Stimulation of adventitious root formation by laser wounding in rose cuttings: A matter of energy and pattern

Abstract

Adventitious root (AR) formation is the basis of vegetative propagation in rose, be it via stem cuttings or via stenting. During this process, wounding plays a pivotal role since cell reprogramming takes place at the tissue adjacent to the wound. We investigated the effects of wounding on AR formation on leafy single-node stem cuttings of the rose rootstock R. canina 'Pfänder' (codes R02-3 and R02-6) and the cut rose cultivar Rosa 'Tan09283' (Registration name 'Beluga'). Laser wounding treatments were based on the assisted removal of tissue layers located in the bark. The positioning of wounding was studied based on two marking directions: along the cutting base (strip pattern) and around the cutting base (ring pattern). Additionally, the effects of external supply of indole-butyric acid (IBA 1 mg L^-1) on rooting were analyzed. Results showed that in order to remove specific tissue layers, the calculation of the laser energy density (J cm^-2) in terms of cutting diameter was necessary. Interestingly, the application of energy densities from 2.5 J cm^-2 up to approximately 8.5 J cm^-2 were sufficient to expose the tissue layers of epidermis up to regions of phloem. Regarding AR formation for R. canina 'Pfänder', characterized by a low rooting response, an increase in the rooting percentage was registered when the laser treatment eliminated the tissue up to phloem proximities. Analysis of the nodal position showed that bud location was a preferential place for AR formation independently of wounding treatment. In case of Rosa 'Tan09283', laser treatments did not reduce its high rooting capacity, but an apparent reduction in rooting quality due to an investment in tissue healing was observed when wounding reached deeper layers such as parenchyma and sclerenchyma. Results also showed a strong AR formation directly from wounded regions in case of Rosa 'Tan09283' specifically when the wound was located below the axillary bud. In conclusion, wounding by assisted-elimination of layers by laser can induce positive effects on AR formation of single-node stem cuttings of the rose if energy applied is able to expose phloem proximities, a longitudinal orientation, and relative position to the axillary bud are considered.

Keywords: Rosa canina; adventitious root formation; histology; laser ablation; stem cutting; wounding.

Figure 1

Power, beam size and beam profile determination. (A) Power determination of the laser equipment measured by a calorimeter J22201, a Mahoney, and Dohicky power meter. An enlarged section from 0 to 12% laser power is included at the corner of the diagram. (B) Beam size of a laser dot on the surface of a R. canina ‘Pfänder’ cutting. (C) Performance of a flap top beam profile (yellow marked) on a PMMA cylinder 7 mm outside and 5 mm inside.

Figure 2

Rose stem anatomy depending on the diameter. (A) Partial cross section of a 5 mm rose stem of Rosa ‘Tan09283’. f: fibers, p.r.: parenchyma radial, t: tracheid, v: vessel. (B, C) Different stem diameters representing different positions on the shoot for Rosa ‘Tan09283’ (B) and R. canina ‘Pfänder’ (C).

Figure 3

Histological analysis of laser wounding applied as strip and ring pattern on Rosa ‘Tan09283’ cuttings under four different energy densities (2.5 J cm^-2, 3.8 J cm^-2, 5.9 J cm^-2 and 8.5 J cm^-2). Arrows indicate sclerenchyma (black), phloem (red), vascular cambium (white) and xylem (yellow).

Figure 4

AR formation depending on the different wounding treatments of R. canina 'Pfänder' cuttings in presence of exogenous IBA (1 mg L-¹) after 4 week of culture. (A) Rooting percentage of R. canina 'Pfänder' R02-3 and R. canina 'Pfänder' R02-6 classified by the treatment (three laser intensities and two laser patterns): C- control treatment, B- blade (manual wounding) treatment, R- ring pattern, S- strip pattern. Columns represent the mean values with its confidence intervals (n=20). Columns that do not share a common letter represent significantly different levels (Tukey test, p ≤ 0.05). (B) Pearson`s correlation between rooting percentage and cutting diameter classified by treatment. (C) Pearson`s correlation between rooting percentage and cutting diameter classified by cultivar. **, *** Significant at P ≤ 0.01, 0.001, respectively.

Figure 5

AR formation of Rosa ‘Tan09283’ cuttings depending on the treatment (three laser intensities and two laser patterns) in the presence or absence of exogenous IBA (1 mg L^-1) after 4 weeks of culture. C- control, B- blade (manual) treatment, R- ring pattern, S- strip pattern. (A) Rooting percentage classified as basal and acrobasal rooting: Number (B) and total root fresh mass (C) per rooted cutting. Mean values and standard errors are shown at treatment level (n = 40). Columns indicated by the same letter, do not represent significantly different levels (Tukey test, p ≤ 0.05). ns, non-significant.

Figure 6

Adventitious root distribution regarding bud position by ring (A, B) and strip (C, D) patterns under the effect of exogenous auxin on Rosa ‘Tan09283’ cuttings. Left side: Stem cuttings are divided by vertical dashed white lines describing the division between the bud side and the opposite side of the cutting. Right side: Comparison of root mass ratio in presence and absence of exogenous auxin for Rosa ‘Tan09283’, R. canina ‘Pfänder’ R02-3 and R. canina ‘Pfänder’ R02-6. Columns and bars represent the mean values and SE (n= 20 for R. canina ‘Pfänder’ R02-3 and R. canina ‘Pfänder’ R02-6, n= 40 for Rosa ‘Tan09283’). Gray and blue sections in each bar represent the proportion of root mass on the cutting opposite to the bud or below the bud, respectively. Letters represent significantly different levels for each genotype (Tukey test, p ≤ 0.05).

Figure 7

Laser penetration relative to the bark thickness in terms of energy applied for R. canina ‘Pfänder’ R02-6 cuttings depending on stem diameter. The different tissue layers that compose rose bark are represented in different colors and arranged in proportion to their original dimension in the bark. The vertical broken black line describes the laser induced damage threshold (LIDT) determined at 2.5 J cm^-2. Dots represent the penetration mean values with SE, n = 5.

Figure 8

Comparison of ring and strip pattern laser treatments reaching defined tissue layers regarding rooting percentage and root distribution. AR formation of R. canina ‘Pfänder’ (A) and Rosa ‘Tan09283’ (B) cuttings when phloem and sclerenchyma layer were wounded without exogenous auxin. C- control treatment, R- ring pattern, S- strip pattern. Columns represent mean values and confidence intervals (n= 50 for R. canina ‘Pfänder’ R02-6 and n= 40 Rosa ‘Tan09283’). Root spatial distribution of the treatments for R. canina ‘Pfänder’ R02-6 (C) and Rosa ‘Tan09283’ (D). Red shaded region represents the laser treatment position in relation to the axillary bud. Columns that do not share a common letter represent significantly different levels (Tukey test, p ≤ 0.05); ns, non-significant.

Figure 9

Wounding and bud position influence AR formation. (A) Root spatial distribution of Rosa ‘Tan09283’ cuttings when the sclerenchyma layer was reached applying a strip pattern on the side of the axillary bud and the opposite side in presence of 1 mg L^-1 IBA. Red lines represent position of the laser strip treatment. (B) Dynamics of AR formation over time (days after planting) expressed as mean values with SE. (C) Root number and fresh mass distribution depending on the treatments. Columns represent the mean values and SE (n= 25). Columns that do not share a common letter represent significantly different levels (Tukey test, p ≤ 0.05); ns, non-significant.