Biology of Adventitious Root Formation, pp. 100-103 (2013)
Tim D. Davis, Bruce E. Haissig

CHARACTERISTICS OF ETIOLATION-INDUCED CHANGE IN ROOTING COMPETENCE

A general or localized exclusion of light from shoot tissue during the early stages of growth from apical or axillary buds can influence the competence for adventitious root formation in cuttings taken from the treated shoots (Gardner, 1936; Frolich, 1961; Stoutemyer, 1961; Herman and Hess, 1963; Harrison-Murray, 1982; Maynard and Bassuk, 1988). Gardner (1936), working with the difficult-to-root apple variety McIntosh, used black tape wrapped around young shoot tissue to exclude light. Subsequently, the cuttings were taken with the basal cut in the blanched area of the shoot, then placed in the propagation bench without the application of exogenous auxin. The effect of this blanching treatment on increased rooting potential was greatest when the shoot was wrapped with tape as near the shoot tip as possible, with up to 70% of the cuttings rooting. In comparison, when the expanding shoots were taped at greater distance basipetal to the shoot tip, the formation of adventitious roots by cuttings taken from these shoots was progressively reduced with the greater distance. These data indicate that the exclusion of light was most effective in changing or maintaining competence for rooting in the less differentiated, acropetal tissue of the growing shoot tip. Gardner also combined complete exclusion of light during the initial 7.5 cm of growth of McIntosh shoots in the spring, with the taping of the basipetal portion of the shoots at the time of transfer to daylight conditions. This approach allowed testing the rooting potential of tissue at the base of the cutting that had not undergone light-grown greening processes either prior to or after dark treatments. When these shoots were used as hardwood cuttings the following March, 97% of the cuttings rooted, an apparent increase in rooting as compared to the response to blanching described above. These results indicate that when the etiolation treatment is maintained over many months, competence for root formation is maintained.

Howard and coworkers have extensively studied the influence of dark treatments on adventitious root formation in softwood cuttings of the apple rootstock variety M.9. The average treatment response over four years of studies demonstrated that there was a seven-fold increase in rooting percentage for auxin treated cuttings that were etiolated initially, but acclimated in light for two weeks (78%), as compared to auxin treated, light-grown controls (11%) (Harrison-Murray, 1982). With the continued exclusion of light imposed by taping the basal portion of these etiolated shoots during the two week light acclimation period, there was an additional, small increase in the average rooting to 85%. When tape was applied to blanch non-etiolated shoots (ca. 4-cm-long), there was only a three-fold increase in rooting of the blanched softwood cuttings as compared to non-etiolated controls (Howard, 1981). These data indicate that for M.9, blanching light-grown shoot tissue's significantly less effective than etiolation, with or without taping, in changing or maintaining competence. However, the initial growth of shoots under heavy shading has been shown to he as effective as complete darkness in influencing the rooting potential of M.9 cuttings (Howard, 1984, 1985).

To test the effect of the time of imposition of dark treatment, relative to the stage of shoot growth, dark treatments were applied to shoots that had already been growing for three weeks or applied to newly breaking buds (Howard, 1982). After the dark treatments were imposed, there was a gradual change in the newly formed shoot tissue to the typical, etiolated phenotype. Two weeks after the termination of the dark treatment, distal cuttings from the dark-grown part of shoots rooted at a level comparable to basal cuttings that were etiolated beginning at bud-break (75% vs 73%, respectively). Thus, we can conclude that chronologically-young shoot tissue which is undergoing growth and differentiation is sensitive to dark treatment, whether given early or late in the growth from buds.

To assess the persistence of the effect of light exclusion on the maintenance of competence, etiolated shoots were acclimated in light for different periods of time, up to over three weeks, prior to taking cuttings (Howard, 1980). There was no effect of the length of light acclimation on rooting; all cuttings rooted at a high percentage. It is interesting to note that these shoots became green after a few days of exposure to light, indicating that the effect of etiolation on competence for rooting persisted subsequent to the expression of processes induced by light. In a later study (Howard, 1982), hardwood cuttings taken from shoots that were etiolated and subsequently allowed to green nine months earlier, rooted at a rate of 53% compared to 12% for the non-etiolated controls. This level of rooting for the etiolated hardwood cuttings was significantly less than the response obtained with softwood cuttings taken two weeks afire etiolation the previous summer, however, it does demonstrate that an intermediate level of competence persisted nine months after the etiolation treatment. Furthermore, when cuttings were taken from shoot tissue, which extended after the termination of the etiolation treatment, they rooted at a rate of 41% compared with 12% of equivalent, distal cuttings of non-etiolated shoots (Howard, 1981). The newly formed leaves that developed in the weeks immediately following the onset of light treatment showed signs of juvenile morphology. These observations suggest that the influence of etiolation persisted in tissue which was present during the etiolation treatment, as well as in tissue formed afire exposure to light. However, this subsequently formed tissue was derived from activity of the apical meristem that had been exposed to the etiolation treatment.

One important factor in determining the degree of persistence of the effect of etiolation is the duration of the dark treatment. For instance, when cuttings were removed from Hass avocado shoots grown in the dark to a length of 7.5 cm, they had a high proportion (92%) of adventitious root formation (Frolich, 19b1). However there was a rapid decline in the rooting of cuttings of these initially etiolated shoots as a result of each day of light exposure, with only 33% of the cuttings forming roots by day seven of light treatment. This leads to the conclusion that the influence of etiolation is short lived in avocado. However, when the basal portions of the initially etiolated shoots were maintained in darkness for up to five additional weeks, followed by exposure to light for seven days prior to taking cuttings, there was an increase in the rooting of cuttings. The cuttings that received four weeks of additional darkness rooted at a rate of 88%, even though they were exposed to light for seven days. Thus, competence for rooting was maintained at a high level in the light when the duration of the dark treatment was sufficiently long.

In total, these experiments with apple and avocado indicate that the effect of etiolation is not ephemeral, but persists up to many months subsequent to the exposure to light, as well as during the further growth and differentiation of the shoot tissue derived from apical meristems exposed to the dark treatment.

Two important questions arise from these observations about the influence of light exclusion on competence for adventitious root formation. First, is there an effect of light exclusion in other species? and, if so, is it a general response that permits the manipulation of rooting potential in mature-phase tissues of a number of species? Maynard and Bassuk (1987) tested the effect of light exclusion on the rooting of softwood cuttings of 21 difficult-to-root taxa. Light exclusion significantly improved adventitious root formation of cuttings in 18 of the 21 taxa. Furthermore, in a review of this subject, they list at least 42 taxa that have been reported to have increased rooting potential in response to fall or partial light exclusion (Maynard and Bassuk, 1988). Therefore, it appears that the influence of light exclusion on competence for adventitious root formation is a general response.

Second, what is the basis of the difference in rooting competence between dark-treated and light-grown control tissue of difficult-to-root species? A considerable amount of research has focused on the effect of light exclusion on physiological factors, such as the level of endogenous aunin and rooting cofactors (Herman and Hess, 1963; Kawase, 1965; Kawase and Matsai, 1980; Maynard and Bassuk, 1988). However, the persistent effect of etiolation with subsequent exposure to light on the rooting of M.9 hardwood cuttings nine months later (Howard, 1982), suggests there is a fundamental change that is not related to the level of endogenous aunin or rooting cofactors, or other transient metabolic effects. Furthermore, the maintenance of competence in tissue formed by meristematic activity after the exclusion of light, suggests there is either a transmissible factor produced in the initially etiolated tissue that influences rooting in later-formed tissue or, alternatively, a persistent effect on the apical meristem and cells derived from the meristem (Howard, 1981). There is little evidence (Kawase and Matsai, 1980; Howard, 1983) or no evidence (Frolich, 1961; Doss et al., 1980) for a factor that is transmissible over a significant distance from the region exposed to dark treatment.

*Cybe|Rose note: I am reminded of Michurin's "layering tube", which seems to have awakened genes that allowed rooting even years after. "With cuttings from the second vegetative generation, i.e., when rooting cuttings not from seedlings but from specimens previously rooted by cutting, the entire process of development of the roots takes place much more easily and quickly."

What influence, then, could etiolation have on the apical meristem that persists with cell divisions, growth and differentiation in light? Perhaps the exclusion of light from the apical meristem of chronologically young, undifferentiated shoot tissue results in a persistent change (but not a change in the nucleotide sequence) in the ability of cells to express genes that are required for rooting or that prevent rooting.* For instance, it can be hypothesized that etiolation might result in a change in the chromatin which permits or prevents the expression of key regulatory or structural genes, and that this altered chromatin is mitotically heritable in newly formed tissue. To test this hypothesis or alternative hypotheses, phase-dependent genes related to rooting or other phase characteristics first must be isolated and characterized with respect to the level at which they are regulated (i.e., transcriptionally or post-transcriptionally), and then tested in light exclusion experiments,

Finally, one obvious effect of etiolation or early blanching treatment is on the histology of the treated stem tissue. The blanching of Phaseolus vulgaris hypocotyls and Hibiscus rosasinensis stem tissue resulted in decreased cell watt thickness (i.e., reduced deposition of cellulose and hemicellalose) and decreased lignification of phloem and pericyclic fibers, relative to non-blanched controls (Herman and Hess, 1963). Additionally, a greater proportion of cells remained parenchymatous. Similar observations have been made in other species in regard to reduced sclerification (fibers or sclereids) is response to light exclusion (Stoutemyer, 1961; Doud and Carlson, 1977). It is interesting to note that Stoutemyer (1937) states is a review of adventitious root formation is Malus spp. that "...Practically the only distinction which can be pointed out is that the mature phase stem of Virginia Crab contains more pericyclic fibers than are evident in stems of the juvenile form. ...".  Similar observations have been made in juvenile- and mature-phase stem tissues of other Malus spp. (Beakbane, 1961). Therefore, light exclusion with mature-phase stems of apple (Doud and Carlson, 1977) results in stem tissue histology that is at least initially similar to the juvenile-phase characteristic of reduced fiber formation. However, we are not aware of any studies that tested the persistent effect of light exclusion on the histology of stem tissue subsequent to prolonged light treatment. If this altered histology per se is the primary determinant of altered competence for rooting, it would be expected that the characteristics of the cell types would persist after exposure to light. In lieu of altered histology, differential competence for the expression of rooting-related genes might persist after exposure to light. The following discussion provides insight into the possibility of differential gene expression of rooting-related genes in the juvenile- and mature-phase of English ivy.

Maynard, B.K. and N.L. Bassuk. 1985. Etiolation as a tool for rooting cuttings of difficult-to-root woody plants. Comb. Proc. Int. Plant Prop. Soc. for 1985, 35: 488-494.

The stockplant pretreatment techniques of etiolation and banding were used with success in the cutting propagation of 13 woody ornamental species. Each pretreatment alone was noted to have a significant effect on rooting while the combination of the two resulted in optimal rooting in most trials. An alternative banding method has been developed, using reusable adhesive bands of Velcro, which allows for the addition of root promoting chemicals as a part of the banding procedure. Substantial improvements in rooting response were obtained in a number of species previously considered difficult to root.

Bassuk, N.L., Diane Miske and B. K. Maynard. 1985. Stock plant etiolation for improved rooting of cuttings. Comb. Proc. Int. Plant Prop. Soc. for 1985, 35: 543-550.

The practice of stock plant etiolation, whereby dormant plants are grown under severely restricted light levels and then allowed to green up while shoot bases remain etiolated, using a covering of black adhesive tape, produced significantly better rooting of cuttings. Rooting was improved from 5% to 68.5% for Fagus sylvatica, from 15% to 42.5% for Carpinus betulus, and from 53.3% to 83.3% for Pinus strobus. Cuttings from 6 hybrid lilac cultivars also showed improved rooting with prior etiolation and, moreover, the period over which lilac cuttings could be propagated successfully was lengthened considerably.

Maynard, B.K. and N.L. Bassuk. 1987. Stock plant etiolation and blanching of woody plants prior to cutting propagation. J. Amer. Soc. Hort. Sci. 112:273–276.

A modification of the traditional technique of etiolation and blanching, using Velcro adhesive fabric strips as the blanching material, was used with success in improving cutting propagation of a wide range of difficult-to-root woody species. Stockplants were etiolated under black cloth at budbreak, followed by banding for a period of 4 weeks, to produce a cutting with an etiolated base. Rooting of softwood cuttings from 18 of 21 species tested was improved significantly by these stockplant pretreatments. The use of Velcro as the banding material facilitated blanching, permitted the application of rooting hormone as a part of the blanching procedure, wounded underlying stem tissues, and resulted occasionally in the formation of adventitious roots on intact shoots.

Maynard, B.K. and N.L. Bassuk. 1988. Etiolation to improve softwood cutting propagation: Aspects of hormone application and timing of taking cuttings. Comb. Proc. Intl. Plant. Prop. Soc. 37: 420–427.

The research presented here focuses on the factors affecting hormone application and timing of taking etiolated cuttings, with the goal of developing etiolation and banding into practical methods for use by the plant propagator. Etiolation was found to be more beneficial than banding when applied to 4 cultivars of Fagus sylvatica and 2 of 4 cultivars of Acer saccharum. In experiments with 2 cultivars of Syringa reticutala, banding, with or without etiolation, was most beneficial in stimulating rooting. The treatment of S. reticulata cuttings with 4000 ppm IBA was of little additional benefit. Conversely, cuttings from etiolated shoots of Stewartia pseudocamellia rooted best, the effect still evident after 2 months greening. Hormone applied at sticking was of a significant benefit to the rooting of Stewartia cuttings.