Introduction

Plants have remarkable plasticity and adaptability which makes it possible for them to form new organs such as lateral roots, shoots and flowers including meristems (Benková et al. 2003). Meristems enables plant growth and development in these organs which are affected by numerous external and internal factors (Dello Loio et al. 2008). These meristems coordinate the initiation of new organs, continuously provide development of new cells for plant and are all regulated by a dynamic system (Heisler and Jonsson 2007).

However, previous studies suggest that succulents have a more peculiar way of developing new plants through its leaves. This is due to the presence of intercalary meristems attached to the leaf tissues which common for other plants. Due to abundant moisture in leaves of succulents, leaf cuttings are able to survive for longer periods until new organs appear (Donnelly et al. 1999; Gorelick 2015; von Willert et al. 1990).

Succulents are growing in popularity and demand due to their minimum maintenance and drought-tolerant characteristic as an indoor and landscape plant. Aside from their minimal care, succulents have attractive leaf formation with geometric shapes which are unique among ornamental plants (Nyffeler et al. 2008). Nowadays, these succulents are commercially produced and have been increasing in popularity for plant collectors, landscapers and in households (Altman 2001).

The use of Echeveria group among research experiments is popular for propagation studies due to its large group encompassing greater commonality for morphological structures amongst succulent species. Despite its demand, there is slow production of new plants thus, plenty of studies are conducted using the use of tissue culture demands sophisticated techniques and higher finances (Lee and Park 2013; Lee et al. 2012).

The use of leaf cuttings is a cheaper option for plant growers which require simple methods but may take longer time to develop individual plants (Heisler and Jonsson 2007; Zinkan 2010).

Leaves and stem cuttings or any method of vegetative propagation in general have naturally occurring substances that are able to stimulate and produce necessary hormones for root and shoot formation, however, most cuttings require additional stimulation (Altman and Waisel 1997; Carlson 1950). Bio-stimulators were identified in 1930’s as natural forming substances which are now called ‘plant growth regulators’ (PGRs). These PGRs are molecules which affects numerous plant growth responses even in extremely low concentrations. These plant functions include seed growth, flowering, fruit development and ripening, plant longevity and, vegetative development (Brown 2006; Davis and Haissig 1990).

The use of auxin, such as indole-3-butyric acid (IBA), has been studied as rooting hormones for stem cuttings while cytokinin use is reported to help promote adventitious bud and shoot formation in leaf and root cuttings (Abdullateef and Osman 2012; Kassahun and Mekonnen 2011). Both plant hormones have synergistic or antagonistic effect on several important developmental processes in growth in plants which includes the formation and maintenance of meristems for plant propagation (Davies 2002; Su et al. 2011).

Studies of Paterson and Rost (1978) reported that the use of auxin alone had only minimal stimulating effect. Their study also concluded that the use of regenerating new plants using leaf cuttings took longer periods. However, they recommended that the use of these exogenous substances, auxin in combination with cytokinin, to quicken the shoot and root initiation should be explored for study. Thus, this study aims to determine the effects of auxin, cytokinin and their combination on the propagation of selected Echeveria species using leaf cuttings.

Materials and Methods

Planting Materials and Growth Conditions

The experiment was conducted in the greenhouse of the Department of Environmental Horticulture, Sahmyook University for duration of two months. Two Echeveria species (E. subsessilis and E. runyonii) were utilized as experimental units of the study.

Whole leaves located at the base of the mother plant were carefully removed close to the petiole of the stem. Leaves were reselected to obtain a uniform size, weight (2 - 3 g) and stage of development. These mother plants completed 80 - 85% of their apical and marginal expansion and reached their optimal maximum thickness of leaves.

These leaves were procured a week before planting to provide enough time for drying (Raju and Mann 1971). These leaves were placed in a dry and well-aerated room in the conditions of an average temperature of 20°C and average relative humidity (RH) of 70% for 5 - 10 days in order to induce callusing before any application or contact with aqueous substances which will prevent rotting of leaves.

After application of treatments, the leaf succulents were planted in their respective growing trays and were grown inside the greenhouse with an average temperature of 24°C and 75% RH and an average daylight intensity of 15 μmol·m^-2·s^-1.

Experimental Design and Treatments

The study was done in a 3 x 3 factorial arrangement in completely randomized design (CRD) with three replications having 10 leaf cuttings per replication with a total of 30 leaves per treatment for each species. Three levels of auxin as represented by the use of IBA (0, 100, 500 ppm), cytokinin represented by Kinetin (0, 100, 200 ppm) and their combination served as treatments.

Preparation and Application of Hormones

Powder formulations were prepared by mixing the growth hormones in required quantities to be adjusted to their respective concentrations following the procedure of Hartmann and Kester (1983). Different levels of concentration were made by weighting 0.5 g (500 ppm) and 0.1 g (100 ppm) of the hormone and were added with 1 - 2 drops of ether for IBA and NaCl for Kinetin to freely dissolve substances. Hormone combinations were diluted to 1 L distilled water and were stirred together inside an Erlenmeyer flask. Treatment combinations composed of two hormones were added together and also separately prepared.

The nodal part of the leaves was dipped into their respective solutions for 10 - 15 minutes based on the studies of Carpenter and Cornell (1992) on the application of auxin or other hormones for rooting cuttings. In higher concentrations (e.g. 8,000 - 1,000 ppm), cuttings or planting materials should be dipped in a shorter time (between 2 - 6 min) and at lower concentrations (lower than 1,000 ppm) should have at least 10 - 15 min. Within the allotted 15 min of treatment, optimum absorption of hormones was considered to be most effective.

Planting of Leaf Cutting

Leaf cuttings were laid carefully in 60 x 30 cm growing trays using commercial growing media (Nursery Bed Soil, Seoul Bio Inc., South Korea) without any addition of fertilizers. Growing trays were equipped with tiny holes on its base to facilitate drainage of excess water. These were laid carefully in an upright position with a planting distance of 1 cm away from each other. Each treatment had been separately placed in one growing tray. These were also grouped and properly labelled based on their corresponding treatments, treatment combinations and replications.

Watering and Management

Watering was carefully done by drenching the growing medium and ensuring that no water droplets stay contained in the ridge of the leaves which would promote rotting and attract fungi. Growing conditions were recorded including temperature and relative humidity by using a data logger (Center 342 Temperature Humidity Recorder, Center Technology Corp, Taiwan). Rotten leaf cuttings that did not develop any roots and/or buds were removed from the growing trays to avoid fungal growth and spread towards other growing leaf cuttings.

Data Collection

Shoot height and diameter, the number of leaves and shoots, shooting and rooting rate, the number of days to rooting, and percent mortality were measured as parameters to evaluate the response of leaf cuttings to treatments.

Statistical Analysis

The data were analyzed using SPSS ver. 22. Duncan Multiple Range Test (DMRT) was conducted to test the significant differences between groups.

Results and Discussion

Echeveria subsessilis

Also known as ‘Morning Beauty’, margins of this species have touches of pink in the fully rounded by gray-blue rosettes which is native to Mexico (Zinkan 2010). E. subsessilis has been found to be very distinct aside from those of E. peacockii which are grown with short stem or none at all and accessorized with numerous and crowded leaf formation as slightly dusty and crumbly. This species is known to quickly leach away water from its roots and could not tolerate submergence in water (Walter 1972).

Results of the application of auxin and cytokinin as IBA and Kinetin for this species is shown on Table 1. Plant height of this species was affected by auxin levels alone. Application of 500 ppm IBA significantly gave the tallest shoots (11.27 mm) which was followed by descending levels of IBA, 100 ppm and 0 ppm, respectively. This was also similar for the shoot diameter which had 14.97 mm (500 ppm IBA) which significantly differed from those of 100 ppm (12.07 mm) and 0 ppm (11.35 mm). These individual hormone responses are evident in species response shown in Fig. 2 and Fig. 1 for the control.

Despite its reputation to inhibit shoots, the positive response of this species did exhibit unorthodox response. Auxin substances in different forms at high concentrations are already classified as toxic and often retards protoplasmic streaming which may also inhibit the morphological growing parts of the plant where it was applied (Bonner and Bandurski 1952; Thimann 1939). However, findings of Zhao (2010) concluded that auxins, exogenous or biosynthesis, are also responsible for seedling growth and vascular patterning which are mainly involved in developmental processes. Thus, this level of concentration or the use of higher auxin levels, specifically IBA, has been used for treating apple, camellia and rose cuttings (Sun and Bassuk 1991; Susaj et al. 2012; Wazir 2014).

Interaction of auxin and cytokinin significantly affected the number of leaves for this species. More number of leaves were found in plants that were pretreated with 100 ppm IBA and 100 ppm Ki (26.94 leaves) which significantly differed from higher levels: 500 ppm IBA + 500 ppm Ki (15.17), 100 ppm IBA + 500 ppm Ki (14.56) and 500 ppm IBA + 100 ppm Ki (14.33) that were statistically did not differ from each other (Fig. 3).

Adjusted according to species, the formation of leaves is flexible and changes based on certain environmental circumstances and developmental circumstances as well as the initiation of shoot apical meristem with variable sizes (Bar and Ori 2014). These leaf developments are regulated by internal and external cues which includes certain hormones like auxins and their interaction with other plant hormones, transcription regulators that affects its mechanical properties of certain leaf tissues (Kaplan 2001; Zimmerman 1952). Cleland (2001) concluded that in localizing the application of the auxin hormone would result to the wall-loosening protein expansion which is sufficient to induce the primordium and provide more mature leaves.

Treatments did not give a significant difference on the number of shoots of E. subsessilis leaf cuttings. However, the root length had a significant difference between application of auxin and cytokinin as separate factors. Among treatments the control (Fig. 1) gave the highest root length with 25.83 mm for auxin levels which had similar effects with those of 100 ppm (21.88 mm) while application of 500 ppm gave the shortest roots with 16.26 mm. This case was also similar to the IBA levels having the control (24.27 mm) as the plants with highest roots which were also similar to those of 100 ppm IBA (23.30 mm) and 500 ppm IBA (16.26 mm) having the shortest roots as well.

Su and Zhang (2011) stated that shoot meristems would give rise to above-ground parts of the plant while the root meristems make below-ground parts wherein auxin-cytokinin interactions are responsible for both meristem developments and in the excess of its levels, it may avert the effects of its supposedly its intended purpose. This seems to be the case for E. subsessilis.

It was observed that the plants applied with 500 ppm gave tallest plants and largest shoot diameter, however, it caused a decline on the root growth (16.26 mm) and also a larger mortality (36.67%) of leaf cuttings. It was observed that the use of hormones increased the mortality rate around 3 - 5% compared to the control. However, without exogenous application of hormones, there were evident growth and quality differences of shoots produced.

After 500 ppm having the highest percent mortality, this was then followed by plants treated with 100 ppm IBA (28.36%) and the control (24.45%) which significantly differed within the IBA or auxin application. The application of 100 ppm Ki gave the highest percent mortality with 33.89% which was followed by those of 500 ppm Ki (27.78% which were similar with those of the control. Based on the hormone interactions, application of 500 ppm IBA + 500 ppm Ki gave the lowest mortality rate of 21.67 % followed by other combinations which did not statistically differ from each other.

Echeveria runyonii

Native to Mexico, numerous cultivars have been described and cultivated with this species and also belongs, like that of E. subsessilis, to the Crassulaceae family (Zinkan 2010). These species are described to have glaucous pinkish-white color of leaves that are spatulate to oblong in shape and has similar leaves with those of E. peacokii in terms of its glaucous feature. It is distinctively different due to its wedged shape tips (Rose 2011).

Results on the application of auxin and cytokinin hormones, and their combinations are shown on Table 2. Upon the analysis of these results, the application of auxin using IBA significantly affected the shoot height, diameter and percentage mortality of leaf cuttings. Leaf cuttings treated with 500 ppm IBA gave the tallest shoots with 9.22 mm which significantly differed from those of the 100 ppm IBA (11.96 mm) and control (11.07 mm), however, this also resulted in minimal growth of its shoots (diameter) with the smallest shoots with 14.40 mm compared to 100 ppm IBA and the control. The application of this IBA level also gave the highest percent morality of 42.22%. The application of 100 ppm IBA significantly gave largest shoots (diameter) with 16.77 mm and the lowest percent mortality of 31.11%. Although not statistically significant, it may be noted that the application of 100 ppm IBA also gave the highest number of leaves (23.27) and shoots (3.05) and the longest roots (28.47 mm).

Mahonen et al. (2014) reported that the application of 500 ppm IBA or higher concentrations of auxins rapidly inhibited cell division and expansion but does not include cell differentiation. This concept is the application of the development of auxin herbicides such as 2 - 4 D in the markets which has been an effective weed control in modern production systems (Grossmann 2007). It was also concluded besides increasing and stimulating production of ethylene and gibberellin, auxin herbicides are able to directly trigger gene activation of NCED.

Terminal buds or growing shoots and their development may also be inhibited but are variable on the quantity applied. Application of its appropriate concentrations may also increase developing shoot growth which includes lateral bud development and quantity of leaves (Thimann 1939). This is true to the results of leaf cuttings applied with 100 ppm IBA.

Shoot height, diameter, number of leaves and percent mortality were significantly affected by the application of cytokinin and their different levels of concentration. Application of 100 ppm Ki gave the tallest (11.90 mm), largest shoots (17.09 mm) and more abundant leaves (24.62), however the soot height and diameter did not significantly differ from those of the control or 0 ppm with 11.07mm (shoot height) and 16.08 mm (shoot diameter). Although it did not significantly affect the treatments, results revealed that the highest number of leaves (3.19) and shoots (27.52) were observed from plant cuttings that were previously treated with 100 ppm Ki. However, the use of 500 ppm Ki did have the lowest percent mortality of 24.44% compared to 100 ppm Ki (36.67%) and the control which had the highest mortality (44.44%) for E. runyonii species. Sole application of hormones IBA and Ki on E. runyonii species are shown on Fig. 3.

Cytokinins are important key players in the cell cycle and influences numerous developmental programs in plant development including cell division, shoot initiation and growth, gametophyte, embryonic development, leaf senescence, apical dominance, sink/source relationships, nutrient uptake, phyllotaxis, and vascular and plant responses to biotic and abiotic factors (Kieber and Schaller 2014; Werner et al. 2001). Thus, its deficiency effects may include stunted shoots and smaller apical meristems as studied in tobacco plants (Werner et al. 2001). However, in higher concentrations it may limit the production of other hormones and may be also be detrimental for shoot development and enlargement (Brzobohaty et al. 1994).

The combination of both hormones significantly affected shoot height and diameter including percent mortality. Among interaction treatments, the use of lower combinations (100 ppm IBA + 100 ppm Ki) with 10.90 mm which was followed by leaf cuttings applied with 500 ppm IBA + 100 ppm Ki (10.06 mm), 100 ppm IBA + 500 ppm Ki (8.69 mm) and 500 ppm IBA + 500 ppm Ki (8.71 mm), respectively. Largest shoots (diameter) were also observed from those of 100 ppm IBA + 100 ppm Ki with 16.23 mm and had the lowest percent mortality rate of 20.0% which significantly differed from other treatments. Similar results were observed with those of the other species. Combination of hormones auxin and cytokinin on E. runyonii species are shown on Fig. 4.

Several studies concluded from genetic and biochemical studies of the development of these plants have very intricate relationship for hormone and are both required for differentiation and maintenance of meristems as well as for continuous growth of plants which are all grounded in the interaction of auxin and cytokinin as well as their levels of application (Dettmer and Helariutta 2009; Su et al. 2011; Zhao 2010). Fig. 5.