Timing of root dormancy and tolerance to root waterlogging in clonal Sitka spruce

 Actively growing root tips of Picea sitchensis (Bong.) Carr. plants are highly susceptible to damage if waterlogged, but they are known to have some tolerance after they stop growing in the autumn. This paper describes the selection of clones on the basis of root dormancy timing and the corresponding responses of their roots to over-winter waterlogging. Sitka spruce transplants of Alaska, Queen Charlotte Islands (QCI), and Washington provenances were screened for early or late root dormancy over 2 successive years. Cuttings were propagated from the selected plants and after growing on for 2 years, they were planted in transparent acrylic tubes within outdoor ‘root observation chambers’. Extension of main roots and the timing of onset of root dormancy was recorded on the clonal plants. The tubes were flooded in November and maintained with a water table 280 mm below the soil surface until March of the next year. Waterlogging caused most main root tips to die back, but within 2 months of draining regeneration occurred on the main roots below the waterlogging level. This regeneration was most commonly the growth of existing lateral tips or production of new lateral roots. Roots of early-dormant Washington plants died back on average 129 mm less than late-dormant Washington plants, and early-dormant Alaska plants had 173 mm less dieback than late-dormant Alaska plants. Differences between the clones of the QCI provenance were not significant. The 40% and 52% increases in survival depth of roots in early-dormant Washington and Alaska clones respectively indicates a potential for improving the rooting depth of Sitka spruce on seasonally waterlogged soils by planting clones selected on the basis of root dormancy. Received: 14 July 1997 / Accepted: 15 September 1997     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated transformation of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> and production of flavonoids in hairy roots

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A₄GUS, R1000 LBA 9402 and ATCC11325. The A₄GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B₅ medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A₄GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).     

Variations in the Phenolic Contents of Cocoyam Clones in Correlation to Resistance to Pythium Myriotylum

Phenolic compounds from leaves and roots of infected and healthy cocoyam clones resistant (RO1075), tolerant (RO1043), and susceptible (RO1157) to Pythium myriotylum were quantified and tested for their in vitro fungitoxicity on the causal agent of the cocoyam root rot disease. All clones, infected or not, have phenolic compounds showing fungitoxic activity. The phenolic content of the tolerant and susceptible clones is less than that observed in the resistant one meanwhile in the resistant clone RO1075, a large increase in phenolic content is observed particularly in the roots during attack by pathogen.     

Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots

Transgenic hairy roots were induced from petiole and root segments of in vitro plant Aralia elata, a medicinal woody shrub, after co-cultivation with A. rhizogenes ATCC 15834. The percentage of putative hairy root induction from root segments was higher (26.7%) than petiole explants (10.0%). Hairy roots showed active production of lateral roots with vigorous elongation. Transgenic plants were regenerated from hairy roots via somatic embryogenesis. These plants had wrinkled leaves, short petioles and numerous lateral hairy roots. The RT-PCR analysis showed the expression of rol A, B, C, D, aux 1 and 2 genes differed between the transgenic lines. Endogenous IAA level was higher in transgenic than non-transgenic plants. Conclusively, transgenic hairy roots were developed for first time in A. elata and the transgenic hairy root lines showed distinct morphological growth pattern and gene expression.     

Development of a potent <Emphasis Type="Italic">in vitro</Emphasis> source of <Emphasis Type="Italic">Phylanthus amarus</Emphasis> roots with pronounced activity against surface antigen of the hepatitis B virus

Summary In vitro culture of hairy roots of Phyllanthus amarus induced by Agrobacterium rhizogenes was established. Their growth and ability for in vitro inactivation of hepatitis B virus surface antigen was studied and compared with adventitious roots grown in vitro. The selected hairy root clone HR-1 was capable of growing at a very fast rate, and an approximately 900-fold increase in weight of root biomass was achieved after 4 wk of culture in hormone-free quarter-strength liquid Murashige and Skoog medium with continuous agitation. Non-transformed roots cultured in the presence of 1.0 mg l⁻¹ (5.71 μM) indole-3-acetic acid increased by 330-fold. The immuno-inactive property of roots was maximal in the crude extract. The hairy roots were shown to possess 85% inhibition (in contrast to 15% in the control) in binding of hepatitis B surface antigen (HBsAg) to its antibody (anti-HBs) after 24 h of incubation with HbsAg-positive sera in vitro at 37°C. Out of three fractions selected on the basis of molecular weight components of the extract, the Fraction III containing comparatively lower molecular weight substances (≤3500) yielded the highest activity. The extract from non-transformed roots was found to possess similar efficiency (87% inhibition). The levels of activity in both types of in vitro-raised roots were higher than those of naturally occurring roots and leafy shoots. The ability of P. amarus hairy root cultures to yield high biomass with the anti-viral property at high levels may provide an alternative source of raw material for more detailed study in the field of pharmaceutical research.     

Lateral roots affect the proteome of the primary root of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Lateral roots are initiated from the pericycle cells of other types of roots and remain in contact with these roots throughout their life span. Although this physical contact has the potential to permit the exchange of signals, little is known about the flow of information from the lateral roots to the primary root. To begin to study these interactions the proteome of the primary root system of the maize (Zea mays L.) lrt1 mutant, which does not initiate lateral roots, was compared with the corresponding proteome of wild-type seedlings 9 days after germination. Approximately 150 soluble root proteins were resolved by two-dimensional electrophoresis and analyzed by MALDI-ToF mass spectrometry and database searching. The 96 most abundant proteins from a pH 4–7 gradient were analyzed; 67 proteins representing 47 different Genbank accessions were identified. Interestingly, 10 (15/150) of the detected proteins were preferentially expressed in lrt1 roots that lack lateral roots. Eight of these lrt1-specific proteins were identified and four are involved in lignin metabolism. This study demonstrates for the first time the influence of lateral roots on the proteome of the primary root system. To our knowledge this is the first study to demonstrate an interaction between two plant organs (viz., lateral and primary roots) at the level of the proteome.     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

The relationships between static and dynamic variables in the description of root growth. Consequences for field interpretation of rooting variability

Field root investigations are often limited by the static nature of classical observations, resulting in the need to develop alternative methodologies that allow dynamic interpretation of root architecture variability on the basis of static measurements. The objectives of this work were (i) to evaluate the use of selected morphological indicators, namely root apical diameter (Da) and the length of the apical unbranched zone (LAUZ), in predicting primary and lateral root growth patterns in banana trees, (ii) to propose a field methodology for the assessment of root dynamics based on static measurements. Banana trees (Musa acuminata cv `Grande Naine') were grown in 5 rhizotrons as well as in field conditions, respectively on pouzzolane and Mollic Andosols. In rhizotrons, root growth analysis was carried out by reporting root elongation, Da and LAUZ, three times a week. In field conditions, 4 series of excavations were made at three-week intervals. Apart from root growth rate, measurements were the same as those in the rhizotrons. LAUZ was confirmed as a stable and good predictor of root growth rate for the different types of roots. In the rhizotrons, the root growth of lateral roots was found to be well correlated to the product of Da and the growth rate of the bearing root. Evaluation in field conditions from static observations attested consistent relationships between measured and predicted root length for lateral roots (slopes close to 1:1). The apical diameter can be considered as a good indicator of root growth potential, while actual lateral root growth depends on the bearing root elongation rate. Morphological static indicators calibrated from growth dynamics in rhizotrons are of major interest in explaining growth variability in field conditions. Especially the `growth rate-LAUZ' relationship can be considered a useful tool in interpreting field patterns of growing roots in relation to various soil conditions.     

Use of gentian violet to differentiate in vitro and ex vitro-formed roots during acclimatization of grapevine

The dye gentian violet was added to culture medium in order to distinguish in vitro and ex vitro-formed roots during acclimatization of micropropagated plantlets. Shoots of the grapevine rootstock Kober 5BB were rooted on media containing the dye (0.3 and 0.15 mg·l^–1) for 3 weeks. The dye coloured the roots. Root length was reduced by the presence of the dye, but root number and shoot growth were not affected. Most in vitro-formed roots continued to grow during acclimatization, and 3 weeks after the transfer to soil the root system was 60% composed of in vitro-formed roots. Our results suggest that in grapevine Kober 5BB, the in vitro-formed roots contribute to plantlet growth at least during acclimatization.     

STRUCTURE OF CLONES OF NORTHERN PRICKLY ASH (XANTHOXYLUM AMERICANUM)

Clones of northern prickly ash (Xanthoxylum americanum) were excavated to expose their horizontal root structure and pattern of vegetative spread; stems and horizontal roots were mapped. Most of the root system was very shallow (< 10 cm deep). Stems and roots were aged from annual rings. The four clones studied ranged in age from 26 to 33 yrs. All root connections between shoots were maintained for the life of the clone. Clones were found to be single individuals in a physical as well as a genetic sense. There was virtually no overlap of adjacent clones; horizontal roots turned away or stopped growing when the border of another clone was encountered. No differences were found between male and female clones in above- or below-ground morphology. There was, however, a great deal of intrasexual variation among clones.     

Symptomatic Evidence for Differential Root Invasion by Fusarium Crown and Root Rot Pathogens Between Common Tomato Lycopersicon esculentum and Its Varieties

The pathogenic isolates (Kin2001a, Kin2001b and Kin2003) of Fusarium oxysporum f. sp. radicis‐lycopersici were obtained from hydroponically cultured seedlings of pear tomato (Lycopersicon esculentum var. pyriforme) infected at different times and their pathogenicity examined in an in vitro assay system on cotyledonal seedlings of pear tomato, cherry tomato (L. esculentum var. cerasiforme) and common tomato (L. esculentum). With the in vitro assay, infection and subsequent disease progress could be microscopically observed. Pear and cherry tomatoes suppressed invasion by all isolates at the junctions of epidermal cells along the root, comparable with the resistant cultivars of common tomato. The pathogen entered pear and cherry tomatoes at the tips of lateral roots and tap roots, in contrast to infection of susceptible cultivars of common tomato. In Kin2003‐inoculated roots, the top of the lateral rootlets first became discoloured, followed by the cortical parenchyma, central xylem vessel and finally the crown. This dark‐brown discolouration expanded rapidly and severe rot developed in the discoloured regions. In contrast, the dark‐brown discolouration in Kin2001b‐infected roots expanded into the cortical parenchyma cells abutting the originally infected lateral rootlets and at a much slower rate. Kin2001a was in a new group that entered via the cortical cleavage formed by the emergence of lateral rootlets, in addition to the tips of taproots and lateral roots. In this in vitro assay system, the Japanese pathogenic isolates collected from different districts of Japan were characterized and classified by the mode of host invasion. Of 13 isolates, four were placed with Kin2003, six with Kin2001a and three with Kin2001b.     

Lateral root development and saponin accumulation as affected by IBA or NAA in adventitious root cultures of <Emphasis Type="Italic">Panax ginseng</Emphasis> C.A. Meyer

Summary The effect of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) on lateral root formation was investigated in adventitious root culture of Panax ginseng. Lateral root formation was affected by IBA (24.6 μM) or NAA (9.8 μM). Lateral root primordia emerged from the explant root pericycle after about 7 d of culture when the roots were cultured on Schenk and Hildebrandt (SH) medium supplemented with 24.6 μM IBA or 9.8 μM NAA. However, no changes were observed in the explant root pericycle on auxin-free medium. The IBA treatment was more effective for lateral root induction and root growth compared to NAA. In morphological and histological aspects, the lateral roots formed under IBA treatment developed normally, while NAA-treated roots exhibited abnormal growth. The accumulation of total saponin was greater in roots treated with IBA than with NAA.     

Root porosity and oxygen movement in waterlogging-tolerant Trifolium tomentosum and -intolerant Trifolium glomeratum

Trifolium tomentosum and T. glomeratum are small (< 0·5 mg) seeded pasture legumes which are considered to be waterlogging tolerant and intolerant, respectively. The root porosity of the two species was compared for plants raised for 10 d in aerated nutrient solution and then transferred to either aerated (0·25 mol O₂ m^–3) or ‘hypoxic’ (0·031–0·069 mol O₂ m^–3) solutions for a further 7 and 21 d. After 21 d, T. tomentosum developed a root porosity of 11·2% in ‘hypoxic’ solution, which was significantly higher than the 6·1% developed by T. glomeratum. When grown in aerated solution, T. tomentosum also had a larger constitutive porosity (6·7%) than T. glomeratum (3·9%). Cylindrical root-sleeving O₂ electrodes were used to measure the rates of radial O₂ loss (ROL) from roots of the two species when in an O₂-free medium. In general, roots previously grown in ‘hypoxic’ solution had higher rates of ROL than roots grown in aerated solution. Moreover, the rates of ROL along the main root of T. tomentosum were ≈ 5-fold faster than from equivalent locations along roots of T. glomeratum. Manipulations of the shoot O₂ concentration resulted in rapid changes in ROL near the root tip of T. tomentosum plants raised in aerated or ‘hypoxic’ solutions, whereas for T. glomeratum ROL only increased for roots of plants raised in ‘hypoxic’ solution. Thus, the cortical air spaces in roots of both species raised in ‘hypoxic’ solution formed a continuous, low resistance pathway for O₂ diffusion from the shoots to the roots. ROL from the lateral roots was also evaluated and it was 3-fold faster from T. tomentosum than from T. glomeratum. Moreover, ROL from lateral roots of T. tomentosum was 10–20-fold higher than from a position on the primary root axis the same distance from the root/shoot junction. Relatively, high rates of ROL were also recorded for young (40 mm in length) lateral roots of T. glomeratum which were previously grown in ‘hypoxic’ solution, but the ROL was low for the older lateral roots of this species. The substantial amounts of ROL from the lateral roots may limit O₂ supply to the lower parts of the primary root axis, so that the laterals probably become the main functional root system in waterlogged soils.     

Effects of the Temperature of the Rooting Zone on the Growth and Development of Roots of Potato (Solanum tuberosum)

In solution culture the effect of optimal (20 °C) and supra-optimal(30 °C) root-zone temperatures on root growth and root morphologyof six potato clones (Solanum tuberosum L spp) was studied Growthwas compared with sweet potato (Ipomoea batatas L) and cassava(Manihot esculenta Crantz) Significant genotypical differencesin the responses of potato roots to supra-optimal temperatureswere observed, indicating the potential for selecting heat tolerantpotato clones In both heat tolerant and heat sensitive clones,the size of the root system was reduced by supra-optimal root-zonetemperature This was principally a result of decreased numberand length of lateral roots The first symptom of heat damagewas a reduction in the rate of cell division, followed by cessationof root elongation Bending of the apical root-zone togetherwith the formation of root hairs on the inner (concave) andlateral roots on the outer (convex) side were other symptoms,these coincided with the loss of starch granules in the root-cap Potato, genotypical differences, root morphology, root growth, temperature, Solanum tuberosum L     

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     

Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of <Emphasis Type="Italic">Alyssum murale</Emphasis> Waldst et Kit

Shoot cultures of nickel hyperaccumulating Alyssum murale were established from epicotyl explants of seedlings aseptically germinated on hormone-free MS medium. They were further maintained on media with 0–0.92 μM kinetin. Optimal shoot multiplication was at 0.46 μM kinetin. Inoculation by shoot wounding was performed with overnight suspension of A. rhizogenes A4M70GUS which contains GUS gene cointegrated in pRiA4. After 30 days hairy roots were produced at the wounding site in 31 explant (25% out of 124). Hairy roots were excised and further propagated on hormone-free medium as separate clones. In the first passage clones 3 and 6 could be distinguished by fast growth and spontaneous shoot regeneration. In other clones (12, 23 and 25) shoot regeneration required presence of cytokinins. The five shoot culture clones regenerated from hairy roots were further cultured on media with 0.46 μM kinetin. These shoots were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus all plants regenerated from hairy root cultures manifested the characteristic Ri syndrome phenotype. They all had a strong positive GUS reaction. PCR analysis confirmed presence of uidA sequence from the gus construct. They were also tolerant to nickel accumulating up to 24,700 μg g⁻¹ dry weight.     

In vitro Regeneration and Transformation of Blackstonia perfoliata

In vitro root culture of yellow wort (Blackstonia perfoliata (L.) Huds.) was initiated on Murashige and Skoog (MS) medium. In the presence of benzylaminopurine (BAP) numerous adventitious buds formed, which developed into shoots. Presence of indole-3-butyric acid (IBA) in media significantly decreased number of buds, but increased development of lateral roots. On hormone-free medium shoots successfully rooted and developed flowers and viable seeds that formed another generation. Shoot cultures of B. perfoliata inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS developed hairy roots at 3 weeks and they were cultured on hormone-free MS medium. Spontaneous shoot regeneration occurred in 3 clones.     

Micropropagation of Ficus benjamina clones

In vitro culture of eight Ficus benjamina clones was initiated from shoot tips four times from January to June 1988. Shoot formation and growth in vitro were followed during eight subsequent subcultures, whereafter the developed shoot clusters were rooted in vitro. Significant differences among clones in proliferation rate and time to emergenece of first root in vitro were observed. A superior clone Cleo, previously selected for fast growth as a potted plant, also proved to have the highest proliferation rate and the shortest time until emergence of first root in vitro. The proliferation rate was nearly stabilized after five subcultures. A negative correlation between proliferation rate and time to emergence of first root in vitro was found.Abbreviations C clone - S subculture - SP(C) stock plant within clone - T time of initiation     

Lateral root formation in pine seedlings

The role of assimilates in lateral root development was studied in Pinus pinea seedlings grown in a nutrient solution. Seedlings were treated with ¹⁴CO₂ for 2 h following removal of the tap root tip at various times prior to the application of ¹⁴CO₂ or removal of a different number of cotyledons at one time. In seedlings with intact root systems most of the radioactivity accumulated in the lower section of the root containing the tap root apex. When the tap root tip was removed, the pattern of radioactivity accumulation along the root was affected by the presence and the stage of lateral root development. Removing the tap root tip of young seedlings (with no lateral roots) resulted in an almost equal distribution of radioactivity along the root. About 50% of the total radioactivity was found in the section showing the highest lateral root growth. Removing the tap root tip of mature seedlings (with lateral roots in the upper section) resulted in an immediate increase in the radioactivity accumulation in the upper section. When lateral roots appeared in the middle section, the pattern of radioactivity distribution was similar to that found in root decapitated young seedlings. Removal of cotyledons of mature seedlings somewhat increased the transport of radioactivity to the lower root section at the expense of the radioactivity in the lateral roots of the upper section. The present study suggests that competition within the root system between the tap root apex and the lateral roots may play an important role in determining the morphology of the root system.