet survival. Seed embryos were not ideal explants for multiple shoot induction. Shoot tips and axillary buds of in vitro-germinated seedlings showed an optimal multiple shoot formation on B5 with 8.9 μM BA, double the optimal BA level for greenhouse axillary buds. Using axillary buds to propagate C. acuminata plants in vitro is feasible for mass propagation of desired clonal lines high in camptothecin concentrations.     

Micropropagation of candelilla,Euphorbia antisyphilitica Zucc

Axillary shoot proliferation was induced in vitro from shoot explants of greenhouse grown candellila (Euphorbia antisyphilitica Zucc). Optimum shoot proliferation was obtained by supplementing a modified Murashige and Skoog [7] medium with 0.13 M naphthalene-acetic acid and 4.44 M 6-benzylaminopurine. Rooting occurred on 100% of shoots transferred to a medium containing half strength salts supplemented with 0.49 M indole-3-butyric acid. Fully rooted plants were transferred to potting soil and established under greenhouse conditions without special acclimatization techniques.     

Micropropagation ofGmelina arborea

Multiple shoots were obtained from single node explants of matureGmelina arborea Roxb. on MS medium supplemented with 6-benzyladenine (BA). Seven to nine shoots were formed whenin vitro-derived single node explants were subcultured on MS medium supplemented with 1.1 M BA. For root initiation the cut ends of microshoots were pulsed for 5 min with 246 M indole-3-butyric acid and transferred to a plastic cup containing sterile vermiculite. The shoots were covered with polyethylene bag and maintained in a culture room. After hardening, plantlets were transferred to earthen pots containing a mixture of garden soil: compost and have been established in the field.     

Micropropagation of Eucalyptus tereticornis Smith.

Axillary shoot bud multiplication has been achieved in Eucalyptus tereticornis Smith. using explants from different regions of 8–10 years old elite trees, growing in the field. Results showed that addition of NAA at 0.1 mgl^-1 and BAP at 1.0 mgl^-1 to modified MS medium induced maximum number of shoot buds. For inducing axial growth in regenerated bud promordia, the hormone concentration of the medium was lowered. The addition of charcoal and gibberellic acid to the medium were beneficial. Rooting was best in Knop's medium containing 1.0 mgl^-1 IBA. The key factor in root induction was primarily a dark incubation for a short period. The percentage of both rooting of shoots and survival of the rooted shoots was 60–80.Continuous trials using explants from the elite trees throughout the year showed that the period between July–September was the best season for the explant source for rapid and increased multiplication of axillary buds. Phenolic exudation was also minimum at this period. The experiments were repeated using 50 populations from different plantations. It was observed that during culture, genotypically different populations responded differently in spite of optimal growth conditions.     

Micropropagation of mature siberian elm in two steps

A simple and reliable two-step micropropagation system was developed for mature Siberian elm (Ulmus pumila L.). Shoot-tip cultures were initiated and multiplied on a modified Murashige and Skoog medium supplemented with 1 M benzyladenine, B5 vitamins, solidified with 0.22% Gelrite, and subcultured weekly. Proliferated shoots 2–3 cm in length rooted successfully in sterile and non-sterile soil mix at 100% efficiency. Healthy plants were acclimatized to the ambient environment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IBA indole-3-butyric acid - MS Murashige & Skoog medium (1962)     

Micropropagation of the rare species Stylidium coroniforme and other Stylidium species

The number of plants in the gazetted rare species Stylidium coroniforme was increased through micropropagation. A method was first developed using the common species S. brunonianum. It was found that for both species, rapid propagation could be obtained by excising shoots from sterile seedlings and inducing shoot proliferation on Murashige and Skoog medium supplemented with 1 M BAP. Rooting was achieved using 1 M IBA and over 100 plants of each species were successfully established in soil. Leaf pieces could also be used to initiate cultures. In media with 20–25 M BAP and 1–5 M IBA, leaf pieces of S. brunonianum, S. piliferum, S. caricifolium and S. crassifolium produced adventitious buds, thus providing another method of micropropagation.     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Micropropagation by explants ofGracilaria chilensisBird, McLachlan and Oliveira

Regeneration studies were carried out on four morphotypes ofGracilaria chilensisfound on the coast of Chile (36–41°S). Vegetative explants were obtained from sections of apical and medial origin and were cultured in five media: filtered autoclaved seawater (FAS), Provasoli enriched seawater (PES), PES + indole-3-acetic acid (PIAA), PES + kinetin (PK) and PK + IAA (PKIAA). Mature female gametophyte and tetrasporophyte explants were obtained from sections of medial origin and were cultured in FAS and PES. Bud differentiation and/or callus formation were the morphogenetic responses to the wounding of the explants in all culture media. Plantlet regeneration was obtained from excised buds and calluses cultured separately.     

Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

Micropropagation ofTribulus terrestris L., an important medicinal plant

Direct regeneration of shoots and roots through juvenile expiants has been achieved inTribulus terrestris. Cotyledonary leaves along with epicotyl segment from young seedlings were cultured on MS medium containing various concentrations of auxin with cytokinin and glutamine. A combination of 0.2 mgL⁻¹ NAA, 0.5 mgL⁻¹ BAP and 50 mgL⁻¹ glutamine induced high frequency of shoot and root differentiation in 10 weeks. The callus also could be induced on the above medium from the cut end of radical segments. Morphogenic response such as per cent shoot and root differentiation was recorded at regular intervals.