Introduction

Often associated with semi-arid, regular but limited precipitation, succulents provide a mechanism to avoid drought as storing plenty of water and placing these deposits in leaves and other plant parts which is considered as a physiological tolerance (Edwards and Ogburn 2013). Nowadays, succulents are commercially produced and have been increasing in popularity for plant collectors, landscapers and in households (Altman 2001). Demands of succulents may have been because of certain characteristics which are both practical and efficient as they are drought resistant and can survive even in minimal light making it best for indoors (Alaspa 2016; Bell 2001). Succulents form architectural, sculptural and geometric shapes providing clear and simple lines that are delved into bizarre, eye-catching and collectible arrangements (Baldwin 2013).

Propagating succulents have been commonly done by leaf cutting and stem cuttings especially for, but not limited to Haworthia, Sanseveria, Crassula, Sedum, Graptoptalum and Kalanchoe (Neisen 2013; Tuttle 2012). Succulents may be propagated from seed but are not commonly practiced due to lengthy germination period, thus the use of cuttings and offsets are made to root first in nursery flats filled with potting mixes before being placed in gardens or replication area (Baldwin 2013). A member of the Crassulaceae Family, Echeveria has increased its popularity and demand leading to an increase of cultivation area (Christenhusz and Byng 2016). These plants, like any other succulents, maybe propagated using seeds but most of its hybrids and cultivated varieties are more conveniently propagated by leaf cuttings (Neisen 2013).

Plant propagation is purposely done to perpetuate species, multiply these species and maintain its youthfulness and can be done through sexual and asexual reproduction of plants (McNeilan and Gorman 2013; Sorensen 2015). With the use of vegetative propagation, the plant multiplied will be identical to those of its parents unlike those of plant propagated by seeds (Lei 2010). In the case of succulents, the use of offsets, suckers, leaf and stem cuttings are common meanwhile offsets and suckers take longer periods to develop compared to the leaves which are readily available (Baldwin 2013; Neisen 2013). Although common to succulents, leaf cuttings among most plants are not capable of producing an independent plant and usually produces roots, and decay due to lack of auxiliary buds that are capable of forming adventitious buds (Evans and Blazich 1999; Gorelick 2015).

Success of propagating succulents have been reported to be unpredictable and some growers and enthusiast call the multiplication of plants using leaf cuttings as a game of chance (Artichoker 2014; Tuttle 2012). Thus, this study aims to identify impacts on the growth and development of shoots with respect to the common and basic among practices in propagating leaf cuttings of succulents, the leaf cutting type (tip or base) and its position upon planting (upright or faced-downward). Tip leaves are composed of young cells that are highly merisimatic and are capable of raid cell division while base or older leaves are already differentiated cells which are mature and also capable of production (Hartmann and Kester 1983). Thus, both leaf cutting types have their respective capabilities to produce a new plant. Planting positions have never been considered as a factor for improving quality plants. Propagators often depend on the kind of space or tray they use on how these leaf cuttings may be planted and thus, no suggested specific placement has been recommended.

There have been only few studies conducted for practices and factors of leaf propagation of succulents including rooting media, cutting part, rooting promoters, drying period (Jeong 1999; Mihaela et al. 2011; Paterson and Rost 1978). Limited information is available and only few research studies devoted on improving quality of this high-valued ornamental crop especially on basic planting positions and location of leaves to procure when using leaf cuttings.

Materials and Methods

Planting Materials

The experiment was conducted at the greenhouse at the Department of Environmental Horticulture, Sahmyook University for a duration of two months starting at the 2nd week of March with an average daily temperature of 25°C (minimum of 15°C and maximum of 29°C recorded temperature) and a relative humidity of 60 ± 5 %. Mother plants with more or less the same size per species were purchased from a succulent farm grower. Four (4) Echeveria succulent species were chosen to evaluate the growth and development in response to location of leaves namely: E. ‘A Grimm One’, E. ‘Momorato’, E. pulvinata ‘Frosty’ and E. pulidonis (Fig. 1a – 4d) while planting position of leaf cuttings were investigated with Graptoveria opalina species (Fig. 1e). The leaf cuttings were placed in a 60 cm x 30 cm planting tray filled with 1 : 1 ratio of commercial soil growing media (Seoul Bio, Korea) and sand.

Treatments and Experimental Design

Two individual factors (leaf cutting types and planting position) were individually investigated in this study. Each experimental set-up was done in a completely randomized design with three replications with an approximate 15 leaf cuttings per treatment species.

Leaf cutting type was separated into two treatments, the tip and the base. The conventional method of propagating through leaf cuttings makes no regard what type of leaf or where the leaves are located from the mother plant. The tip leaves were taken from the first two whorls of leaves from the apex of the plant while the base leaves were collected from the lower leaves toward the next higher whorl (Fig. 2a). Evident differences of the tip and base are the extent of edge colors and the size of leaves. The base leaves of G. optalina species were used and placed in two planting positions, upright and faced-downward (Fig. 2b). Each experimental replication set was placed in separate growing trays.

Planting of Leaf Succulents

Healthy and undamaged leaves were tagged and carefully removed from the mother plant as to include the nodal attachment and prevent damaging or creating injuries to the leaves. Succulent leaves were removed from the mother plants carefully to make sure that the nodes of the leaves were not damaged and scarred. These were cleaned properly to remove the debris.

After the leaves were removed from the mother plant of four Echeveria and Graptoveria succulent species, the leaves were laid out in a drying tray with a 60 x 60 cm dimension lined with two layers of clean paper. These were orderly arranged in order to expose the wounds and prevent from any added moisture thus, these trays were placed in an area with good ventilation with an average temperature of 20°C and average relative humidity (RH) of 70% for 5 - 10 days to induce callusing which will prevent rotting of leaves until browning and drying of the wounds. The removal of the leaves is considered as a wound, stimuli to develop unorganized cell masses or callus (Ikeuchi et al. 2013).

Data Collection

To determine the shoot growth and development, the following vegetative plant parameters were set and collected 60 days after treatment (DAT) for the location of leaves and planting position: shoot diameter, shoot and root length and number of developed leaves and roots according to the methods specified by Jeong (1999).

Two months after planting leaf cuttings, the overall quality of Graptoveria succulent species only from two treatments were graded visually based on a 1 - 5 scale based on the studies of Wang et al. (2005) with a few modifications where, leaves are chlorotic, with very poor root and foliage quality = 1; leaves are light green, substandard quality of root and foliage, unsalable = 2; leaves are green, good quality of roots and foliage, salable = 3; leaves are evenly green, very good quality of roots and foliage = 4; and leaves are dark green, excellent quality of root and foliage = 5. CIELAB scale was determined by placing the optical lens of the Konica Minota Spectrophotometer CM2600d on the center of the shoot whorls. This color scale makes use of the L* a* b* values indicating color space to indicate lightness, hue and saturation of colors (Hunterlab 2012).

Data Analysis

Data analysis were performed using statistical software SPSS v. 22. Results were subjected to One-way Analysis of Variance (ANOVA) and Duncan Multiple Range Test (DMRT) was used to compare treatment means. Differences were considered to be significant at P < 0.05 and highly significant at P < 0.01 and standard deviation of means were also determined and presented.

Results and Discussion

The limiting factor in leaf cutting propagation is the formation of adventurous buds and not solely on the roots. Diameter and height of shoots were significantly influenced by location of leaves taken from the mother plant for all selected species of Echeveria (Fig. 3a). Shoot diameter taken from the base leaf cuttings of E. ‘A Grimm One’ gave the largest size with 9.01 mm compared to those of the tip with 2 .3 5 mm (P < 0.01). Consistent results were also observed for E ‘Momorato’ (base – 17.54 mm; tip – 2.95), E. pulvinata (base – 9.16 mm; tip – 0.10) and E. pulidonis (base – 8.70 mm; tip – 0.55) where the base leaf cuttings had bigger shoots compared to the tips (P < 0.01).

Matching the results of shoot diameter, the shoot height was highly influenced by different location sources of leaf cuttings (Fig. 3B) of all selected succulent species. Bases with 6.22 mm, 11.61 mm, 6.31 mm and 6.17 mm had highly differed (P < 0.01) with the stunted shoots from tip leaf cuttings with 1.45 mm, 2.29 mm, 0.10 mm and 0.41 mm, from E. ‘A Grimm One’, E. ‘Momorato’, E. pulvinata and E. pulidonis, respectively.

Number of leaves were also influenced by the treatments (Fig. 3c). The use of the base cuttings gave the highest number of leaves consistently to all selected succulents. E. ‘A Grimm One’, E. ‘Momorato’ and E. pulidonis base leaf cuttings had significantly differed thrice as much as those of the tip (P < 0.01) while those of E. pulvinata was also significantly differed (P < 0.05) with 0.20 from the tip and 8.22 leaves from the base.

It was observed that the shoot formation of base leaves provided bigger, taller and healthier shoots compared to the tip (Fig. 4). These results are explained by Sadhu(1989) that lateral or lower cuttings taken from any mother plants have increased level of stored food in the form of carbohydrates. The development and growth of shoots rests on the case that these accumulated carbohydrates at the basal portion provide a better immunity against attacks of microorganism especially bacterial and fungal infections on the wounds of the cuttings despite the curing process (Moghaddam and Van den Ende 2012). The availability of food assimilates on the base nourishes the developing plants and tends to provide more to the developing buds. After these assimilates have been used, the older leaves will dry out (Brown 2008). Most vegetative propagated plants often use mature portions of the plant parts (Ibironke 2013; Leal and Krezdorn 1964; Saifuddin et al. 2013).

The planting of positions of succulents have not been studied and have various recommendations from different growers and hobbyists. Position of planting highly influenced the shoot growth of G. optalina specifically those of shoot diameter and height, and number of leaves, expect for the number of buds (Table 1). The use of upright position gave the largest shoots (16.59 mm) compared to those planted in a faced-downward position (13.46 mm) (P < 0.01). This result was also similar to those of the shoot height which succulents planted uprightly had the tallest plants (21.53 mm) compared to those planted in a faced-downward manner (17.93) (P < 0.05). However, position of planting did not significantly affect the growth of shoots of succulents.

The shoots were significantly affected by the faced-downward planting position. It was observed that the formation or growth of shoots were patterned to how the leaves are formed from the mother plant. Abnormal growth was observed from those of the succulents planted faced-downwardly. Most of the shoot growth had twisted stem shoots as it goes away from the covered leaf portions (Fig. 5).

The development of the roots during were the first part of the establishment of the new plant. In producing roots, the outer injured cells die and form a necrotic plate or scales the wound which are called suberin or cork and gum block xylem (Gregory, 2006).

The length of roots on E. ‘A Grimm One’, E. ‘Mamaorato’ and E. pulidonis was not significantly affected by the use of treatments (Table 2). However, E. pulvinata was significantly affected having the base leaf cuttings with the longest root length of 37.78 mm compared to those of the tip with 9.99 mm (P < 0.05). However, number of roots had no significant differences between treatments.

In agronomic crops, such as wheat, rice and soybean, starches are mobilized at the base leaves before they are transported to phloem vessels for storage in sink compartments within the plant. These starches provide and gives off sturdy tissues and food reserves (Bird 2014; Hartmann and Kester 1983). Mature leaves are capacitated wit pre-bud initials which produce endogenous hormones that induces faster and more rapid root and bud formations (Brown 2008).

Based on the hedonic rating scale, the leaf cutting planted in an upright manner gave saleable and high quality shoots (4.61 shoots) which highly differed from those of the succulents planted in a faced-downward manner with a rating of 1.64 described as shoots with light green leaves with not saleable. This result is supported by the Hunter’s CIELAB color reading which indicated a darker color (L* = 30.84) and hues and saturation indicating richer tones (a* = 0.17 and b*= 9.62) which highly differed to those shoots grown in faced-downward position (P < 0.01).

Severe damages resulted from the opposition of gravitropic nature of nodal growth included stem and leaf etiolation, twisted and abnormal stem and leaf growth leading to lower quality of shoot growth (Table 2). Geotropic responses at the nodes of G. optalina leaf cuttings may be attributed by its position on the mother plant oriented horizontally facing upwards. This case is similar to studies of Bridges and Wilkins (1973) in the wheat where by growth of nodes were attributed to initiation of growth of the horizontal leaf sheath base with the outer epidermis facing downwards creating an interaction with hormones and protons of tissues. Morphological studies at a cellular level may be done to determine the development of nodes and its capability to produce not only one plant but more buds. It was observed that under the stress of growing buds under the faced-downward planting position separate buds sprouted from the other side of the leaf creating more than one shoot per leaf cutting. Aside from efficacy to gravity, other tropic growth patterns may also be considered such as phototropism (Raven and Johnson 1999).