Long-term culturing of plants with aseptic roots Determination of rape root exudates

Abstract A plant culture system allowing growth of aseptic roots has been designed. One version of the system comprises vessels developed for plant root-microorganism interaction studies in series. A second version has been built for measurements of different physiological parameters of the shoot and root system during growth periods of at least 2 months. The system was tested by determining soluble organic carbon glucose and sucrose in the root exudates of rape plants (Brassica napus L.) grown for 3 weeks in the culture system. The plants were cultivated with a sterile or a non-sterile root system, or with a root system infected with Verticillium dahliae Kleb.     

Increase of Scopolamine Production by High Density Culture of Duboisia myoporoides Roots

A circulation culture system was established for the high density root culture of Duboisia roots. It consists of a vessel for root culture, an aeration tube, a medium reservoir, and a peristaltic pump. Medium saturated with pure oxygen was circulated continuously through the culture vessel and the medium reservoir, and was pumped back into the aeration tube by the pump. Duboisia roots could be cultured at densities of up to 120g dry weight (DW) dm^?3 with no decrease in scopolamine content, this density being about 20 times the amount that can be used in ordinary flask culture. The final scopolamine yield at the end of 3 weeks was 1350mgdm^?3.     

Continuous production of scopolamine by a culture of Duboisia leichhardtii hairy root clone in a bioreactor system

The scopolamine-releasing hairy root clone DL47-1 of Duboisia leichhardtii was cultured in an Amberlite XAD-2 column-combined bioreactor system for continuous production of scopolamine. The medium used was continuously exchanged during culture to maintain the electrical conductivity of the medium constant. After culturing the hairy roots in the system for 11 weeks, 0.5 g/l of scopolamine was obtained in the column. When the roots were cultures in the reactor system containing polyurethane foam or stainless-steel mesh to support the hairy roots, scopolamine recovery was increased. Thereafter, a two-stage culture, the first stage in the medium for hairy root growth and the second stage in the medium for scopolamine release, was carried out in this system by using a turbine-blade reactor with stainless-steel mesh as a support. Under these conditions, 1.3 g/l of scopolamine was recovered during 11 weeks of culture in the medium for scopolamine release. This bioreactor system seems applicable for the production of various plant metabolites by cultures of hairy roots. Correspondence to: T. Muranaka     

Induction and Characterization of Adventitious Roots Directly from the Explants of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

Adventitious roots from leafstalks and lateral roots were obtained directly from explants of Panax notoginseng. The lateral root explants were more sensitive to the induction of adventitious roots using indole-3-butyric acid. HPLC analysis of saponins extracted from the adventitious roots indicated that several protopanaxatriol saponins were present but ginsenoside Rd was missing, compared with the saponins extracted from the raw herbs. The dry weight of primary adventitious root culture of Panax notoginseng increased 5.25 times during multiplication in a classical shaking-flask system, suggesting that it is a culture system with great potential for scale-up. Revisions requested 13 July 2005; Revisions received 1 September 2005     

QTLs and epistasis for seminal root length under a different water supply in rice (Oryza sativa L.)

To identify the genetic background of seminal root length under different water-supply conditions, a recombinant inbred (RI) population consisting of 150 lines, derived from a cross between an indica lowland rice, IR1552, and a tropical japonica upland rice, Azucena, was used in both solution culture (lowland condition) and paper culture (upland condition). Quantitative trait loci (QTLs) and epistatic loci for seminal root length were analyzed using 103 restriction fragment length polymorphism (RFLP) markers and 104 amplified fragment length polymorphism (AFLP) markers mapped on 12 chromosomes based on the RI population. One QTL for seminal root length in solution culture (SRLS) and one for seminal root length in paper culture (SRLP) were detected on chromosomes 8 and 1, and about 11% and 10% of total phenotypic variation were explained, respectively. The QTL for SRLP on chromosome 1 was very similar with the QTL for the longest nodal root referred to in a previous report; this QTL may be phenotypically selectable in a breeding program using paper culture. Five pairs of epistatic loci for SRLS were detected, but only one for SRLP, which accounted for about 60% and 20% of the total variation in SRLS and SRLP, respectively. The results indicate that epistasis is a major genetic basis for seminal root length, and there is a different genetic system responsible for seminal root growth under different water supply conditions. Received: 26 May 2000 / Accepted: 19 October 2000     

Ara-Rhizotron: An Effective Culture System to Study Simultaneously Root and Shoot Development of Arabidopsis

Studying Arabidopsis thaliana (L.) Heynh. root development in situ at the whole plant level without affecting shoot development has always been a challenge. Such studies are usually carried out on individual plants, neglecting competition of a plant population, using hydroponic systems or Agar-filled Petri dishes. Those both systems, however, present some limitations, such as difficulty to study precisely root morphogenesis or time-limited culture period, respectively. In this paper, we present a method of Arabidopsis thaliana (L.) Heynh. cultivation in soil medium, named “Ara-rhizotron”. It allows the non-destructive study of shoot and root development simultaneously during the entire period of vegetative growth. In this system, roots are grown in 2D conditions, comparable to other soil cultures. Moreover, grouping several Ara-rhizotrons in a box enables the establishment of 3D shoot competition as for plants grown in a population. In comparison to a control culture grown in pots in the same environmental conditions, the Ara-rhizotron resulted in comparable shoot development in terms of dry mass, leaf area, number of leaves and nitrogen content. We used this new culture system to study the effect of irrigation modalities on plant development. We found that irrigation frequency only affected root partitioning in the soil and shoot nitrogen content, but not shoot or root growth. These effects appeared at the end of the vegetative growth period. This experiment highlights the opportunity offered by the Ara-rhizotron to point out tardy effects, affecting simultaneously shoot development and root architecture of plants grown in a population. We discuss its advantages in relation to root development and physiology, as well as its possible applications.     

Scopolamíne Yield in Cultured Roots of Duboisia myoporoides Improved by a Novel Two-stage Culture Method

Localization analysis of scopolamine and hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11) contents in Duboisia root cultures showed that scopolamine biosynthesis was accompanied by root elongation. A new two-stage culture method in which the first stage promotes lateral root induction and the second stage enhances root elongation therefore was devised to obtain high scopolamine production. Consequent development of the most suitable media for each stage of culture increased scopolamine productivity 1.8 times that obtained with a one-stage culture. A total of 2500 mg dm ^?3 of scopolamine was produced when this two-stage culture was combined with our high-density culture method.     

Adventitious root induction inCorylus avellana L. cotyledon slices

Summary An experimental rooting system was developed to study in vitro adventitious root formation in hazelnut cotyledons. Experiments involveda) assay of several culture media,b) use of different developmental status of the seeds (germinated and ungerminated),c) cotyledons subjected to various light regimes, andd) different size of cotyledons slices. It was observed that higher rooting was induced in cotyledonary portions of 5- or 7-mm thickness (250 and 350 mm³, respectively) cultured on half-strength basal medium supplemented with 50 μM indole-3-butyric acid and 5 μM kinetin. Rooting was affected by light and the developmental state of seeds. Preinitiation, initiation, and root manifestation stages were defined according to specific culture periods and in relation with morphologic and histologic changes. The first histologic changes (cell division and root primordia induction) were observed after 12 days in culture. At 30 days of culture in rhizogenic medium root primodia were fully differentiated with well-developed vascular tissues.     

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5±1.13 and 0.34±0.023 mg g⁻¹ dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71±0.043 vs. 6.59±0.56 mg g⁻¹ dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l⁻¹ syringin.     

Micropropagation of the endangered <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis> Baijnath

Summary The species, Kniphofia leucocephala is extant at only one location, Langepan, KwaZulu-Natal in South Africa, where the population is threatened by afforestation and possibly grazing. Consequently, a continuous culture system was established as part of a program for the propagation and re-introduction of plants into the wild. The efficiency of the system in terms of shoot multiplication and, particularly, the frequency and rate of root initiation was strongly influenced by the concentration of benzyladenine in the shoot multiplication medium. The optimum shoot multiplication medium for subsequent root initiation contained 2 mgl⁻¹ (8.9 μM) benzyladenine alone. The shoots were successfully rooted and acclimatized. Approximately 200 shoots can be produced from one shoot after five 4-wk cycles. Thus, large numbers of plantlets can be propagated in this continuous culture system, serving conservation interests.     

Somatic embryogenesis,regeneration and <Emphasis Type="Italic">in vitro</Emphasis> production of glycyrrhizic acid from root cultures of <Emphasis Type="Italic">Taverniera cuneifolia</Emphasis> (Roth) Arn.

Taverniera cuneifolia (Roth) Arn. or Indian licorice is considered to be a substitute for Glycyrrhiza glabra L. owing to an equivalent content of glycyrrhizic acid (GA). GA recognized as the main active ingredient of T. cuneifolia, GA imparts several medicinal properties to these plants. However, research on this plant is scanty with no published record on tissue culture studies. Present study demonstrates a method for (1) induction and successful development of somatic embryos from the root culture, (2) regeneration of plants, and (3) GA production by root cultures of T. cuneifolia. Shoot initiation frequency of cultured roots ranged from 52.57% to 97.71%. Plant regeneration frequency (germination) from somatic embryos was 88.66% in 1/4-strength Murashige and Skoog (MS) medium supplemented with 2% sucrose, as against 68% in half 1/2-strength MS containing 2% sucrose. Glycyrrhizic acid of 1.32 mg/gm of dry weight was obtained in full-strength MS medium supplemented with 3% sucrose, through high-performance thin layer chromatography analysis with standard GA in root cultures. The present study provides an efficient method for the mass production of plants from a single mother plant as well as the potential root culture system to study the GA production pathways in T. cuneifolia.     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.     

Dose-, time-, and tissue-dependent effects of 5-bromo-2′ -deoxyuridine on the in-vitro organogenesis of Arabidopsis thaliana

Vigorous organogenesis can be induced from hypocotyl and root explants of Arabidopsis thaliana using a two-step culture procedure consisting of preculture on callus-inducing medium (CIM) and subsequent culture on shoot-inducing medium (SIM) or root-inducing medium (RIM). With this culture system, we examined the influence of 5-bromo-2′-deoxyuridine (BrdU), a thymidine (dT) analogue, on plant organogenesis in vitro. Treatment with BrdU during SIM or RIM culture had negative effects on shoot and root redifferentiation over a broad range of concentrations. When explants were exposed to low concentrations of BrdU during preculture and then transferred onto BrdU-free SIM, shoot redifferentiation was accelerated significantly. At higher doses, BrdU treatment during the pre-culture inhibited shoot redifferentiation strongly in hypocotyl explants, but not in root explants. This suggests that a target of the BrdU action lies within the process of acquisition of cell proliferation competence specifically involved in hypocotyl dedifferentiation. These effects of BrdU were counteracted by the simultaneous addition of excess dT. BrdU-pretreated and untreated explants did not differ significantly in the phytohormone dependency of shoot redifferentiation. Our results provide a basis for future studies on plant organogenesis combining pharmacological analysis with BrdU as a probe and molecular genetics with Arabidopsis mutants.     

Artificial seed preparation as the efficient method for storage and production of healthy cultured roots of medicinal plants

The technique for the refinement of pRi T-DNA-transformed root cultivation by the root fragment encapsulation in the gel coat, i.e., so-called “artificial seed” (AS) production, was studied. AS were produced from genetically transformed roots of Baikal skullcap (Scutellaria baicalensis Georgi) and common rue (Ruta graveolens L.). The effects of duration of AS storage at 4°C on their subsequent growth activity and a capability for resumption of actively growing root cultures were analyzed. Encapsulation of contaminated Baikal skullcap root culture with the addition of antibiotic and storage during 2–5 weeks at low above-zero temperature resulted in a complete elimination of infection, i.e., obtaining the healthy root culture. Growth activity and total flavone concentration were markedly increased in this culture, so that total productivity of this renewed root culture increased substantially. Using AS produced from the root fragments of common rue, it was shown that, after long-term storage at low above-zero temperature, they are capable of not only root growth resumption but also active shoot formation, which is of interest for plant micropropagation. Long-term retaining growth activity of AS produced from root cultures of valuable medicinal plants permits their usage as a reserve and also, in the case of necessity, for long-distance transport as compact axenic root inocula. The storage of viable root fragments within AS also helps to optimize intervals between numerous subculturings of root cultures required for the maintenance of IPPRAS collection in the active state.     

Turf strength and root characteristics of ten turfgrass cultivars

Ten turfgrass cultivars were grown, singly and in mixtures, in glasshouse culture, to create standard sods. The tearing strength of the sods was measured using an Instron Universal Testing Instrument. Two Poa cultivars were found to be the strongest, followed in order of decreasing strength by three Festuca cultivars, three Lolium cultivars and two Agrostis cultivars. The relationship between the values obtained and shear strength of the sod root mass was investigated with a view to using this latter measurement as an indicator of tearing strength. No correlation between the two was found. Various characteristics of the root mass of individual plants were examined in an attempt to account for differences in turf strength: root system architecture (magnitude, altitude, pathlength and link numbers and lengths), root numbers at different soil depths and root mass. Sod tearing strength was found to be very strongly inversely related to the interior link length and mass of roots. The use of these as indices of strength is discussed.     

Establishment of adventitious root culture of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni in a roller bottle system

Cultures of adventitious roots of Stevia rebaudiana (Bert.) Bertoni were performed in a roller bottle system for the production of both primary and secondary metabolites. Adventitious roots were induced from 1-cm-long root tip explants derived from in vitro regenerated plantlets on solid Murashige and Skoog (MS 1962) media supplemented with 10.7 μM of α-naphthaleneacetic acid. These cultures were successfully maintained in the same medium for 6 months with regular subcultures after 4 weeks. Thereafter, the roots were cut into 1.0- to 1.5-cm-long segments and transferred to the roller bottle system containing a fresh root tissue culture on liquid MS medium supplemented with 10.7 μM NAA. The apparatus consisted of a flask rolling system adjusted to 4g, and 3° of flask inclination. The roots were allowed to grow in the absence of light for adaptation and adventitious root formation. The best conditions for cultivation were investigated, considering culture volume (25 ml), culture period (4 weeks), salt concentrations in the nutrient medium (33%) and optimal initial inoculum (0.2 g) of S. rebaudiana roots. These results could give important information on how to improve the development of adventitious roots of S. rebaudiana for the production of primary and secondary metabolites.     

Compared root system architectures in seedlings and in vitro plantlets of Hevea brasiliensis,in the initial years of growth in the field

In vitro culture of Hevea was undertaken to propagate selected clones on their own roots. The challenge was to overcome the failure of cuttings due to the poor conformity of regenerated root systems. Trees of several juvenile or mature genotypes were propagated either by in vitro microcutting, or by somatic embryogenesis, and planted in the field. Certain static and dynamic components of the root system were observed at different growth stages, from 0 to 3 years, and compared to those of seedlings of the same age used in the trial as a reference. A simple method was designed for measuring the vigour and balance of the root system. The in vitro plantlets had a well-developed taproot and lateral root system, with an architecture similar to that of plants obtained from seed. Moreover, clear differences occurred between selected clones for the relative vigour of the tap roots, lateral roots and trunk. 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.     

不同培育方式对橡胶树实生苗生理指标、养分及长势的影响

橡胶树(Heveabrasiliensis)种子催芽生长一般使用沙床培育,沙子是不可再生资源,为了选择一种适合橡胶树种子培育方式来替代对沙子的依赖,该研究通过水培、悬空培育和传统的沙培比较橡胶树实生苗第1蓬叶稳定时,苗木的生长势、生理指标及养分含量。结果表明,水培实生苗地上部株高、茎粗、叶面积的长势最佳,壮苗指数和生物量的含量最高,但其根太长,根相对较细。水培的叶、茎、根的可溶性糖、丙二醛、游离脯氨酸、超氧化物歧化酶的含量均较低;水培和悬空培育的叶片和茎的叶绿素、类胡萝卜素及根系活力的含量没有显著性差异,均高于沙培。水培的叶、茎、根中的氮和磷含量最低,沙培的最高;而水培实生苗根和茎中钾的含量较高,叶片中含量与悬空培育、沙培均没有显著性差异;悬空培育在叶、茎、根中钾的含量最低。水培促进了苗木的生长,降低干旱胁迫,提高养分利用率,但后续还需调控根系,建设良好根团。悬空培育的苗木长势较弱,还需进一步完善方法。     

Stress-induced changes in the root architecture of white lupin (Lupinus albus) in response to pH, bicarbonate, and calcium in liquid culture

Current agronomic cultivars of white lupin (Lupinus albus) are intolerant of calcareous or limed soils. In these soils, high pH, bicarbonate (HCO₃^?), and calcium (Ca) concentrations are the major chemical stresses to the root system. To determine the responses of the root system to these factors, evaluate root architecture, and compare genotypes for tolerance, a series of liquid culture experiments was completed using root chambers that allowed the study of the root system in two dimensions. Each stress condition caused changes in different parts of the root system and there was no generalised stress response. HCO₃^? (5 mM) had the greatest effect on cultivars intolerant of calcareous soil; it decreased the dry weight of the shoot and caused the highest percentage of tap root deaths. HCO₃^? also discriminated between short (determinate) and long (indeterminate) roots, as it decreased the number and density of the determinate roots only. Calcium (3 mM) affected all parts of the root system. The tap root was shortened and showed an increased tortuousness in its path compared with 1 mM Ca, although no plants suffered tap root death. The numbers and densities of the two lateral root forms were also decreased, as were the lengths of the indeterminate roots. Stress from alkaline pH (7.5) media caused a lower number and density of determinate lateral roots to be produced than at pH 6.5. The experiments demonstrated that each culture condition elicited a definable stress response. Stress conditions altered the root architecture of genotypes reported to be tolerant of calcareous soil less than in intolerant genotypes. Although soil is more complex than liquid culture, it is possible that in a calcareous or limed soil each stress condition examined may affect the overall stress of the plant, and increased tolerance may result from tolerance to a single stress.