Piper betle L., an important species of the Piperaceae family, is an evergreen perennial climber with glossy heart-shaped leaves. It grows abundantly in many countries in the South-east Asian region [1] . It is commonly used as a tropical culinary herb or ulam and is often consumed raw in South-east Asian countries, such as Thailand, Malaysia, and Indonesia. It is assumed that the regular intake of the leaves can assist in preventing degenerative diseases, delaying ageing and improving general health [2] . According to various phytochemical studies, P. betle contains a wide variety of biologically active compounds whose concentrations depend on the diversity of the plant species [3] . These components are valued as a stimulant for their medicinal properties such as antiplatelet, anti-inflammatory effects as well as immunomodulatory, gastro-protective and antidiuretic activities [4] . P. betle is a significant crop in Malaysia, but very few reports are available for this species [5] [6] . P. betle contains compounds active in the biological activities such as anticancer, anti-inflammatory, and immunomodulatory. Hydroxychavicol, allypyrocatechol, methylpiperbetol, as well as piper betel are considered as active compounds separated from leaves and other fractions. In line with this, great antioxidant activities have been exhibited by Phenol-rich leaves of P. betle. The fact is that there is scarcity of studies investigating more effective methods of propagation and cultivation which allow progresses in plant breeding or rapid propagation of an enormous quantity of the plants similar to the species [7] . A momentous phase involved in plant vegetative propagation has been claimed to be adventitious root formation. This could be predominantly controlled by genetic, physiological, chemical or even physical factors [8] . There have been numerous researches conducted on investigating the impacts of plant regulators on rooting among different plant species [8] [9] [10] . To regulate the root development, Phytohormones, particularly auxins, contribute predominantly. Auxins are declared to be engaged in the course of adventitious root formation [11] [12] , as well as the interdependent physiological stages encompassing the rooting process which seem to be accompanying the variations in the endogenous auxin levels [13] . Auxins are efficient inducer of adventitious roots in numerous medicine plants [14] [15] [16] .

In contrast, micropropagation has been considered as an extensively employed practice, commonly to produce plants having economic prospective. The reason why micropropagation or in vitro plant regeneration from the culture of axillary buds has been reported to be paramount is that it is able to create clones which uphold the properties of the mother plant [17] . The most noteworthy element in acquiring a vast quantity of plantlets via micropropagation is using a suitable culture medium along with formulating the plant growth regulators like cytokinins with the aim of inducing and propagating the in vitro axillary buds [18] . Nevertheless, there is lack of surveys investigating the propagation of P. betle so far. For that reason, the current research is undertaken to investigate the effects of IBA on rooting of stem cuttings of P. betle and the development of in vitro propagation protocol of P. betle node explant.

Semi-hardwood cutting resulted in the maximum average dry weight and root number. The reason for this is attributed to the fact that the semi hardwood cutting encompassed more stored carbohydrate in opposition to the softwood. By a rise in the IBA concentrations, there was a corresponding upsurge in the root percentage. This was due to the fact that applying IBA on the cutting accelerated

The Values are the means ± SE. of 5 replications, the Means with the same letter are not significantly different at p ≤ 0.01 according to DMRT.

the root initiation, elevating the rooting uniformity. Moreover, there was a boost in the quantity of the root once there was a rise in IBA concentrations in both experiments in all kinds of cuttings [26] . Likewise, there was a growth in the root percentage corresponding to any rise in IBA concentrations. This is attributed to the use of IBA increased root percentage which speeded the root initiation, elevating the rooting uniformity. Furthermore, there was an upsurge in the root quantity once IBA concentrations underwent a rise in both experiments for all kinds of cuttings. Consistent with [27] , applying IBA could give a rise to the quantity of the roots.

Auxin has been known as an ingredient created in one tissue (shoots) and migrated with the aim of influencing the development of another tissue; in this case the lower portion of stem cuttings. Auxin is able to stimulate cell elongation while having a range of other growth-regulating impacts. It has been asserted by Noor Camellia et al. (2009) the impact of the auxin is exclusive.

Adventitious rooting and shooting in leaf-cuttings of P. betle, examining the role of growth hormones on rooting and shooting responses. This was undertaken under individual and combined treatments of the hormones. We observed that in opposition to their individual impact on rooting, IBA in combination was more efficient. Our results are congruent with the findings on adventitious rooting in stem cuttings of numerous crops [28] .

Different concentrations of IBA and types of cuttings could exert impacts on the roots quantity and length. More roots were produced from semi-hardwood cuttings treated with 2000 mg/L IBA as compared to the roots per plant from semi hardwood and softwood cuttings without any IBA treatments. The IBA is a root promoting the hormone which helps root induction. The increase in the trend in the number of the roots with the increase in the IBA concentrations up to 2000 mg/L shows a positive response. Similar results were also reported by [26] . Most softwood and semi-hardwood cuttings decayed and a high rooting performance was observed. The misting system with intermittent mists was supplied every 15 minutes at 45 minutes intervals from 7:00 am to 7:00 pm daily. It was believed that over watering causing the humidity in the mist house to be very suitable. High humidity caused the problem of disease [29] .

Histological study showed that the IBA treatment stimulated root initiation in P. betle. The meristematic activity in phloem parenchyma cells preceded the formation of the adventitious roots. On day 0, no root primordia were initiated in the non-treated cuttings (Figure 2(a) and Figure 2(b)). The Phloem parenchyma cells adjacent to the vascular cambium divided and produced adventitious roots. These results indicate that phloem parenchyma cells in P. betle were able to dedifferentiate and became meristematic (Figure 2(c)). This observation is consistent with the previous studies [30] [31] [32] [33] . Three days were required to observe the development of root primordia after the lag phase in P. betle treated with 1000 mg/L IBA, which demonstrated that primordia developed rapidly once cell division was initiated (Figure 2(d)). On day 6, root primordia were present in the cuttings treated with 1000 mg/L IBA, suggesting that this treatment accelerated the primordia formation. On day 3, all the cuttings treated with the IBA had primordia that emerged through the phloem fibres. By day 6, root primordia were absent in the non-treated cuttings, which explain why the non-treated cuttings were difficult to root before six days [34] (Figure 2(e) and Figure 2(f)). This may be due to the higher carbohydrate content of the semi hardwood cutting providing the developing cells with greater supply of energy [35] .

In the tissue culture study, sterilization of the explants is one of the major steps for effective in vitro micropropagation. The step can be undertaken by using different agents like calcium hypochlorite, sodium hypochlorite, ethanol, mercuric chloride, hydrogen peroxide or silver nitrate [36] . Generally, the type of the explants showed the main component where internodes were more preferred compared to the shoots apex. The survival rate reached 35% for the internodes compared to the shoots apex which was only 5%. The successful sterilization was essential for micropropagation and obtaining reduced or contamination free explants [37] . However, selection of the sterilizing agent depends on the type of the explants depending on the morphological characteristics like softness or hardness of the tissue [38] . In this study, using the shoots apex was not suitable due to the softness that could injure the tissue, where the explants would become necrotic and die. [39] showed the same result was obtained for shoots initiation, where internodes cultured on MS medium encompassing 1 mg/L BAP give the highest at 20% Clorox 20 min, respectively (Figure 5). It was higher compared to the time when nodes were used as explants. The browning problem is a result of phenolic compounds secretion which inhibits micropropagation. Almost the explants showed 21% browning problem in 20% Crolox 10 min (Table 4). However, the stem of Pelargonium radula explants performed the best on shoot initiation. Manickavasagam et al. (2004) reported that with the 1 mg/L BAP produced the highest number of shoot from stem explants. The explants produce axillary buds then converted into seedlings [40] . As reported by [41] , the node explants of gypsophila originating from the first internodes regenerated more shoots at ranges 74% - 100%. They also found that the best shoots per explants up to 19 regenerated from the top internodes. As a result, the BAP (1.0

The values are the means ± SE. of 5 replications, the means with identical letter do not significantly differ at p ≤ 0.01 according to DMRT.

mg/l) showed the optimum plant growth regulator compared to 0.5, 2.0 mg/L of the BAP in shoot initiation at the early stage.MS medium supplemented with 1.0 mg/l BAP was more efficient in promoting the shoot in propagation of the explants [42] [43] . [44] also underscored the importance of the BAP for regeneration of in vitro plant. Furthermore, [43] studied the medicinal herb and reported that MS supplemented with 1.0 mg/L BAP showed the best results (100%) with multiple shoots formed.

The results of this investigation indicated that the stem cuttings of P. betle cultivar significantly affected the percentage of rooting and the roots number. The fresh and dry weights of the roots, the length of the roots diameter, the surface area of the root and the roots volume in the softwood cuttings, and the concentrations of the auxin significantly affected (IBA 1000 and 1500, 2000 mg/L) the rooting percentage in the semi-hardwood cuttings and only at 2000 mg/L IBA in the semi-hardwood cuttings.

In addition, the best development of the meristematic activity and the phloem parenchyma cells was initiated appearing on 6 days of semi-hardwood showing subsequent development of root initials organized root primordial. P. betle is a sensitive-to-root piper species. Lack of regeneration of mature tissues in the stem cuttings reduces the rooting ability of the betel plants. However, the present study showed that 1000 - 2000 mg/L of IBA treatments were effective in increasing the rooting percentage of the P. betle cuttings. For the adventitious roots initiation and development of betel semi-hardwood cuttings, root primordial was found developed from root initials occurring near the vascular bundle, the phloem and besetting by fiber layers which could hinder its emergence. Nonetheless, IBA applications promote cell expansion and proliferations which are able to break the fiber layers and allow the root primordial to arise.

The results of the tissue culture study showed that the percentage of Clorox with immersing time played a considerable role in inducing shoot sterilization from P. betle explants. The use percentage of Clorox with immersing time in an appropriate ratio was the most effective for the shoot induction. The immersing nodes explants in 20% Clorox for 20 minutes significantly reduced the contamination, and the percentage of the plant survival was the highest (70.00%). On the other hand, the BAP at 1.0 mg/L was the best concentration to induce shoot multiplication from P. betle explants. The highest explants with new leaves and the number of axillary buds were attained using the medium containing BAP (1.0 mg/L). The percentage of the axillary buds was the highest (22.29%). Therefore, the medium at 1.0 mg/L is recommended for the growth of P. betle explants during the development and growth of the explants.

Muttaleb, Q.A., Abdullah, T.L., Rashid, A.A. and Hassan, S.A. (2017) Rooting of Stem Cuttings with Different Indole 3 Butyric Acid (IBA) Treatments and Development of Micropropagation Protocol for Piper betle L. Node Culture. American Journal of Plant Sciences, 8, 3084-3100. https://doi.org/10.4236/ajps.2017.812208