In vitro propagation of Halesia carolina L. and the influence of explantation timing on initial shoot proliferation

Halesia carolina L., a small, ornamentally valuable tree, is difficult to propagate due to the complexity of seed propagation and the unavailability of propagules for conventional vegetative propagation. A micropropagation system was developed to facilitate easy propagation of this species. Actively growing shoot tips achieved optimum shoot proliferation from axillary buds when placed on Woody Plant Medium supplemented with 1.0 to 2.5 mg/l benzyladenine. The addition of 0.1 mg/l naphthaleneacetic acid had little effect on culture performance. Murashige and Skoog medium was incapable of supporting vigorous shoot proliferation. Non-sterile rooting conditions provided better rooting and subsequent plantlet growth, when compared to an in vitro rooting method. The seasonal fluctuations in the stock plant dramatically affected the shoot proliferating potential of the explants in vitro. Rapidly elongating shoots formed shoot proliferating cultures more slowly than explants taken either before or after the rapid elongation phase.     

In vitro propagation of pummelo (Citrus grandis L. osbeck)

Summary Several experiments were carried out to develop protocols for the in vitro propagation of pummelo (Citrus grandis L. Osbeck) using shoot-tip explants from seedlings. Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BA) and thidiazuron (TDZ), singly or in combination with α-naphthaleneacetic acid (NAA), was used to determine the rate of shoot proliferation. The response of explants to all concentrations of TDZ was very poor. After 6 wk culture, the most adventitious shoots per explant (average 5.2) were obtained on medium supplemented with 1.8 μM BA. NAA with cytokinin in the medium did not improve the rate of shoot multiplication significantly. Addition of 5.8 μM gibberellic acid in shoot-proliferation medium during the second subculture improved shoot elongation significantly. Shoot multiplication increased 3.5-fold in each successive subculture. NAA was superior to indolebutyric acid for in vitro root induction. Over 75% of the shoots developed roots when transferred to half-strength MS medium with 1.3, 2.7, or 5.4 μM NAA.     

Initiating cultures ofHalesia andMalus: influence of flushing stage and benzyladenine

Shoot tips ofHalesia carolina L. andMalus domestica Borkh. Golden Delicious collected at various times during the spring growth flush varied considerably in their ability to initiate shoot proliferating cultures. Shoot tips collected during, or at the end of, the period of most rapid shoot elongation exhibited weak shoot proliferation potential, while shoot tips collected before or after this period were capable of strong shoot proliferationin vitro. Benzyladenine concentrations in the culture media above 22.5 µM (Halesia) or 44.5 µM (Malus) were inhibitory during the period between bud break and rapid shoot elongation. Benzyladenine concentrations of 22.5 or 44.5 µM were useful in enhancing shoot proliferation potential in shoot tips collected after the period of rapid shoot elongation, but before the onset of summer dormancy. Benzyladenine concentration did not affect the shoot proliferation potential of shoot tips collected during the rapid shoot elongation phase of the spring growth flush.Halesia andMalus shoot tips behaved similarly in this study. For deciduous woody perennials, the time of explant collection for culture initiation could be refined on the basis of these results.     

Natural light as an alternative light source for the in vitro culture of banana (Musa acuminata cv. ‘Grande Naine’)

The concept of using sunlight for micropropagation systems is proposed as a way of reducing tissue culture costs. Shoot tips of Musa acuminata cultivar ‘Grande Naine’ were cultured in a non-controlled natural light environment at the IAEA Laboratories, Austria during summertime. Significantly more shoots were produced by plantlets cultivated in a sunlit room with photosynthetic photon flux densities (PPFD) fluctuating up to 570 μmol m-2 s-1, temperatures between 23 and 30 °C and photoperiods of 12 to 16-h, than by plantlets under artificial light in a growth chamber providing controlled conditions of a constant PPFD of 65 μmol m-2 s-1, temperatures ranging from 23 to 29 °C and a 16-h photoperiod. Highest multiplication rates were achieved in a greenhouse with PPFD reaching 860 μmol m-2 s-1 and temperatures of 18 – 43 °C, but browning of leaves and loss of turgor occurred. Nevertheless, rooted plantlets showed 100% survival during acclimatisation and normal development. Photoperiods of 12 – 16 h did not affect the multiplication rates. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro induction of multiple shoots and plant regeneration from shoot tips of mung bean (Vigna radiata (L.) Wilczek)

Conditions for plant regeneration from excised shoot tips of Vigna radiata were studied. Complete plants were regenerated directly without an intervening callus phase from shoot tips on basal medium (MS salts+B₅vitamins). Regeneration frequency varied with genotype, explant size and growth regulator combinations in the medium. Addition of cytokinins induced a variable amount of callus at the base of the shoot tip, followed by multiple shoot formation. Benzyladenine (BA), kinetin and zeatin at 5×10^-6 M each induced multiple shoots in 100% of the explants but the highest number of regenerants per explant (9) was produced with BA. The efficacy of BA for shoot multiplication was not improved when it was supplemented with naphthaleneacetic acid (NAA) or indoleacetic acid (IAA). NAA or adenine sulphate, when applied alone, induced complete plantlets. The growth regulator requirement of explants for the induction of multiple shoots varied with explant size. The shoot tip explants maintained proliferation ability on subculture. None of the treatments was effective in inducing shoot bud differentiation from callus. Regenerated shoots were rooted on MS basal medium and MS supplemented with either IAA or indolebutyric acid. The rooted plants were transferred to the field; 60% subsequently survived and grew.Abbreviations BM basal medium [MS (Murashige & Skoog 1962) salts+B₅ (Gamborg et al. 1968) vitamins] - BA 6-benzyladenine - AdS adenine sulphate - IAA indole-3-acetic acid - NAA-1 naphthaleneacetic acid - IBA indolebutyric acid     

Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration

The effect of addition of silver nitrate (AgNO₃) on organogenesis of proximal and distal cotyledon and hypocotyl explants of five cucumber (Cucumis sativus L.) cultivars was investigated. Distal cotyledon and hypocotyl were unresponsive while only poor shoot regeneration was observed in proximal cotyledon and hypocotyl explants of all cucumber cultivars. The addition of different concentrations of AgNO₃ (10, 30 and 50 μM) to the medium, however, induced shoot regeneration in distal cotyledon except Suyo Long cultivar and effectively increased shoot regeneration response as well as the number of shoots per explant in proximal cotyledon and hypocotyl of all cucumber cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro axillary shoot proliferation and somatic embryogenesis of yellow pitaya Mediocactus coccineus (Salm-Dyck)

Yellow pitaya (Mediocactus coccineus) seeds were sown on Murashige and Skoog (1962) mineral salt medium. After germination, epicotyls were placed on media enriched with a combination of naphthaleneacetic acid (NAA) (0.05, 0.27 or 0.54 M) and benzyladenine (BA) (2.2 or 4.4 M). The apical tip was excised from half of the shoots and the other half were kept intact. Different values for proliferation rate, shoot length and thickness were observed on each medium. The cotyledons and roots were placed on MS medium supplemented with NAA (2.7 or 5.4 M) and embryogenic calluses were formed. Somatic embryos were induced on these media and then they normally developed on a growth regulator-free medium.Abbreviations BA benzyladenine - MS Murashige and Skoog - NAA -naphthalenacetic acid     

A tissue culture of the Ostrich fern Matteuccia struthiopteris (L.) Todaro

Multiple bud formation was induced from shoot apices of Matteuccia struthiopteris cultured on semi-solid Knudson's medium supplemented with 10^-5 and 10^-6 M kinetin. The effect of kinetin, naphthaleneacetic acid and gibberellic acid on shoot and root development is discussed and a three-part tissue culture system was devised for micropropagation and rooting.     

杭白菊茎尖组织培养及试管苗繁殖技术研究

采用茎尖组织培养技术,建立了杭白菊中大洋菊(Chrysanthemum morifolium Ramat.)的无菌试管苗体系.通过基本培养基和激素配比实验,筛选出杭白菊试管苗快速繁殖的最佳培养基组成.结果表明:最适宜的外植体为直径0.3 mm的茎尖;诱导丛生芽的最适培养基为:MS 6-BA 0.1 mg*L-1 IAA 0.02 mg*L-1;诱导试管苗生根的最适培养基为:1/2MS IAA 0.7 mg*L-1.用电子显微镜进行病毒检测后,筛选出2个脱病毒株系,脱病毒试管苗可作为今后提供优质种苗的种源.     

Anatomy of normal and hyperhydric leaves and shoots of in vitro grown Simmondsia chinesis (link) schn

Summary The anatomy of normal and hyperhydric in vitro shoots and leaves from micropropagated simmondsia chinensis (Link.) Schn. (jojoba) was compared with that of seedlings (control plants). In vitro normal plantlets displayed good development and survived during the acclimatization stage. In vitro hyperhydric plantlets presented numerous anatomical defects, such as hypertrophy of the mesophyll and of the stem cortex, malformed non-functional stomata, epidermal discontinuity, and xylem hypolignification; they did not survice acclimatization. The study of the anatomical features of in vitro jojoba shoots and leaves allowed determination of the structural condition of the plantlets and prediction of which plantlet would survive the critical acclimatization stage.     

Regeneration of roots, shoots and embryos: physiological, biochemical and molecular aspects

When the proper stimuli are given, somatic plant cells may form adventitious embryos, roots or shoots. The three pathways of regeneration show apparent similarities. They consist of three analogous phases: 1) dedifferentiation (during which the tissue becomes competent to respond to the organogenic/embryogenic stimulus), 2) induction (during which cells become determined to form either a root, a shoot or an embryo), and 3) realization (outgrowth to an organ or an embryo). The first phase may involve a period of callus growth (indirect regeneration), but often cells present in the explant become competent without cell division or without cell division at a large scale (direct regeneration). In an explant, only very few cells show the organogenic/embryogenic response. In direct regeneration, the three regenerative pathways start from cells in different tissues. This is most obvious when the different types of regeneration occur in the same explant. The hormonal trigger for the dedifferentiation phase is a general one, probably auxin. During the induction phase, each pathway requires specific hormonal triggers. During the realization phase, hormones should be absent or at low concentration. The successive steps in the regeneration process coincide with events on the molecular and biochemical levels, but so far no coherent picture has emerged. In particular during the early stages of regeneration, research on these levels is hampered by a technical problem, viz., the very low proportion of cells that participate in the process of regeneration. New methods may overcome this problem. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro propagation of Ancistrocladus abbreviatus Airy Shaw (Ancistrocladaceae)

Ancistrocladus abbreviatus Airy Shaw (Ancistrocladaceae), a West African liana producing naphthylisoquinoline alkaloids, was successfully raised from seeds in vitro. Clonal propagation was best achieved growing nodal stem segments on 1/5 Linsmaier and Skoog medium with full strength organics and supplemented with 0.02 μM thidiazuron, 4.44 μM 6-benzylaminopurine and 0.05 μM 1-naphthaleneacetic acid. Detached axillary shoots were grown on Anderson's Rhododendron medium devoid of phytohormones and rooted within one month when dipped in 4.92 μM indole-3-butyric acid. Rooted plants became acclimatized to nonsterile greenhouse conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of Pelecyphora aselliformis Ehrenberg and P. strobiliformis Werdermann (cactaceae)

Summary Development of efficient in vitro propagation, systems for Pelecyphora aselliformis Ehrenberg and P. strobiliformis Werdermann, two endangered Mexican species of cacti, are described. Multiple shoot formation from areoles of in vitro-germinated plantlets was achived in two types of explants (apical and transversal) cultured in Murashige and Skoog (MS) basal media supplemented with 30 or 50 gl⁻¹ sucrose, 10 gl⁻¹ agar and various treatments with cytokins. Shoot production in these proliferation media was evaluated after one (60 d) and three (180 d) culture cycles. In P. asellifornis 13.7 shoots per explant were produced after the first cycle using apical explants in medium with 8.8 μM 6-benzylaminopurine (BA) and 30 gl⁻¹ sucrose. In P. strobiliformis the highest proliferation rate (12.4 shoots per explant) was reached using 8.8 μM Ba and 50 gl⁻¹ sucrose with shoot transverse segments as explants. After the third proliferation cycle, 128.1 and 136.3 shoots per explant were obtained in P. aselliformis and P. strobiliformis, respectively. The shoots were clongated in MS basal medium with 3 gl⁻¹ activated charcoal and rooted in MS basal media indoleacetic acid (2.85 or 5.71 μM) or indolebutyric acid (2.46 or 4.90 μM). On averge, rooting efficiency was 89% for P. aselliformis and 87% for P. strobiliformis. The survival frequency, of the plants once transferred, to soil was on average 88%.     

Introduction and establishment of apricot in vitro through regeneration of shoots from meristem tips

Summary In this study different aspects of the in vitro introduction and establishment of apricot cultivars were investigated through meristem tip culture. The best time to introduce the meristems of ‘Canino’ was when buds were starting to swell. Various plant growth regulators were used at different concentrations on four distinct apricot cultivars to promote the development of the meristems to shoots which could then be micropropagated. Very diverse results were obtained depending on the genotype. In general, meristems did not survive without N⁶-benzyladenine. Concentrations of gibberellic acid from 2 to 4 mg 1⁻¹ (5.8–11.4 μM) promoted explant elongation. This step was critically important to obtain apricot shoots large enough to be transferred to proliferation medium.     

Influence of nutrient salts,auxins and cytokinins on the in vitro growth of Salvia greggii

Experiments were designed to determine the optimal MS salt concentration and the best auxin and cytokinin to use for shoot growth of Salvia greggii A. Gray. Full or 1/2 MS salts were superior to 1/4 MS salts based on number of shoots produced. There were no differences in the various auxins tested (IAA, NAA or IBA) as to their abilities to stimulate shoot production or increased fresh weight. BA, and BA + Kin stimulated the greatest shoot number among the cytokinins tested. A final experiment was designed to determine optimal BA and NAA concentrations for shoot growth. A medium containing 11.1M BA and no NAA produced the best growth of Salvia greggii in vitro. Shoots produced in vitro rooted and acclimatized readily in the green-house.Abbreviations MS Murashige and Skoog salts - IAA indoleacetic acid - IBA indolebutyric acid - NAA napthaleneacetic acid - BA benzyladenine - Kin kinetin - 2iP isopentenyl adenine     

Change in shoot proliferation with repeated in vitro subculture of shoots of woody species of Rosaceae

Shoots of 6 ornamental species and cultivars of Rosaceae were repeatedly subcultured in vitro for 9 generations on Linsmaier and Skoog (1965) medium with the addition of BA. Shoot proliferation increased over the first few generations and then gradually declined in all species and at all BA concentrations tested with the exception of Chaenomeles japonica in which a decline in shoot formation occurred only at 5.0 mg 1^-1 BA. A decrease in shoot length and leaf size and an increase in the incidence of callus formation was observed after several subcultures. This apparently irreversible decline could be due to either genetic or epigenetic change resulting from repeated fluxes in cytokinin, nutrient status or sucrose, or to elimination of seasonal environmental fluctuations.     

In vitro propagation of Clianthus formosus (Sturt's desert pea) an Australian native legume

Hypocotyl and cotyledon segments of Clianthus formosus were cultured on a modified deFossard medium M supplemented with cytokinins. 62% of cultures on medium with 20 μM BA produced callus which subsequently gave rise to shoots. 40% of shoots excised from callus produced roots after transfer to auxin-rich media (20 μM NAA or 10 μM IBA+10 μM NAA). Root production was enhanced following a 7-day dark treatment. 32% of nodes from mature plants produced multiple shoots on 2 μM BA+2 μM KIN. 30% of these shoots rooted on medium without hormones. 70% of rooted plantlets were successfully transferred to potting medium and glasshouse conditions. A period of cold treatment (10 days at 5°C) reduced vitrification from 68 to 22% of cultures.     

Protective enzymatic systems against activated oxygen species compared in normal and vitrified shoots of Prunus avium L. L. raised in vitro

Vitrification of shoots of Prunus avium L. L. was induced and expressed in a four week in vitro multiplication cycle simply by replacing agar by gelrite. The first vitrification symptoms were visible from the 7th day on. Enzymatic antioxidants were compared weekly in crude extract of normal (on agar) and vitrifying (on gelrite) shoots. The activity of superoxide dismutase was higher in vitrifying shoots. The other enzymes (gaîacol-peroxidase, catalase, ascorbate peroxidase, mono- and dehydro-ascorbate reductases, glutathione reductase) had lower activities. Increased superoxide dismutase activity might mean hydrogen peroxide accumulation and decreased activities of the other enzymes, deficiency in its detoxification. The question therefore is raised whether the hyperhydric morphological abnormalities result from the accumulation of toxic oxygen forms. Vitrification is often considered as a morphological response to several stresses. Contrary to most plants which adapt themselves to stresses by increasing all the above defence enzymes, in vitro shoots under vitrifying conditions appear unable to react in a similar manner.Abbreviations Apx ascorbate peroxidase - Gpx gaîacol peroxidase - CAT catalase - H₂O₂ hydrogen peroxide - SOD superoxide dismutase - MDHAR monodehydroascorbate reductase - DHAR dehydroascorbate reductase - GR glutathione reductase - MS Murashige and Skoog (1962) - IBA indolebutyric acid - BAP benzyladenine - GA₃ gibberellic acid     

Regeneration of Elaeagnus angustifolia from leaf segments of in vitro-derived shoots

Shoot regeneration was achieved from in vitro-produced leaves of Elaeagnus angustifolia L. Half-leaf explants from the terminal part of the shoot produced more shoots than explants from the basal part of the in vitro-derived shoots on agar-solidified WPM medium supplemented with 1 M benzyladenine (BA). In liquid medium of the same formulation, compact shoots that did not elongate were formed on the explants. Leaf cross-section explants (1 mm thick) produced shoots both on solid and liquid medium with 1 M BA, whereas again compact shoots were formed with 10 M BA. Further shoot development on these explants was promoted by their transfer to fresh solid medium containing 1 M BA and 1 M gibberellic acid (GA₃).Abbreviations BA benzyladenine - GA₃ gibberellic acid - WPM woody plant medium     

Toxicity of ethanol during proliferation and adventitious root formation in apple microcuttings in vitro

We have examined the toxicity of ethanol in tissue culture of the apple rootstock ‘Jork 9’. During proliferation through axillary branching, 0.2% (v/v) ethanol slightly stimulated proliferation whereas significant inhibition occurred at concentrations of 0.4 % or higher. In adventitious root formation, significant inhibition occurred at concentrations of 0.1 % or higher. The effect of ethanol was stage-dependent: during the induction period (i.e. from 24 to 72 h after the start of the rooting treatment), there was little or no inhibition. During autoclaving, ethanol evaporated to ca. 50 %. This revised version was published online in July 2006 with corrections to the Cover Date.