Distinct modes of adventitious rooting in Arabidopsis thaliana

The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species.     

Role of basipetal auxin transport and lateral auxin movement in rooting and growth of etiolated lupin hypocotyls

The involvement of polar auxin transport (PAT) on the growth of light-grown seedlings and rooting is generally accepted, while the role of auxin and PAT on the growth of dark-grown seedlings is subject to controversy. To further investigate this question, we have firstly studied the influence of NPA, a known inhibitor of PAT, on the rooting and growth of etiolated Lupinus albus hypocotyls. Rooting was inhibited when the basal ends of de-rooted seedlings were immersed in 100 micro m NPA but was partially restored after immersion in NPA + auxin. However, NPA applied to de-rooted seedlings or the roots of intact seedlings did not inhibit hypocotyl growth. It was taken up and distributed along the organ, and actually inhibited the basipetal transport of ((3)H)-IAA applied to isolated hypocotyl sections. Since the apex is the presumed auxin source for hypocotyl growth and rooting, and the epidermis is considered the limiting factor in auxin-induced growth, the basipetal and lateral auxin movement (LAM) after application of ((3)H)-IAA to decapitated seedlings were studied, in an attempt to evaluate the role of PAT and LAM in the provision of auxin to competent cells for growth and rooting. Local application of ((3)H)-IAA to the stele led to the basipetal transport of auxin in this tissue, but the process was drastically reduced when roots were immersed in NPA since no radioactivity was detected below the apical elongation region of the hypocotyl. LAM from the stele to the cortex and the epidermis occurred during basipetal transport, since radioactivity in these tissues increased as transport time progressed. Radioactivity on a per FW basis in the epidermis was 2-4 times higher than in the cortex, which suggests that epidermal cells acted as a sink for LAM. NPA did not inhibit LAM along the elongation region. These results suggest that while PAT was essential for rooting, LAM from the PAT pathway to the auxin-sensitive epidermal cells could play a key role in supplying auxin for hypocotyl elongation in etiolated lupin seedlings.     

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.     

Juvenility and endogenous rooting substances inCastanea sativa Mill

The rooting response to exogenous auxin of cuttings in a juvenile phase of growth from plants ofCastanea sativa Mill. was determined and simultaneously the rooting potential of the water extracts was evaluated in presence of IAA by a bean rooting test. The level of the extractable rooting promoters was high in the cuttings which exhibited the highest percentage of rooting. An inhibition of the effect of IAA on rooting was detected in the cuttings which showed the lowest rooting response, the histogram differing not much from that of the adult plant. The results indicate that in chestnut the juvenile condition, easy rooting, is associated with high levels of endogenous rooting promoters.     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

Differences in endo/exogenous auxin profile in cuttings of different physiological ages

The process of physiological ageing in woody plants is a very important factor influencing adventitious rooting. However, there is a lack of knowledge of biochemical backgrounds triggering ageing and consequently, rhizogenesis. Experiments with Prunus subhirtella ‘Autumnalis’ leafy cuttings of three different physiological ages (adult (over 40-year-old stock plants), semi-adult (5-year-old cutting plants) and juvenile (5-year-old in vitro plants)) were conducted in 2009. Half of the cuttings were banded ca. 3 cm above the bottom of the cutting with aluminum wire prior to insertion into the substrate to block the polar auxin transport. IBA, which was exogenously applied to the cuttings, could only be detected in the base of the cuttings on the first day after severance. Juvenile cuttings tended to have the highest values, but the effect was age specific. Later, the detection was not possible, regardless of the age. The IAA profile in cutting bases was similar for all physiological ages, reaching the peak on the first day after severance. Juvenile cuttings, in which the stems had been banded before insertion, contained more IAA in their bases on day 1 compared to the stems, which were not banded. These cuttings presumably transported absorbed auxin mainly via phloem, and not via mass flow like semi-adult and adult cuttings, where IAA concentrations were similar or even greater in non-banded cuttings compared to banded ones. These cuttings also tended to exhibit the best rooting results. The IAA-Asp accumulation was especially strong in adult cuttings, which contained significantly more aspartate on the first and third days after severance when compared with semi-adult and juvenile cuttings.     

Root Formation in Ethylene-Insensitive Plants

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.     

Rooting of microcuttings: Theory and practice

Summary Poor adventitious root formation is a major obstacle in micropropagation and in conventional propagation. This paper reviews recent progress in the understanding of adventitious root formation as a developmental process focusing on the role of plant hormones and on the effect of rooting conditions on plant performance. Since the discovery of the rhizogenic effect of auxin ca. 70 yr ago, no new broadly applicable rooting treatments have been developed. Recent research, though, may lead to new rooting procedures. Application of wounding-related compounds may be effective in difficult-to-root crops. Furthermore, by adapting conditions during the propagation phase, microcuttings with an enhanced capability to root may be produced. These conditions include elongation of stems (by etiolation or double-layer culture) and repeated subculture (rejuvenation; i.e. transition from adult to juvenile). Data are presented that show that during tissue culture maturation (transition from juvenile to adult) also occurs. The conditions during the in vitro rooting treatment may have a tremendous effect on performance after transfer ex vitro. In particular, accumulation of ethylene during in vitro rooting may have a devastating effect. Addition of stress-protecting compounds during propagation or rooting in vitro may enhance the performance ex vitro. Based on a presentation at the Plant Symposium ‘Rooting of Micropropagated Plants’ at the 2001 Congress of In Vitro Biology held at St. Louis, MO, June 16–20, 2001.     

Constitutive expression of rice MADS box gene using seed explants in hot pepper (Capsicum annuum L.).

Two transgenic pepper plants were obtained from 255 seed explants that were infected with Agrobacterium LBA4404 (pGA1209). One of them (PT2) showed morphological change, such as dwarfism and early flowering by the constitutive expression of the rice OsMADS1 gene. The in vitro condition of the plant regeneration has been optimized from hypocotyl explants on a MS medium that was supplemented with zeatin 3 mg/L, IAA 0.3 mg/L for shoot induction. The optimal rooting condition was at NAA 0.3 mg/L. The transformation frequency was 0.8% from the total hypocotyls. DNA and RNA hybridization analyses showed that the introduced gene was integrated and stably expressed in regenerated plants.     

Etiolation and flooding of donor plants enhance the capability of <Emphasis Type="Italic">Arabidopsis</Emphasis> explants to root

Rooting of cuttings depends not only on the rooting treatment and the genotype, but also on the condition of the cuttings at the time of excision. The physiological and developmental conditions of the donor plant may be decisive. We have examined in Arabidopsis the effect of two donor plant pre-treatments, etiolation and flooding, on the capability of flower stem and hypocotyl segments to root. For etiolation, plantlets were kept in the dark, hypocotyls up to 12 days and plantlets for 12 weeks. Flooding was applied as a layer of liquid medium on top of the semi-solid medium. This procedure is also referred to as “double layer”. Both pre-treatments strongly promoted rooting and we examined possible mechanisms. Expression of strigolactone biosynthesis and signaling related genes indicated that promotion by etiolation may be related to enhanced polar auxin transport. Increased rooting after flooding may have been brought about by accumulation of ethylene in the cutting (ethylene has been reported to increase sensitivity to auxin) and by massive formation of secondary phloem (the tissue close to which adventitious roots are induced). Both pre-treatments also strongly lowered the endogenous sucrose level. As low sucrose favors the juvenile state and juvenile tissues have a higher capability to root, the low sucrose levels may also play a role.     

Effect of genotype on rooting of hypocotyls and in vitro-produced shoots of Pinus radiata

Significant genotypic variation at both the family and clone-within-family levels was seen for hypocotyl rooting and the rooting of adventitious shoots produced in vitro for Pinus radiata D. Don. High rooting frequencies for hypocotyls were obtained in the absence of exogenous auxin; auxin greatly stimulated the rooting of adventitious shoots. No correlation was seen between the rooting of hypocotyls and shoots; families whose hypocotyls rooted at high frequencies did not necessarily produce shoots that rooted at high frequency. No correlation was seen between adventitious shoot production and subsequent rooting at either the family or clone level. The lack of a negative correlation indicated that selecting families or clones for high levels of shoot production will not automatically select for low rooting ability, obviating a possible bottleneck for commercial propagation of Pinus radiata. Significant family by replication interaction suggests that rooting protocols could be optimized through manipulations of the rooting environment for each family.     

Micropropagation of juvenile and adult Annona squamosa

A micropropagation system for Annona squamosa L. (Sugar Apple) using hypocotyls of seedlings and nodal cuttings from 3-year-old plants was developed. Shoot proliferation was achieved with Woody Plant Medium supplemented with BA. Silver thiosulphate was added at 0.5 mg l^–1 to control leaf abscission. Rooting was obtained when subcultured shoots were preconditioned for 2 weeks in medium with 10 g l^–1 activated charcoal before treatment with 43 µm NAA or 39 µm IBA. Rooting was improved when galactose was used instead of sucrose in the rooting medium. The rooted plantlets were acclimatised successfully.Abbreviations NAA naphthaleneacetic acid - IBA indolebutyric acid - MS Murashige & Skoog Medium - WPM Woody Plant Medium - NN Nitsch Medium - Juv juvenile explant - Adu adult explant     

The Role of Endogenous Auxins and Ethylene in the Formation of Adventitious Roots and Hypocotyl Hypertrophy in Flooded Sunflower Plants (Helianthus annuus)

The role of ethylene and auxins in flood-induced adventitious root formation and hypocotyl hypertrophy in sunflower (Helianthus annuus L. cv. Russian) plants was studied. Flooding without aeration (F) resulted in a steady increase in ethylene in hypocotyls, and flooding with aeration (FA) caused a transient increase. Low light intensity increased ethylene levels but decreased adventitious root formation. Treatment of shoots with benzyladenine (BA) increased ethylene content in non-flooded (NF) but not in F or FA shoots. Twenty-four hours of flooding brought about a rise of endogenous indole-acetic acid (IAA) in hypocotyls. ¹⁴C-IAA applied to the shoot accumulated more in F and FA hypocotyls than in NF hypocotyls, and BA reduced this accumulation. There was less IAA metabolism in F and FA than in NF hypocotyls. Tri-iodo benzoic acid (TIBA) applied to the hypocotyls of F plants inhibited root production. Benzyladenine (BA) applied to the leaves had similar effect but was not effective when supplied to the shoot apex. BA did not inhibit flood-induced hypocotyl hypertrophy. Ethrel did not affect adventitious root formation in NF plants but did increase hypocotyl thickening. It is concluded that flood-induced adventitious root formation is stimulated primarily by an accumulation of auxins in the hypocotyls. Increases in ethylene might cause this auxin build up. Hypocotyl hypertrophy is presently thought to be the result of an interaction of auxin and ethylene with ethylene being the major factor.     

Distribution of indole-3-acetic acid in relation to the growth of etiolated Lupinus albus hypocotyls

The free indole-3-acetic acid (IAA) in methanolic extracts of etiolated hypocotyls of lupin ( Lupinus albus L., from Bari, Italy) was determined by fluorimetry. The distribution of IAA along the hypocotyls was parallel to the growth, but when growth ceased oscillations occurred in the auxin level. These oscillations could be related to processes of differentiation mediated by IAA. The oscillations did not obey any impulses from the apex, since the application of [1-¹⁴C]-IAA to decapitated plants gives a distribution of radioactivity which also presents an undulatory pattern. Our results support the hypothesis that morphogenesis can be regulated by information transmitted by the translocation of waves of auxin.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole-3-acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography-mass spectrometry with [¹³C₆]-IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h. Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N-1-naphthylphthalamic acid and 2,3,5-tri-iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5-tri-iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.     

Comparison of the effects of white light and the growth retardant paclobutrazol on the ethylene production in bean hypocotyls

At a concentration of 17 µmol·L^–1, paclobutrazol (PP), a triazole plant growth retardant, effectively reduced the elongation and increased the thickness of hypocotyls in 6-day-old Phaseolus vulgaris L. cv. Juliska seedlings, both in the light and in the dark. PP treatment did not increase the cell number in transverse sections of hypocotyls. The diameter of hypocotyls was uniform from the zone of intensive elongation along the whole hypocotyl in etiolated plants, but those grown in the light exhibited an additional lateral expansion at the base. Ethylene evolution was not reduced by PP in etiolated hypocotyls, and did not differ significantly in the elongating apical and fully grown basal zones. PP reduced the ethylene release by the growing zones in green hypocotyls, but not in the basal parts, which resulted in an increasing ethylene gradient towards the hypocotyl base. The level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, was much higher in retardant-treated hypocotyls than in the controls, which was due in part to the reduced malonylation. The swelling of the hypocotyl bases could be eliminated by inhibitors of ethylene biosynthesis or action, or could be induced by 10 µmol·L^–1ACC in control plants in the light. None of these treatments had a significant effect on the lateral expansion of hypocotyls in etiolated seedlings. PP treatment induced a similar effect to that of white light in etiolated seedlings, and amplified the effect of light in green plants with respect to the ACC distribution, and consequently, the ethylene production in the hypocotyls of 6-day-old bean seedlings. It can be concluded that the lateral expansion of hypocotyl bases in PP-treated green plants is controlled by ethylene.     

The role of hormones on morphogenesis of thin layer explants from normal and transgenic tobacco plants

The organogenic potential of thin layer stem explants of non-reproductive tobacco plants was tested on a hormone-free medium and under various hormonal conditions. A comparison was made between thin layers excised from normal and transgenic plants at the same developmental stage. The transgenic plants were transformed by insertion of TR- and TL-DNA from Agrobacterium rhizogenes 1855 root-inducing plasmid. The aim was to identify hormonal conditions capable of stimulating the expression of the flowering competence present in the differentiated stem tissues at the induced stage before any visible sign of transition to reproductive development. Flower neoformation, observed at the end of the culture period (day 25), occurred on untransformed thin layers only with kinetin treatment. Explants from transgenic plants showed flower bud regeneration on hormone-free medium, indoleacetic acid alone (1 μ M ), kinetin alone (1 μ M ), and most abundantly on indoleacetic acid plus kinetin (1 μ M each). No flower formation was observed on indolebutyric acid plus kinetin (10 μ M and 0.1 μ M , respectively) in both normal and transgenic explants. The latter treatment enhanced rooting instead, above all in the transgenic explants. On hormone-free medium vegetative bud formation was well expressed both by untransformed and transgenic explants, and enhanced by the combined, equimolar concentrations of indoleacetic acid and kinetin.
The results show that cytokinin allows flowering in florally determined stem explants from normal plants. In the transgenic explants, the flowering response increases when indoleacetic acid is added to cytokinin, thus suggesting a role for auxin in enhancing the expression of the florally determined state in thin cell layers of non-reproductive plants.     

Reproductive competence from an annual and a perennial perspective

Plants at early stages of development undergo a juvenile phase during which they are not competent to flower in response to environmental stimuli. The length of this phase varies among species and is extended in perennial plants particularly. In annuals, temporal changes in expression of microR156 (miR156), miR172, and their targets are correlated with the transition from the juvenile to the adult phase and flowering. This developmental transition in perennials is probably more complex than in other plants and the molecular mechanisms are less well understood. In addition, once perennials become adult and capable of reproduction they still keep some meristems in the vegetative state that contribute to their polycarpic growth habit. Juvenility and polycarpy, although considered as two different processes in perennials, might be related.     

Is the Root Effect on Flowering of Chenopodium rubrum Mediated by Cytokinins?

Root removal enhances flowering in the short day plant Chenopodium rubrum. The extent of this effect depends on the de-rooting time with respect to photoperiodic induction. The largest promotive effect is observed when de-rooting coincides with the start of the inductive treatment or, to a lesser extent, when performed before it. De-rooting 24 h after induction has no effect on flowering. The flower-inducing action of de-rooting 24 h before the start of induction is increased by benzylaminopurine (BAP), whether applied simultaneously with de-rooting or 24 h later. At the beginning of darkness, BAP inhibits flowering slightly when applied simultaneously with de-rooting but inhibits it strongly when applied 24 h later. Flowering in plants de-rooted 24 h after induction is inhibited strongly by BAP. Root removal at the beginning of inductive darkness does not change the level of endogenous cytokinins in induced shoot explants, but under continuous light the level of cytokinins in shoot explants decreases during the same period compared with the level in the shoots of intact plants. BAP does not affect the level of endogenous cytokinins in light but causes an apparent increase in induced segments. Thus, two phases of the de-rooting effect and cytokinin treatment may be distinguished: one in which flowering is enhanced by both treatments and which is linked directly to photoperiodic flower induction, and the other in which both treatments are inhibitory to flowering and which is related to morphogenetic events following induction. The time courses of the effectiveness of de-rooting and BAP treatment differ slightly, suggesting that the effect of de-rooting cannot be attributed solely to cytokinin deprivation. Received February 27, 1998; accepted March 3, 1998     

Response to auxin changes during maturation-related loss of adventitious rooting competence in loblolly pine (Pinus taeda) stem cuttings

Hypocotyl cuttings (from 20- and 50-day-old Pinus taeda L. seedlings) rooted readily within 30 days in response to exogenous auxin, while epicotyl cuttings (from 50-day-old seedlings) rarely formed roots within 60 days. Responses to auxin during adventitious rooting included the induction of cell reorganization and cell division, followed by the organization of the root meristem. Explants from the bases of both epicotyl and hypocotyl cuttings readily formed callus tissue in response to a variety of auxins, but did not organize root meristems. Auxin-induced cell division was observed in the cambial region within 4 days, and later spread to the outer cortex at the same rate in both tissues. Cells at locations that would normally form roots in foliated hypocotyl cuttings did not produce callus any differently than those in other parts of the cortex. Therefore, auxin-induced root meristem organization appeared to occur independently of auxin-induced cell reorganization/division. The observation that N-(1-naphthyl)phthalamic acid (NPA) promoted cellular reorganization and callus formation but delayed rooting implies the existence of an auxin signal transduction pathway that is specific to root meristem organization. Attempts to induce root formation in callus or explants without foliage were unsuccessful. Both the cotyledon and epicotyl foliage provided a light-dependent product other than auxin that promoted root meristem formation in hypocotyl cuttings.