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.     

Temporary immersion systems in plant micropropagation

Temporary immersion systems for plant micropropagation have been described and grouped into 4 categories according to operation: tilting and rocker machines; complete immersion of plant material and renewal of the nutrient medium; partial immersion and a liquid nutrient renewal mechanism; complete immersion by pneumatic driven transfer of liquid medium and without nutrient medium renewal. The positive effects of temporary immersion on micropropagation are indicated for shoot proliferation and microcuttings, microtuberization and somatic embryogenesis. Immersion time, i.e. duration or frequency, is the most decisive parameter for system efficiency. Optimizing the volume of nutrient medium and the volume of the culture container also substantially improves efficacy, especially for shoot proliferation. Temporary immersion also generally improves plant material quality. It results in increased shoot vigour and in the frequency of morphologically normal somatic embryos. Hyperhydricity, which seriously affects cultures in liquid medium, can be eliminated with these culture systems or controlled by adjusting the immersion times. Plant material propagated by temporary immersion can perform better during the acclimatization phase than material obtained on semi-solid or in liquid media. Successful regeneration of plants, after direct sowing on soil of Solanum tuberosum microtubers and Coffea arabica somatic embryos produced in temporary immersion bioreactors, has been demonstrated. As could be expected when using liquid medium for micropropagation, several estimations confirm large gains in efficacy from temporary immersion. The parameters most involved in reducing production costs include: (1) the drastic reduction in work; (2) reduction in shelving area; (3) reduction in the number of containers used; (4) better biological yields. Scaling-up somatic embryogenesis and shoot proliferation procedures involving temporary immersion systems in order to commercialize this process are now taking place.     

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.     

Practical applications of in vitro propagation: Present situation and future prospects

Abstract

This article surveys the techniques and approaches used in the in vitro propagation of economically important plants. The current state of the art for each major class of plants, namely ornamentals, vegetable and agronomic crops, temperate fruits and forest trees is described. The advantages of vegetative propagation in general and the specific advantages which micropropagation offers in the domestication, breeding and conservation of plants are listed. Specific problems associated with in vitro propagation such as juvenility vs maturation, vitrification, rooting and morphological or physiological variations are discussed.     

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.     

Development of a new culture medium and efficient protocol for in vitro micropropagation of Ceratonia siliqua L.

A new basal culture medium was developed and tested using a rapid and efficient protocol of in vitro axillary shoot bud proliferation of Ceratonia siliqua L., an important Mediterranean Fabaceae plant species. In a first experiment, the new formulated ‘LA’ mineral composition significantly improved shoot growth and proliferation as compared with Murashige and Skoog medium (MS, 1962) in both solid and liquid culture media. However, the liquid culture system proved to be the most suitable for shoot induction, shoot length (about fourfold higher), and multiplication rate (about two-fold higher), the difference being significant. The measured growth and proliferation parameters were further improved when LA mineral composition was optimized, in a second experiment. The highest multiplication rate (6.3) was achieved during the second subculture using the optimized ‘LAC’ medium. Noticeably, hyperhydricity and shoot-tip necrosis symptoms were absent in both formulated LA and LAC compositions when using the liquid culture system. In vitro rooting in solid medium showed 41.7 to 46.3% response on a solid medium which was more suitable than the liquid culture system, the difference being significant. In contrast, pretreated microcuttings with 3 μM IBA (indole-3-butyric acid) were successfully rooted ex vitro, showing significantly higher response (91.7%), average root number (8.3), and root length (31.5 mm). The plantlets were successfully acclimatized showing more than 90% survivability and normal morphology. The present study is a first cost-effective protocol for carob micropropagation combining the use of the newly formulated LAC basal medium, a liquid culture system, and ex vitro rooting.

     

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method

Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid‐gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ‐induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor‐derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin‐containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.     

An efficient protocol for micropropagation of <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis> Cheel

Melaleuca alternifolia is cultivated for the production of an essential oil useful in the cosmetic and pharmaceutical industries. Despite the economic importance of this species, there is little knowledge about its in vitro propagation. The aim of this study was to establish an efficient protocol for micropropagation of M. alternifolia. With the goal of in vitro multiplication by axillary shoot proliferation, both solid and liquid MS and WPM media were tested with supplementation with BA at 0, 0.55, 1.11, 2.22, 3.33, and 4.44 μM. The best result for shoot multiplication was obtained when either 0.55 μM BA was added into solid MS medium or 1.11 μM BA was added into liquid MS medium, with 5.6 and 11.8 shoots per explant generated, respectively. On solid or liquid WPM medium supplemented with 0.55 μM BA, the proliferation rates were 5.5 and 4.7, respectively. Three auxins (NAA, IAA, and IBA) were tested at 0.53 and 2.64 μM during the rooting stage. Several sucrose concentrations (15, 30, and 45 g L⁻¹) were compared to a sucrose-free medium. Rooting performances on four culture media were then compared: MS, half-strength MS (MS/2), MS + activated charcoal (AC), and MS/2 + AC. The results showed that auxin addition to culture medium is not necessary for in vitro rooting. Rooted microcuttings from different culture media were acclimatized in a greenhouse, and the survival percentage was evaluated. All shoots cultured in an auxin-free MS medium supplemented with sucrose (30 g L⁻¹) produced roots, and all plants survived during acclimatization. Activated charcoal added in rooting medium reduced rooting rates.     

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.     

Micropropagation and β-ecdysone content of the Brazilian ginsengs <Emphasis Type="Italic">Pfaffia glomerata</Emphasis> and <Emphasis Type="Italic">Pfaffia tuberosa</Emphasis>

The aim of this study was to investigate both a mass in vitro propagation system and the β-ecdysone content in roots and aerial parts of Pfaffia glomerata and Pfaffia tuberosa. Nodal segments of two genotypes (BRA and JB-UFSM) of P. glomerata, originated from aseptically grown plants, were cultivated on hormone-free Murashige and Skoog medium. For the proliferation of P. tuberosa shoots, nodal segments, originated from aseptically grown plants, were either cultivated on hormone-free Murashige and Skoog (MS) medium or were supplemented with 1.0 μM thidiazuron (TDZ); the elongation and rooting of these plants were carried out on MS medium without TDZ. Plantlets of both species were acclimatized and transferred to field conditions. The β-ecdysone content in the plants was determined by high performance liquid chromatography. The BRA genotype showed a greater in vitro proliferation rate and β-ecdysone content than that of the JB-UFSM genotype. The culture of nodal segments of P. tuberosa on medium with 1.0 μM TDZ with subsequent subcultivation of shoots on hormone-free medium was shown to be a suitable method for micropropagation due to the high multiplication rate and good plant development. Both species showed good adaptation to ex vitro conditions. The β-ecdysone content in micropropagated P. tuberosa was similar to that found in field-grown plants. For both species, the aerial parts accumulated higher β-ecdysone content than roots. These results reveal that micropropagation is a successful, alternative method for rapid plant multiplication of both species of Brazilian ginseng. Furthermore, this study demonstrates that these two species have a potential for cultivation that is associated with high β-ecdysone production.     

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.     

Use of bioreactor systems in the propagation of forest trees

Plant biotechnology can be used to conserve the germplasm of natural forests, and to increase the productivity and sustainability of plantations. Both goals imply working with mature trees, which are often recalcitrant to micropropagation. Conventional in vitro culture uses closed containers and gelled medium with sugar supplementation. Bioreactor culture uses liquid medium and usually incorporates aeration. The increased absorption of nutrients via the liquid medium together with the renewal of the air inside the bioreactors may improve the physiological state of the explants. In this review, we will explore the feasibility of using bioreactors to overcome the recalcitrance of many trees to micropropagation and/or to decrease the cost of large‐scale propagation. We will focus on the recent use of bioreactors during the multiplication, rooting (plant conversion in the case of somatic embryos), and acclimation stages of the micropropagation of axillary shoots and somatic embryos of forest trees (including some shrubs of commercial interest), in both temporary and continuous immersion systems. We will discuss the advantages and the main obstacles limiting the widespread implementation of bioreactor systems in woody plant culture, considering published scientific reports and contributions from the business sector.     

The usefulness of semi‐solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka,Japan

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi‐solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira‐positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi‐solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi‐solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.     

Effect of growth regulators on rapid micropropagation and psoralen production in <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L.

A rapid and efficient micropropagation system was developed for Psoralea corylifolia, an endangered, valuable medicinal plant. Multiple shoot buds were obtained in half-strength liquid Phillips–Collins (L2) medium supplemented with 5 μM benzylaminopurine (BA) and 5 μM thidiazuron (TDZ) from apical bud explants of 1-week-old cultures. The shoot buds were subcultured on enriched solid L2 medium supplemented with different concentrations and combinations of BA, kinetin (KIN), 2-isopentenyladenine (2iP), TDZ, bavistin (BVN) and trimethoprim (TMP). Enriched solid L2 medium supplemented with 2 μM BA, 1 μM TDZ and 100 mg l⁻¹ BVN were more effective in producing greater number of shoots per explant (85.2 ± 0.9 shoots/explant) after 4 weeks of culture. The regenerated shoots (40–50 mm in length) rooted and accompanied by hardening upon transfer to 50 μM indole-3-butyric acid (IBA) for 15 min and followed by planting in sterile soil mixture and vermiculate (3:1 v/v), with 50 ml of one-eight strength L2 basal salt solution devoid of sucrose and inositol, supplemented with 5 μM IBA and 100 mg l⁻¹ BVN. The plants achieved 100% rooting with hardening. Subsequently the rooted plants were successfully established in the field. The survival percentage differed with seasonal variations. The concentration of psoralen was evaluated in different tissues of ex vitro and in vivo grown plants by high-performance liquid chromatography (HPLC). Psoralen content was increased in leaves (2.97%), roots (2.38%), stems (5.40%) and seeds (1.63%) of ex vitro plants than the in vivo plants. This system facilitates for commercial and rapid propagation of P. corylifolia for conservation strategies and phytomedicine production.     

Improving micropropagation of Mentha × piperita L. using a liquid culture system

In the current study, in vitro shoot proliferation and plant regeneration of Mentha × piperita L. (peppermint) cultivar ‘Black Mitcham’ was compared in semi-solid and liquid culture systems. Shoot tips from field-grown plants were used as explants to study shoot proliferation response on either Murashige and Skoog (MS) or Chee and Pool (C2D) medium containing varying levels of 6-benzylaminopurine (BAP), kinetin, and 6-γ,γ-dimethylallyl aminopurine (2iP). Differences in leaf ultrastructure and antioxidant capacity of greenhouse-grown and micropropagation-derived plants were studied to identify potential changes occurring during in vitro culture. Among the various media treatments tested, the maximum number of shoots was produced on the C2D medium with 4.0 μM BAP (40.7) followed by the MS medium with 4.0 μM BAP (32.2). Among the rooting treatments, shoots on the MS medium with 1.0 μM indole-3-butyric acid (IBA) produced the maximum number of roots (14.4). The number of shoots produced in Liquid Lab Rocker® (LLR) vessels containing liquid C2D medium with BAP (103.4) was significantly higher than that produced on semi-solid medium (40.7). No differences were observed in the leaf ultrastructure and antioxidant capacity of leaf extracts obtained from greenhouse-grown and micropropagation-derived plants. The study indicates that the liquid culture system under the described conditions can enhance peppermint micropropagation, with plant material being potentially valuable for use in herbal supplements and essential oil production.

     

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     

In vitro culture of the endangered plant Eryngium viviparum as dual strategy for its ex situ conservation and source of bioactive compounds

Different Eryngium species have been used with ornamental, agricultural and medicinal purposes, as a consequence of their chemical constituents. In the southwest Europe the endemic Eryngium viviparum, presents a high risk of extinction and ex situ strategies are high recommended for efficient conservation and re-introduction program. The objective of this study was to satisfy a dual objective: (i) to develop an ex situ conservation strategy through micropropagation and (ii) taking advantage of the extraordinary potential of plant tissue culture, produce a considerable amount of plant material to carry out a preliminary phytochemical study, based on the accumulation of phenolic compounds and their associated antioxidant activity. First a factorial design was conducted in order to study the effect of two cytokinins (6- benzylaminopurine, BAP, and kinetin, KIN), at three levels (0, 1 and 2 mg L^?1), on shoot multiplication. Later another factorial design was applied, by using three levels of MS medium salt strength (full, half and quarter- strength) and four sucrose levels (0, 1, 2, and 3%) for improving shoot elongation and rooting. In parallel, a preliminary quantification of total phenolic and flavonoid contents from E. viviparum aerial parts was determined. The simple micropropagation protocol designed allowed obtaining a high rates of shoot multiplication (5.1–5.8 new shoots), rooting (100%) with healthy long roots (3.1–3.5 cm) and plantlet acclimatization (96%). Moderate antioxidant activity was recorded in hydromethanolic extracts from E. viviparum aerial parts. High correlation between total phenolic content and BAP levels in the culture media was found. In conclusion, the micropropagation procedure described here for the endangered E. viviparum can be used as new and very efficient ex situ conservation strategy, and as potential source of antioxidants, conferring an added-value to this plant.

     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

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.