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Improving rooting of Lobostemon fruticosus L. cuttings with delayed auxin treatment
South African Journal of Botany 105 (2016) 111–115
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Improving rooting of Lobostemon fruticosus L. cuttings with delayed auxin treatment K.E. Lodama a,b, E.S. du Toit a,⁎, J.M. Steyn a, H.T. Araya b, G. Prinsloo c, C.P. du Plooy b, P.J. Robbertse a a b c
Department of Plant Production and Soil Science, University of Pretoria, Private Bag X20, Hatfield 0028, South Africa ARC-Roodeplaat, Vegetable and Ornamental Plant, Private Bag X 293, Pretoria 0001, South Africa Department of Agriculture and Animal Health, University of South Africa, Florida 0170, South Africa
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Article history: Received 28 May 2015 Received in revised form 27 December 2015 Accepted 4 January 2016 Available online xxxx Edited by SO Amoo Keywords: Growth medium Rooting hormone Semi-hardwood cuttings Vegetative propagation
a b s t r a c t Lobostemon fruticosus is a semi-woody shrub used medicinally for treating wounds, blood poisoning, ringworms, skin diseases and syphilis. The material used is mostly wild harvested, leading to decline in natural populations. Propagation and cultivation methods to establish commercial production can assist in conservation of the species. The objective of this study was to determine the effect of delaying rooting hormone application on the success of vegetative propagation of L. fruticosus using basal stem cutting, cut at an angle of 30° at the base. It was done in a mist bed at the Agricultural Research Council-Vegetable and Ornamental Plant Institute (ARCVOPI) (Pretoria, South Africa) in a factorial randomized complete block design with two growth media (cocopeat and cocopeat + potting soil1:1; v/v), rooting hormone (Seradix No. 1 and control) and six application times, with five replications. The parameters assessed were callus development, rooting percentage, shoot and root length, survival rate and stem cutting anatomical analysis. Rooting percentage significantly increased with delaying the application of rooting hormone for one to two weeks from planting, as compared to the control (without hormone) and hormone application at the time of planting (week zero). Cocopeat medium gave the highest rooting percentage (67.1%) and lowest mortality rate (18%), whereas potting soil + cocopeat medium gave the lowest root development (31.5%). Anatomical observations in this study showed that with delayed auxin application up to two weeks after planting, the callus tissue started to develop from the vascular cambium close to the cut end of the cutting. Parenchyma gaps in the phloem fiber ring close to the cut end of the cuttings was also observed. This study concludes that delaying hormone application for two weeks after planting improved rooting of L. fruticosus cuttings. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Lobostemon fruticosus (Boraginaceae) is among the most important perennial shrubs that are medicinal species in the Western Cape Province of South Africa (Buys, 2011). It is one of the species that has been looked at more closely with regard to its medicinal properties. It is also known as “agtdaegeneesbos” (Afrikaans; meaning eight day healing bush) due to its apparent ability to heal a condition in eight days. Decoctions are used to treat wounds, skin disease, ringworm and ulcers while infusions are used for general internal problems and purifying the blood (Van Wyk et al., 1997; VanWyk and Gericke, 2000). Traditional health practitioners also believe that this plant has anti-HIV properties. The plant is gaining more attention and popularity due to the high demand for medicinal extracts from the leaves used by traditional healers (Van Wyk et al., 1997). Lobostemon plants are currently harvested from the wild and this can lead to a decline in natural populations or even extinction from its ⁎ Corresponding author: Tel.: +27 12 420 3227; fax: +27 12 420 4120. E-mail address: [email protected] (E.S. du Toit).
http://dx.doi.org/10.1016/j.sajb.2016.01.005 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
natural habitats. Unfortunately, due to difficulty of vegetative propagation, L. fruticosus is rarely grown in the nursery trade. The development of successful vegetative propagation methodology, followed by cultivation can therefore help to minimize the wild harvest pressure on the species. Poor rooting ability in some plant species has been attributed to the presence of growth inhibitors (Barlow et al., 1961), a lack of or imbalance of hormones or rooting cofactors (Raviv et al., 1986). The most common technique to promote rooting in difficult to root plant species is to use exogenous rooting hormones (Muhammad et al., 2006; Owuor et al., 2009; Jordan et al., 2010). Rooting hormones such as indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) are more effective than naturally occurring phytohormones like indole-3acetic acid (Henrique et al., 2006) to optimize rooting of cuttings. In most of the cases best results were obtained when rooting hormones were applied directly before planting, especially with easy-to-root species such as Rosa damascenae Mill. (Muhammad et al., 2006); Lippia javanica (Soundy et al., 2008) and Guindilia trinervis Gillies (Jordan et al., 2010). However, Luckman and Menary (2002) observed that delaying the application of indole-3-butyric acid (IBA) for six weeks after
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K.E. Lodama et al. / South African Journal of Botany 105 (2016) 111–115
planting cuttings, many roots formed on Eucalyptus nitens. According to Hartmann et al. (1997) and Ofori-Gyamfi (1998), rooting performance further depends on the type of medium used in the propagating program as well as the type of rooting hormone used to improve root initiation. Auxins often hasten root initiation, increase the number of cuttings rooted, as well as improve the quality and uniformity of rooting cuttings (Newton et al., 1992; Al-Saqri and Alderson, 1996). Several anatomical studies have suggested a correlation between difficulty in rooting and the presence of a pericyclic sclerenchyma layer (Goodin, 1965; Beakbane, 1969; Edwards and Thomas, 1980). Amissah et al. (2008) also suggested that the presence of a continuous sclerenchyma layer might act as a physiological barrier to adventitious root initiation or as a mechanical barrier to root emergence. Roots arising from the cambium and phloem regions in Vaccinium corymbosum hardwood cuttings were reportedly impeded by a continuous layer of lignified pericyclic fibers and by the epidermis (Mahlstede and Watson, 1952). There is hardly any information on rooting of cuttings of L. fruticosus and an urgent need therefore exists to develop appropriate techniques, including the best rooting substrate to ensure good root initiation and shoot formation from stem cuttings that can easily be adopted by start-up nursery enterprises as well as rural communities. The objectives of this study were (1) to determine the effect of delaying rooting hormone application on the success of vegetative propagation of stem cuttings of L. fruticosus; (2) identifying the site(s) of primordial initiation of adventitious roots and recording the progress of root formation with the view of (3) establishing whether the sclerenchyma ring can be interpreted as a barrier to the initiation and emergence of adventitious roots that can be broken by delayed hormone application. 2. Materials and methods 2.1. Study area and plant material collection The experiment was carried out at the Agricultural Research Council Vegetable and Ornamental Plant Institute (ARC-VOPI) (latitude 25°9′S, longitude 28°35′E and altitude 1200 m.a.s.l), Pretoria, Republic of South Africa, during the 2013 winter (May to July) and spring (September to November) seasons. The experimental unit was a glasshouse supplemented with 24 h a day misting which worked automatically based on the humidity of the greenhouse. Throughout the experimental period, the temperature of the greenhouse was recorded using a data logger (Tinytag View 2, TV-1500, UK). Mean maximum and minimum temperatures during the study period were 25 and 13 °C, respectively. Shoots containing secondary xylem were obtained from three year old stock plants of L. fruticosus at the ARC-VOPI medicinal plant nursery. The shoots were collected early in the morning and were immediately placed in a bucket filled with water in order to keep them cool and turgid until taken to the working area, following the protocol given by Soundy et al. (2008). The immature top parts of the shoots were removed before cutting them into 8 cm cuttings, using sterile pruning shears. Bottom leaves were removed from the base of the cuttings, leaving only one pair of leaves at the top node with the basal end cut at an angle of 30°. 2.2. Rooting medium preparation Seradix No. 1 hormone rooting powder was used for this experiment. It contains 4-(indol-3-yl)-butyric acid and is an Auxin class plant growth regulator (PGR) which is used to promote and accelerate root formation of plant clippings and to reduce transplant shock of non-food ornamental nursery stock (Al-Saqri and Alderson, 1996). Two commercial rooting substrates were tested in this study. The first was a commercial rooting substrate (M1) made up of a 1:1(v/v) mixture of potting soil (90% composted bark and 10% sand) and cocopeat. The
second rooting substrate (M2) was cocopeat obtained from coconut husk fibers which is a by-product of the coconut, also known as coir. The moist rooting substrates were used to fill seedling trays with 98 cavities each, measuring 4 × 4 × 9 cm (width, breadth and depth). After planting the cuttings were kept at 80% RH in a mist bed system which was set to spray for 15 min at 1 h intervals.
2.3. Experimental design and treatment details The layout of the experiment was a factorial randomized complete block design with 16 treatment combinations and five replications. The design consisted of 2 rooting substrates × 8 treatments of delayed Seradix No. 1 rooting hormone containing 4-(indol-3-yl)-butyric acid); 0.1% IBA) applications (2 × 8). For each treatment 14 cuttings were used. The eight hormone (Seradix No. 1) treatments consisted of a control (no Seradix applied at all), or Seradix applied immediately before planting (week zero), one week after planting (week 1), two weeks after planting (week 2), three weeks after planting (week 3) four weeks after plating (week 4), five weeks after planting (week 5) and six weeks after planting (week 6). The latter six treatments were applied once only on the specific week by removing the cuttings from the medium, rinsing the base of the cuttings with water, followed by quick dipping (3–4 s.) in Seradix No. 1 solution at a concentration of 1 mg/10 ml of distilled water. After hormone treatments, the cuttings were re-planted in the same cavity in the tray. Cuttings which had already started root initiation after week five were also treated carefully. The idea behind treating cuttings which had already started root development was to increase root initiation, number of roots, and uniformity of rooting and callus formation. Callusing, root initiation, shoot number, shoot length and survival were observed at the end of the experiment (week 9). The amount of callus formation at the bases of the cuttings was assessed using a four point rating scale where 0 = no visible callus and 4 = callus mass formation at the whole cut end of the cutting or greater (Luckman, 1996). 2.4. Stem anatomy Three cuttings of each of the eight treatments were investigated, the basal 1 cm of the cuttings (including the slanted cut) were removed and fixed for two days in formalin:alcohol:glacial acetic acid (FAA) solution (1:9:1; v/v/v) using 70% (v/v) ethanol in distilled water, after which they were dehydrated using a series of 30%, 50%, 70% and 100% (2 ×) ethanol. The ethanol was substituted by means of a series of 30%, 50%, 70% and 100% (2×) xylol, followed by infiltration of wax (O'Brien and McCully, 1981). Transverse sections of about 10 μm thick were made from the bottom part of the cuttings, using a rotary microtome (Reichert-Jung). Sections were mounted on slides, stained with safranin and counter stained with fast green and photographed using a digital camera (DXC900; Sony Corp., Tokyo) mounted on an optical microscope (BX 60; Olympus Optical Co., Tokyo). 2.5. Data collection and statistical analysis Cuttings were finally assessed for root number, root length, shoot length and cutting survival nine weeks from planting. The survival percentage was calculated as the total number of cuttings with shoot growth divided by the total number of cuttings planted per treatment. Rooting parameters were measured by gently separating rooted cuttings from the seedling trays, followed by washing the root zone with water. The data collected were transformed using log transformation, which is normally used for measurable data such as length, whereafter S.A.S. (Statistical Analysis System version 9.1) was used to perform analysis of variance (ANOVA).
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3. Results and discussion Interactions between the winter 2013 (n = 16) and spring 2013 (n = 16) trials for rooting substrate, rooting hormone and delaying hormone application were not significantly different (P ≤ 0.05) and data were therefore pooled (n = 32) and subjected to statistical analysis.
3.1. Callusing rating and percentage rooting of cuttings The mean callus ratings varied between 1.25 and 2.75. There were significant differences between delayed IBA hormone treatments as well as growing media in relation to callusing success (Fig. 1a). Callus development on cuttings planted in cocopeat medium and where rooting hormone application was delayed with one or two weeks, was significantly higher (P b 0.05) than that of the control and other treatments. The results in general showed that delaying rooting hormone application beyond two weeks after planting had no significant effect on callus development of the cuttings. Similar results were reported by Luckman and Menary (2002) in Eucalyptus nitens, where callus production was high when hormone treatment was applied during the first two weeks after planting. However, there were no significant
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differences in callusing between treatments planted in the mixed growing medium. Many studies have reported the benefits of rooting hormone application in rooting success of cuttings, root length and uniformity of rooting on different plant species (Arya et al., 1994; Aminah et al., 1997; Hartmann et al., 1997; Araya et al., 2007). In most of these studies, the application of rooting hormone was before planting. However, in this study cuttings were treated after planting (Fig. 1b). Cuttings which were grown in the cocopeat medium and received rooting hormone treatment one to two weeks after planting had significantly higher rooting percentage as compared to the control (without hormone) (Fig. 1b). Delaying the application of the rooting hormone longer than two weeks caused a decline in the rooting percentage of the cuttings. Similar findings were reported by Luckman and Menary (2002), where Eucalyptus nitens cuttings rooted at a higher percentage when the rooting hormone application was delayed for four to five weeks from planting, in comparison to no hormone application, or application at the time of planting. The variation between results of the present study and that of Luckman and Menary (2002) regarding the best time of delayed hormone application could be due to the differences in nature of the plant species used, such as differences in stem anatomy and morphology.
Fig. 1. (a) Mean callusing rating; (b) rooting percentage; (c) relationship between callus rating and rooting percentage; (d) root length (cm); (e) shoot length (cm); (f) survival percentage of Lobostemon fruticosus stem cuttings as influenced by delayed application of Seradix No. 1 rooting hormone and growing medium. Error bars data indicate significant (P b 0.05) differences between treatments according to Fisher's protected test.
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Callus developed due to delaying of rooting hormone application showed a strong positive linear relationship with the percentage rooting of the cuttings, regardless of the applied treatment (Fig. 1c). Based on this relationship (P b 0.05), it is possible to conclude that callusing was the precursor of rooting. Root initiation is a developmental process that is dependent on the availability of auxin (Davies and Hartmann, 1988) and differentiation of the first root initial cells is dependent on auxin, either in the form of endogenous or exogenous sources (Hartmann et al., 2002). In addition, the variation in sensitivity to auxin during different phases of adventitious root development has also been documented (Mohammed and Eriksen, 1974). Therefore, there might be a particular stage where auxin is needed for optimum root initiation and this might be the reason why the cuttings responded to a specific period of delayed rooting hormone application (Luckman and Menary, 2002).
cuttings, three weeks after planting and two weeks after Seradix treatment. The basal parts of the cuttings had a slanted cut surface where the Seradix was applied. The stem shows the typical dicotyledonous stem configuration (Beck, 2010) consisting (Fig. 2a and b), from the outside to the inside, of the epidermis (Fig. 2a) with sharply pointed trichomes (not shown in the figure), small cortex cells, larger specialized cortex cells with slightly thickened walls and tanniniferous contents, a few layers of phloem fibers, one to three layers of active phloem, the vascular cambium, secondary xylem, primary xylem and pith. Fig. 2b is a higher magnification of a part of Fig. 2a to emphasize the region around the vascular cambium and the absence of callus tissue. All three figure panels (Fig. 2a–c) are from the same section but panels A and B
a
3.2. Root length per cutting The mean root length of cuttings was significantly affected by treatment with Seradix No. 1 and delaying the hormone treatment (Fig. 1d). The mixture (1:1; v/v) of cocopeat and potting soil tended to produce slightly longer roots as compared to the cocopeat medium alone. Root number followed the same pattern as root length and there was no significant response to the different rooting hormone treatments (data not presented). According to our knowledge there is no published information about how delaying hormone application can affect root length. However, in other studies where IBA was applied at the time of planting, the number and length of roots were lower as compared to the control (Murat and Elmas, 2008; Shereen and Aly, 2011). 3.3. Shoot length of cuttings The data pertaining to shoot length per treatment are presented in Fig. 1e. The delayed hormone treatments as well as growing medium had no significant effect on shoot length, and there was also no significant difference between the interaction of hormone treatment and growth media, except for the treatment where auxin application was delayed for six weeks. The maximum shoot length (3.5 cm) was observed in control cuttings, which were growing in cocopeat + potting soil medium, followed by the treatment where hormone application was delayed for four weeks (3.36 cm). The minimum shoot length (1.4 cm) was recorded for the six week delayed treatment in cocopeat + potting soil medium.
p
pf sx
ep
c sc
50 µm
xv
b
vc pf sc 50 µm
3.4. Cutting survival Applying rooting hormone either immediately before planting or delaying application had no significant effect on mortality rate of the cuttings, with the exception of treatment at weeks five and six, where there were significant differences between the growth mediums. The lowest survival rate (38.6%) was found for the mixture of potting soil and cocopeat medium, where hormone application was delayed until week six, while the same hormone treatment with cocopeat medium had a 74.2% survival rate. Although the difference was not significant, the highest survival of 98.6% was observed for the control treatment followed by treatments where hormone was applied at weeks 0, 1, 2, 3 and 4 (Fig. 1f). There is no published information about how delaying hormone application can affect survival of semi-hardwood cuttings. In other studies where IBA hormone was applied immediately before planting such as in Ficus roxburghii. Alikhani et al. (2011) and Rana and Sood (2012) reported highest survival percentage. 3.5. Stem anatomy The following description of the stem anatomy of L. fruticosus is based on transverse sections that were made from the basal part of
c
cal
100 µm
Fig. 2. Different positions of the same transverse section of a L. fruticosus stem cutting that was treated with Seradix treatment two weeks after planting. a: Part of section opposite the slanting cut surface, showing no callusing; b: magnification of part of A, emphasizing the vascular cambium (vc) and phloem fibers (pf); c: part of section next to the upper end of the slanting cut showing callus tissue (cal) produced by the vascular cambium and gap in phloem fiber ring (arrow).; cal = callus tissue; ep = epidermis; p = pith; sc = specialized cortex cells; sx = secondary xylem; vc = vascular cambium; xv = xylem vessel. Bar scale = 50 and 100 μm.50 μm and 100 μm.
K.E. Lodama et al. / South African Journal of Botany 105 (2016) 111–115
1 cm Fig. 3. Secondary root development from the callus tissue of semi-hardwood cutting of Lobostemon fruticosus, nine weeks after planting and hormone treatment two weeks after planting. Bar scale = 1 cm.
(Fig. 2a and b) represent the opposite side of the slanted cut surface of the cutting, while Fig. 2c represents the part next to the cut surface. An interesting fact is that there are hardly any active phloem cells visible in Fig. 2a and b and most of the phloem consists of sclerenchyma fibers. From Fig. 2c it is clear that the callus tissue started to form from the cambium cells close to the cut surface, especially along the slanted cut surface of the cutting. Fig. 2c also shows gaps in the fiber ring (yellow arrow) to allow adventitious roots developing in the callus tissue, to penetrate. Most roots were found around the slanted cut end of the cuttings where callus tissue was formed (Fig. 3). Very little or no callus tissue was observed in sections made from cuttings from the control and week zero cuttings or cuttings that received the Seradix treatment at later stages. The high correlation between callus and root formation (Fig. 1c) is clear evidence that adventitious roots in Lobostemon cuttings develop from callus tissue. The slanted cut end of the cuttings also allows better exposure of the vascular cambium to the Seradix and therefore may have resulted in more callus and root formation. 4. Conclusions L. fruticosus can be successfully propagated by stem cuttings. This study concludes that delaying rooting hormone applications for one to two weeks after planting significantly increased rooting of the cuttings that were planted in peat. Delaying applications may have played some role in stimulating the vascular cambium to produce callus tissue from which root initials developed. Most roots appeared on the slanted cut end of the cuttings (Fig. 3), suggesting that the slanted cut made a larger part of the vascular cambium accessible to the rooting hormone and also opened the possible barrier caused by the ring of phloem fibers. References Alikhani, L., Ansari, K., Jamnezhad, M., Tabatabaie, Z., 2011. The effect of different mediums and cuttings on growth and rooting of pomegranate cuttings. Iranian Journal of Plant Physiology 1, 199–203.
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Hartmann, H.T., Kester, D.E., Davies, F.T., Geneve, R.L., 1997. Plant Propagation: Principles and Practices. sixth ed. Prentice-Hall International Editions. Eaglewood Cliffs, New Jersey, USA, p. 880. Hartmann, H.T., Kester, D.E., Davies, F.T., Geneve, R.L., 2002. Plant Propagation: Principles and Practices. seventh ed. Prentice-Hall International Editions, Eaglewood Cliffs, New Jersey, USA, pp. 280–594. Henrique, A., Campinhos, E.N., Ono, E.O., de Pinho, S.Z., 2006. Effect of plant growth regulators in the rooting of Pinus cutting. Brazilian Archives of Biology and Technology 49, 189–196. Jordan, M., Doris, P., Marlene, G., Jorge, N., Gloria, M.P., Juan, V., Ricardo, S.M., 2010. Adventitious root initiation in adult and juvenile cuttings of Guindilia trinervis, an endemic plant of Chile suitable for biodiesel production. Bosque 31, 195–201. Luckman, G.A., 1996. 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Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Improving rooting of Lobostemon fruticosus L. cuttings with delayed auxin treatment K.E. Lodama a,b, E.S. du Toit a,⁎, J.M. Steyn a, H.T. Araya b, G. Prinsloo c, C.P. du Plooy b, P.J. Robbertse a a b c
Department of Plant Production and Soil Science, University of Pretoria, Private Bag X20, Hatfield 0028, South Africa ARC-Roodeplaat, Vegetable and Ornamental Plant, Private Bag X 293, Pretoria 0001, South Africa Department of Agriculture and Animal Health, University of South Africa, Florida 0170, South Africa
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Article history: Received 28 May 2015 Received in revised form 27 December 2015 Accepted 4 January 2016 Available online xxxx Edited by SO Amoo Keywords: Growth medium Rooting hormone Semi-hardwood cuttings Vegetative propagation
a b s t r a c t Lobostemon fruticosus is a semi-woody shrub used medicinally for treating wounds, blood poisoning, ringworms, skin diseases and syphilis. The material used is mostly wild harvested, leading to decline in natural populations. Propagation and cultivation methods to establish commercial production can assist in conservation of the species. The objective of this study was to determine the effect of delaying rooting hormone application on the success of vegetative propagation of L. fruticosus using basal stem cutting, cut at an angle of 30° at the base. It was done in a mist bed at the Agricultural Research Council-Vegetable and Ornamental Plant Institute (ARCVOPI) (Pretoria, South Africa) in a factorial randomized complete block design with two growth media (cocopeat and cocopeat + potting soil1:1; v/v), rooting hormone (Seradix No. 1 and control) and six application times, with five replications. The parameters assessed were callus development, rooting percentage, shoot and root length, survival rate and stem cutting anatomical analysis. Rooting percentage significantly increased with delaying the application of rooting hormone for one to two weeks from planting, as compared to the control (without hormone) and hormone application at the time of planting (week zero). Cocopeat medium gave the highest rooting percentage (67.1%) and lowest mortality rate (18%), whereas potting soil + cocopeat medium gave the lowest root development (31.5%). Anatomical observations in this study showed that with delayed auxin application up to two weeks after planting, the callus tissue started to develop from the vascular cambium close to the cut end of the cutting. Parenchyma gaps in the phloem fiber ring close to the cut end of the cuttings was also observed. This study concludes that delaying hormone application for two weeks after planting improved rooting of L. fruticosus cuttings. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Lobostemon fruticosus (Boraginaceae) is among the most important perennial shrubs that are medicinal species in the Western Cape Province of South Africa (Buys, 2011). It is one of the species that has been looked at more closely with regard to its medicinal properties. It is also known as “agtdaegeneesbos” (Afrikaans; meaning eight day healing bush) due to its apparent ability to heal a condition in eight days. Decoctions are used to treat wounds, skin disease, ringworm and ulcers while infusions are used for general internal problems and purifying the blood (Van Wyk et al., 1997; VanWyk and Gericke, 2000). Traditional health practitioners also believe that this plant has anti-HIV properties. The plant is gaining more attention and popularity due to the high demand for medicinal extracts from the leaves used by traditional healers (Van Wyk et al., 1997). Lobostemon plants are currently harvested from the wild and this can lead to a decline in natural populations or even extinction from its ⁎ Corresponding author: Tel.: +27 12 420 3227; fax: +27 12 420 4120. E-mail address: [email protected] (E.S. du Toit).
http://dx.doi.org/10.1016/j.sajb.2016.01.005 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
natural habitats. Unfortunately, due to difficulty of vegetative propagation, L. fruticosus is rarely grown in the nursery trade. The development of successful vegetative propagation methodology, followed by cultivation can therefore help to minimize the wild harvest pressure on the species. Poor rooting ability in some plant species has been attributed to the presence of growth inhibitors (Barlow et al., 1961), a lack of or imbalance of hormones or rooting cofactors (Raviv et al., 1986). The most common technique to promote rooting in difficult to root plant species is to use exogenous rooting hormones (Muhammad et al., 2006; Owuor et al., 2009; Jordan et al., 2010). Rooting hormones such as indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) are more effective than naturally occurring phytohormones like indole-3acetic acid (Henrique et al., 2006) to optimize rooting of cuttings. In most of the cases best results were obtained when rooting hormones were applied directly before planting, especially with easy-to-root species such as Rosa damascenae Mill. (Muhammad et al., 2006); Lippia javanica (Soundy et al., 2008) and Guindilia trinervis Gillies (Jordan et al., 2010). However, Luckman and Menary (2002) observed that delaying the application of indole-3-butyric acid (IBA) for six weeks after
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planting cuttings, many roots formed on Eucalyptus nitens. According to Hartmann et al. (1997) and Ofori-Gyamfi (1998), rooting performance further depends on the type of medium used in the propagating program as well as the type of rooting hormone used to improve root initiation. Auxins often hasten root initiation, increase the number of cuttings rooted, as well as improve the quality and uniformity of rooting cuttings (Newton et al., 1992; Al-Saqri and Alderson, 1996). Several anatomical studies have suggested a correlation between difficulty in rooting and the presence of a pericyclic sclerenchyma layer (Goodin, 1965; Beakbane, 1969; Edwards and Thomas, 1980). Amissah et al. (2008) also suggested that the presence of a continuous sclerenchyma layer might act as a physiological barrier to adventitious root initiation or as a mechanical barrier to root emergence. Roots arising from the cambium and phloem regions in Vaccinium corymbosum hardwood cuttings were reportedly impeded by a continuous layer of lignified pericyclic fibers and by the epidermis (Mahlstede and Watson, 1952). There is hardly any information on rooting of cuttings of L. fruticosus and an urgent need therefore exists to develop appropriate techniques, including the best rooting substrate to ensure good root initiation and shoot formation from stem cuttings that can easily be adopted by start-up nursery enterprises as well as rural communities. The objectives of this study were (1) to determine the effect of delaying rooting hormone application on the success of vegetative propagation of stem cuttings of L. fruticosus; (2) identifying the site(s) of primordial initiation of adventitious roots and recording the progress of root formation with the view of (3) establishing whether the sclerenchyma ring can be interpreted as a barrier to the initiation and emergence of adventitious roots that can be broken by delayed hormone application. 2. Materials and methods 2.1. Study area and plant material collection The experiment was carried out at the Agricultural Research Council Vegetable and Ornamental Plant Institute (ARC-VOPI) (latitude 25°9′S, longitude 28°35′E and altitude 1200 m.a.s.l), Pretoria, Republic of South Africa, during the 2013 winter (May to July) and spring (September to November) seasons. The experimental unit was a glasshouse supplemented with 24 h a day misting which worked automatically based on the humidity of the greenhouse. Throughout the experimental period, the temperature of the greenhouse was recorded using a data logger (Tinytag View 2, TV-1500, UK). Mean maximum and minimum temperatures during the study period were 25 and 13 °C, respectively. Shoots containing secondary xylem were obtained from three year old stock plants of L. fruticosus at the ARC-VOPI medicinal plant nursery. The shoots were collected early in the morning and were immediately placed in a bucket filled with water in order to keep them cool and turgid until taken to the working area, following the protocol given by Soundy et al. (2008). The immature top parts of the shoots were removed before cutting them into 8 cm cuttings, using sterile pruning shears. Bottom leaves were removed from the base of the cuttings, leaving only one pair of leaves at the top node with the basal end cut at an angle of 30°. 2.2. Rooting medium preparation Seradix No. 1 hormone rooting powder was used for this experiment. It contains 4-(indol-3-yl)-butyric acid and is an Auxin class plant growth regulator (PGR) which is used to promote and accelerate root formation of plant clippings and to reduce transplant shock of non-food ornamental nursery stock (Al-Saqri and Alderson, 1996). Two commercial rooting substrates were tested in this study. The first was a commercial rooting substrate (M1) made up of a 1:1(v/v) mixture of potting soil (90% composted bark and 10% sand) and cocopeat. The
second rooting substrate (M2) was cocopeat obtained from coconut husk fibers which is a by-product of the coconut, also known as coir. The moist rooting substrates were used to fill seedling trays with 98 cavities each, measuring 4 × 4 × 9 cm (width, breadth and depth). After planting the cuttings were kept at 80% RH in a mist bed system which was set to spray for 15 min at 1 h intervals.
2.3. Experimental design and treatment details The layout of the experiment was a factorial randomized complete block design with 16 treatment combinations and five replications. The design consisted of 2 rooting substrates × 8 treatments of delayed Seradix No. 1 rooting hormone containing 4-(indol-3-yl)-butyric acid); 0.1% IBA) applications (2 × 8). For each treatment 14 cuttings were used. The eight hormone (Seradix No. 1) treatments consisted of a control (no Seradix applied at all), or Seradix applied immediately before planting (week zero), one week after planting (week 1), two weeks after planting (week 2), three weeks after planting (week 3) four weeks after plating (week 4), five weeks after planting (week 5) and six weeks after planting (week 6). The latter six treatments were applied once only on the specific week by removing the cuttings from the medium, rinsing the base of the cuttings with water, followed by quick dipping (3–4 s.) in Seradix No. 1 solution at a concentration of 1 mg/10 ml of distilled water. After hormone treatments, the cuttings were re-planted in the same cavity in the tray. Cuttings which had already started root initiation after week five were also treated carefully. The idea behind treating cuttings which had already started root development was to increase root initiation, number of roots, and uniformity of rooting and callus formation. Callusing, root initiation, shoot number, shoot length and survival were observed at the end of the experiment (week 9). The amount of callus formation at the bases of the cuttings was assessed using a four point rating scale where 0 = no visible callus and 4 = callus mass formation at the whole cut end of the cutting or greater (Luckman, 1996). 2.4. Stem anatomy Three cuttings of each of the eight treatments were investigated, the basal 1 cm of the cuttings (including the slanted cut) were removed and fixed for two days in formalin:alcohol:glacial acetic acid (FAA) solution (1:9:1; v/v/v) using 70% (v/v) ethanol in distilled water, after which they were dehydrated using a series of 30%, 50%, 70% and 100% (2 ×) ethanol. The ethanol was substituted by means of a series of 30%, 50%, 70% and 100% (2×) xylol, followed by infiltration of wax (O'Brien and McCully, 1981). Transverse sections of about 10 μm thick were made from the bottom part of the cuttings, using a rotary microtome (Reichert-Jung). Sections were mounted on slides, stained with safranin and counter stained with fast green and photographed using a digital camera (DXC900; Sony Corp., Tokyo) mounted on an optical microscope (BX 60; Olympus Optical Co., Tokyo). 2.5. Data collection and statistical analysis Cuttings were finally assessed for root number, root length, shoot length and cutting survival nine weeks from planting. The survival percentage was calculated as the total number of cuttings with shoot growth divided by the total number of cuttings planted per treatment. Rooting parameters were measured by gently separating rooted cuttings from the seedling trays, followed by washing the root zone with water. The data collected were transformed using log transformation, which is normally used for measurable data such as length, whereafter S.A.S. (Statistical Analysis System version 9.1) was used to perform analysis of variance (ANOVA).
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3. Results and discussion Interactions between the winter 2013 (n = 16) and spring 2013 (n = 16) trials for rooting substrate, rooting hormone and delaying hormone application were not significantly different (P ≤ 0.05) and data were therefore pooled (n = 32) and subjected to statistical analysis.
3.1. Callusing rating and percentage rooting of cuttings The mean callus ratings varied between 1.25 and 2.75. There were significant differences between delayed IBA hormone treatments as well as growing media in relation to callusing success (Fig. 1a). Callus development on cuttings planted in cocopeat medium and where rooting hormone application was delayed with one or two weeks, was significantly higher (P b 0.05) than that of the control and other treatments. The results in general showed that delaying rooting hormone application beyond two weeks after planting had no significant effect on callus development of the cuttings. Similar results were reported by Luckman and Menary (2002) in Eucalyptus nitens, where callus production was high when hormone treatment was applied during the first two weeks after planting. However, there were no significant
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differences in callusing between treatments planted in the mixed growing medium. Many studies have reported the benefits of rooting hormone application in rooting success of cuttings, root length and uniformity of rooting on different plant species (Arya et al., 1994; Aminah et al., 1997; Hartmann et al., 1997; Araya et al., 2007). In most of these studies, the application of rooting hormone was before planting. However, in this study cuttings were treated after planting (Fig. 1b). Cuttings which were grown in the cocopeat medium and received rooting hormone treatment one to two weeks after planting had significantly higher rooting percentage as compared to the control (without hormone) (Fig. 1b). Delaying the application of the rooting hormone longer than two weeks caused a decline in the rooting percentage of the cuttings. Similar findings were reported by Luckman and Menary (2002), where Eucalyptus nitens cuttings rooted at a higher percentage when the rooting hormone application was delayed for four to five weeks from planting, in comparison to no hormone application, or application at the time of planting. The variation between results of the present study and that of Luckman and Menary (2002) regarding the best time of delayed hormone application could be due to the differences in nature of the plant species used, such as differences in stem anatomy and morphology.
Fig. 1. (a) Mean callusing rating; (b) rooting percentage; (c) relationship between callus rating and rooting percentage; (d) root length (cm); (e) shoot length (cm); (f) survival percentage of Lobostemon fruticosus stem cuttings as influenced by delayed application of Seradix No. 1 rooting hormone and growing medium. Error bars data indicate significant (P b 0.05) differences between treatments according to Fisher's protected test.
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Callus developed due to delaying of rooting hormone application showed a strong positive linear relationship with the percentage rooting of the cuttings, regardless of the applied treatment (Fig. 1c). Based on this relationship (P b 0.05), it is possible to conclude that callusing was the precursor of rooting. Root initiation is a developmental process that is dependent on the availability of auxin (Davies and Hartmann, 1988) and differentiation of the first root initial cells is dependent on auxin, either in the form of endogenous or exogenous sources (Hartmann et al., 2002). In addition, the variation in sensitivity to auxin during different phases of adventitious root development has also been documented (Mohammed and Eriksen, 1974). Therefore, there might be a particular stage where auxin is needed for optimum root initiation and this might be the reason why the cuttings responded to a specific period of delayed rooting hormone application (Luckman and Menary, 2002).
cuttings, three weeks after planting and two weeks after Seradix treatment. The basal parts of the cuttings had a slanted cut surface where the Seradix was applied. The stem shows the typical dicotyledonous stem configuration (Beck, 2010) consisting (Fig. 2a and b), from the outside to the inside, of the epidermis (Fig. 2a) with sharply pointed trichomes (not shown in the figure), small cortex cells, larger specialized cortex cells with slightly thickened walls and tanniniferous contents, a few layers of phloem fibers, one to three layers of active phloem, the vascular cambium, secondary xylem, primary xylem and pith. Fig. 2b is a higher magnification of a part of Fig. 2a to emphasize the region around the vascular cambium and the absence of callus tissue. All three figure panels (Fig. 2a–c) are from the same section but panels A and B
a
3.2. Root length per cutting The mean root length of cuttings was significantly affected by treatment with Seradix No. 1 and delaying the hormone treatment (Fig. 1d). The mixture (1:1; v/v) of cocopeat and potting soil tended to produce slightly longer roots as compared to the cocopeat medium alone. Root number followed the same pattern as root length and there was no significant response to the different rooting hormone treatments (data not presented). According to our knowledge there is no published information about how delaying hormone application can affect root length. However, in other studies where IBA was applied at the time of planting, the number and length of roots were lower as compared to the control (Murat and Elmas, 2008; Shereen and Aly, 2011). 3.3. Shoot length of cuttings The data pertaining to shoot length per treatment are presented in Fig. 1e. The delayed hormone treatments as well as growing medium had no significant effect on shoot length, and there was also no significant difference between the interaction of hormone treatment and growth media, except for the treatment where auxin application was delayed for six weeks. The maximum shoot length (3.5 cm) was observed in control cuttings, which were growing in cocopeat + potting soil medium, followed by the treatment where hormone application was delayed for four weeks (3.36 cm). The minimum shoot length (1.4 cm) was recorded for the six week delayed treatment in cocopeat + potting soil medium.
p
pf sx
ep
c sc
50 µm
xv
b
vc pf sc 50 µm
3.4. Cutting survival Applying rooting hormone either immediately before planting or delaying application had no significant effect on mortality rate of the cuttings, with the exception of treatment at weeks five and six, where there were significant differences between the growth mediums. The lowest survival rate (38.6%) was found for the mixture of potting soil and cocopeat medium, where hormone application was delayed until week six, while the same hormone treatment with cocopeat medium had a 74.2% survival rate. Although the difference was not significant, the highest survival of 98.6% was observed for the control treatment followed by treatments where hormone was applied at weeks 0, 1, 2, 3 and 4 (Fig. 1f). There is no published information about how delaying hormone application can affect survival of semi-hardwood cuttings. In other studies where IBA hormone was applied immediately before planting such as in Ficus roxburghii. Alikhani et al. (2011) and Rana and Sood (2012) reported highest survival percentage. 3.5. Stem anatomy The following description of the stem anatomy of L. fruticosus is based on transverse sections that were made from the basal part of
c
cal
100 µm
Fig. 2. Different positions of the same transverse section of a L. fruticosus stem cutting that was treated with Seradix treatment two weeks after planting. a: Part of section opposite the slanting cut surface, showing no callusing; b: magnification of part of A, emphasizing the vascular cambium (vc) and phloem fibers (pf); c: part of section next to the upper end of the slanting cut showing callus tissue (cal) produced by the vascular cambium and gap in phloem fiber ring (arrow).; cal = callus tissue; ep = epidermis; p = pith; sc = specialized cortex cells; sx = secondary xylem; vc = vascular cambium; xv = xylem vessel. Bar scale = 50 and 100 μm.50 μm and 100 μm.
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1 cm Fig. 3. Secondary root development from the callus tissue of semi-hardwood cutting of Lobostemon fruticosus, nine weeks after planting and hormone treatment two weeks after planting. Bar scale = 1 cm.
(Fig. 2a and b) represent the opposite side of the slanted cut surface of the cutting, while Fig. 2c represents the part next to the cut surface. An interesting fact is that there are hardly any active phloem cells visible in Fig. 2a and b and most of the phloem consists of sclerenchyma fibers. From Fig. 2c it is clear that the callus tissue started to form from the cambium cells close to the cut surface, especially along the slanted cut surface of the cutting. Fig. 2c also shows gaps in the fiber ring (yellow arrow) to allow adventitious roots developing in the callus tissue, to penetrate. Most roots were found around the slanted cut end of the cuttings where callus tissue was formed (Fig. 3). Very little or no callus tissue was observed in sections made from cuttings from the control and week zero cuttings or cuttings that received the Seradix treatment at later stages. The high correlation between callus and root formation (Fig. 1c) is clear evidence that adventitious roots in Lobostemon cuttings develop from callus tissue. The slanted cut end of the cuttings also allows better exposure of the vascular cambium to the Seradix and therefore may have resulted in more callus and root formation. 4. Conclusions L. fruticosus can be successfully propagated by stem cuttings. This study concludes that delaying rooting hormone applications for one to two weeks after planting significantly increased rooting of the cuttings that were planted in peat. Delaying applications may have played some role in stimulating the vascular cambium to produce callus tissue from which root initials developed. Most roots appeared on the slanted cut end of the cuttings (Fig. 3), suggesting that the slanted cut made a larger part of the vascular cambium accessible to the rooting hormone and also opened the possible barrier caused by the ring of phloem fibers. References Alikhani, L., Ansari, K., Jamnezhad, M., Tabatabaie, Z., 2011. The effect of different mediums and cuttings on growth and rooting of pomegranate cuttings. Iranian Journal of Plant Physiology 1, 199–203.
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