Excised Root Culture Research Articles

Identification of Early Salt-Stress-Responsive Proteins in In Vitro Prunus Cultured Excised Roots.

Fruit-tree rootstock selection is a challenge under a scenario of growing environmental stresses in which the soil and climate are greatly affected. Salinization is an increasing global process that severely affects soil fertility. The selection of rootstocks with the ability to tolerate salt stress is essential. Excised root cultures may be an excellent experimental approach to study stress physiology and a predictive tool to assess possible tolerance. In this study, we show how protein changes in response to salt stress evaluated in excised root cultures of Prunus cerasus (moderate salt-sensitive cultivar) could be representative of these changes in the roots of whole plants. The 2D electrophoresis of root extracts and subsequent spot identification by MALDI-TOF/TOF-MS show 16 relevant proteins differentially expressed in roots as a response to 60 mM NaCl. Cytoplasmic isozyme fructose 1,6-bisphosphate aldolase shows relevant changes in its relative presence of isoforms as a response to saline stress, while the total level of enzymes remains similar. Ferredoxin-NADP+ reductase increases as a response to salinity, even though the measured activity is not significantly different. The observed changes are congruent with previous proteomic studies on the roots of whole plants that are involved in protection mechanisms against salt stress.

An efficient protocol for clonal regeneration and excised root culture with enhanced alkaloid content in Thalictrum foliolosum DC.— an endemic and important medicinal plant of temperate Himalayan region

Thalictrum foliolosum DC. is an endemic herbaceous plant known for its various medicinal properties. Due to the presence of valuable alkaloids they are uprooted leading to a threat to their numbers and existence. In this study an efficient protocol has been developed for plant regeneration using hypocotyl explant. Hypocotyl explants formed multiple shoots via direct shoot organogenesis on Murashige and Skoog’s (MS) medium containing 0.25–2.0 mg l−1 BAP with NAA (0.1 mg l−1). Maximum shoot formation in hypocotyl regenerated shoots was achieved in BAP 1 mg l−1and NAA 0.1 mg l-1 with 0.3% activated charcoal and ascorbic acid 20 mg l−1. The regenerated shoots formed roots on MS medium (half strength) containing IBA (0.2 mg l−1). The rooted plantlets were established in the glasshouse after acclimatization and gave 72% survival. Genetic stability assessment of in vitro regenerated plants was done using simple sequence repeat (SSR) markers and no difference was observed when compared to the mother plants. Also, flow cytometric analysis confirmed that their ploidy level was similar to the mother plant. Additionally, establishment of excised root culture was also done and quantification by HPLC revealed that excised cultured roots were accumulated higher benzylisoquinoline alkaloids (BIAs).

Chitosan versus yeast extract driven elicitation for enhanced production of fragrant compound 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in root tuber derived callus of Decalepis salicifolia (Bedd. ex Hook.f.) Venter

Decalepis salicifolia (Bedd. ex Hook.f.) Venter is a new source of the commercially important flavor compound 2-hydroxy-4-methoxybenzaldehyde (2H4MB), a structural isomer of vanillin, with high pharmacological value and used as a flavoring agent at industrial level for making soft drink and bakery products. The natural availability of this high-valued plant is not able to meet the ever-increasing demands due to limited studies. Considering the importance of suspension culture over excised root culture, present communication provides a protocol for the suspension culture of root tuber derived callus of D. salicifolia for the first time. Maximum frequency (84.8%) callus induction was obtained on Murashige and Skoog (MS) medium augmented with (1.0 µM) thidiazuron (TDZ) + (1.0 µM) α-naphthalene acetic acid (NAA). Two important biotic elicitors, chitosan (CH) and yeast extract (YE) at different concentrations (50, 100, 200 and 300 µM) were used as elicitor to investigate the effects on biomass content, 2H4MB content, total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity in cell suspension culture with different contact periods i.e. 24, 48 72 and 96 h. The results showed the supremacy of CH over YE and maximum biomass was 9.7 DW g/l at optimum contact period of 72 h when suspension culture was treated with 200 µM CH. Maximum content of 2H4MB was14.8 µg/g at 200 µM CH and it was found to be 1.4-fold increase in relation to the control (10.8 µg/g). The maximum TPC and TFC were 4.8 mg/g and 4.0 mg/g respectively at 200 µM CH and 72 h of the contact period. The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity (RSA) had also been evaluated and most of the suspension culture showed the correlation with TPC and TFC. The maximum RSA (63.8%) and hydroxyl radical scavenging activity (HRSA) (55.2%) were revealed the ability of plant as a potent antioxidant agent. Thus, a new protocol has been developed for the elicitors-induced enhancement of metabolites and antioxidant activity in suspension culture of D. salicifolia.

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l−1 6-benzyladenine (BA) and 0.1 mg l−1 α-naphthaleneacetic acid and excised root cultures were maintained on ½ MS medium with 0.5 mg l−1 indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.

Influence of cytokinins, basal media and pH on adventitious shoot regeneration from excised root cultures of Albizia lebbeck

A highly reproducible and efficient in vitro shoot regeneration system was developed in a potential medicinal plant, Albizia lebbeck using root explants. Root explants from 15 day-old-aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5 and 10.0 μM) of 6-Benzyladenine (BA), Kinetin (Kn), 2-Isopentenyl adenine (2-iP) singly as well as in combination with α-Naphthalene acetic acid (NAA) (0.1, 0.5, 1.0, 1.5 and 2.0 μM). The highest rate of shoot multiplication (16.0 ± 1.87 for the average shoot number and 5.16 ± 0.38 cm for shoot length) was achieved on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA. The effects of medium type, medium strength, pH and subculture on shoot induction and proliferation were also tested. An average of 21.6±2.87 shoots per explants could be obtained following this protocol. Rooting was achieved on microshoots using half strength MS medium with 2.0 μM Indole-3-butyric acid (IBA) after four weeks of culture. The in vitro raised healthy plantlets were successfully established in earthen pots containing garden soil and grown in greenhouse with >80% survival rate.

Early Detection of Salt Stress Tolerance of Prunus Rootstocks by Excised Root Culture

Salt tolerance varies between species and genotypes of plants, but evaluation of these differences is cumbersome, because whole plants that are highly complex systems show a variety of responses depending on the applied methodology. However, focusing on plant roots, which are in direct contact with the soil, could offer a simpler and more efficient model for analyzing salt stress tolerance in different species. This study explores whether root growth under salt stress is associated with genotypic differences in Prunus species with different degrees of salt tolerance. Excised root cultures were grown in vitro under increasing salt concentrations (0, 20, 60, and 180 mm NaCl). Root tips taken from in vitro-rooted shoots of Prunus species with different salt tolerance were measured after 3 weeks of culture in a shaker, and changes in their anatomy were examined. Both growth and starch content of in vitro root cultures were affected by salt concentration. Root length increments were related to salt stress tolerance at 60 mm NaCl, in which significant differences were also found between species. A significant inverse correlation was found between salt tolerance and starch accumulation in the maturation zone of root tips. Genotypic differences were observed in agreement with species' salt stress tolerance in vivo. These results suggest the use of excised root cultures for rapid, early detection of salt stress tolerance in plants. Chemical names: sodium chloride (NaCl).

Root in vitro cultures of six Gypsophila species and their saponin contents

A simple and rapid method of excised root cultures from six Gypsophila species was performed allowing continuous growth without phytohormones. Established on MH3 medium from solid-grown seedlings these roots were subcultured for 1 year on a solid medium before being transferred in a liquid medium to obtain substantial biomass for saponin content analysis. Morphologically, the different root lines presented different growth behaviors and different physical aspects: some have linear growth by the tip of the main axial root from the original seedling; others have additional lateral root initiations producing a hairy root-system more or less dense. A marked increase of biomass was observed in the light by comparison with dark conditions. Significant growth for Gypsophila glomerata was achieved within 3 weeks on liquid medium; biomass grew up to 50-fold in batch cultures reaching 10 g DW. The fingerprints of the saponin HPLC profiles of the six Gypsophila species were drastically different with at least up to 30 different saponins detected for some of them. The roots of Gypsophila elegans accumulated saponins up to 65 mg/g DW. These amounts were higher than in Gypsophila paniculata roots classically found as the best producing ones. On the contrary the root lines of G. glomerata showed a smaller quantitative amount of saponins (between 1.3 and 7.10 mg/g DW) than those of G. elegans but nearly the same HPLC profiles as for root extracts of G. paniculata plants grown directly in the fields.

Micropropagation through excised root culture of Clitoria ternatea and comparison between in vitro–regenerated plants and seedlings

AbstractAn efficient protocol has been developed for high‐frequency shoot regeneration and plant establishment of Clitoria ternatea – a potential medicinal legume. Adventitious shoots were regenerated from young excised root segments of aseptic seedlings on Murashige and Skoog (MS) medium supplemented with various concentrations of 6‐benzyladenine (BA), kinetin, α‐naphthalene acetic acid (NAA) or 2,4‐dichlorophenoxy acetic acid (2,4‐D) either singly or in various combinations. The highest frequency (100%) of shoot regeneration and maximum number (16.4 ± 0.24) of shoots per explant was obtained on MS medium supplemented with 20 μm BA and 2.0 μm NAA. Organogenic calli were produced on a medium containing 2,4‐D (10 or 20 μm) and BA (5.0 μm). The calli were differentiated into multiple shoots on MS medium supplemented with 2.5–10 μm BA and 2.0 μm NAA. The microshoots were rooted on half‐strength MS medium supplemented with 5.0 μm indole‐3‐butyric acid and transplanted successfully in field conditions. After 12 months of transfer to ex vitro conditions, the performance of micropropagated plants were evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo–grown plants of the same age. The sodium dodecyl sulphate polyacrylamide gel electrophoresis protein profile was same between regenerated and naturally growing shoots. Total soluble protein in aerial part as well as in seeds of in vitro–regenerated and in vivo–grown plants was almost the same. The mitotic study showed normal chromosomal movement and numbers (2x = 16).

Agrobacterium rhizogenes-mediated transformation of Brassica oleracea var. sabauda and B. oleracea var. capitata

Agrobacterium rhizogenes A4M70GUS-mediated transformation of Savoy cabbage (Brassica oleracea L. var. sabauda) and two local lines of cabbage (B. oleracea L. var. capitata) was obtained using hypocotyl and cotyledon explants. The percentage of explants which formed roots was very high in all genotypes: 92.3 % in Savoy Gg-1, 64.4 % in cabbage P22I5, and 87.2 % in P34I5. Spontaneous shoot regeneration of excised root cultures grown on the hormone-free medium occurred in all three genotypes. In cabbage lines P22I5 and P34I5 shoot regeneration was higher (9.3 and 2.6 % respectively) than in Savoy cabbage Gg-1 (1.3 %). Transgenic nature of hairy root-derived plants was evaluated by GUS histological test and PCR analysis. All the tested cabbage shoots were GUS positive whilst in a Savoy cabbage GUS expression was registered only in 55 % of tested clones. PCR analysis demonstrated the presence of the GUS gene in regenerated shoot clones and in T1 progeny.

Enhanced colchicine production in root cultures of Gloriosa superba by direct and indirect precursors of the biosynthetic pathway

Excised root cultures of Gloriosa superba reached 7.5 g dry wt l−1 and accumulated 240±40 μg colchicine g−1 cell dry wt after 4 weeks growth. While all precursors (except trans-cinnamic acid) enhanced colchicine content of root cultures without adversely affecting root growth, treatment with p-coumaric acid + tyramine (each at 20 mg l−1) increased colchicine content to 1.9 mg g−1 cell dry wt.

UDP-Glucuronosyltransferase activity is correlated to saponin production in Gypsophila paniculata root in vitro cultures

UDP-Glucuronosyltransferase (UGT, EC 2.4.1.17) activity was detected in excised root cultures of Gypsophila paniculata. The UGT activity (up to 0.8 nkat mg−1 protein) correlated to the total saponin content (2 mg g−1 dw) during the exponential phase of the batch culture. This gives rise to the hypothesis of a direct relationship in the biosynthetic pathway regulation between the enzyme activity and the saponin biosynthesis.

Excised rootstock roots cultured in vitro.

Root cultures have been established successfully for the Prunus rootstocks Adafuel and Adarcias (Prunus×amygdalopersica), A843 (P. armeniaca), Mariana 2624 (P. cerasifera×munsoniana) and Myrobalan 605AD (P. cerasifera), and for the apple rootstock Jork 9 (Malus×domestica). High percentages of root tips grew during the first 15 days and then decreased. Root growth was affected by culture conditions and the composition of the culture medium. Liquid medium was preferable as increasing agar concentrations reduced root growth. Darkness, instead of a 16-h photoperiod, was beneficial for Adafuel, but not the other genotypes. Sucrose at 3% was better than higher concentrations. Full Murashige and Skoog salts sustained better root growth than dilutions to 1/2 or 1/4. The addition of various organic supplements such as coconut water, casein hydrolysate or malt extract did not improve root growth, and sucrose was the best carbon source tested. Data presented here support the notion that excised root culture is an efficient experimental model to study the response to various factors, since controlled variations in the culture medium, such as those studied here, had a very noticeable effect on root length.

Analysis of Timecourse Changes in Root System Morphology of Rice in Excised Root Culture.

The development of cultured seminal root system of rice was analyzed to determine the most appropriate culture period for the characterization of root system morphology. Excised seminal root tips of Taichung Native 1 (TN-1, indica type) and Yukara (japonica type), were cultured for six weeks with the conventional tissue culture method, and their growth was evaluated with developmental, topological and fractal analyses. Developmental root growth parameters, such as the number and length of each root system component, reached almost maximum values at the fourth week in TN-1, and the third week in Yukara. On the other hand, it was possible to characterize the root system morphology by the branching densities of L- and S-type first order lateral roots and the average length of S-type first order lateral roots at the third week. In the timecourse changes of a topological parameter (log a/log μ), which indicates the branching pattern, the difference between cultivars became clear at the third week. The fractal dimension (D), which indicates the complexity of root morphology, was higher in Yukara than in TN-1 throughout the experimental period. This suggested the possibility for characterizing the root system morphology of each cultivar by the D value. Therefore, we concluded that the most appropriate sampling time for the morphological research of cultured seminal root system was three weeks when the conventional excised root culture method is used.

Regeneration of plants from long-term root culture of lime, Citrus aurantifolia (Christm.) Swing.

Excised root culture of lime, C. aurantifolia, was established in Murashige & Skoog medium containing 3% sucrose. De novo shoot bud initiation was recorded in basal medium at a low frequency during three years of continuous culture. The effect of indole-3-acetic acid and benzyladenine (BA) were investigated on root growth and shoot morphogenesis. Inclusion of BA in the culture medium had an important effect on root growth and shoot elongation: the frequency of shoot regeneration was only slightly improved by the growth regulator treatment. Shoot bud differentiation occurred at the proximal region of the root at or just distal to the cut end. Histology of shoot regeneration revealed the endogenous origin of the shoot buds from the pericycle tissue of the root. The regenerated plants contained normal diploid chromosome number (2n=18) and have been established in soil.

In vitro clonal multiplication of the actinorhizal plant Comptonia peregrina

Micropropagation of the actinorhizal plant Comptonia peregrina of the Myricaceae was achieved successfully by the induction of root buds in excised root culture with cytokinin (1.0 μM benzyladenine). Excised root segments with initiated root buds were subcultured in Woody Plant Medium (WPM) lacking growth regulators, developing extensive callus which subsequently gave rise to multiple adventitious buds. Shoot elongation was facilitated by transfer of calluses to more aerated conditions. Root initiation was induced on shoots by brief treatment with auxin (<1 μM indolebutyric acid) and transfer to WPM for plantlet development. Controlled light and aeration in liquid medium were critical conditions for successful micropropagation.

Production of emetine and cephaeline from cell suspension and excised root cultures of Cephaelis ipecacuanha

Production of the ipecac alkaloids, emetine and cephaeline was studied in cell suspension and excised root cultures of Cephaelis ipecacuanha. A two-stage cell suspension culture was developed for enhanced accumulation of the alkaloids. In the first-stage, suspension cultures were established in Murashige and Skoog's (MS) medium containing 2,4-D and NAA which was suitable for cell growth and the second-stage culture system was composed of MS medium containing IBA, IAA and 6% sucrose which favoured alkaloid production. The production of emetine and cephaeline was greatly increased in the two-stage culture method compared to the single-stage culture. Optimal alkaloid synthesis was obtained in excised root culture of the plant in medium composed of half-strength MS salts, IBA (0.25 mgl −1) and 2% sucrose. A discernible higher accumulation of cephaeline in two-stage cell suspension culture as well as in excised root culture in comparison to that of the three-year-old roots was a

Resistance of wheat genotypes to boron toxicity is expressed at the cellular level

The effects of toxic boron (B) concentrations on the growth of wheat genotypes at organ level and cellular level were investigated using excised root culture techniques. At the organ level, wheat genotypes differed for root elongation and lateral root development in response to toxic B concentrations. Genotypes classified as resistant from field studies had longer root axes and more lateral roots than sensitive or moderately sensitive genotypes, but there was no difference in axis elongation between sensitive and moderately sensitive genotypes. At the cellular level, callus production of root explants among genotypes still different at toxic B concentrations: resistant genotypes could produce more callus than sensitive or moderately sensitive genotypes. These results suggest that differences among genotypes in resistance to toxic B concentrations may be related to cell membrane permeability to B since they were also expressed in undifferentiated callus cells. The distinct and consistent differences among genotypes in response to B toxicity both at the organ level and at the cellular level could serve as a basis for selection in a breeding program. The methods were precise and efficient.

Excised tomato root culture as a tool for testing root-knot nematode resistance

The excised root culture inoculation technique proved to be an accurate method for evaluating resistance to root-knot nematodes in tomato (Lycopersicon esculentum). Root cultures of seven tomato cultivars growing in an incubator at 25°C were inoculated with egg masses ofMeloidogyne javanica. Infection severity was determined 5 weeks after inoculation. Nematode-susceptible cultivars were characterized by high galling indices and resistant cultivars by low ones. The excised root culture technique is simple, allows results to be obtained rapidly, and no greenhouse space is needed.

Effect of different kinds of sugars on nodule formation in leguminous plants as examined by excised root culture technique

Abstract It is well known that the nodule formation in leguminous plants is affected by various kinds of environmental factors including the nutrient status in the soils and the aerial conditions surrounding the plants (1, 2). The light intensity and day length have been shown to affect nodule formation remarkably (3, 4), and the effect was ascribed to photosynthesis through which the level of carbohydrates in plants is regulated. Foliar application of sugars also was shown to stimulate nodule formation in plants (3, 5).

Direct Evidence of Sucrose Effect on Nodule Formation and Symbiotic Nitrogen Fixation in Leguminous Plants as Revealed by the Use of Excised Root Culture Method and Acetylene Reduction Assay Method

Direct Evidence of Sucrose Effect on Nodule Formation and Symbiotic Nitrogen Fixation in Leguminous Plants as Revealed by the Use of Excised Root Culture Method and Acetylene Reduction Assay Method