In Situ Clonal Propagation of Stevia ( Stevia rebaudiana , Bertoni) Using Hormones

The sweet taste of Stevia leaves makes it a potential substitute for table sugar which can be used to sweeten foods and beverages. However, the limited planting materials become a constrained to large production; hence the experiment aims to investigate the different part and methods of propagation for stevia specifically use of different rooting hormones. The experiment was laid out in 3 × 4 factorial arranged in Randomized Complete Block Design. It consists of 3 types of cutting (shoot tips, intermediate stem and basal stem part) and four kinds of commercial hormones (miracle gro, rootech gel, NAA and control). Results showed that the highest percentage survival of stevia was obtained from shoot tips (93.92%) which differed statistically from those intermediate (91.00%) and basal stem cuttings (85.51%). On the other hand, basal stem cutting significantly has the lowest percent survival. Results revealed that shoot tip cuttings treated with Rootech Gel developed roots early (6.92 days), with most number of roots (13.70), longer roots (3.33 cm), and 96.38% survival.

The sweet taste of Stevia leaves makes it a potential substitute for table sugar. Stevia can be used to sweeten foods and beverages and is a potential export winner [5].
The demand and production of this plant in the Philippines would likely increase due to its various uses and consumption especially as the population becomes increasingly diabetic [6]. However, there are some drawbacks on the production and utilization of this plant. Poor seed germination, unavailability or lack of planting materials and limited production technology (such as nutrient requirement and proper period of harvesting) and few product development derived from Stevia are major limiting factors in production and utilization of stevia.
Stevia can be propagated by stem cuttings as the cheapest way to multiply the plant. However, propagation by cuttings utilizes only the shoot tips while the lower parts of the stem are not used as planting materials. This limits the number of explants obtained from a single plant; hence, a suitable alternative method for plant production is to utilize stem cuttings from the lower parts of the stem as planting materials to obtain fast production of rooted stem cuttings or planting material.

Materials and Methods
The experiment was conducted at Western Mindanao State University College of Agriculture, San Ramon, Zamboanga City from August 2018 to January 2019.
The study used Stevia (Stevia rebaudiana Bertoni) mother plants as sources of experimental materials purchased from a private nursery in Cagayan de Oro City. Commercial rooting hormones such as naphthalene acetic acid, Rootech gel and Miracle Gro were purchased from agricultural outlets. The experiment was laid in 3 × 4 factorial arrangement in Randomized Complete Block Design (RCBD), replicated three times. Factor A consist of 3 types of cutting (shoot tip, intermediate stem and basal stem) while Factor B consist of three rooting hormones (Miracle Gro, Rootech Gel and NAA) and a control. Miracle Gro contained 0.1% indole-3 Buteric Acid (IBA) [7], Rootech Gel contained 0.55% IBA and 0.0054 Vitamin B 1 [8], and NAA contained 0.10% alpha naphthalene acetic acid and 0.09% B 1 [9]. The said rooting hormones were selected base on their availability in the community.
Stem cuttings were obtained from mother plants early in the morning. Stem cuttings were cut according to treatments: 1) shoot tips; 2) middle stem; and, 3) basal stem with at least two nodes per cutting. The cuttings were dipped in their respective rooting hormone per treatment prior to planting. The cuttings were planted to a germination try consist of mixture of 1:1:1:1 ratio of garden soil, sawdust, vermicast and carbonized rice hull. The seedlings were covered with rooting bed to minimized adverse effects of environmental factors such as wind, heavy rain and pest on seedling viability. Watering using mist sprayer was done every morning or as often as necessary. The experiment was monitored regularly for insect pest, diseases and weeds. Data on number of days to rooting, number of root, length of root, number of leaves and percent survival were taken.
The data were analyzed using analysis of variance (ANOVA) to determine the significant effects of the treatments. Differences among treatment means were analyzed using Tukey's HSD (Honestly Significant Difference) test. Table 1 shows that the number of days to rooting of stevia was highly influenced by the independent and interaction effects of both factors employed. It revealed that shoot tip of stem cuttings of stevia highly exhibited the earliest rooting of 7.93 days which differed significantly from the intermediate stem cuttings which took 8.51 days while the basal stem cuttings had delayed rooting of 9.14 days.

Results and Discussion
Among the rooting hormones, stem cuttings applied with Rootech Gel significantly rooted early at 6. 92 days and differed significantly from those treated with Miracle Gro and NAA which formed roots more or less at the same duration of 7.88 and 7.82 days respectively. The Control had significantly delayed rooting of 11.47 days.
The early rooting of shoot tip stem cuttings of stevia can be due to the highly meristematic tissues of the shoot tips. According to [10]  and lesser meristematic activity of differentiated cells already. It was reported by [11], that although the basal cuttings have accumulated more stored food which can be utilized for growth and rooting of stem cuttings, however plants differ in their response to other various factors.
Results also showed that Rootech Gel highly induced the early rooting of cuttings. According to Technoflora Plant Product Ltd, Rootech Gel contains 0.55% NAA and 0.0054 Vitamin B 1 which are essential for rooting of stem cuttings. It was further reported by [12], that using hormones of the auxin family induced the roots to develop quickly, resulting in early development of roots. Vitamin B 1 contained in Rootech Gel.
Inter-action effects of both factors showed that the use of shoot tip stem cuttings treated with Rootech Gel had significantly induced the early rooting of stevia cuttings (Table 1). This may be attributed to the highly meristematic tissues of shoot tips and as pointed out by [10] that the apex as also the loci of hormone production; hence lead to early rooting of shoot tips stem cuttings.
The number of roots of stevia in response to types of stem cutting and kinds of rooting hormone is presented in Table 1. However, this parameter was highly influenced by the rooting hormones. The types of stem cuttings failed to affect the number of roots. No significant interaction effects between these two factors were noted. Means within the same column followed by a common letter are not significantly different at 5% level of significance based on Tukey's HSD test (**-highly significant, ns-not significant).
It was found that the number of roots of stevia plants regardless of types of stem cuttings were more or less similar which ranged from 10.63 to 11.42 roots per cutting.
Results however showed that regardless of types of stem cuttings, those treated with Rootech Gel significantly developed the most number of 13.70 roots/cuttings. This was followed by those applied with Miracle Gro (11.70 roots) which was significantly higher than cuttings treated with NAA 4ppm with a mean of 10.19 roots. The Control obtained significantly the least number of 8.69 roots per cutting.
The most number of roots developed in stem cuttings applied with Rootech Gel may be due to indole butyric acid (IBA) and Vitamin B 1 contents compared to the rest of the rooting hormones. Based on the manufacturer's label it contains 0.55% IBA and Vitamin B 1 . The hormone IBA belongs to the group of auxins which induces cell division and cell elongation and consequently induced root formation hence more number of roots. According to [13] NAA is an auxin type similar to IBA which induced the development of more leaves, longer and more roots and higher dry weight of roots.
The Miracle Gro ranked second with 11.70 roots. This rooting hormone contains 0.10% IBA of concentration that of Rootech Gel. The treatment NAA on the other hand contained 0.10% Alpha Naphthalene Acetic Acid and 0.09% vitamin B 1 . According to [14], IBA has shown to produce a higher number of roots compared to other auxins and IBA is the most commonly used auxin for root formation [15].

E. V. Galo American Journal of Plant Sciences
The mechanism of IBA action can be attributed to its conversion into IAA through a similar process to B-oxidation of fatty acids. The conversion of IBA to IAA then suggests that IBA works as a storage sink for IAA in plants [16]. However, [17] suggested that IBA is not converted to IAA but acts as an auxin on its own. Furthermore, according to [18] and [19], IBA can be synthesized from IAA by a microsomal membrane preparation from maize seedlings and by an enzyme preparation from Arabidopsis seedlings in the presence of acetyl-CoA and ATP. These results only show that IBA works in similar manner or IAA which can induce cell division, cell enlargement and to some degree cell differentiation in combination with other substances like Vitamin B 1 .
The length of root in response to rooting hormones and types of cuttings and showed that this parameter was highly affected by the independent effects of both factors but no interaction effects were observed (Table 1). Among the types of cuttings, the shoot tip cuttings developed the longest roots with a mean of 3.31 cm, which differed from the rest of types of stem cuttings. This was followed by the basal cuttings with a mean of 3.05 cm and the shortest root length was observed from intermediate cutting with a mean of 2.39 cm.
Among the rooting hormones, Rootech Gel significantly exhibited longest root of 3.33 cm but differed from the rest of the treatments. However, cuttings treated with NAA had relatively longer roots (3.01 cm) but statistically similar with cuttings treated with Miracle Gro which developed root length of 2.92 cm the shortest root length of 2.39 cm was observed in Control which differed significantly from the rest of treatment.
Studies of [13] revealed that the longest roots of stevia was obtained from treated with 500 ppm IBA. This result was contrary to the current commercial hormones like Rootech Gel and Miracle Gro whose IBA content is about 10% -55% only. Similar results was obtained by [20] in rose cuttings where maximal number of roots and root length and healthier seedlings were recorded by using 500 ppm IBA. Rootech Gel however contained higher concentrate of IBA (0.55%) than Miracle Gro (0.10%) and (0.09%), respectively.
The number of stevia leaves in response to the types of stem cuttings and rooting hormone is shown in Table 1. Results revealed that this parameter was significantly affected by the types of stem cuttings but the rooting hormones failed to influence the number of leaves. No significant interaction effects were observed.
It was found that intermediate stem cuttings developed the highest number of leaves (4.67) followed by the basal cuttings with a mean of 4.54 leaves, while the shoot tip cuttings have significantly the least number of (3.86) leaves. Regardless of types of stem cuttings and hormone treatments, all rooted cuttings developed a more or less similar number of 4.0 to 4.7 leaves per cutting. The most number of leaves that developed from the intermediate and basal stem cuttings may be due to the two shoots that arise at the nodal axils of the intermediate and basal stem cuttings. Each shoot formed fully developed leaves which were higher than that of the shoot tip because the shoot tip has only one growing point the leaves arise only from the tip of the stem cuttings.
The percent survival of stevia as influenced by types of stem cutting and rooting hormones is shown in the Table. Statistical analysis on these data revealed that this parameter was highly influenced by the independent and interaction of both factors employed.
Results showed that the highest percentage survival of stevia was obtained from shoot tips (93.92%) which differed statistically from those intermediate (91.00%) and basal stem cuttings (85.51%). On the other hand, basal stem cutting significantly has the lowest percent survival.
Comparison among the rooting hormones revealed that stem cuttings treated with Rootech Gel significantly exhibited the highest survival of 96.28% which differed from the rest of the treatments. This was followed by those stem cuttings treated with Miracle Grow with a mean of 93.48. The lowest percent survival was observed in cuttings treated with NAA. Differences among treatment means on percentage survival were highly significant.
Interaction effects of both factors revealed that the use of shoot tips treated with Rootech Gel significantly exhibited the highest survival of 98.33% but relatively similar to those shoot tip cuttings regardless of rooting hormone which ranged from 83.33% to 98.17%.
The high percent survival of stevia shoot tip stem cuttings regardless of rooting hormones may be due to its meristematic activity, and its hormone production at the apex which induce cell division, cell enlargement that leads to survival and growth. Likewise, these results are best supplied by the studies of [21] which revealed that shoot tip cuttings for the propagation of stevia gave the best results and high percentage survival. Moreover, [11] reported that shoot tip stem cuttings with three nodes, have demonstrated significantly higher values on number of shoots, most number of leaves/branch and high percentage survival.

Conclusions
Generally it was observed that the use of shoot tip stem cuttings treated with Rootech Gel is the best method for in situ clonal propagation of stevia for early rooting of stem cuttings with more developed number of roots, leaves and for high percentage survival; Results showed that the highest percentage survival of stevia was obtained from shoot tips (93.92%) which differed statistically from those intermediate (91.00%) and basal stem cuttings (85.51%). On the other hand, basal stem cutting significantly has the lowest percent survival.
It can be concluded that the shoot tip is the best propagating part stevia plant and Rootech Gel shows the best result among the rooting hormones.

Conflicts of Interest
The author declares no conflicts of interest regarding the publication of this paper.