Removal of the stem terminal and application of auxin change carbohydrates in Pinus banksiana cuttings during propagation

This study determined how surgical removal of the stem terminal, with indole-3-butyric acid (IBA) treatment, influenced concentrations and partitioning of carbohydrates in Pinus banksiana Lamb, cuttings during propagation. Seedlings and cuttings that originated from 90-day-old stock plants were untreated or treated by removing the stem terminal, followed by application of IBA to the severed apical or basal (cuttings only) stem. Fresh and dry weights of the basal 1-cm stems of cuttings were determined daily for the first 10 days of propagation (i.e., before roots were visible). In addition, basal 1-cm stems, upper (ca 9-cm) stems and needles of seedlings and cuttings were analyzed for sucrose, soluble reducing sugar and total non-structural carbohydrate. Net concentrations of each carbohydrate in cuttings were obtained by subtracting corresponding concentrations for similarly treated seedlings, yielding data directly related to only the physiology of rooting. Data for cuttings indicated that presence of the stem terminal combined with applied IBA positively influenced rooting through processes that increased basal stem fresh and dry weights before root emergence. Removal of the stem terminal influenced accumulation of net total carbohydrate in cuttings, but the major effect was on carbohydrate partitioning. Either type of IBA treatment after removal of the stem terminal usually resulted in different net carbohydrate concentrations in each tissue source of cuttings, compared with only removal of the terminal. Neither basal nor apical IBA treatment of cuttings without stem terminals yielded results for carbohydrate accumulation and partitioning like those obtained with intact cuttings. Removal of the stem terminal, even if followed by IBA treatment, may have lessened rooting potential of cuttings because it resulted in greater reducing sugarstarch concentration ratios in basal stems compared with those in intact cuttings.     

Carbohydrate accumulation and partitioning in Pinus banksiana seedlings and seedling cuttings

Tests were conducted to identify possible relations between carbohydrates and callusing-rooting of Pinus banksiana Lamb, cuttings. Terminals, upper stems, and basal (1 cm) stems of 90-day-old untreated seedlings and seedling cuttings were analyzed for sucrose, total soluble reducing sugar, starch and total non-structural carbohydrate during propagation. Seedlings were evaluated in order to determine whether data for cuttings alone properly described carbohydrate-callusing-rooting relations under conditions where stock plants and cuttings were propagated in different environments. Results indicated that seedling terminals and upper stems, but not basal stems, accumulated the measured carbohydrates much like cuttings, though to lesser concentrations. Thus, carbohydrate accumulation by cutting terminals and upper stems would have been overestimated, based on cutting data alone. In terms of rooting, results indicated that: 1) Total carbohydrate accumulation in cutting basal stems was related to callusing-rooting, but a cause-effect relation was not established; 2) The positive relation between callusing-rooting and total carbohydrate accumulation was primarily due to accumulation of reducing sugar and starch, with reducing sugar predominant. 3) Reducing sugar/starch concentration ratios were the most sensitive and convenient indicators of specific carbohydrate differences within and between seedlings and cuttings.     

Effects of auxin and irradiance on the rooting of cuttings of Pinus sylvestris

Abstract Seedlings of Pinus sylvestris L. were grown under controlled conditions (temperature 20°C, photoperiod 17 h) at two irradiances, 8 or 40 W m^-2. Hypocotyl cuttings were excised and rooted at different irradiances in tap water solutions of indolebutyric acid (IBA). The fastest rooting and highest rooting percentage were obtained with cuttings from stock plants grown at 8 W m^-2 and treated with 10^-5M IBA for 21 days. The concentration of 10^-4M IBA inhibited root formation. In comparable treatments rooting was always better in cuttings from stock plants grown at 8 W m^-2 than in cuttings from stock plants grown at 40 W m^-2. The irradiance during the rooting period had only a minor influence on rooting. When cuttings from plants irradiated with 40 W m^-2 were treated with 10^-5M IBA for 21 days the rooting percentage almost reached the same level as in untreated cuttings from stock plants given 8 W m^-2. In cuttings treated with IBA during the whole rooting period, rooting was depressed in comparison to untreated cuttings. Aeration of the 10^-4M IBA solution increased the rooting percentage, but aeration had no effect on untreated cuttings and on cuttings treated with lower IBA concentrations.     

Effects of nitrogen limitation on water relations of jack pine (Pinus banksiana Lamb.) seedlings

The water relations of shoots of young jack pine (Pinus banksiana Lamb.) seedlings were examined 6 and 15 weeks after the initiation of four different dynamic nitrogen (N) treatments using a pressure-volume analysis. The N treatments produced a wide range of needle N concentrations from 12 to 32 mg g^?1 dry mass and a 10-fold difference in total dry mass at 15 weeks. Osmotic potential at full turgor did not change over the range of needle N concentrations observed. Osmotic potential at turgor-loss point, however, declined as N concentrations decreased, indicating an increased ability of N-deficient jack pine plants to maintain turgor. The increase could be attributed largely to an increase in cell wall elasticity, suggesting that elasticity changes may be a common, significant adaptation of plants to environmental stresses. Dry mass per unit saturated water almost doubled as needle N level dropped from 32 to 12 mg g^?1 and was inversely correlated to the bulk modulus of elasticity. This suggests that cell wall elasticity is determined more by the nature of its cross-linking matrix than by the total amount of cell wall material present. Developmental change was evident in the response of some water relation variables to N limitation.     

Differential expression during drought conditioning of a root-specific S-adenosylmethionine synthetase from jack pine (Pinus banksiana Lamb.) seedlings

Differential screening of a cDNA library constructed from root mRNA from jack pine (Pinus banksiana Lamb.) seedlings exposed to two cycles of drought conditioning identified a S-adenosylmethionine synthetase (sam-s) cDNA. A cDNA encoding the entire open reading frame of SAM-S was identified and characterized. Analysis of the full-length sam-s cDNA revealed that it was 1675 bp, encoded an open reading frame of 393 amino acids and had a predicted protein mass of 43 kDa. Jack pine sam-s was found to be highly similar to several other plant sam-s genes. RNA gel blot analysis showed that sam-s mRNA abundance increased following two cycles of drought conditioning and remained abundant after 3 d of rewatering. Expression of this gene appears to be root-specific. Quantitative slot blot analysis showed that two cycles of drought conditioning caused a 6-fold increase in sam-s mRNA abundance whereas heat shock, cold stress and anoxia did not result in the accumulation of sam-s mRNA. SAM-S enzyme activity increased 2-fold following two cycles of drought conditioning. The increase in the rate of SAM-s enzyme activity was also correlated with changes in rates of ethylene and betaine synthesis, biosynthetic pathways that utilize SAM as a substrate. Ethylene evolution and betaine abundance increased following two cycles of drought conditioning.     

Acclimation of respiratory temperature responses in northern and southern populations of Pinus banksiana

Temperature acclimation of respiration may contribute to climatic adaptation and thus differ among populations from contrasting climates. Short-term temperature responses of foliar dark respiration were measured in 33-yr-old trees of jack pine (Pinus banksiana) in eight populations of wide-ranging origin (44-55 degrees N) grown in a common garden at 46.7 degrees N. It was tested whether seasonal adjustments in respiration and population differences in this regard resulted from changes in base respiration rate at 5 degrees C (R(5)) or Q(10) (temperature sensitivity) and covaried with nitrogen and soluble sugars. In all populations, acclimation was manifest primarily through shifts in R(5) rather than altered Q(10). R(5) was higher in cooler periods in late autumn and winter and lower in spring and summer, inversely tracking variation in ambient air temperature. Overall, R(5) covaried with sugars and not with nitrogen. Although acclimation was comparable among all populations, the observed seasonal ranges in R(5) and Q(10) were greater in populations originating from warmer than from colder sites. Population differences in respiratory traits appeared associated with autumnal cold hardening. Common patterns of respiratory temperature acclimation among biogeographically diverse populations provide a basis for predicting respiratory carbon fluxes in a wide-ranging species.     

Effects of glutamine and K-glutamate on assimilation of [15N]-nitrate during auxin treatment for root formation in vitro (Pinus pinaster)

In order to establish the relationship of nitrogen nutrition to root formation in vitro, efficiency of various forms of nitrogen supply was investigated in micropropagated shoots of Pinus pinaster. Nitrate (3.3 m M as N) containing medium supplemented with glutamine (2 m M as N) resulted in optimal rooting (control medium) whereas the rooting response was inhibited by the presence of glutamate (2 m M as N) in the medium. Study of ¹⁵NO⁻₃ behaviour in shoots cultured on the control medium showed a marked increase of ¹⁵N incorporation during the second half of a period of auxin treatment (total duration 16 days). The majority of the nitrogen incorporated from the medium was accumulated into protein in the rooting zone. Autoradiography of [³H]-thymidine in this reacting zone showed strong DNA synthesis at this time. Addition of glutamate both prevented the root formation process and resulted in a marked slowdown of nitrate uptake and decrease of protein synthesis.     

The northern limit of Pinus banksiana Lamb. in Canada: explaining the difference between the eastern and western distributions

Aim Present northern distribution limit of jack pine (Pinus banksiana Lamb.) follows the northern limit of continuous open boreal forest in western Canada, but not in eastern Canada where it is located further south. We tested the hypothesis that fire plays a more important role than climate in explaining the present position of the northern distribution limit of jack pine. Location An experimental jack pine plantation was set up in 1992, c. 300 km north of the present distribution limit of the species, in the Boniface river area of northern Québec (57°43′ N, 76°05′ W). Methods Climate and fire data were used to compare sites at and north of the present distribution limit of jack pine. In 2001, surviving individuals from the plantation were measured (total height, annual shoot elongation, basal diameter, and presence/absence of cones). Results Climate data from the ten weather stations used in this study did not show major differences. The northern limit of jack pine distribution is closely associated with the occurrence of fires larger than 200 ha. Survival of the planted jack pines was 31%. About 25% of the surviving pines qualified as normal, single‐stem individuals; the others were slightly uprooted and/or showed marks of erosion or foraging. Cones were produced, although no viable seeds were found. Main conclusions The low number of degree‐days above 5 °C at the plantation site could explain why the seeds were not viable. However, such climate conditions are not sufficient to prevent growth, as was shown by annual shoot elongation measurements. Most of the surviving jack pines from the Boniface river plantation are relatively healthy and follow a normal developmental programme. Low fire frequency and small fire size are amongst the main factors that prevented P. banksiana from migrating further north or east following deglaciation in northern Québec and Labrador.     

Coupling of respiration, nitrogen, and sugars underlies convergent temperature acclimation in Pinus banksiana across wide-ranging sites and populations

Patterns and mechanisms of short‐term temperature acclimation and long‐term climatic adaptation of respiration among intraspecific populations are poorly understood, but both are potentially important in constraining respiratory carbon flux to climate warming across large geographic scales, as well as influencing the metabolic fitness of populations. Herein we report on leaf dark respiration of 33‐year‐old trees of jack pine (Pinus banksiana Lamb.) grown in three contrasting North American common gardens (0.9, 4.6, and 7.9 °C, mean annual temperature) comprised of identical populations of wide‐ranging geographic origins. We tested whether respiration rates in this evergreen conifer acclimate to prevailing ambient air temperatures and differ among populations. At each of the common gardens, observed population differences in respiration rates measured at a standard temperature (20 °C) were comparatively small and largely unrelated to climate of seed‐source origin. In contrast, respiration in all populations exhibited seasonal acclimation at all sites. Specific respiration rates at 20 °C inversely tracked seasonal variation in ambient air temperature, increasing with cooler temperatures in fall and declining with warmer temperatures in spring and summer. Such responses were similar among populations and sites, thus providing a general predictive equation regarding temperature acclimation of respiration for the species. Temperature acclimation was associated with variation in nitrogen (N) and soluble carbohydrate concentrations, supporting a joint enzyme and substrate‐based model of respiratory acclimation. Regression analyses revealed convergent relationships between respiration and the combination of needle N and soluble carbohydrate concentrations and between N‐based respiration (R_N, μmol mol N^{? 1} s^{? 1}) and soluble carbohydrate concentrations, providing evidence for general predictive relationships across geographically diverse populations, seasons, and sites. Overall, these findings demonstrate that seasonal acclimation of respiration modulates rates of foliar respiratory carbon flux in a widely distributed evergreen species, and does so in a predictable way. Genetic differences in specific respiration rate appear less important than temperature acclimation in downregulating respiratory carbon fluxes with climate warming across wide‐ranging sites.     

Adventitious root formation in cuttings of loblolly pine (Pinus taeda L .): developmental sequence and effects of maturation

 Adventitious root formation in cuttings from fascicular shoots in loblolly pine (Pinus taeda L.) consists of four more or less discontinuous stages: (1) proliferation of cells at the base of the cutting, (2) differentiation of wound vascular tissue and periderm, (3) dedifferentiation of a zone near the wound cambium and wound phloem to form a root initial, and (4) formation of a root meristem. Anatomical changes during adventitious root initiation are described in cuttings from donors of different types and ages. Cuttings from seedlings and 3- to 7-year-old hedged stock plants rooted better than cuttings from 3-year-old tree form donors. It is concluded that the loss of rooting capacity in loblolly pine can be arrested by shearing loblolly pine stock plants to low hedges. The process of root initiation, however, was similar in cuttings from all sources and is apparently not the cause for the rapid decline of rooting potential with increasing age of the donor plant. Received: 3 June 1997 / Accepted: 15 August 1997     

A relationship between irradiation, carbohydrates and rooting in cuttings of Pisum sativum

Rooting ability was studied for cuttings derived from pea plants ( Pisum sativum , L. cv. Alaska) grown in controlled environment rooms. When the cuttings were rooted at 70 μmol m⁻² s⁻, ¹ (photosynthetic photon flux density) or more, a stock plant irradiance at 100 μmol m⁻² s⁻¹ decreased rooting ability in cuttings compared to 5 μmol m⁻², s⁻¹, However, cuttings rooted at 160 μmol m⁻² s⁻¹ formed more roots compared to 5 (μmol m⁻² s⁻¹. Although a high irradiance increased the number of roots formed, it could not overcome a decreased potential for root formation in stock plants grown at high irradiance. Light compensation point and dark respiration of cuttings decreased by 70% during the rooting period, and the final levels were strongly influenced by the irradiance to the cuttings. Respiratory O₂ uptake decreased in the apex and the base of the cutting from day 2 onwards, whereas a constant level was found in the leaves. Only the content of extractable fructose, glucose, sucrose and starch varied during the early part of the rooting period. We conclude that the observed changes in the cuttings are initiated by excision of the root system, and are not involved in the initiation of adventitious roots.     

Maturation-related loss in rooting competence by loblolly pine stem cuttings: The role of auxin transport, metabolism and tissue sensitivity

A comparison of rooting ability of stem cuttings made from hypocotyls and epicotyls from 50-day-old seedlings of loblolly pine ( Pinus taeda L. ) reveals a dramatic decline by epicotyl cuttings, which do not root at all in 20–30 days in the presence or absence of auxin. In contrast, almost all the hypocotyls root during this time, but only in the presence of exogenously applied auxin. The failure of epicotyls to root does not appear to be due to differences in [¹⁴C]-labeled auxin uptake, transport, metabolism, or tissue distribution in the two types of cuttings. At the cellular level, initial responses to auxin, such as differentiation of the cambium into parenchyma, occur in both types of cuttings, but localized rapid cell division and root meristem organization are not observed in epicotyls. Autoradiography revealed that radioactivity from a -naphthalene acetic acid is bound in the cortex but not concentrated at sites of root meristem organization prior to the organization of the meristem in hypocotys. During the development of the epicotyl. cellular competence to form roots appears to be lost. Although this loss in competence is not associated with a concurrent loss in ability to transport auxin polarly, the latter process appears to play a key role in rooting other than to move auxin to the site of root formation. The phytotropin N-(1-naphthyl)phthalamic acid inhibits rooting if applied during the first 3 days after the cutting is made, but does not affect auxin concentration or metabolism at the rooting site.     

Adventitious root initiation in hypocotyl and epicotyl cuttings of eastern white pine (Pinus strobus) seedlings

The present paper reports results of experiments to develop a system for studying adventitious root initiation in cuttings derived from seedlings. Hypocotyl cuttings of 2-week-old eastern white pine (Pinus strobus L.) seedlings were treated for 5 min with 0, 100, 200, 300, 400, 500 or 600 mg l^?1 (0, 0.54, 1.07, 1.61, 2.15, 2.69 or 3.22 mM) 1-naphthaleneacetic acid (NAA) to determine the effect on root initiation. The number of root primordia per cutting was correlated with NAA concentration and the square of NAA concentration. Thus, the number increased from less than one per cutting in the 0 NAA treatment to approximately 40 per cutting at 300 mg l-1 NAA, above which no substantial further increase was observed. The larger number of root primordia formed in response to increasing concentrations of NAA was due to the formation of primordia over a larger proportion of the hypocotyls. Histological analysis of the timing of root primordium formation in hypocotyl cuttings revealed three discernible stages. Progression through these stages was relatively synchronous among NAA-treated hypocotyl cuttings and within a given cutting, but variation was observed in the portion of different cuttings undergoing root formation. Control-treated hypocotyl cuttings formed root primordia at lower frequencies and more slowly than NAA-treated cuttings, with fewer primordia per cutting. Epicotyl cuttings from 11-week-old seedlings also formed adventitious roots, but more slowly than hypocotyl cuttings. NAA treatment of epicotyl cuttings caused more rapid root initiation and also affected the origin of adventitious roots in comparison with nontreated cuttings. NAA-treated epicotyl cuttings formed roots in a manner analogous to that of the hypocotyl cuttings, directly from preformed vascular tissue, while control-treated epicotyl cuttings first formed a wound or callus tissue and subsequently differentiated root primordia within that tissue. This system of inducing adventitious roots in pine stem cuttings lends itself to studying the molecular and biochemical steps that occur during root initiation and development.     

Early changes in auxin and ethylene production in vine cuttings before adventitious rooting

Node cuttings of in vitro cultured grapevine were rooted in absence of any growth regulator, before the onset of the axillary bud. There were two peaks of ethylene production at 2 and 10–12 h, well marked in the top and bottom portions of the cuttings for the former. The level of IAA increased in the basal portions of the cuttings only, from the 4th hour, and culminated at the 24th hour. The wound ethylene of the first rise might be initiating the sequence of reactions leading to root formation. The second ethylene rise might result from the beginning of the increase of the IAA level.     

Role of ethylene and auxin in regenerative differentiation and orientation of tracheids in Pinus pinea seedlings

A low auxin concentration (0.1% naphthalene acetic acid) induced tracheids with longitudinal polarity parallel to the hypocotyl axis in young Pinus pinea seedlings. Application of 0.1% ethrel laterally and 0.1% naphthalene acetic acid apically disturbed axial tracheid polarity and promoted the differentiation of tracheids with a lateral orientation. Ethrel by itself, with no auxin background, did not affect tracheid differentiation. Apical application of 1% gibberellic acid with the low auxin, reversed the polarity disorder induced by ethrel. Disturbance of axial tracheid polarity was observed under a high auxin concentration (0.5% naphthalene acetic acid) which was similar to the combined effect of ethrel and auxin. The high auxin concentration increased tracheid number significantly. This effect was curtailed following treatment with inhibitors of ethylene formation (Co²⁺; 1-aminoethoxy- vinylglycine) and action (Ag²⁺). The role of ethylene in controlling the differentiation of radial tracheids, which characterize the vascular rays of pines, is discussed.     

Role of auxin and gibberellin in regenerative differentiation of tracheids in Pinus pinea seedlings

A new experimental system was developed for studying the hormonal mechanisms which control tracheid differentiation. In this system the tracheids redifferentiated from parenchyma cells in the hypocotyl of young Pinus pinea L. seedlings. The experimentally induced tracheids have unique shapes and patterns, and are therefore easily distinguished from the primary and secondary tracheids formed before the experiments. Auxin (0·1–1% NAA) alone sufficed to cause the redifferentiation of short tracheids, usually in discontinuous patterns across the hypocotyl. Gibberellin by itself did not induce redifferentiated tracheids. Combinations of auxin with gibberellin (0·1% NAA+0·1–1% GA₃) promoted the differentiation of long tracheids (up to threefold greater than those induced by auxin) in continuous patterns along the stem axis. Gibberellin in the presence of auxin promoted tracheid elongation by stimulating intrusive growth of both the upper and lower ends of the differentiating tracheids. The role of auxin and gibberellin in controlling the evolution of tracheary elements, from tracheids to vessels and fibres, is discussed.     

The role of light and polar auxin transport in root regeneration from hypocotyls of tomato seedling cuttings

A relationship between light conditions, auxin transport and adventitious root formation by hypocotyls of tomato seedling cuttings was demonstrated. Effective rooting of tomato seedling cuttings was observed under continuous white light (WL) irradiation. However, root formation was reduced in darkness or under red (RL) or blue light (BL). At least 3/4-day-long irradiation treatment with (WL) was necessary to increase the number of roots formed in comparison with control cuttings grown in darkness. Light was most effective if applied during the first half of the 13-day-long rooting period. The role of photoreceptor-dependent light perception in the light-regulation of rooting was tested using tomato photomorphogenic mutants: aurea (au) and high pigment (hp). When exposed to WL both mutants generated fewer roots then their isogenic wild type (WT). In darkness or under BL and RL less roots were formed on all plants and no difference was observed between mutants and WT plants. TIBA (2,3,5-triiodobenzoic acid) inhibited rooting in a dose-dependent manner both in darkness and under WL. However, although rooting was suppressed by 0.75 M TIBA in the dark, 8 M TIBA was necessary to block root formation in continuous WL. Inhibition of rooting by TIBA was most efficient when applied at the initial period of rooting, a 1-day-long treatment with TIBA being sufficient to suppress rooting if given during the first 2 days of culture. Later treatment had much less effect on the root formation.     

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 μM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 μM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.     

Changes in oxidation‐reduction state and antioxidant enzymes in the roots of jack pine seedlings during cold acclimation

The role of glutathione and the response of components of the ascorbic acid‐glutathione cycle in cold acclimation and the acquired freezing tolerance of jack pine ( Pinus banksiana Lamb.) seedlings were investigated. An increase in the reduced to oxidized glutathione mole ratio was correlated with the increase in root soluble and membrane‐bound protein thiol concentrations during cold acclimation and after a freeze and thaw event. All the enzymes involved in the ascorbic acid‐glutathione cycle were regulated by low temperatures and increased activities of ascorbic peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were observed after the conditioning of the seedlings to low temperatures. Our results suggest that these enzymes play a protective role following the exposure of the seedlings to freezing temperatures.     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.