JARS v45n4 - The Application of Stock Plant Etiolation and Stem Banding to the Softwood Cutting Propagation of Indumented Rhododendron Species

The Application of Stock Plant Etiolation and Stem Banding to the Softwood
Cutting Propagation of Indumented Rhododendron Species

Brian K. Maynard & Nina L. Bassuk
Department of Floriculture and Ornamental Horticulture, Cornell University
Ithaca, New York

A study was conducted to evaluate the stock plant treatments of etiolation and stem banding on subsequent adventitious root formation on softwood stem cuttings of Rhododendron smirnowii Trautv. and R. yakushimanum Nakai. Stock plants were etiolated in a greenhouse by promoting new growth under black cloth. Following etiolation, shoots were banded with Velcro for ~6 weeks before cuttings were collected. Cuttings were treated with 40mM IBA and rooted under mist for up to 240 d. The rooting of R. smirnowii was improved by stock plant etiolation more than by stem banding. Cuttings from shoots of R. smirnowii which had been both etiolated and banded rooted best. The rooting of R. yakushimanum increased following banding but was unaffected by etiolation. Stem banding reduced the number of roots formed on cuttings of R. smirnowii , but had no effect on the root number of cuttings of R. yakushimanum . After potting, the bud break of R. yakushimanum was improved in cuttings from etiolated and banded shoots, with 53% growing actively within 80 d as compared to 29% of control cuttings. However, stem banding actually appeared to reduce bud break and shoot growth in cuttings of R. smirnowii . Stock plant etiolation and stem banding substantially improved the propagation success of both of these indumented Rhododendron species. The relative benefit of each treatment was species-dependent, and the effects of the initial stock plant treatments on subsequent bud break were apparent into the establishment phase of propagation.

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Significance to the Nursery Industry
Tissue culture methods have been used with success in the vegetative propagation of numerous Rhododendron species, particularly azaleas. However, the establishment of pubescent and indumented rhododendrons in aseptic culture remains a problem due to difficulties in the surface sterilization of explants. Stock plant etiolation and stem banding prior to softwood cutting propagation offer effective, easily-implemented means of increasing the rooting capacity and quality of Rhododendron cuttings. This study shows that either etiolation, banding, or a combination of the two may be used to substantially improve the cutting propagation of two indumented Rhododendron species, R. smirnowii and R. yakushimanum .

Stock plant etiolation is a technique which involves excluding light during new shoot growth. Stem banding during stem development is an additional stock plant treatment which excludes light from that section of stem which will become the cutting base. A modification of stem banding makes use of a reusable self-adhesive fabric known as Velcro, and has been shown, along with stock plant etiolation, to yield increased rooting percentages, more roots per rooted cutting and faster rooting in numerous difficult-to-root woody plant species (5, 6, 7). These stock plant treatments have not been extensively tested on the propagation of rhododendrons or azaleas. Rhododendrons are notoriously slow to root and typically require high concentrations of applied rooting hormones (2, 11), while many azalea cultivars are being cloned on a commercial scale via tissue culture. Species of Rhododendron which possess a dense indumentum on the lower leaf surface, such as R. yakushimanum and R smirnowii , are reportedly difficult to establish in vitro because of problems with contamination by pathogens retained by the indumentum. The extreme measures necessary to surface sterilize ex-plants of indumented rhododendrons, such as removal of the indumentum, often result in injury to the explant tissue (B. Briggs, personal communication).
A study by Davis and Potter (1) demonstrated that localized etiolation (blanching with aluminum foil) was beneficial to the rooting of 2 rhododendron cultivars tested, R. 'Corsage' and R. 'Ramapo'. The treatment lasted for 5 weeks and resulted in faster rooting and larger root balls ( R. 'Ramapo'). It has been suggested that etiolation of the whole stock plant may have resulted in even greater success with this wide range of Rhododendron cultivars (T.D. Davis, personal communication). Pierik (8) demonstrated the benefit of excluding light in promoting the rooting, in vitro , of isolated stem segments of R. 'Catawbiense Album' and R. 'Pink Pearl'.
Shading stock plants is a common nursery practice for improving the rooting of Rhododendron and azalea cuttings (8, 11). This may result from the production of thinner cutting wood, which gives better rooting than strong, vigorous terminal growths (2). In a study with 6-year-old stock plants of R. 'Roseum Elegans', Johnson and Roberts (4) found that 95% shading increased the rooting of leaf-petiole cuttings. French (3) found that shading 3-year-old stock plants of R. ' Anna Rose Whitney' by 80% significantly increased root ball diameters and rooting depths. Rooting percentages were relatively unaffected, increasing from 90 to 97% following shading.
This study was undertaken to examine the usefulness of stock plant etiolation and stem banding with Velcro in improving the softwood cutting propagation of two indumented Rhododendron species.

Etiolated and banded shoots of 
R. smirnowii
Etiolated and banded shoots of etiolated
and banded shoots of R. smirnowii
Photo by author

Materials and Methods
Thirty 25-year-old field-grown stock plants of Rhododendron yakushimanum Nakai and four 25-year-old field-grown seedling stock plants of R. smirnowii Trautv. (provided by Dr. Gus Mehlquist, Storrs, CT) were potted into 40 liter plastic containers in a medium of 1 sandy loam soil: 4 sphagnum peat moss: 1 perlite (by volume) and placed in a greenhouse maintained at an average day/night temperature of 20°C. Incandescent lamp (60W), suspended about 3 m above the stock plants and spaced 1 m apart, were used from 4:00 PM to 12:00 PM to extend the natural photoperiod to 16-hr. Stock plants were fertilized weekly with 200 mg/liter 20N-10P-20K. Stock plants of both species began to break bud between 15 May and 21 May, 1989. Etiolation was applied to 2 plants of R. smirnowii and 6 plants of R. yakushimanum beginning at bud break and proceeding for 7 to 11 d, respectively. Etiolation involved forcing new growth to develop under black cloth (>98% shade) to produce typical etiolated growth (lack of green color, lengthened internodes, succulence). In addition, stock plants of R. yakushimanum were forced under intermediate shade levels of 50, 75 and 95% irradiance. Remaining stock plants of both species were grown without shade. Following the etiolation treatment, shoots from half of the light-grown, shaded and etiolated plants were basally banded using 2.5cm wide strips of Velcro coated with 0.8% indole butyric acid (IBA) in talc, the standard procedure for stem banding developed by Maynard and Bassuk (5). Etiolated plants were then acclimated to full sunlight over 4 d by first removing the cover on the north side, and then gradually rolling the cloth back each day until all plants were in full sun. Bands remained in place while the stock plants greened for ~6 weeks before cuttings were harvested for propagation (July 6, 1989). Basal shoot cuttings were taken from positions throughout the stock plant crown, and prepared to a length of 7-10 cm with 3-4 terminal leaves. Banded shoots were cut immediately below the Velcro bands and the bands removed. Four types of softwood cuttings were taken: (1) light-grown, non-banded shoots (controls); (2) light-grown, banded shoots (banded); (3) etiolated shoots which had greened for 6 weeks (etiolated); and (4) etiolated and banded shoots, which yielded a cutting with an etiolated base (etiolated and banded). The cuttings receiving each treatment were then distributed randomly into 8 groups of 8 cuttings each prior to insertion into the rooting medium in a completely randomized design. The cuttings of each group were then treated with a 5-second dip in 39.4 mM (8,000 ppm) IBA (in 50% aqueous ethanol), allowed to dry for 15 minutes and inserted to a depth of ~2 cm in a medium of 1 perlite: 2 sphagnum peat moss (by volume) and rooted under mist (6 seconds every 12 minutes from 6:00 AM to 8:30 PM) and a 16-hr photoperiod (see above) provided by 60W incandescent lamps suspended 1 m above the medium and spaced 1 m apart. Cuttings were evaluated for rooting after 80, 160 and 240 d and were considered rooted if they possessed one or more roots > 1 mm in length. Rooting percentage, the number of roots per rooted cutting and the length of the longest root or root ball diameter, in mm were recorded. Data were analyzed using the General Linear Means procedure of SAS (9); percentage data were transformed to arcs in square root before analysis (10). All cuttings were potted into 6" plastic pots in a medium identical to the rooting medium for evaluation of establishment and bud break. Bud break and new shoot length were evaluated 80 d after potting.

R. smirnowii and R. yakushimanum in 
rooting bench
R. smirnowii and R. yakushimanum in rooting bench
Photo by author

Table 1. Stock plant etiolation and stem banding effects on rooting percentages, root number per rooted cutting (rrc) and longest root (lr) lengths of Rhododendron smirnowii and R. yakushimanum after 80, 160 and 240 d rooting.
stock plant treatment
light-grown etiolated statistical significance z
- bane + band - band + band etiolation banding interaction
R. smirnowii
rooting %: 80 d 33 45 56 73 ** NS NS
160 d 45 53 60 77 ** NS NS
240 d 53 57 62 77 * NS NS
rrc: 80 d 7 3 4 3 NS ** *
160 d 7 3 4 4 NS * *
240 d 7 3 4 4 NS * *
lr (mm): 80 d 12 15 19 15 NS NS NS
160 d 10 13 19 14 NS NS NS
240 d 10 14 19 14 NS NS NS
R. yakushimanum
rooting %: 80 d 17 31 22 31 NS * NS
160 d 22 42 22 48 NS * NS
240 d 27 45 25 53 NS ** NS
rrc: 80 d 4 3 3 3 NS NS NS
160 d 4 3 3 3 NS NS NS
240 d 4 3 3 3 NS NS NS
lr (mm): 80 d 9 5 4 6 NS NS NS
160 d 10 6 4 7 NS NS *
240 d 9 6 4 7 NS NS *
z Significance determined by ANOVA: at the 0.05 (*), 0.01 (**), or 0.001 (***) level, or non-significant (NS).

Results and Discussion Rhododendron smirnowii
The etiolation of shoots of R. smirnowii significantly increased the percentage of softwood stem cuttings which rooted between 80 and 240 d (Table 1). Initially, etiolation increased rooting by 25% over the control response; after 240 d by 14%. Banding initially increased rooting by 15%, though this benefit fell to ~10% by 160 and 240 d. Etiolation and banding appeared to act independently (i.e., no statistically significant interaction) and together increased rooting by 40% over the control after 80 d, 30% after 160 d and 23% after 240 d rooting time.
In contrast to its effect on rooting percentages, stock plant etiolation had no effect on root number, which averaged 4 roots per rooted cutting at each date (Table 1). Banding without prior etiolation, however, decreased the number of roots formed on light-grown cuttings. The length of the longest root produced averaged 16 mm and was unaffected by either stock plant treatment. Cuttings possessing root balls were not included in the estimation of root length. Of 60 rooted cuttings 31 possessed root balls, averaging 4.6 cm diameter.
Fifty-five percent of those cuttings which rooted broke bud within 80 d of being potted. New shoot growth on these cuttings averaged 3.2 cm. Stem banding appeared to reduce both bud break percentage (39% vs. 66% of non-banded cuttings) and subsequent shoot elongation (2.5 cm vs. 3.4 cm on non-banded cuttings). Etiolation did not affect either growth response. At the time of potting, the average number of roots on those cuttings which broke bud was 5, while on those cuttings which remained dormant the number of roots at potting averaged 3 (p = 0.06). Root-system size appeared to promote bud break; the root ball diameters of cuttings which broke bud averaged 5.2 cm, while those of cuttings which did not break bud averaged 3.7 cm (p = 0.09). The presence of the root ball on the cutting apparently had no influence on subsequent bud break; 16 cuttings with root balls broke bud, 15 did not.

Rhododendron yakushimanum .
After 80 d, the rooting of cuttings of R. yakushimanum averaged 25%, with no significant differences between light-grown and etiolated stock plant treatments (Table 1). Regardless of the initial stock plant treatment, banding increased rooting from 20% (non-banded) to 31 % (banded) in this time. After 160 d the banding response doubled the rooting of non-banded shoots, from 22% to 45% (p = 0.001). No etiolation effect was apparent at this time. No differences were apparent in the rooting responses observed at 160 and 240 d. Banding improved rooting at 240 d, from 26% to 49%, while etiolation had no effect on rooting percentages.
Softwood stem cuttings of R. yakushimanum produced 3 roots per rooted cutting on average, regardless of stock plant treatment or rooting time. Similarly, the length of the longest root averaged 6 mm. Root balls were present on 12 of 69 cuttings which rooted, and averaged 3.3 cm diameter.

Table 2. Stock plant shading effects on rooting percentages, root number per rooted cutting and longest root lengths of Rhododendron yakushimanum after 250 d rooting.
stock plant treatment
light-grown 50% shade 75% shade 95% shade statistical significance z
parameter - band + band - band + band - band + band - band + band shading banding interaction
rooting % 27 45 16 28 6 22 2 23 *** *** NS
roots/rooted cutting 4 3 4 2 4 7 8 12 * NS NS
longest root (mm) 9 6 6 7 7 5 19 11 NS NS NS
cutting mortality (%) 5 0 11 2 2 5 69 52 *** y *** ***
z Significance determined by ANOVA: at the 0.05 (*), 0.01 (**), or 0.001 (***) level, or non-significant (NS).
y Significance determined by Chi-Square analysis: at the 0.01 (**), or 0.001 (***) level.

Continuous shading of the stock plants using either 50, 75, or 95% Saran dramatically reduced rooting (Table 2). Despite this reduction in rooting, stem banding independently improved rooting in all shading treatments. The mortality of cuttings during the rooting period increased with shade level. Banding also reduced cutting mortality in all shading treatments. Virtually none of the light-grown or initially etiolated cuttings (data not presented) died during the rooting phase. Interestingly, the shoots harvested from the heaviest shading treatments produced up to 12 roots per rooted cutting with an average longest root length of 10-19 mm, versus an average of 3 roots per rooted cutting and 6 mm among other stock plant treatments.
Within 80 d of potting, bud break commenced on 60% of the shoots of R. yakushimanum which had rooted. Bud break percentages ranged from 30% for control shoots, to 53% for those collected from etiolated and banded stems. Shoot length averaged 2 cm with 3-4 new leaves produced on each plant. At the time of potting, the number of roots per rooted cutting for cuttings breaking bud averaged 6, significantly higher (p = 0.002) than the 2 roots per rooted cutting recorded on average for dormant cuttings. The root-ball diameters were similar (~3.5 cm) for both growing and dormant cuttings. The presence of a root ball did not affect bud break (4 cuttings with root-balls broke bud, 6 did not).

A striking result is that stock plants of R. smirnowii and R. yakushimanum showed a differential response to the two pre-treatments evaluated. This underlines the importance of separately evaluating the etiolation and banding responses of each species. Etiolation was primarily beneficial to the propagation of R. smirnowii while banding was beneficial for R. yakushimanum . Together, etiolation and banding improved the rooting of R. smirnowii by up to 40%, and by up to 27% in R. yakushimanum . The reduction of root number, bud break percentage and shoot growth in cuttings of R. smirnowii which had been stem banded might reflect an inhibitory effect of auxin applied with the band on subsequent root and shoot growth. Further studies are underway to characterize auxin effects on the subsequent growth of rooted cuttings.
The bud break of rooted cuttings of both species was associated with a higher number of roots per rooted cutting. The presence of a root ball had no effect on bud break, however. Although not utilized as a technique in the present study, heavy stem wounding is recommended for the development of root balls on rhododendron cuttings, and would likely have increased the proportion of rooted cuttings which possessed root balls (11).
Banding was applied with 0.8% IBA in talc on the band, though higher concentration of IBA in talc are commercially available. Future studies of this methodology could investigate the effects of higher auxin concentrations applied to the shoot at the banding stage. This might result in a more significant effect of banding on the rooting of R. smirnowii .

Literature Cited
1.  Davis, T.D., and J.R. Potter. 1983. Effect of localized etiolation of stock plants on the rooting of Rhododendron cuttings. J. Environ. Hort. 1:96-98.
2.  Dirr, M.A. and C.W. Heuser, Jr. 1987. The reference manual of woody plant propagation: From seed to tissue culture. Varsity Press, Athens, GA.
3.  French, C.J. 1990. Rooting of Rhododendron 'Anna Rose Whitney' cuttings as related to stem carbohydrate concentration. HortSci. 25:409-411.
4. Johnson, C.R. and A.N. Roberts. 1971. The effect of shading Rhododendron stock plants on flowering and rooting. J. Amer. Soc. Hort. Sci. 96:166-168.
5.  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.
6.  Maynard, B.K. and N.L. Bassuk. 1987. Etiolation to improve softwood cutting propagation: aspects of hormone application and timing of taking cuttings. Proc. Int. Plant Prop. Soc. 37:420-427.
7.  Maynard, B.K. and N.L Bassuk. 1990. Rooting Acer griseum softwood cuttings: Promotion by stockplant etiolation, inhibition by catechol. HortSci. 25(2):200-202.
8.  Pierik, R.L.M. 1969. Factors affecting adventitious root formation in isolated stem segments of Rhododendron . Neth. J. Agric. Sci. 17:203-208.
9.  SAS Institute. 1985. SAS user's guide: Statistics. 5th ed. SAS Institute Inc., Cary, N.C.
10. Snedecor, G.W. and W.G. Cochran. 1980. Statistical methods. 7th ed. The Iowa State University Press, Ames. IA.
11.  Wells, J.S. 1985. Plant Propagation Practices. American Nurseryman Publishing Co., Chicago, IL.

Dr. Brian Maynard received his doctorate from Cornell University in 1990. He recently completed a revision of the Cornell University Information Bulletin 136, "Culture of Rhododendrons and Selected Ericaceae in the Northeast." He is employed as a Plant Materials Specialist with the National Plant Materials Center, Beltsville, MD.

Nina Bassuk is currently an Associate Professor in the Department of Floriculture and Ornamental Horticulture and Program Leader of the Urban Horticulture Institute at Cornell University, Ithaca, New York. Dr. Bassuk received her undergraduate degree in horticulture at Cornell and her Ph.D. from the University of London while carrying out her research at the East Mailing Research Station in Kent, England. Her work in Cornell's Urban Horticulture Institute focuses on the physiological problems of plants grown in urban environments, using New York City and Ithaca as test areas. She is also Vice-chair of the International Society for Horticultural Sciences' Commission for Urban Horticulture.