The Effect of Actinomycin D and Cycloheximide on the Dedifferentiation of Cotyledon in Phaseolus radiatus L. and Its Nucleic Acid and Protein

Calli were induced from cotyledon segment of mung bean (Phaseolus radiatus L.) in Miller medium supplemented with NAA 4 mg/l, kinetin 10 mg/L. The callus formation was completely prevented by the addition of actinomycin D 15 μg/mL or cyclo- heximidc 0.5 μg/mL at 0 hour. The inhibitory effect of actinomycin D or cycloheximide was increased with the increment of concentration but decreased when the inhibitory agents were added a few hours later. If actinomycin D or cycloheximide was added at 24 hour culture it inhibits neither the induction of callus formation nor the proliferation. The content of RNA, DNA and protein were determined. RNA in each segment increased obviously in the early stage of callus formation, but DNA and protein increased slightly afterward. It is suggested that a large increase of RNA is the characteristic of dedifferentiation of cotyledon in P. radiatus. In addition, it has also been shown that an actinomycin D or cycloheximide-sensitive process in the early stage of dedifferentiation is crucial for the callus formation. Both RNA and protein synthesis are required for the initiation of dedifferentiation.     

Influence of indole-3-acetic acid on adventitious root primordia of brittle willow

Summary Removal of the stem apex and certain leaves and axillary buds of brittle willows (Salix fragilis) was employed to limit the supply of endogenous auxin to adventitious root primordia during their formation, which occurs at predetermined sites. Limiting endogenous auxin by this surgical treatment resulted in reduced primordium initiation and, to a lesser degree, primordium growth in cell number. Root primordium cells in surgically treated plants differentiated into mature parenchyma after losing their meristematic character. Application of indole-3-acetic acid (IAA) to surgically treated plants partially overcame the effects of the surgical tretament, increasing root primordium initiation and growth by cell division. When IAA-2-¹⁴C was applied to surgically treated plants, label was detected in root primordium cells by means of autoradiography. Root primordium cells took up more label during the earliest stage of initiation than during a later stage of growth. The data indicate that the initiation of these primordia is more dependent on a supply of auxin than is their subsequent development. Further, the auxin apparently acts directly in the cells which initiate primordia.This investigation was supported in part by Public Health Service Research Grant No. UI 00110-07 (now 5R01 FD 00074-09) from the National Center for Urban and Industrial Health. Paper No. 7138, Min nesota Agricultural Experiment Station.     

Influence of auxin and mycorrhizal fungi on thein vitro formation and growth ofPinus pinaster roots

Summary Experiments, performed withPinus pinaster cloned shoots submitted to an auxin treatment (NAA 10^–6 M, 18 days), demonstrated that rooting abilityin vitro persists over 5 successive induction cycles (through out a 9-month period). Rooting ability needs a permanent synthesis of auxin synergists which activate the metabolism of cell dedifferentiation and root primordium initiation. Agar culture permitted intense meristem initiation, but prevented active root elongation. In the presence of a mycorrhizal fungus,Pisolithus tinctorius orHebeloma cylindrosporum, roots resumed growth and short lateral root formation was stimulated. These two phenomena induced by fungal association improve the quality of the root systems required to facilitate successful transplantation from test-tubes to field conditions.     

Influence of Aryl Esters of Indole-3-acetic and Indole-3-butyric Acids on Adventitious Root Primordium Initiation and Development

Synthetic aryl esters of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) greatly enhanced adventitious root primordium initiation in bean (Phaseolus vulgaris L. cv. Top Crop) and jack pine (Pinus banksiana Lamb.) cuttings, respectively. Bean cuttings produced 95 to 154% more macroscopically visible root primordia in 2 days when treated with phenyl indole-3-acetate (P-IAA), in comparison with an equal concentration of IAA. Substantial but lesser increases occurred when treatment was done with 3-hydroxyphenyl indole-3-acetate (3HP-IAA). On a molar basis, either P-IAA or 3HP-IAA were 10 or more times as efficient as IAA in inducing adventitious root primordium initiation in bean cuttings. Methyl indole-3-acetate was no more effective than IAA in these tests. Phenyl indole-3-butyrate (P-IBA) consistently enhanced the number of rooted jack pine seedling cuttings by 11 to 12% in comparison with a 27% higher concentration of IBA. The number of elongated roots (2 mm or more) after 5 days was 165 to 276% greater for P-IAA than for IAA-treated bean cuttings. Similar but lesser increases occurred as a result of 3HP-IAA treatment. P-IBA in comparison with IBA treatment did not influence either the number of roots or length of the longest root per rooted jack pine cutting. Enzymes in bean and jack pine cuttings hydrolyzed the aryl esters. However, check experiments showed that initial integrity of the esters was required for enhanced activity in inducing root primordium initiation. Treatment of bean cuttings with hydrolysates of P-IAA, or with IAA and phenol, alone or combined, did not influence root primordium initiation or development in a manner different from treatment with IAA alone.     

Light and decapitation effects on in vitro rooting in maize root segments

The effects of white light and decapitation on the initiation and subsequent emergence and elongation of lateral roots of apical maize (Zea mays L. cv LG 11) root segments have been examined. The formation of lateral root primordium was inhibited by the white light. This inhibition did not depend upon the presence of the primary root tip. However, root decapitation induced a shift of the site of appearance of the most apical primordium towards the root apex, and a strong disturbance of the distribution pattern of primordium volumes along the root axis. White light had a significant effect neither on the distribution pattern of primordium volumes, nor on the period of primordium development (time interval required for the smallest detectable primordia to grow out as secondary roots). Thus, considering the rooting initiation and emergence, the light effect was restricted to the initiation phase only. Moreover, white light reduced lateral root elongation as well as primary root growth.     

欧美杂种山杨微扦插不定根发生过程的解剖学研究

采用石蜡切片技术,以欧美杂种山杨插穗基部茎段为实验材料,连续解剖观察插穗不定根发生发育过程,分析根原基发生部位与扦插生根的关系。结果显示:欧美杂种山杨插穗不定根的发生过程分为4个时期,为根原基诱导期,不定根起始期、表达期和伸长生长期。根原基诱导期维管形成层产生具有分生组织特点的薄壁细胞;不定根起始期,维管形成层及附近的薄壁细胞脱分化,形成不定根原基发端细胞;不定根表达期,根原基发端细胞不断分裂成具有方向性的根原基,根原基穿过韧皮射线和皮层,向皮孔方向发展;不定根伸长生长期,根原基从皮孔伸出,其内部的维管系统开始发育,形成不定根。研究认为,欧美杂种山杨为皮部诱导生根类型,不定根原基起源于维管形成层区,起源部位单一,扦插难生根。     

The Influence of Colchicine on Initiation and Early Development of Adventitious Roots

The first sign of adventitious root formation in the petiole of the primary leaves of Phaseolus vulgaris after treatment with IAA was the dedifferentiation of mature parenchyma cells next to strands of sieve elements and companion cells. Colchicine strongly inhibited this dedifferentiation. Treatment with colchicine 3 days after treatment with IAA, caused the groups of meristematic cells formed to grow by cell enlargement only. Groups of more than about 30 meristematic cells changed into recognizable root primordia during this growth. Groups with a smaller number of meristematic cells extended also in size but did not form a recognizable root primordium.     

Effects of Actinomycin D and 2,4-Dinitrophenol on the Development of Root Primordia in Azuki Bean Stem Cuttings

In the rooting of disbudded azuki bean stem cuttings, actinomycinD and 2,4- dinitrophenol (DNP) acted as auxin synergists. Althoughcuttings treated with actinomycin D or DNP alone formed almostthe same number of roots as water-treated cuttings, cuttingstreated with actinomycin D or DNP for 24 hr then with auxinfor another 24 hr formed more roots than cuttings treated onlywith auxin during the second 24 hr. Both actinomycin D and DNPincreased the number of root primordia with longitudinally dividedcells, but they acted differently on the first transverse celldivision which led to root primordium formation. ActinomycinD delayed the start of the first transverse cell division, butDNP hastened it. (Received July 7, 1981; Accepted September 21, 1981)     

TIME-LAPSE PHOTOGRAPHIC OBSERVATIONS OF MORPHOGENESIS IN ROOT NODULES OF COMPTONIA PEREGRINA (MYRICACEAE)

Seedlings of the sweet fern Comptonia peregrina (L.) Coult. were grown aeroponically with their roots bathed in a nutrient mist lacking nitrogen except for 10 ppm N at the outset. The initiation and early development of root nodules capable of fixing atmospheric nitrogen were recorded with time-lapse photography through early development to the establishment of highly branched, roughly spherical nodules. In Comptonia multiple primary nodule lobes are formed at or near the site of infection with as many as 10 primary lobes occurring together. On the shoulders of the swollen primary lobes new primordia develop, forming secondary nodule lobes, which may persist without nodule root elongation, giving a coralloid appearance. The tips of the lobes may elongate, forming nodule roots which grow vertically upward, or, if disturbed, in random orientation. Nodule roots occasionally form lateral roots. The root axis upon which the nodule forms undergoes secondary thickening on the proximal side of the nodule attachment; the distal portion of the root shows no secondary thickening and later atrophies. Thus, nodules are perennial structures on a woody root system. The endophyte infects and occupies the basal cortical tissues of the primary nodule lobes and successive nodule lobes as they are formed, being restricted to the swollen bases and not infecting the elongate nodule roots. Development of the nodule is interpreted in terms of complex host-endophyte interactions involving the initiation of multiple primordia forming nodule lobes, the active inhibition of nodule lobes and finally nodule root elongation. Anatomical evidence for the endogenous origin of nodule primordium formation substantiates the view obtained from time-lapse photomacrography.     

Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants

 In walnut (Juglans regia L.), an otherwise difficult-to-root species, explants of cotyledons have been shown to generate complete roots in the absence of exogenous growth regulators. In the present study, this process of root formation was shown to follow a pattern of adventitious, rather than primary or lateral, ontogeny: (i) the arrangement of vascular bundles in the region of root formation was of the petiole type; (ii) a typical root primordium was formed at the side of the procambium within a meristematic ring of actively dividing cells located around each vascular bundle; (iii) the developing root apical meristem was connected in a lateral way with the vascular bundle of the petiole. This adventitious root formation occurred in three main stages of cell division, primordium formation and organization of apical meristem. These stages were characterized by expression of LATERAL ROOT PRIMORDIUM-1 and CHALCONE SYNTHASE genes, which were found to be sequentially expressed during the formation of the primordium. Activation of genes related to root cell differentiation started at the early stage of primordium formation prior to organization of the root apical meristem. The systematic development of adventitious root primordia at a precise site gave indications on the positional and biochemical cues that are necessary for adventitious root formation. Received: 30 July 1999 / Accepted: 16 February 2000     

Effect of Photoperiod and Chlormequat on Apical Development and Growth in a Spring Wheat (Triticum aestivum) Cultivar

Spring wheat, cv. Timmo, was grown under three photoperiod regimes(16, 13 and 11 h) with and without treatment with the plantgrowth regulator chlormequat (applied at the glume primordiumstage of apical development) and the relationships between apicaldevelopment, primordium initiation and growth stage examined The effects of photoperiod were generally similar to those reportedfrom other studies; shorter photoperiod slowed the rate of apicaldevelopment, increased the duration of the primordium initiationphases and reduced the rate of primordium initiation. The finalnumber of spikelets was increased, but there was no effect onnumber of floret primordia per spikelet The number of tillersproduced was also higher in the shorter photoperiods. Chlormequattreatment had a similar effect to imposing short-days: floweringwas delayed and tiller production increased There were strong correlations between certain development eventsand the phasing of primordium initiation and growth stages andthese were not affected by photoperiod or chlormequat treatments.For example, the end of spikelet primordium initiation, i.e.terminal spikelet (TS) formation, coincided with the floret-stamenprimordium stage (of the most advanced spikelet) and the endof floret primordium initiation with the stigma tic branchesand hairs on ovary wall elongating stage. Similarly, rapid stemextension growth always started at TS formation while spikeextension and spike growth commenced at TS formation and thestigmatic branches stage, respectively. Tiller production alsoceased at TS formation, when rapid stem growth started Although the timing of the phases of primordium initiation andcertain growth events were linked to apical development, therate of apical development did not determine either the rateof spikelet primordium initiation or the rates of stem and eargrowth. However, there was a strong relationship between rateof development and rate of floret primordium initiation. Therewas also a strong relationship between spike length and apicaldevelopment stage Triticum aestivum, spring wheat, photoperiod, chlormequat, apical development, primordium initiation, stem and spike growth     

DNA SYNTHESIS AND MITOSIS IN MERISTEMS: REQUIREMENTS FOR RNA AND PROTEIN SYNTHESIS

Following provision of sucrose to starved, stationary phase pea root meristems, G₁ and G₂ cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G₁ and G₂ cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8-12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.     

The seminal root primordia in barley and the participation of their non-meristematic cells in root construction

In addition to the primary seminal primordium, the so-called secondary seminal root primordia are also initiated in a barley embryo. The primary root primordium is developmentally most advanced. It is formed by root meristem covered with the root cap, and by a histologically determined region with completed cell division. On germination, the restoration of growth processes begins in this non-meristematic region of root primordium by cell elongation, with the exception of the zone adjacent to the scutellar node, the cells of which do not elongate but continue differentiating. In the root primordia initiated later, the zone with completed cell division is relatively shorter, in the youngest primordia the non-meristematic cells may be lacking. The root meristem is reactivated after the primary root primordium has broken through the sheath-like coleorrhiza and emerges from the caryopsis as the primary root. The character of root meristem indicates a reduced water content at the embryonic development of root primordium. With progressing growth the root apex becomes thinner, the meristematic region becomes longer, and the differences in the extent of cell division between individual cell types increase. — The primary root base is formed of cells pre-existing in the seminal root primordium. Upon desiccation of caryopsis in maturation, and subsequent quiescent period, their development was temporarily broken, proceeding with the onset of germination. The length of this postembryonically non-dividing basal zone is different in individual cell types. The column of central metaxylem characteristic of the smallest number of cell cycles, has, under the given conditions, a mean length of about 22 mm, whereas the pericycle, as the tissue with most prolonged cell division, has a mean length of about 6 mm. In the seminal root primordia initiated later the non-dividing areas are relatively shorter. The basal region of seminal roots thus differs in its ontogenesis from the increase which is formed “de novo” by the action of root meristem upon seed germination.     

Chimeras and the Origin of Lateral Root Primordia in Zea mays

The difficulty of determining the contribution made by the pericycleand endodermis of mother roots to lateral primordia in the Gramineaehas been solved by inducing polyploid chimeras at initiation. The endodermis forms a layer covering the primordium, but thislayer does not form the epidermis of the lateral. It does formthe root cap of the young primordium, but this is replaced ata variable stage of development by the quiescent centre donatinga new set of cap initials of pericyclic origin. Reasons forthe previous diversity of interpretations are presented. chimera, lateral root primordia, Zea mays     

The position and growth of lateral roots on cultured root axes of tomato,Lycopersicon esculentum (Solanaceae)

Root axes of tomato (Lycopersicon esculentum) were cultured in vitro in three different concentrations of sucrose in order to vary their growth rate. Lateral root growth and the initiation of lateral root primordia were studied on each group of axes. Various aspects of primordium initiation, positioning, and emergence were quantified with a view to discovering variable and constant features of these processes. Variable parameters were the rate and frequency of root primordium emergence. Constant parameters, at least under the prevailing conditions, were the spacing between successive laterals and primordia, and the position of the primordia in relation to the vascular system. A model of primordium initiation is presented which combines controls determined by the divisional history of the potential primordium cell and by the vascular pattern.Dedicated with great respect to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday in recognition of her distinguished contributions to cytology.