Innovative protocol for “<Emphasis Type="Italic">ex vitro</Emphasis> rooting” on olive micropropagation

The commercial micropropagation of olive trees is currently limited by the production cost. An ex vitro method for olive microshoot rooting could reduce both the production cost per plant and the propagation time. In this study a successful ex vitro rooting protocol tested on seven olive cultivars is reported. The explants of cv. Maurino were collected from fifth, sixth, and seventh proliferative subcultures carried out on MSM medium, while for the other cultivars the explants were collected from only seventh proliferative subculture. Continuous light during the rooting phase was a prerequisite for the success of the ex vitro protocol. The best source of microshoots for a high rooting percentage was the seventh proliferative subculture. Cvs. Coratina, Maremmano, Maurino, Picholine, and S. Francesco showed high rooting percentages with a range of 62–76%; whereas for cvs. Correggiolo and Frantoio the experimental conditions need to be optimised. Up to 90% of the rooted microplants survived, and continuous growth of shoots was subsequently observed. The proposed protocol can be easily applied to several different olive cultivars to produce microplants by commercial laboratories. The approach makes olive micropropagation in the nursery industry both possible and profitable.     

Ex Vitro Rooting of Micropropagated Shoots of Stackhousia Tryonii

Micropropagated shoots of Stackhousia tryonii were exposed (individually or in combination) to indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthalene acetic acid (NAA) at concentrations 1, 2 or 4 g dm^–3 with the view to induce rooting under ex vitro conditions. The treated microshoots were grown in a mist room for four weeks and assessed for survival, rooting percentage, number of roots and root length. The results showed that IBA at 2 g dm^–3 was most effective in inducing roots. Mixing of two or more auxins markedly reduced rooting percentage indicating antagonistic effects. The results demonstrated the potential of combining ex vitro rooting and hardening in one step, with view to reducing costs of multiplying plants via micropropagation.     

Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots

Buds and shoot tips of wild bilberry (Vaccinium myrtillus L.) and lingonberry (V. vitis-idaea L.) plants were cultured on a modified MS medium containing N⁶-isopentenyladenine (2iP), 9.8–78.4 μM, in order to study the effect of the 2iP-concentration on the initiation of growth. The experiment was first performed in the autumn and repeated in the spring to determine the influence of season on growth initiation. To optimise rooting, three different rooting treatments were tested for the bilberry and lingonberry microshoots. Shoots were rooted either in vitro with 0.49 μM IBA (indole-3-butyric acid) or ex vitro, incubating microshoots in 2.07 mM KIBA-solution (potassium salt of IBA) before planting, or microshoots were planted directly on peat without exogenous auxin. The best 2iP concentration for the initiation of the growth for bilberry was 49.2 μM and for lingonberry 24.6 μM. It was observed that increasing the 2iP concentration at the growth initiation stage increased the number of brownish explants both in bilberry and in lingonberry microcultures. Spring was a considerably better time than autumn for the initiation of new growth, for both species. The results of the rooting test showed that the KIBA-treatment before planting on peat increases rooting efficiency in both bilberry and lingonberry. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of cytokinin levels on <Emphasis Type="Italic">in vitro</Emphasis> propagation of shy suckering chrysanthemum “Arka Swarna” and activation of endophytic bacteria

In an effort to develop a sustainable protocol for the micropropagation of a shy suckering elite chrysanthemum cv. Arka Swarna (yellow pompon type), in vitro cultures were established using surface-sterilized nodal microcuttings (1–1.5 cm) from polyhouse-grown plants on MS medium containing 3% sucrose, 0.25% phytagel, and 5 μM benzyl adenine (BA) or kinetin. Microbial contamination in the range of 6–24% was encountered during the first in vitro passage. Apparently clean cultures after one passage on MS basal medium were transferred to medium with BA or kinetin (0, 1, 5, 10, or 20 μM) in culture bottles, and were monitored for eight in vitro passages (1 mo. each) for growth and microbial contamination. Plant growth regulator (PGR)-free medium was the best for sustainable micropropagation over successive in vitro passages yielding a single shoot from cultured microcuttings. Higher cytokinin levels inhibited rooting and induced one or more shorter shoots with close nodes resulting in low propagation rates. All apparently clean stocks revealed covert endophytic bacteria during tissue-indexing using bacteriological media. Three distinct bacterial morphotypes were isolated from such stocks, identified based on 16S rRNA gene sequence analysis as different morphotypes of Curtobacterium citreum. The endophytes tended to show obvious growth on chrysanthemum culture medium with increase in cytokinin levels (5–20 μM), but such growth was not noticed in inoculations on MS medium without plants. Sustainable micropropagation of cv. Arka Swarna for more than 2 yr with the resident endophytic bacteria in covert form was realized on PGR-free MS medium giving a net propagation rate of three to four times over a subculture cycle of 2–3 wk.     

Application of a novel disposable film culture system to photoautotrophic micropropagation of <Emphasis Type="Italic">Eucalyptus uro-grandis (Urophylia x grandis)</Emphasis>

Summary To overcome various disadvantages of conventional culture vessls for plant micropropagation, we previously developed the photoautotrophic micropropagation technique, with special mention for the first practical film culture system, the ‘Miracle Pack’ (MP), which was made of fluorocarbon polymer film (Neoflo^? PFA film) and supported by a polycarbonate frame. While the PFA film has superior thermal stability, high light transmittance and high gas permeability, making the MP system (MP-PFA) superior to conventional culture vessels for the micropropagation of various plant species, its high cost is a disadvantage. In this study, a possible alternative of lower-cost OTP^? film made of TPX (4-methyl-1-pentane polymer) and CPP (a polypropylene), which possesses similar characteristics to PFA film, is evaluated to develop a novel disposable film culture vesel, termed ‘Vitron’, for culturing Eucalyptus (urophylla x grandis), plantlets. The three film culture systems, MP-PFA, MP-OTP (MP with OTP film), and Vitron, were placed under CO₂ enrichment, low photosynthetic photon flux density (PPFD; 45 μmol m⁻² s⁻¹), and sugar-free medium, using phenol resin foam (Oasis^?) as a substrate. In vitro and ex vitro growth and development of Eucalyptus shoots from the four-leaf stage to the rooting stage were compared for all three culture systems. The effects of the duration and concentration of CO₂ enrichments on in vitro growth of Eucalyptus cultured in the Vitron film system were also examined. The best growth and quality of Eucalyptus plantlets was obtained for the Vitron vessel placed in 3000 ppm CO₂ enrichment for 24 hours per day at low PPFD with sugar-free liquid medium and Oasis as substate. Results of this study suggest that the novel Vitron culture system is suitable for the photoautotrophic micropropagation of Eucalyptus. These authors contributed equally to the research results.     

In vitro studies on Eucalyptus globulus rooting ability

Summary Micropropagation has the potential to quickly introduce selected genotypes of adult Eucalyptus globulus clones and it is now widely used in Portugal as a part of genetic improvement programs. Several clones have been established and multiplied in vitro. The different clones have individual requirements for successful rooting. Rejuvenation was achieved at different periods after culture initiation for the different clones. Subculturing preceding rooting in multiplication medium supplemented with riboflavin and cholene chloride allowed the increase of rooting ability for several clones tested. Removal of boron from the rooting medium increased rooting by 10%. Indolebutyric acid (IBA) dipping before transfer to the rooting medium resulted in a rooting percentage of 80–95% for the best clones tested. Acclimatization was performed without difficulties (90–95% success) and the rooted plants were either planted directly or used as mother plants for further cutting production, depending on the needs. The results described in this paper increase the commercial feasibility of the micropropagation system for E. globulus.     

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.     

Use of sugarcane bagasse as an alternative low-cost support material during the rooting stage of apple micropropagation

Summary Studies were carried out to evaluate sugarcane bagasse as an alternative to agar for micropropagation of apple clones to reduce the cost of micropropagation and improve the quality of the propagules. Significant improvement in the in vitro rooting process, coupled with cost reduction, were obtained by the use of sugarcane bagasse as a substitute for the traditionally used agar-gelled medium. The tests were undertaken with micro-cuttings of the apple rootstock Marubakaido (Malus prunifolia Borkh.) using a rooting medium composed of half-strength Murashige and Skoog salts and vitamins, 3% (w/v) sucrose, and 0.49 μM indole-3-butyric acid. The plants grown on sugarcane bagasse yielded a 22% increase in root length, 20% increase in plant length, and 63% increase in the number of roots, compared with agar-grown micro-cuttings. Particle size of the sugarcane bagasse had a significant impact on all those parameters, and the best results were obtained with bagasse comprising particles smaller than 0.18 mm. The results demonstrated that the sugarcane bagasse could be used effectively as a substitute for agar during rooting of apple shoots.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

Micropropagation of sweet viburnum (<Emphasis Type="Italic">Viburnum odoratissimum</Emphasis>)

A micropropagation protocol for shoot culture of sweet viburnum (Viburnum odoratissimum) is described. Nodal explants, initially established on MS medium, were transferred to WPM supplemented with combinations of BA and GA₃. Maximum shoot multiplication was observed on explants cultured on medium supplemented with BA concentration higher than 1.1 μM, and 14 μM GA₃. Although Stage II medium supplemented with BA concentration higher than 1.1 μM resulted in increased shoot multiplication, it also caused a decrease in shoot length. A negative carry over effect of GA₃ on rooting was observed in subsequent Stage III cultures. The presence of GA₃ in Stage II medium promoted shoot elongation, but it also caused a decrease in microcutting rooting. For this reason, 0.5 μM BA and 14 μM GA₃ were selected for optimum Stage II shoot multiplication. Although 100% microcuttings formed roots when cultured on medium containing 6.0 μM NAA, significant callus formation was observed and ex vitro survival rate was low (49%). Rooting was achieved after 3 weeks with 82% of microcuttings on medium supplemented with 3 μM IBA. The survival rate of plantlets under ex vitro conditions was 100% after 3 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 30 plants.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.     

In vitro propagation ofSalix tarraconensis Pau ex Font Quer,an endemic and threatened plant

Summary Salix tarraconensis Pau ex Font Quer, an endemic willow species from northeast Spain, was micropropagated with nodal segments. Shoot multiplication was obtained with different cytokinins, either on Murashige and Skoog medium or woody plant medium. Best results for shoot formation were obtained on Murashige and Skoog medium containing 4.9 μM of 6-γ-γ-dimethylallylaminopurine. Shoots showed strong apical dominance, and some cultures displayed apical necrosis. Benzyladenine gave the worst results; shoots displayed very slow growth, deformed leaves, and hyperhydrity. Good rooting of shoots was obtained with different auxins or without plant growth regulators on woody plant medium. The best results (90-100%) were obtained within 20 d. On rooting media with indole-3-butyric acid or indoleacetic acid, shoot elongation was good (35-40 mm length). Apical necrosis was observed in elongating shoots on rooting medium, but this disturbance favored axillary bud sprouting and formation of new shoots. Shoot length and quality of roots decreased gradually as the concentration of naphthaleneacetic acid increased. Plant survival was 90% 4 weeks after removal fromin vitro conditions.     

Rooting of microcuttings: Theory and practice

Summary Poor adventitious root formation is a major obstacle in micropropagation and in conventional propagation. This paper reviews recent progress in the understanding of adventitious root formation as a developmental process focusing on the role of plant hormones and on the effect of rooting conditions on plant performance. Since the discovery of the rhizogenic effect of auxin ca. 70 yr ago, no new broadly applicable rooting treatments have been developed. Recent research, though, may lead to new rooting procedures. Application of wounding-related compounds may be effective in difficult-to-root crops. Furthermore, by adapting conditions during the propagation phase, microcuttings with an enhanced capability to root may be produced. These conditions include elongation of stems (by etiolation or double-layer culture) and repeated subculture (rejuvenation; i.e. transition from adult to juvenile). Data are presented that show that during tissue culture maturation (transition from juvenile to adult) also occurs. The conditions during the in vitro rooting treatment may have a tremendous effect on performance after transfer ex vitro. In particular, accumulation of ethylene during in vitro rooting may have a devastating effect. Addition of stress-protecting compounds during propagation or rooting in vitro may enhance the performance ex vitro. Based on a presentation at the Plant Symposium ‘Rooting of Micropropagated Plants’ at the 2001 Congress of In Vitro Biology held at St. Louis, MO, June 16–20, 2001.     

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.     

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions. Received: 12 January 1998 / Revision received: 10 October 1998 / Accepted: 28 October 1998     

Induction,growth and direct rooting of adventitious shoots of Begonia x hiemalis

The efficiency of commercial micropropagation programs for Begonia x hiemalis depends on the production of large adventitious shoots for easy handling and on effective rooting and acclimatization procedures. Maximum induction of adventitious buds on petiole segments occurred in response to NAA (0.1 mg, l^-1) and BA (0.5 mg l^-1), but continued shoot growth was limited. With a lower concentration of BA (0.1 mg l^-1) fewer shoots were produced but shoot growth was enhanced. With a combined agar/liquid culture program the low BA (0.1 mg l^-1) medium produced 50 percent more shoots larger than 1 cm than did the high BA (0.5 mg l^-1) medium. In vitro rooted explants developed weak root systems and acclimatization losses occurred during adaptation to greenhouse conditions. Adventitious shoots treated with commercial rooting powder and placed directly in mist frames produced much stronger root systems and could be adapted to greenhouse conditions without loss. The elimination of the in vitro rooting stage also simplifies the micropropagation program.Contribution No. 743     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

Rapid Axillary Bud Proliferation and ex vitro Rooting of Eupatorium triplinerve

Effective protocol was established for micropropagation of the medicinal plant Eupatorium triplinerve Vahl through rapid axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium fortified with 8.87 M benzylaminopurine (BAP) and 2.46 M indole-3-butyric acid (IBA) was the best for axillary bud proliferation and developed a mean of 8.1 shoots per node. Excision and culture of the node segments of the in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 d. Shoot multiplication did not exhibit decrease in the number of shoots even at 7^th subculture. Dipping of the basal end of shoots in 2.46 M IBA solution for 10 d induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100 %). Ex vitro rooting by direct transfer of the shoots from multiplication medium showed 92 % survival.     

Comparison of gene expression under <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">ex vitro</Emphasis> conditions during rooting of grape cuttings through mRNA differential display

An mRNA differential display (DD) analysis during rooting in grape cuttings was carried out to determine whether gene expression patterns differed under in vitro and ex vitro conditions. The four tissue samples for differential display and subsequent Northern hybridization analyses included control stem tissue from in vitro and ex vitro sources, microcuttings planted in MS based in vitro rooting medium and softwood cuttings planted in ex vitro soil medium, both collected 48 h after planting. DD autoradiographs showed gross similarity in banding pattern between in vitro and ex vitro stem tissue, whether constitutive or induced. Northern blot analysis of a few bands that appeared to be differentials did not indicate them as true positives. The results suggested that gene expression pattern during physiological processes such as rooting may be identical under in vitro and ex vitro conditions.