Micropropagation of ‘Durondeau’ pear in modified-gelled medium

Summary The influence of partial substitution of agar by galactomannans (GMs) in culture media was studied in pear (Pyrus communis L. cv. ‘Durondeau’) micropropagation. GMs. extracted from seeds of Cassia fastuosa (cassia) or Cyamopsis tetragonolobus (guar gum, a commercial GM), were mixed in equal proportions with agar to a final concentration of 0.3% (w/v) for each type of gelling agent. The production of multiple shoots and the formation of roots from shoots were compared with the control solidified with agar alone at a concentration of 0.6% (w/v). In the media solidified with the mixtures of agar/guar and agar/cassia GMs, an, increase of 32 and 17%, respectively, was obtained in the number of regenerated shoots. The modified media promoted a higher number of roots and increased the rooting percentage. A maximum of 91% rooting was obtained in the medium solidified with the agar/cassia GM and containing 9.80 μM indole-3-butyric acid. Less callus formation at the base of the shoot was also observed on this medium. The improved in vitro performance of shoot formation and rooting, combined with a significantly lower cost, suggests a potential use of agar/GM gels in plant tissue culture.     

四种添加物对铁皮石斛原球茎生长及多糖含量的影响

为探讨铁皮石斛(Dendrobium officinale)培养基中添加物的作用,在1/2MS培养基中加入椰肉、甘蔗渣、香蕉皮和麦麸等4种添加物,研究不同浓度添加物和培养时间对原球茎生长和多糖含量的影响。结果表明,4种添加物对铁皮石斛原球茎的增殖、分化和多糖含量均有一定影响,其中添加15.0 g L–1甘蔗渣,培养60 d能明显促进铁皮石斛原球茎的增殖与分化(146.1%);而添加20.0 g L–1甘蔗渣,培养40 d能显著提高铁皮石斛原球茎多糖含量(50.4%)。这说明甘蔗渣是培养铁皮石斛原球茎的适宜添加物,既能促进铁皮石斛原球茎的生长发育,还能降低生产成本。     

Micropropagation of a <Emphasis Type="Italic">Casuarina</Emphasis> hybrid (<Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> L. × <Emphasis Type="Italic">Casuarina glauca</Emphasis> Sieber ex Spreng) following facilitated seed germination

A suitable protocol for micropropagation of Casuarina hybrid, Casuarina equisetifolia L. × Casuarina glauca Sieber ex Spreng (C. e. × C. g.), was developed. When seeds without seed coats were cultured on 4 germination media, the optimal seed germination percentage (91%) was obtained on 0.8% agar solidified water medium. Shoot multiplication was achieved by culturing 2-cm long epicotyls, excised from germinated seedlings, on MS (Murashige and Skoog 1962) basal medium supplemented with BA (6-benzylaminopurine) at 4.4, 8.8, 17.8 and 35.6 μM. The greatest percentage of axillary bud sproutings (87.5%), mean number of sprouts per explant (3.8), and shoot length (3.2 cm) were achieved on MS medium supplemented with 17.8 μM BA. MS medium supplemented with 4 different concentrations of IBA (indole-3-butyric acid) (4.3, 8.7, 13.0 and 17.4 μM) were used for rooting of in vitro grown shoots. The highest rooting percentage (65.6%), mean number of roots per explant (2.5) and mean length of roots per explant (1.6 cm) was achieved at 13.0 μM IBA. Rooted shoots grew well after transfer to a substrate of peat and pinebark (7:3) in the greenhouse.     

Sago: An Alternative Cheap Gelling Agent for Potato In Vitro Culture

Sago, a processed (gelatinized) edible starch, was successfully used as a gelling agent in culture medium. The efficacy of sago-gelled (80 g dm^–3) medium was studied in ten potato (Solanum tuberosum L.) genotypes during micropropagation and minimal growth conservation. Sago starch provided a firm gelling surface throughout the entire culture period, and fostered optimum plantlet growth in terms of shoot height, number of nodes per plant, number of leaves and fresh mass. No softening of the sago-gelled medium occurred over prolonged (six months) storage. The study showed that sago starch could be used as a substitute to agar in culture medium to substantially reduce the medium cost.     

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.     

Direct organogenesis from leaf explants of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> and cultivation in bioreactor

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 μM of N ⁶-benzylaminopurine and kinetin ranging from 4.65 to 6.98 μM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm⁻³ sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 μM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm⁻³ sucrose resulted in a high biomass yield of 50.68 g dm⁻³ (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 μM, 15 g dm⁻³ sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.     

Low-cost alternatives for the micropropagation of banana

A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose and Gelrite in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun light instead of artificial light. The micropropagation of Musa `Grande Naine' by shoot tip culture was used as model. Thirteen commercial sugars from different countries were tested. Best results were achieved using white and light brown sugars with low electrical conductivity. Sugars of cane or sugar beet origin were suitable. Starches of corn or potato could partially substitute for Gelrite and agar. In all experiments, micropropagation rates under natural light conditions were equal to or higher than under the controlled conditions of a growth room with PPFD of 65 μmol m⁻² s⁻¹ and a 16-h photoperiod. Plants were exposed to average PPFD levels of 58–96 μmol m⁻² s⁻¹ and photoperiods ranged from 8–16 hours. This revised version was published online in June 2006 with corrections to the Cover Date.     

茄子野生近缘种托鲁巴姆试管微扦插繁殖技术研究

嫁接栽培是茄果类蔬菜防治土传病害和提高产量的重要措施之一。茄子野生近缘种托鲁巴姆(Solanums torvum)因综合抗性强,成为茄子和番茄嫁接的常用优良砧木。但是,由于托鲁巴姆种子的发芽率、发芽势和发芽指数较低,苗龄较长,限制了其在工厂化育苗中的大规模应用,因此迫切需要开发其他方法及相应技术体系提高托鲁巴姆的育苗效率,降低育苗成本。为优化托鲁巴姆微扦插技术,该研究探索并优化了试管内微扦插繁殖托鲁巴姆技术,以无菌播种获得初代无菌苗的茎段为外植体,通过在培养基中添加植物生长调节剂,对比不同浓度植物生长调节剂对托鲁巴姆微扦插繁殖过程中的影响。结果表明:(1)托鲁巴姆在不同培养基中,腋芽诱导、继代增殖和生根培养的效果存在显著差异,初代芽诱导的最佳培养基为MS+KT 0.5 mg·L^-1+IBA 0.1 mg·L^-1,出芽率达90%。(2)继代扦插最佳培养基为MS+IBA 0.4 mg·L^-1,培养30 d的增殖系数达6.11,植株长势健壮。(3)最佳生根培养基为1/2MS+IBA 0.2 mg·L^-1,生根培养30 d,单株一级根数4.56条,最长根长125.80 mm、根粗0.50 mm,根系发达。采用试管内微扦插技术繁殖托鲁巴姆种苗,操作简单,增殖系数较高,可满足快速繁育种苗的要求。该研究结果为托鲁巴姆的工厂化规模育苗提供了新途径。     

Micropropagation of photinia employing rhizobacteria to promote root development

An alternative protocol was developed for in vitro propagation of photinia (Photinia × fraseri Dress), an ornamental shrub, using the plant growth-promoting rhizobacteria (PGPR) Azospirillum brasilense and Azotobacter chroococcum during rhizogenesis. Shoot tips from four-year-old mature plants, cut in spring and summer, were used as initial explants. They were cultured on Murashige–Skoog (MS) medium with Gamborg’s vitamins, N⁶-benzyladenine (BA: 11.1 μM) and gibberellic acid (GA₃: 1.3 μM), obtaining 63% of established explants. The highest shoot length (22.9 mm) and multiplication rate (4.3) was achieved by cultivating for four weeks in the same basal medium supplemented with 4.4 μM BA. Both auxin induction and bacterial inoculation were used for rooting. Elongated shoots were treated with two concentrations of indole-3-butyric acid (IBA: 4.9 or 49.2 μM) during 6 days for auxin induction. Then, the shoots were transferred to an auxin-free medium and inoculated with A. brasilense Cd, Sp7 or A. chroococcum (local strain). Bacterial inoculation induced earlier rooting of photinia shoots. A. brasilense Cd with 49.2 μM IBA pulse showed a significant increase (P ≤ 0.05) in root fresh and dry weight (105%, 137%), root surface area (65%) and shoot fresh and dry weight (32%, 62%). A. brasilense Sp7 enhanced the root fresh weight (34%) and root surface area (41%) while no significant differences with A. chroococcum inoculation were detected. The PGPR inoculated micro-cuttings in combination with auxin induction pulses may play a useful role in root organogenesis of micropropagated plants.     

An efficient plant regeneration system for Mucuna pruriens L. (DC.) using cotyledonary node explants

Summary The purpose of this study was to develop an efficient micropropagation system for Mucuna pruriens, an important medicinal plant in India. A range of cytokinins was investigated for multiple shoot regeneration with cotyledonary node explants from 7-d-old aseptic seedlings. Of all the cytokinins, 6-benzyladenine (BA), kinetin (KIN) and 2-isopentenyl adenine (2-iP) tested in Murashige and Skoog medium (MS), BA was the most effective and 5.0 μM was found to be optimum for inducing maximum shoots. Medium types, medium strength and pH were also investigated for induction and proliferation of shoots. The highest efficiency of shoot proliferation was observed in 5.0 μM BA and 0.5 μM α-naphthalene acetic acid (NAA) in half-strength MS medium at pH 5.8. The best condition for rooting was half-strength MS medium solidified with agar and with 2.0 μM indole-3-butyric acid (IBA). After rooting, the plantlets were transferred to plastic pots filled with sterile soilrite where 90% grew and all exhibited normal development.     

Micropropagation and germplasm conservation of Central Park Splendor Chinese elm (<Emphasis Type="Italic">Ulmus parvifolia</Emphasis> Jacq. ‘A/Ross Central Park’) trees

Micropropagation offers opportunities to propagate, preserve and ship tree germplasm. It also reduces the risk of moving pathogens and insects with the germplasm due to built-in pathogen detection capabilities of aseptic cultures. For the past few decades, our laboratory has been involved in a project to preserve and restore a large, cold hardy, and historically important Chinese elm (Ulmus parvifolia Jacq. ‘A/Ross Central Park’) tree. Here we present three simple and efficient systems for its micropropagation, germplasm conservation and distribution: (1) in vitro plant formation from meristematic nodules (MNs), (2) plantlet generation from axillary buds, and (3) in vitro rooting of micro-cuttings from 20-years-old hedged stock plants. Newly flushed nodal segments were used as explants. WPM with 0.5 mg/l BA was found to be the best medium for meristematic shoot development and WPM supplemented with 2.0 mg/l 4-CPPU and 0.5 mg/l TDZ was best for meristematic nodule formation. Rhizogenesis of regenerants and micro-cuttings was best achieved on WPM with 1.0 mg/l NAA and 2% sucrose. Rooted plants were readily acclimatized to the greenhouse ambient environment and continued to grow well under greenhouse conditions. The survival rate of acclimatized plantlets under ex vitro conditions was 100% after 4 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 1,000 plants. Cycling of shoot explants and MNs through repetitive cultures was effective in scaling-up propagules.     

Protein enrichment of lignocellulose resulting from the growth of two Streptomyces strains

Two Streptomyces strains were grown on sugarcane bagasse and groundnut hulls lignocelluloses in semi-solid state culture at 37°C for 12 weeks. Best results gave a 45% depletion of sugarcane bagasse lignocellulose with a 21% crude protein content of final material. The possibility of using S. viridosporus to improve the protein content of both lignocelluloses for use as an animal feedstock supplement is discussed.At the time of this research the authors were with the Department of Biological Sciences, Rivers State University of Science & Technology, PMB 5080, Port Harcourt, Nigeria and the Department of Biological Sciences, University of Calabar, PMB 1115, Calabar, Nigeria. Dr lyo is now with the Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.     

Efficient rooting for establishment of papaya plantlets by micropropagation

A low cost micropropagation protocol to produce high quality root systems which are easy and economical to acclimatize is essential for large-scale micropropagation of papaya (Carica papaya L.). In this study, individual shoots (>0.5 cm) with 2_∼3 leaves from in vitro papaya multiple shoots were cultured on MS agar medium containing 2.5 μM IBA under dark conditions for 1 week for root induction. They were then transferred to agar or vermiculite media, containing half strength MS medium, under aerated or non-aerated conditions, for root development. Rooting percentage of shoots cultured for 2 weeks in aerated vermiculite was 94.5%, compared with 90.0% in non-aerated vermiculite, 71.1% in aerated agar, and 62.2% in non-aerated agar. Shoots with roots were acclimated in vermiculite under 100% RH for 1 week and then under ambient conditions for 2 weeks in a temperature-controlled growth chamber (28 °C). The survival rates of the plantlets were 94.5% from aerated vermiculite, 87.8% from non-aerated vermiculite, 42.2% from aerated agar, and 35.6% from non-aerated agar. Thus, root induction in low-concentration IBA agar medium followed by root development in vermiculite containing half strength MS medium under aerated conditions results in efficient rooting of in vitro papaya shoots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

Micropropagation of Ilex aquifolium L.

Summary Two procedures were tested for micropropagation of Ilex aquifolium (English holly), one in which shoots proliferated on solid medium and another one using liquid medium. Different growth regulator treatments and supports were analyzed for optimizing in vitro rooting, showing that indolebutyric acid and agar or cellulose plugs gave the best results. The surival percentage of successfully in vitro or ex vitro rooted plants did not differ significantly between the best treatments. However, the efficiency with ex vitro rooting was 80%, while for in vitro rooting, the final efficiency was 64%. The results show that a correct manipulation of Murashige's stage II of micropropagation and eliminating or decreasing stage III are useful tools to reduce the requirements of acclimatization.     

Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues

The production of an alkali-stable xylanase, with dual pH optima, from haloalkalophilic Staphylococcus sp. SG-13 has been enhanced using agro-residues in submerged fermentation and a biphasic growth system. The agro-residues such as wheat bran, sugarcane bagasse, corncobs and poplar wood when used as sole carbon source, improved the xylanase yield by five-fold as compared to xylose and xylan. Staphylococcus sp. SG-13 also produced equally good amounts of xylanase when grown simply in deionized water (pH 8.0) supplemented with agro-residues as sole carbon source. In the biphasic growth system (lower layer containing agricultural residue set in agar medium with liquid medium above it), the prime substrate, wheat bran (1% w/v), resulted in maximum xylanase production of 4525 U l^–1 (pH 7.5) and 4540 U l^–1 (pH 9.2) at an agar: broth ratio of 4.0 after 48 h of incubation at 37 °C under static conditions. In general, the cost-effective agro-residues were found to be more suitable inducers for xylanase production over expensive substrates like xylan.     

β-Methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. β-Methyl-xyloside (β-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with β-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus β-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus β-mx as substrate. To our knowledge, this is the first report on the effect of β-mx on the induction of xylanase in C. flavigena.     

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.     

Relations between auxin and cytokinin contents and in vitro rooting of tree Peony (Paeonia suffruticosa Andr.)

In this work, a combined HPLC-ELISA technique was used to associate in vitro rooting capacity of tree peony micro-cuttings with contents of cytokinin and auxin; the cytokinin mainly detected corresponded to the N⁶-benzyladenine which had been added to the multiplication medium. Rooting capacity of explants was favoured by a preliminary accumulation of endogenous IAA only when levels of the BA absorbed from the multiplication medium had decreased. Main shoots coming from a 5-weeks subculture fulfilled these hormonal conditions and were the best microcuttings for rooting (87% rooting). Main shoots coming from shorter cycles or axillary shoots coming from a 5-weeks cycle always contained high benzyladenine levels and had a low rooting capacity (25–55% rooting). Root induction was associated with an early peak of indole-3-acetic acid followed by a 10-fold lower peak of endogenous ribofuranosyl-isopentenyladenine. Only a low and transitory accumulation of isopentenyladenine occurred during root development, and this could explain the lack of shoot development. Root development was efficient, especially in a medium containing activated charcoal, which led to an almost 3-fold decrease of IAA contents in roots.Abbreviations AC activated charcoal - BA N⁶-benzyladenine - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - IBA indole-3-butyric acid - iP N⁶-(²-isopentenyl)adenine - RDM root development medium - RIM root induction medium - 9RIP 9--d-ribofuranosyl-iP - 9RZ 9--d ribofuranosyl-zeatin - Z zeatin     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.