Geert-Jan De Klerk

Review the formation of adventitious roots: New concepts, new possibilities

SummaryConsiderable progress has recently been made in understanding adventitious root formation using physiological studies. It is recognized that rooting is a process consisting of distinct phases, each with its own requirements. In this review, the successive phases in the rooting process are described and the possible roles of wounding-related compounds, auxin, ethylene and phenolic compounds during these specific phases are discussed. Recent results are assisting the development of advanced rooting treatments. Molecular studies on rooting are underway and will be essential in revealing the mechanisms underlying adventitious root formation.

Rooting of microcuttings: Theory and practice

SummaryPoor 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.

Effectiveness of indoleacetic acid, indolebutyric acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus ‘Jork 9’

We have examined in vitro rooting of apple ‘Jork 9‘ shoots exposed for three weeks to each of the three auxins commonly used for ex vitro rooting: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). During the initial five days of the rooting treatment, the cultures were incubated in darkness. In this period, the root initials are formed. Then, the cultures were moved to the light. NAA resulted in a low (ca. 8 roots), and IAA or IBA in a high (ca. 15 roots) maximal root number. The maximal root number was reached at a wide range of IAA concentrations (10-100 μM) but at only one concentration of IBA (10 μM) or NAA (3 μM). With NAA and IBA, growth of roots and shoots was much more inhibited than with IAA. For these reasons, IAA is the preferable auxin for in vitro rooting of apple ‘Jork 9’ shoots.

Plant growth regulators II: Cytokinins, their analogues and antagonists.

Hormones in plants differ from most of those in animals by having pleiotropic effects; that is, they are involved in the control of a wide range of developmental processes. At the same time the effect of a hormone on any developmental process depends on the species. For example, ethylene inhibits growth in dicotyledons and most monocotyledons but is promotory in deepwater rice and other hydrophytes. Moreover, two or more hormones can interact synergistically or antagonistically in many circumstances. Equally, any given hormone may affect the biosynthesis or metabolism of another, thus affecting endogenous levels. The issue is further complicated by the fact that environmental factors e.g. light, water status, wounding, pathogens may modify responses and indeed hormone levels themselves. The reason for this appears to be that hormones (and growth regulators) and environmental factors share many components in their transduction chains (i.e. the very early events which occur after the signal abiotic or biotic is perceived by the plant tissue). These transduction chains interact to produce an integrated response.

Effect of sucrose on adventitious root regeneration in apple

We have examined the effect of sucrose on adventitious root formation in apple microcuttings and in 1-mm stem slices cut from apple microcuttings. The sucrose concentration influenced the number of adventitious roots, but at a broad range of sucrose concentrations (1–9%) the effect was small. In addition, there was an interaction between sucrose and auxin: increasing the sucrose concentration shifted the dose–response curve of auxin to the right. When slices were cultured on medium without sucrose for the initial period (0–48 h), rooting was reduced whereas 48-h culture without application of sucrose had hardly any effect or even a slight promotive effect in a later period (48–120 h). The results show that during adventitious root formation, applied sucrose is used as a source of energy and building blocks but they are also in accordance with a possible regulatory role of sucrose.

The development of dormancy in bulblets of Lilium speciosum generated in vitro. 1. The effects of culture conditions.

We have studied the effect of various in-vitro conditions on dormancy of bulblets generated on scale explants of Lilium speciosum Thunb. cv. ‘Rubrum’ nr. 10. The bulblets were harvested after 11 weeks of culture. Dormancy was measured by determining the percent emergence in soil of viable, non-cold-treated bulblets. A study of the physical conditions showed that temperature had a strong effect on the induction of dormancy (15°C induced hardly any dormancy; 25°C induced a high level of dormancy), whereas short or long day and light or dark had no effect. Of the medium components, a low concentration of sucrose (1 gl−1 or less) or a high concentration of gibberellic acid (1 mg 1−1) reduced the level of dormancy. Application of various concentrations of abscisic acid, 6-benzylaminopurine, α-naphthaleneacetic acid, indole-3-acetic acid, 2,3,5-triiodobenzoic acid or a Murashige and Skoog macro- and microelement mixture did not affect the dormancy status.

Effects of phenolic compounds on adventitious root formation and oxidative decarboxylation of applied indoleacetic acid in Malus ‘Jork 9’

Stem slices (1-mm thick) cut from apple microshoots were cultured on a modified Murashige-Skoog medium with indole-3-acetic acid (IAA) or α-naphthaleneacetic acid (NAA), and increasing concentrations of various phenolic compounds. Both auxins were added at a concentration suboptimal for rooting. Indole-3-acetic acid is metabolized through oxidation and conjugation but NAA through conjugation only; which might have affected the results. With IAA, all tested orthodiphenols, paradiphenols and triphenols promoted adventitious root formation from the stem slices. Ferulic acid (FA, a methylated orthodiphenol) had the largest effect and increased the number of adventitious roots from 0.9 to 5.8. With NAA there was little or no promotion after addition of phenolics. Phloroglucinol (a triphenol) and FA were examined in detail. Their effects on the dose–response curve of IAA and the timing of their action indicated that both acted as antioxidants protecting IAA from decarboxylation and the tissue from oxidative stress. Experiments with carboxyl-labelled IAA showed that IAA was massively decarboxylated by the slices and that decarboxylation was strongly reduced by phenolics. Decarboxylation was to a great extent attributable to the wound response and did not occur to such an extent in non-wounded plant tissues. In shoots, FA promoted little rooting. Slices were cultured on top of the medium and shoots were stuck into the medium. Possibly, the anaerobic conditions in the medium near the basal part of the stem of shoots reduced the wound response and consequently decarboxylation of IAA. The monophenolic compound salicylic acid (SA) promoted IAA decarboxylation. Accordingly, SA reduced rooting when added during the initial days of the rooting process (the period during which auxin enhances rooting), and promoted outgrowth of root primordia later on (the period during which auxin inhibits rooting).

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.

The role of cytokinins in rooting of stem slices cut from apple microcuttings

ABSTRACT We examined the role of cytokinins in rooting of 1-mm stem slices cut from microcuttings of the apple rootstock ‘Jork 9˚s. Various types of cytokinins inhibited the rooting of apple stem slices to different extents. Highest inhibition was obtained with thidiazuron and benzylaminopurine. Remarkably, isopentenyladenine and isopentenyladenosine enhanced rooting at low concentration (at the optimal concentration of 0.1 μM by 53 and 19%, respectively). We also examined the effect of lovastatin and simvastatin. These drugs are putative cytokinin-synthesis inhibitors. Both inhibited rooting and inhibition was partially reversed by simultaneous addition of zeatin. Moreover, in the presence of lovastatin a higher concentration of zeatin had to be applied to achieve inhibition of rooting than in the absence of the drug. This data indicates that these compounds indeed inhibited cytokinin synthesis. One-day pulses with lovastatin strongly blocked rooting when given just after cutting the slices but had no effect after that. Adding zeatin simultaneously reversed inhibition completely. In conclusion, our data confirm that cytokinins may strongly inhibit rooting but they also show that at low concentration, certain cytokinins enhance rooting. Moreover, synthesis of cytokinin is essential during root formation. We hypothesise that cell division initiated by a relatively high endogenous level of cytokinins just after cutting the slices is a necessary, initial step in adventitious root formation.

Stem segments of apple microcuttings take up auxin predominantly via the cut surface and not via the epidermal surface

In conventional cuttings, auxin applied to achieve rooting is taken up predominantly via the cut surface and not via the epidermal surface of the stem. Even though in tissue-cultured plants the cuticle is poorly developed and the stomata do not function properly, stem segments of apple microcuttings took up labelled indoleacetic acid also predominantly via the cut surface. Stem segments with an epidermis with gaps caused by excision of the petioles required a lower exogenous concentration of auxin to achieve rooting than segments with an intact epidermis, indicating that the gaps facilitated uptake of auxin from the medium. This was confirmed in an experiment on uptake of labelled indoleacetic acid.