The effect of rooting media, plant growth regulators

South African Journal of Botany 110 (2017) 75–79

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The effect of rooting media, plant growth regulators and clone on rooting potential of honeybush (Cyclopia subternata) stem cuttings at different planting dates G.S. Mabizela a,b,⁎, M.M. Slabbert b, C. Bester a a b

ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa Department Horticulture, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa

a r t i c l e

i n f o

Available online 5 March 2016 Edited by J. van Staden Keywords: Honeybush Rooting Vegetative propagation

a b s t r a c t Cyclopia spp. are shrubs indigenous to the Eastern and Western Cape provinces of South Africa that are used for a herbal infusion known as honeybush tea. Currently, a large amount of honeybush is still wild harvested; this imposes a risk of biodiversity loss, shortage of plant material and extinction of natural resources due to unsustainable harvesting. Therefore, in order to improve the plant material and relieve pressure on wild populations, a study aimed at optimising propagation and adventitious rooting techniques of honeybush stem cuttings was conducted. In this study, four clones of Cyclopia subternata were set in three different rooting media, with three concentrations of IBA growth regulators at six planting dates according to a randomised complete block design, to determine the effect on rooting. The data recorded were analysed using an analysis of variance (ANOVA). This study showed significant differences in the rooting potential of the four clones tested. The highest rooting and survival percentage, root number and root length were experienced when cuttings were propagated in rooting media Bark mix and 3mix, treated with growth regulators Seradix® B2 and Seradix® B3. Cuttings grown in rooting medium Peat mix and with growth regulator Dip & Root™ resulted in the lowest rooting and survival percentage. The ideal time to set cuttings was during March and November with clones SGD7 and SGD9 producing the highest rooting and survival percentages. The lowest rooting percentage was recorded during February. Clone selections SGD1 and SGD7 had the highest rooting success followed by SGD9, and clone SGD6 had the lowest rooting percentage. Cyclopia spp. may be successfully propagated vegetatively by stem cuttings at different planting dates in order to improve plant material and quality of honeybush. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Honeybush (Cyclopia spp.) is a shrub of the legume family Fabaceae and is endemic to the fynbos biome, growing along the coastal and mountainous regions of the Eastern and Western Cape provinces of South Africa. Aerial parts of Cyclopia spp. (mainly C. subternata, C. genistoides and C. intermedia) are used to make honeybush tea, which is characterised by a sweet, honey-like aroma and distinctive brown colour (Joubert et al. 2011; Theron et al. 2014). The tea has been reported to contain low levels of tannins, no caffeine and to have health-promoting properties (Joubert et al., 2008). Because of the health benefits, demand for honeybush tea, both locally and internationally, has increased worldwide (Joubert et al. 2011), necessitating the cultivation of honeybush as a commercial crop (Spriggs and Dakora 2009). Harvesting honeybush from the wild in South Africa has shown a significant increase in recent years, which poses a threat to natural ⁎ Corresponding author at: ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa. E-mail address: [email protected] (G.S. Mabizela).

http://dx.doi.org/10.1016/j.sajb.2016.02.200 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

populations (Mbangcolo 2008; Joubert et al. 2011). A major aim of the honeybush research programme of the Agricultural Research Council is to propagate and distribute improved genetic material, especially higher yielding material (Bester 2013). The majority of Cyclopia species can be propagated by seed and vegetatively by stem cuttings (Bester 2013; Mbangcolo et al. 2013). Seed propagation does not ensure the preservation of selected genetic characters (Hartmann et al. 2002) whereas vegetative propagation results in plants genetically identical to the original parent plant and thus are more uniform (Araya 2005). Vegetative propagation is the preferred method to supply the expanding industry and the demand for more uniform product (Spriggs and Dakora 2007; Bester 2013). Rooting success in stem cuttings of Cyclopia spp. depends on a number of factors including the season during which the cuttings are collected, age of the source plant, cutting size, type of rooting substrate, irrigation, temperature, rooting growth regulator and the clone (Hartmann et al. 2002; Soundy et al. 2008). Plant growth regulators (PGRs) have been successfully employed in many plant species to improve the rootability of stem cuttings (Soundy et al. 2008; Singh et al. 2011a; Saǧlam et al. 2014). These include indole-3-acetic acid (IAA),

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naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) (Adekola and Akpan 2012; Sardoei et al. 2013). Another factor which creates a suitable environment for rooting of stem cuttings is the rooting medium (Tchinda et al. 2013). Adugna et al. (2015) reported the highest rooting percentage of Vanilla planifolia stem cuttings grown in fine sand rooting medium (99.3%), followed by cuttings grown in forest soil rooting medium (93.33%). There may also be large differences in rooting ability among clones of many plant species and with different types of cuttings (Baltunis and Brawner 2010; McIvor et al. 2014). Various studies have reported variations in rooting ability within genotypes in the same locations due to natural genetic variation (Husen and Pal 2003; Rambaran 2013). Seasonal timing, the period of the year in which cuttings are taken, can also effect the rooting of stem cuttings. Ayoub and Qrunfleh (2006) reported an increase in rooting percentage of olive cultivar “Nibali” during September (55.6%), while cultivar “Rassei” had the highest rooting percentage during February (92.6). Studies on the effect of season on rooting of stem cuttings have also been conducted in other plant species (Bushal et al. 2001; Agbo and Obi 2008; Khosla and Pant 2009). To date, limited propagation studies have been conducted in the honeybush species C. subternata (Mbangcolo 2008). In order to standardise the methodology for commercial propagation of C. subternata cuttings, this study was conducted to determine the effect of rooting media, plant growth regulators, clone, and season on the rooting of C. subternata cuttings. The standard protocol used by industry served as control. 2. Material and methods 2.1. Experimental site and plant material The experiments were conducted at the nursery of the Agriculture Research Council (ARC) Infruitec-Nietvoorbij, situated in Stellenbosch, South Africa. The experiments took place between the end of May 2012 and beginning of March 2013. Cyclopia subternata cutting materials were sourced from a 2-year-old seed orchard situated at Elsenburg near Stellenbosch. Cuttings were collected during early morning (8:30 to 9:30 am) and kept moist and cool until transported to the working area. 2.2. Experimental design The experiments were organised as a completely randomised block design in a 3 × 4 × 3 factorial arrangement with growth medium, clone and plant growth regulator (PGR) as main effects. For each treatment combination (medium × clone × PGR), eight terminal semihardwood cuttings were treated, and this was replicated three times. Thus, the experiments utilised 72 cuttings per clone and 288 cuttings per treatment combination. The trials were repeated at six planting dates: 30 May, 24 July, 18 September and 14 November (in 2012), and 1 February and 13 March (in 2013). 2.3. Preparation of trays and growth media Polystyrene seedling trays with dimension of 69 cm × 33 cm × 8.6 cm (length × width × depth) were surface-sterilised in a Spore Kill™ (12% didecyl dimethyl ammonium chloride) solution. Seedling trays were filled with three growth media: (1) Canadian peat moss, sterilised river sand and polystyrene balls (3mix) (1:1:1) (v/v/v); (2) fermented pine bark and sterilised river sand (Bark mix) (1:1) (v/v) and (3) Canadian peat moss and sterilised river sand (Peat mix) (2:1) (v/v). The standard growth medium currently used in honeybush propagation, i.e. 3mix, was used as control medium. Trays were placed in a mist bed on top of heated sand beds with a temperature of 25 °C, and only the temperature of the growth media were measured daily, recorded with a thermometer (inserted in each growth medium) during

the rooting period. The thermometers were removed each time when measuring the temperature and inserted again in the growth media for the next measurement. No other temperature was monitored in this study. Misting was scheduled to irrigate for 10 s every 15 min from 8 am to 4 pm delivering ± 3.5 mm of water per day and 30 mm per week. 2.4. Preparation of stem cuttings and application of growth regulators Terminal cuttings 80 mm long were cut from semi-hardwood stems with uniform size of 5 mm width. The lower leaves (50% of total leaves) of each cutting were removed and the base of each cutting was freshly trimmed by 0.5 mm. The cuttings were immersed in a 0.2% fungicide solution (Dithane® with active ingredient mancozeb 750 g/kg), to protect cuttings from fungal infection, prior to dipping in growth regulators. The basal 1 cm of cuttings was dipped in one of each of the growth regulators Dip & Root™ liquid (10 g/L IBA and 5 g/L NAA), Seradix® B2 (3 g/kg IBA) or Seradix® B3 (8 g/kg IBA) before setting in the growth media. Seradix® B2 served as control as it is used by the honeybush industry for propagation of cuttings. Cuttings were examined for root initiation after 63 days in the mist bed. 2.5. Data collection and statistical analysis Data collected were the percentage of rooted cuttings and survival, number of roots and root length. Data were analysed using a twoway analysis of variance (ANOVA) to test for treatment effects using the general linear model (GLM) procedure of SAS statistical software version 9.2. The Shapiro–Wilk test was performed to test for normality (Shapiro and Wilk 1965). Student's test of least significance was calculated at the 5% level of significance to compare treatment means. 3. Results 3.1. Rooting medium 3.1.1. Survival and rooting The rooting medium had a significant effect (p ≤ 0.05) on the percentage rooting and survival of the cuttings (Table 1). There were no significant differences between the Bark mix and 3mix. Approximately 90% of the cuttings planted in these media survived, as measured 63 days after the start of the treatment. The percentage rooting of both treatments was in excess of 70%. Cuttings grown on peat mix showed the lowest survival and rooting, 79.8% and 51.3%, respectively. 3.1.2. Root number and length The rooting medium had a significant effect (p ≤ 0.05) on the mean number of roots per cutting and the mean root length (Table 1). Significantly higher number of roots were recorded on Bark mix (4.7), and 3mix (4.5), compared to Peat mix (2.7). Bark mix also gave the longest roots (182.8 mm), compared to 3mix (160.9 mm) and Peat mix (52.6 mm).

Table 1 Effect of growth medium on rooting of C. subternata stem cuttings 63 days after the start of treatment. Growth medium

Survival percentage (%)

Rooting percentage (%)

Mean number of roots

Mean root length (mm)

Bark mix 3mix Peat mix

90.54a 89.31a 79.77b

76.44a 73.56a 51.27b

4.86a 4.84a 2.74b

182.85a 160.89b 92.67c

Means with the same letter in the same column are not significantly different (p ≤ 0.05).

G.S. Mabizela et al. / South African Journal of Botany 110 (2017) 75–79 Table 2 Effect of growth regulator on rooting of C. subternata stem cuttings 63 days after start of treatment. Growth regulator





Dip & Root™ Seradix® B2 Seradix® B3

84.10b 87.84a 87.67a

63.93b 69.03a 68.31a

3.78b 4.35a 4.31a

128.52b 151.25a 156.64a


3.2. Plant growth regulators (PGRs)

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Table 4 Effect of planting date on rooting of C. subternata stem cuttings 63 days after start of treatment. Planting date (months)





May July September November February March

87.45b 77.83d 83.18c 92.12a 84.60c 93.98a

66.89b 66.66b 63.79b 74.76a 56.13c 74.30a

4.12b 3.59c 4.51a 4.11b 3.78bc 4.76a

174.32b 159.41b 218.70a 139.88c 74.41e 109.11d


3.2.1. Survival and rooting The PGRs significantly affected (p ≤ 0.05) the survival rate and rooting percentage (Table 2), with Seradix® B2 and Seradix® B3 being the more and similarly effective at ca. 88% survival rate and 69% rooting. Dip & Root™ growth regulator was less effective at 84.1% and 63.9%, respectively. 3.2.2. Root number and length The PGRs had a significant effect (p ≤ 0.05) on the number of roots and length, formed on C. subternata cuttings (Table 2). As for survival and rooting, Seradix® B2 and Seradix® B3 treatments were similarly effective in stimulating the formation and growth of roots, while being more effective than Dip & Root™. The mean number of roots for the Seradix® treatments was 4.3 mm compared to 3.8 mm for Dip & Root™. The root length of cuttings treated with Seradix®, irrespective of the concentration of IBA, exceeded 150 mm, while Dip & Root™ delivered roots of 128.5 mm. 3.3. Clonal effect 3.3.1. Survival and rooting There were significant differences in survival of rooted stem cuttings (p ≤ 0.05) (Table 3). The highest survival was noted in clones SGD7 (97.5%) and SGD1 (95%). The survival rate of SGD9 (91.2%) was slightly less, but that of SGD6 (62.3%) substantially lower. The same trend for the clones was observed in terms of rooting, but in this case SDG7 (83.7%) attained a significantly higher rooting percentage than both SGD1 (74.7%) and SGD9 (73.4%). Once again, clone SGD6 performed the poorest with only 36.4% rooting. 3.3.2. Root number and length Cloning had a significant effect (p ≤ 0.05) on root number and length (Table 3). The highest root number was recorded for clone SGD9 (5.4). The number of roots formed by SGD7 (4.8) and SDG1 (4.4) were not significantly different. SGD6 formed substantially less roots (1.9). The longest roots were formed by SGD7 (198.1 mm) and SGD9 (183.0 mm), followed by SGD1 (120.8 mm) and SGD6 (79.9 mm) (p ≤ 0.05). 3.4. Seasonal effect 3.4.1. Survival and rooting Seasonal variation had a significant effect (p ≤ 0.05) on the survival and rooting percentage of C. subternata cuttings (Table 4). A significantly Table 3 Clonal effect on rooting of C. subternata stem cuttings 63 days after start of treatment. Clone





SGD1 SGD6 SGD7 SGD9

95.12a 62.26c 97.53a 91.23b

74.74b 36.49c 83.71a 73.41b

4.38b 1.97c 4.83b 5.40a

120.82b 79.89b 198.10a 183.08a


higher survival rate was observed for cuttings made during November (92%) and March (93%) than during May (87%). The survival rates of cuttings made during September (83%) and February (84.6%) were slightly, but significantly, lower than that observed for November and March. The lowest survival rate was recorded during July (77.9%). The highest rooting percentages (p ≤ 0.05) were recorded for planting dates from November and March (74.7% and 74.3%, respectively), followed by May, July and September (66.8%, 66.6% and 63.7%, respectively), and lastly, February (56%) (Table 4). Propagation in May, July and September would deliver more or less the same results. 3.4.2. Root number and length The number of roots and length were significantly affected (p ≤ 0.05) by the seasonal variation (Table 4). A significantly higher root number was recorded during September (4.5) and March (4.7) compared to other studied planting dates. The root numbers during November (4.1), February (3.8) and May (3.6) were not significantly different from each other. Furthermore, the lowest root number was obtained for cuttings set during July (3.5), although it was not significantly different (p ≤ 0.05) from February (3.8). The longest root length (p ≤ 0.05) was recorded for setting of cuttings during September (218.7 mm), followed by May (171.3 mm) and July (159.9 mm). Setting in November (139.8 mm), March (109.0 mm) and February (74.4) progressively delivered cuttings with shorter roots. 4. Discussion 4.1. Rooting medium Rooting medium is one of the most important factors affecting the rooting of cuttings, and this study provided evidence of the effect of the composition of growth media on the rooting success of honeybush (C. subternata) plants. The highest cutting survival and rooting rate, number of roots and length of roots were achieved on Bark mix and 3mix. Root initiation of C. subternata was considerably lower in the Peat mix. A number of factors interacting within a growth medium are known to affect the success of rooting including oxygen, water and nutrient availability (Alikhani et al. 2011; Bhardwaj 2014). The success in improved root initiation of C. subternata using Bark mix and 3mix could be attributed to the positive interaction of aeration and waterholding capacity, as compared to Peat mix. A well balanced oxygen and water-holding capacity promotes oxygen availability, transpiration, nutrient uptake, growth and aeration during root initiation. Poor aeration is a fundamental problem in waterlogged situations, leading to decay of cuttings before root initiation (Schmitz et al. 2013). Both Bark mix and 3mix might had a lower water-holding capacity based on the composition of the growth media (coarse and semi-coarse texture, respectively) compared to Peat mix (fine-textured and small pores) (Relf 2009). Creating a good aerated environment will increase respiration at the base of the cuttings, as explained by Akakpo et al. (2014),

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who indicated that rice husk growth medium enhanced rooting of Vitellaria paradoxa stem cuttings. Heating of the growth medium may have contributed to the rooting success of stem cuttings. During this study, bottom heating maintained at 25 °C was used and a thermometer was used to determine the temperature of the growth media (only a single hole on a seedling tray was monitored). A high rooting percentage was observed at rooting medium temperature of 22 °C–27 °C (Bark mix and 3mix) and low rooting was observed at growth medium temperature of 17 °C–23 °C (Peat mix). The differences in the composition of the growth media might explain differences in temperature between the growth media. A coarse growth medium retains more heat than a fine-textured growth medium (Al-Kayiem et al. 2015). Growth medium temperature is known to be the critical factor in rooting of cuttings. Keeping the rooting medium extra warm not only increases the speed of rooting but also improves the percentage of rooted cuttings (Runkle 2006). Reuveni and Castoriano (1993) treated semi-hardwood cuttings of mango cultivars with bottom heating of 20 °C, 25 °C, 30 °C and 35 °C and observed higher rooting at 25 °C and 30 °C, while at 20 °C, cuttings took longer to reach a high rate of rooting. Furthermore, Runkle (2006) stated that the rate of root and shoot development is accelerated by growth medium temperature. Given the findings of Runkle (2006) and our observations in terms of temperature, investigation of the effect of bottom heating on rooting percentage during plant propagation of C. subternata via stem cuttings should be investigated in the future. 4.2. Plant growth regulators (PGRs)

and SGD9 more than 70% rooting, and SGD6 less than 40% rooting. Rooting percentage, survival rate, root number and root length were higher in clones SGD1, SGD7 and SGD9, than in SGD6. The clonal differences in rooting indicate large differences in rootability and considerable genetic variation among clones (Prat et al. 1998). Baltunis and Brawner (2010) reported that the genetic variation among clones was statistically significant for rooting of Pinus radiata clonal populations. 4.4. Seasonal effect A higher survival rate, rooting percentage and number of roots were attained from cuttings collected in September (spring) and November (early-summer) and March (autumn) compared to cuttings collected in July (winter) and February (late summer). This result corroborates the findings of other studies showing that time of collection as a major factor affecting vegetative propagation in plant species (Soundy et al. 2008; Haile et al. 2011). Mbangcolo (2008) suggested that the best time for collecting cuttings of C. genistoides and C. intermedia was during summer (December). Increased rooting during spring and summer months may be attributed to higher temperatures and longer illumination/daylight periods which leads to a higher photosynthetic rate and carbohydrate reserve content, thereby stimulating adventitious root development (Rapaka et al. 2005). The carbohydrate accumulation is also expected to be high in cuttings collected during the dry periods (Mediterranean climate) due to high carbohydrate reserves as the result of lower physiological activities of the donor plants than in May cuttings just after leaf flushing. Therefore, cuttings collected during February and March are expected to root easily due to mobilisation of high amounts of carbohydrates and other metabolites (Haile et al. 2011). However, in this study, cuttings collected during February had on average lower rooting percentage, survival rate, number of roots and length of roots. The lower rooting of C. subternata during February could possibly be due to lower growth activity or depletion of growth hormones (probably IBA) or other physiological factors after flowering (thin and soft plant material). Seasonal effect on rooting and shooting of cuttings is high in plants that undergo a typical low growth activity (lean period) during cold months (winter) (Leakey 2004). In the present study, we demonstrated that the standard industry protocol for rooting of cuttings (3mix and Seradix® B2) yielded good results and is still recommended as the preferred protocol. Due to the differences in rooting success of the various C. subternata clones and the effect of growth media, PGRs and planting date has on rooting success, it is therefore recommended that rooting of the different clones be optimised before commercialisation.

There were distinct differences in survival rate, rooting percentage, number of roots and root length due to the different growth regulators applied in the rooting of C. subternata cuttings. PGRs such as IBA, IAA and NAA are known to accelerate the rate of rooting and increase final rooting percentage and number of roots on cuttings (Gehlot et al. 2014; Ibrahim et al. 2015). In the rooting of C. subternata cuttings, the highest rooting percentage was achieved with Seradix® B2 and Seradix® B3 (3 and 8 g/kg IBA respectively), compared to Dip & Root™ (10 g/L IBA and 5 g/L NAA). Successful rooting could possibly be associated with the IBA content in the growth regulators used. In addition, the number of roots and root length was significantly improved by the IBA (Seradix® B2 and B3) as well. In plant species such as in Lippia javanica L. (Soundy et al. 2008) and V. paradoxa C.F. Gaerth (Akakpo et al. 2014), a high rooting percentage was achieved when stem cuttings were treated with IBA. Similar results were also obtained by Carvalho et al. (1995) and Chalapathi et al. (2001) who reported an increased rooting, number of roots per cutting, root length, root thickness, fresh and dry weight of roots using IBA. The application of Dip & Root™ resulted in lower rooting success, which could possibly be attributed to the presence of NAA as root growth regulator. In studies where IAA and NAA were used independently as rooting agent, low percentage rooting in Bougainvillea glaba cuttings (Singh et al. 2011b) and Ficus hawaii (Hassanein 2013) were reported. Nelson et al. (1992), using four different auxins (IAA, IBA, NAA and indole-3-propionic acid) in Pinus taeda, Pinus elliotti var. ellioti and Pinus palustris proved that IAA and IBA assisted in high root initiation, while rooting with NAA and indole-3-propionic acid resulted in poor rooting of cuttings of these species.

The authors acknowledge the National Research Fund (NRF), Department of Science and Technology (DST), Monetary Treasury Economic Fund (MTEF), ARC and Tshwane University of Technology (TUT) for financial support. Special thanks to Dr. Mardé Booyse for her valuable assistance with the statistical design and analysis, Ms. Marlise Joubert for assisting with plant material as well as Ms. Veronica Korkie and Ms. Gaynor Peterson for assisting with the experiments.

4.3. Clonal effect

References

The effect of clonal variation in rooting response has been reported by various research studies. The four C. subternata clone selections from Groendal were selected based on the preliminary results from a previous study indicating that SGD1 and SGD7 had more than 80% rooting, followed by SGD6 with 50% to 70% rooting and, SGD9 with less than 50% rooting (unpublished data). However, the results from this study indicate that clone SGD7 had more than 80% rooting, SGD1

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