In vitro loss of nucleic acids from cells of aseptically cultured excised pea roots

Summary Analysis of pea root tips taken from attached seedling roots and excised roots cultured in vitro has revealed major differences in cell constituents. The cells of cultured roots have only 40% and 13% of the protein and amino acid content of attached root cells. The nucleic acid content of cultured root cells was shown to be only 20% and 27% of the RNA and DNA respectively found in attached roots. It is suggested that there is excess nucleic acid in whole plant tissues above that required for transfer of genetic information necessary for normal growth and differentiation of root cells.     

Nuclear DNA contents of Pisum genotypes grown in vivo and in vitro

Summary The nuclear DNA content of prophase nuclei in root tips of two cultivars and two primitive lines of Pisum sativum and of Pisum fulvum have been determined, using a scanning microdensitometer. The nuclear DNA contents differed significantly between the genotypes investigated but there was no correlation with their supposed phylogenetic positions.A loss of 73% of the DNA from cells of aseptically cultured excised pea roots has been recently reported (Abbott, 1971). In marked contrast to this claim, our measurements of the nuclear 4C DNA content of root tip meristematic cells have shown that there is no significant loss in excised roots compared with attached roots.     

Improved Cytoplasmic pH Regulation,Increased Lactate Efflux,and Reduced Cytoplasmic Lactate Levels Are Biochemical Traits Expressed in Root Tips of Whole Maize Seedlings Acclimated to a Low-Oxygen Environment

We tested the hypothesis (J.-H. Xia and P.H. Saglio [1992] Plant Physiol 100: 40-46) that the enhanced ability of maize (Zea mays) root tips to survive anoxia, elicited by a 4-h exposure to 3% O2 ("acclimation"), is due to less cytoplasmic acidosis early in anoxia. Cytoplasmic pH and fermentation reactions were monitored in excised and intact (attached) maize root tips by simultaneous in vivo 13C- and 31P-NMR spectroscopy. We demonstrate that both excised and intact acclimated root tips have significantly higher cytoplasmic pH values under anoxia. This reduction in cytoplasmic acidosis is greater in intact root tips. Remarkably, cytoplasmic pH does not change when root tips are transferred from 3% O2 to anoxia. The earlier observation of considerable lactate efflux and lowered intracellular lactate in excised, acclimated root tips (ibid.) was extended to intact seedlings. The predominant fermentation end product retained in the cells of acclimated root tips is alanine. We discuss the relationship between cytoplasmic pH and levels of intracellular lactate and alanine in sugar-replete roots, and the role of cytoplasmic pH in determining survival under anoxia.     

Transmembrane electropotential in barley roots as related to cell type, cell location, and cutting and aging effects

Transmembrane electropotential difference (PD) was measured in whole roots of barley (Hordeum vulgare L. cvs. Compana and Himalaya). Seedlings were grown 4 to 5 days in aerated 0.5 mm CaSO(4) or a nutrient solution. Measurements of PD were made with roots bathed in CaSO(4), KCl + CaSO(4), or the nutrient solution. The following results were found. (a) There was a radial PD gradient with epidermal cells being 10 to 58 millivolts less negative than cells in the third layer of the cortex (outside to inside). There was no longitudinal PD gradient in the region 0.5 to 4 cm from the root tip, nor was there any difference between the PD of young root hairs and other epidermal cells. (b) Cell PD in excised whole roots was not detectably different from that found in roots attached to the shoot, and was unchanged for 2 hours from excision. (c) In 1-centimeter sections of root, cell PD at the freshly cut surface was depolarized by 90 millivolts from that in the intact root; cells farther than 1 millimeter from the cut surface were not depolarized. The PD of cells at the cut surface became more negative upon aging the segment in 0.5 mm CaSO(4), eventually becoming greater by -25 millivolts than that in cells of intact roots. Cells in segments to which the root tips were attached had less negative PDs after aging than those in subapical segments, indicating a possible hormonal effect. PDs in aged, excised segments are not equivalent to those in intact roots. (d) Creeping of cytoplasm over electrode tips inserted into the vacuole gave measurements of vacuole-to-cytoplasm PD of + 9 millivolts in 0.5 mm CaSO(4) and + 35 millivolts in 1 mm KCl + 0.5 mm CaSO(4). Most of the cell PD was across the plasmalemma. (e) The reducing sugar content of roots in CaSO(4) solution was greater than that of roots in the nutrient solution in which ion uptake, particularly K(+) occurred.     

Activation of cell division in the quiescent center of excised maize root tip

The phenomenon of activation of cell proliferation in the quiescent center of excised maize roots is described. The root tips were grown on wet filter paper in Petri dishes. This phenomenon was observed in 8 to 14 maize cultivars and was absent in excised Arabidopsis root tips. The distribution of mitoses in meristems greatly varied in roots of individual seedlings from the same seed lot and seedlings of different cultivars. Meristem opening was observed after the removal of small root tips not longer than 3 mm and intact seminal roots. Sucrose (2%) and 10(-6)-10(-8) M indole-3-acetic acid did not prevent meristem opening. These findings indicate that the state of quiescent center is maintained by a system of intercellular and interorgan relations, which are to be clarified.     

The Effects of l-Naphthylacetic Acid on the Growth of Excised Tomato Roots

I-naphthylacetic acid (NAA) enhances main axis extension andlateral initiation of excised tomato roots cultured in 0.5 percent sucrose medium. NAA has a qualitatively similar effectupon the growth of root tips cultured in glucose medium or transferredafter 2 days' previous growth in standard (1.5 per cent sucrose)medium to a glucose medium containing NAA.NAA does not raisethe growth-rate of roots cultured in glucose medium to thatoccurring in presence of sucrose as carbon source. The enhancedgrowth resulting from addition of NAA to glucose medium is notdependent upon enhanced glucose uptake.The unique role of sucrosein the growth of excised tomato roots is discussed in the lightof the above observations and of studies on the ability of glucoseto maintain the growth of excised roots previously establishedby a period of culture in sucrose medium.     

Effect of Auxins (2,4-Dichlorophenoxyacetic acid, Indole-3-Acetic Acid and Naphthaleneacetic Acid) on the Accumulation of {gamma}-Aminobutyric Acid in Excised Rice Root Tips

-Aminobutyric acid (GABA) accumulation occurs in cultured ricecells when ammonium is added to the medium [Kishinami and Ojima(1980) Plant Cell Physiol. 21: 581–589]. Whether thisphenomenon occurs in rice plant tissues was examined with respectto exogenously supplied auxins: 2,4-dichlorophenoxyacetic acid(2,4-D), indole-3-acetic acid (IAA) and naphthalene-acetic acid(NAA). In intact rice plants grown in medium containing ammonium withoutauxin, glutamine first increased, then asparagine graduallyincreased. In both shoots and roots, the asparagine contentbecame the highest among four amino acids after 4 days of cultureperiod. GABA did not increase at all, its level remaining lowin both shoots and roots throughout the culture period. GABA accumulation was observed in excised rice root tips whenthey were incubated in the medium containing ammonium in thepresence of 2,4-D, IAA or NAA. In the absence of auxin, however,excised rice root tips accumulated asparagine and glutamine,but not GABA. Rice root segments obtained from a region in whichroot cells had already developed to maturity did not accumulateGABA but asparagine and glutamine in the presence of both ammoniumand 2,4-D. These results suggest that GABA accumulation occurs in rapidlygrowing and dividing tissue, such as the apical meristem ofrice root in the presence of auxin during ammonium assimilation. (Received June 15, 1987; Accepted March 14, 1988)     

Cell division activation in the quiescent center of excised maize root tip

The phenomenon of activating cell proliferation in the quiescent center of excised maize roots is described. The root tips were grown on wet filter paper in Petri dishes. This phenomenon was observed in 8 to 14 maize cultivars and was absent in excised Arabidopsis root tips. The distribution of mitoses in meristems greatly varied in individual seedlings roots from the same seed lot and seedlings of different cultivars. Meristem opening was observed after the removal of small root tips not longer than 3 mm and intact seminal roots. Sucrose (2%) and 10⁻⁶–10⁻⁸ M indole-3-acetic acid did not prevent meristem opening. These findings indicate that the state of quiescent center is maintained by a system of intercellular and interorgan relations, which are to be clarified.     

Bicarbonate Fixation and Malate Compartmentation in Relation to Salt-induced Stoichiometric Synthesis of Organic Acid

The relationship of malate synthesis to K⁺ absorption from solutions of K₂SO₄ and KHCO₃ was compared in nonvacuolate barley (Hordeum vulgare) root tips and whole excised roots. The comparison has permitted separation of the process which evokes organic acid synthesis from that which leads to stoichiometry between net acid equivalents formed and excess K⁺ absorbed from K₂SO₄, on the one hand, and total K⁺ absorbed from KHCO₃, on the other. Both in tips and in roots K⁺ uptake from 20 mN salt solution exceeds malate synthesis in the first hour. In vacuolate roots the expected stoichiometry is achieved with time. When root tips are transferred to dilute CaSO₄, malate is rapidly metabolized, and K⁺ is lost to the solution. By contrast, in excised whole roots the malate level remains unchanged, the salt-induced organic acid presumably being retained in the vacuole. In excised roots malonate leads to a marked drop in malate levels in untreated roots as well as in roots which have experienced salt-induced net malate synthesis. In consequence, it is contended that malonate makes available normally sequestered vacuolar malate.     

Regulation of root growth by auxin-ethylene interaction

A large portion of indoleacetic acid (IAA)-induced inhibition of excised root tips and virtually all such inhibition of intact roots are the result of IAA-dependent ethylene production. Under certain conditions an additional effect of IAA accounts for a small portion of the inhibition of excised root tips. Ethylene production in response to applied IAA is governed by the level of applied auxin found inside the root. Evidence is presented to confirm the participation of ethylene in the geotropic response of roots.     

Effect of removal of the root tip on the development of enhanced rb absorption by corn roots

Samples of primary root tissue of corn (Zea mays L.) were aged either in CaSO(4) solution or in humid air, after which they were immersed for 10 minutes in a solution containing 0.1 mm(86)RbCl. Aging in solution, but not in humid air, enhanced the subsequent rate of Rb(+) absorption. Excision of roots before aging was followed by greater enhancement than when exicision followed aging. The time course of aging of 1-cm segments from different portions of the root showed decreasing response with increasing distance from the root cap. The aging response of apical segments (5-15 mm from the root cap) could be detected within 10 minutes and usually reached a maximum within 2 hours. Rb(+) absorption by apical segments (5-15 mm) aged without the tip (0-5 mm) was more than double that by apical segments whose tips were left attached until the end of the aging period. When apical segments without the tip were aged for 2 hours in the CaSO(4) solution in which seedlings had previously been grown for 24 hours, the rate of absorption was only 63% of samples aged in fresh solution. When apical segments were aged for 2 hours in fresh solution containing excised tips floating free in the solution, the rate of Rb(+) absorption was 20% less than in samples aged in solution containing no excised tips. The data presented in this study are interpreted to indicate that a water-soluble metabolite, originating in the root tip and translocated basipetally, inhibits Rb accumulation.     

THE DEVELOPMENT OF ISOLATED ROOTS OF COMPTONIA PEREGRINA (MYRICACEAE) IN CULTURE

Seedling roots of the sweet fern Comptonia peregrina (L.) Coult. were excised aseptically and cultured in a modified Bonner-Devirian liquid nutrient medium. Root elongation was very slow in the basic medium which contained inorganic salts, B-vitamins, trace elements and 4 % sucrose. The addition of plant hormones including gibberellic acid, indoleacetic acid, and zeatin, alone or in combinations, had little effect on growth. Myoinositol at 10 or 100 ppm doubled the rate of elongation. The effect of this sugar alcohol could not be replaced by scyllitol, D-sorbitol, D-mannitol or by increasing the sucrose concentration. Subcultured root tips showed progressively less elongation in successive transfers. Secondary thickening of the roots, especially in the basal half, occurred in initial passages and in subcultured roots without added hormones. Root buds also occurred spontaneously especially in the basal portions of cultured roots, both in first and in successive passages. An anatomical analysis showed that these buds were endogenous, arising from a secondary cortex of pericyclic origin.     

Changes in growth and structure of pea primary roots (Pisum sativum L. cv. Alaska) as a result of sudden flooding

Pea (Pisum sativum L. cv. Alaska) primary roots were exposed to flooding after growth for 4 or 5 d at 25 degrees C under relatively dry conditions. Flooding after 4 d growth reduced, but did not stop, primary root growth, and cavities caused by degradation of central vascular cells were typically found from 10-60 mm from the tips. Flooding after 5 d stopped primary root growth and caused cell death in the tips, and vascular cavities formed that typically were 20-60 mm from the tips of the roots. Degradation of root tip cells in 5-day-roots was very rapid and began in the elongation zone and later in the apical zone. Root tips discolored, narrowed or curled before growth arrest. The mitotic indices of 5-day-root tips were suppressed by the flooding treatment. A few mitotic figures were observed in roots treated with flooding after 4 d growth. Affected cells had condensed nuclei, but cytoplasms appeared to be normal in the early stages of cell degradation. Later these cells became very vacuolated. The relationship of flooding to root growth, vascular cavity formation, and the morphology of pea primary roots is described with regard to the ability to resist flooding stress.     

Influence of inoculum morphology on growth of Atropa belladonna hairy roots and production of tropane alkaloids

Summary The specific growth rate of Atropa belladonna hairy roots measured in terms of root length increased by about 25% to 0.49 d^-1 when the inoculum consisted of a root with the primary root tip excised. The biomass dry weight produced after 14 d increased by 28%. In contrast, presence of laterals in the inoculum with or without lateral tips excised did not influence the growth rate. Although hyoscyamine was found to accumulate at higher concentrations in the more mature root tissues (2.1 ± 0.2 mg g^-1) than in the tips (1.1 ± 0.3 mg g^-1), hyoscyamine content in the harvested roots was independent of inoculum morphology.     

Auxin and the response of pea roots to auxin transport inhibitors: morphactin

The auxin transport inhibitor methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (CFM), a morphactin, inhibits negative geotropism, causes cellular swelling, and induces root hair formation in roots of intact Pisum sativum L. seedlings. In excised pea root tips, CFM inhibits elongation more than increase in fresh weight (swell ratio = 1.3 at 20 mum CFM). CFM growth inhibition was expressed in the presence of ethylene. Indoleacetic acid (IAA) prevented the expression of CFM growth inhibition possibly because IAA inhibited the accumulation of CFM into the tissue sections. CFM inhibited the accumulation of IAA and 2,4-dichlorophenoxyacetic acid into excised root tips. Applying Leopold's (1963. Brookhaven Symp. Biol. 16: 218-234) model for polar auxin transport, this result suggests a possible explanation for CFM inhibition of geotropism in pea roots, i.e. disruption of auxin transport by interfering with auxin binding.     

Thiophene accumulation in relation to morphology in roots of Tagetes patula

Roots of marigold (Tagetes patula L.) accumulate thiophenes, heterocyclic sulfurous compounds with strong biocidal activity. In detached roots cultured in vitro, the thiophene content was 5 mol·(g fresh weight)^-1 which is 25-times higher than in roots attached to the plant. In roots derived from tissues transformed by Agrobacterium tumefaciens and A. rhizogenes, the morphology and thiophene content varied with the bacterial strain used. Transformation stimulated the elongation of the root tips and the formation of lateral roots but lowered the thiophene level to 20–50% relative to the concentration in untransformed detached roots. A negative correlation was found between the number of laterals in a root system and the thiophene content. Extensive branching and a decrease in thiophene accumulation was evoked in untransformed roots by indole-3-acetic acid (1–10 mol·l^-1) added to the medium. Within the roots, the highest thiophene concentrations were found in the tips. The results indicate that auxin directly or indirectly plays a role in the regulation of the thiophene level in root tips.Abbreviations B5 Gamborg's B5 medium - IAA Indole-3-acetic acid     

Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

Profiles of Chloride Concentration and PD in the Root of Commelina communis L.

Chloride concentrations in longitudinal files of cells acrossthe root of Commelina communis have been determined. Vacuolarsap was taken from the root using a microsampling techniqueand chloride concentration determined on nanolitre samples byelectrometric microtitration. No radial gradient in vacuolar chloride was observed, eitherfor roots grown in a nutrient solution containing a low level,or for those grown in a solution containing a high level, ofchloride. Vacuolar electrical potentials were also determined,on attached and excised roots. The PD was found not to varysignificantly across the root from epidermis to pericycle despitethe PD in attached roots being 50 mV more negative than thatin excised roots. These results are discussed in relation tothe mechanism of ion transport across the root.     

Cryopreservation of Hyoscyamus niger adventitious roots by vitrification

An auxin-independent adventitious root culture of Hyoscyamus niger was established, and the roots were successfully cryopreserved with a high regeneration rate of 93.3 percnt; by vitrification method. The root tips were cultured for 12 to 14 days in phytohormone-free Murashige and Skoog (MS) liquid medium, and were excised and precultured on Woody Plant (WP) solid medium supplemented with 0.3 mol/L sucrose at 25 °C in the dark. After 1 day, they were treated with MS-based loading solution for 10 min, followed by soaking in MS-based PVS2 for 10 min at 0 °C. The treated root tips were immersed in liquid nitrogen (-196 °C). For recovery, the root tips were thawed rapidly at 40 °C and washed with MS medium containing 1 mol/L sucrose prior to plating onto WP solid medium. The regenerated roots were evaluated by their growth and tropane alkaloid production. The growth and alkaloid content of regenerated roots analyzed using HPLC were found to be almost the same as those of non-treated roots.     

Light-induced inhibitors from intact and cultured caps of Zea roots

Growth inhibitors were assayed from extracts of intact (attached) and of excised (cultured) root caps of Zea mays L., cv. Merit, the roots of which show a positive geotropic response only after exposure to light. If caps are intact at the time of illumination, at least two inhibitory substances are produced, an acid inhibitor and a neutral inhibitor, whereas if caps are detached from roots, placed in culture and then illuminated only the neutral inhibitor is formed. Cycloheximide retards inhibitor production in both intact and cultured caps. When [¹⁴C]mevalonic acid is included in the culture medium and the caps are illuminated, 15–25% of the recoverable ¹⁴C cochromatographs with the neutral inhibitor, whereas in caps cultured in the dark, this radiolabelling pattern is not observed. Cyloheximide in the light reduces the incorporation of ¹⁴C into compounds cochromatographing at the R_f of the neutral inhibitor. It is suggested that the neutral inhibitor may be important in the light-induced bending of roots.Abbreviations ABA abscisic acid - CH cycloheximide