Callus Induction from Root Fragments of Falcataria moluccana Plantlets

Callus induction of F. moluccana (sengon) was still an obstacle to indirect organogenesis regeneration. The purpose of the study was to determine the callus induction formation from root fragments of F. moluccana plantlets. Primary explants (fragments of roots) were cultivated on MS induction basal media and three concentration combination of PGRs (BAP and NAA): 0.5 ml/l BAP; 0,1 ml/l NAA (single PGR); and combination of 0.5 ml/l BAP + 0.1 ml/l NAA (double PGR). When roots were used as explants, high formation rates of callus (more than 70%) were obtained. Highest formation rates of callus were in NAA added at all clones (12 clones), then BAP added (7 clones) and BAP + NAA added (5 clones). The results indicated that BAP and NAA concentrations used in the media were influence the producing callus and affect the amount of callus produced from roots of sengon. The addition of NAA also gives higher callus proliferation results than the addition of BAP or the addition of a combination of the two hormones. The results indicated that BAP and NAA concentrations used in the media were influence the producing callus and affect the amount of callus produced from roots of sengon.

roots may serve as regeneration competent cells, while in rice the bundle sheath and some immature vascular cells in leaves and the phloem-pole pericycle cells in roots may serve as regeneration-competent cells (6). These competent cells are responsible not only for callus initiation but also for root primordial initiation during adventitious root (AR) or lateral root (LR) formation [5]. There have been few reports on the regeneration of plants root culture, as these explants have been found to be recalcitrant with respect to the formation of morphogenic callus. If their recalcitrant nature could be overcome, roots would be an ideal explant source for obtaining regenerated plants.
Roots are plentiful, available at all times, are easy to excise and are also a well-defined source of meristematic tissue. [7] also stated that the un-pigmented nature of root protoplasts made them ideal for use as markers in protoplast fusion work. Due to the uniqueness of these legume roots, callus culture research has never been conducted using roots as explant material.
An important factor reported in several species with regard to callus induction is the explants type, example: nodal segmen of medicinal tree Premna serratifolia L. [8], inter-nodal segments of T. grandis L.f. [9] and young leaves and shoot tips of L. ca- In the present study, in vitro culture of root fragment callus induction from selected vegetative plantlet material were investigated for the establishment of efficient micro propagation protocol of sengon, multi purposes of tropical fast-growing tree, for mass propagation.

Materials and Methods
The study was located at the tissue culture laboratory in Centre for Forest Biotechnology and Tree Improvement, Yogyakarta, Indonesia. Research observations were carried out for 12 months, from January 2018 to January 2019. The equipment used in this study conformed to tissue culture laboratory standards.

Callus induction
The cutting of the roots as explants from the plantlets was aseptically carried out in laminar air flow (LAF). Root pieces were directly transferred to callus media. Callus formation rate = Number of roots with callus x 100%

Number of incubated roots
After 6 months incubation, formation rate, callus proliferation and callus color were observed. The overall study was carried out for 12 months at tissue culture laboratory.

Results
The roots of plantlet used as the material for this study, one of the successes of sengon tissue culture that Putri [10] has done in previous research, were derived from vegetative micro cutting (Figure 1.A). These plantlets were sub-cultured 3 times, 4 weeks each sub-culture since the transfer of explants.  During experiment we observed formation of seven callus color ( Figure 2). BAP has a browner (B) effect on callus, followed by NAA, while the effect of the combination of BAP and NAA is more diverse in color and all textures were friable and different stages.  Microscopic observation showed callus development at week 4 and week 6 of incubation (Figure 3). Globular friable callus develops to a larger size to form the next callus phase. PGRs stimulated cell elongation by increasing of plasticity of the cell wall to become loose, causing water to easily flow to the inner cell by osmosis, causing the cell to become elongated [12].

Discussion
Macro and micro vegetative propagation of sengon has never been successful before, one of the obstacles to the propagation of macro sengon is that it does not form roots, which occurs in vitro (Figure 1.B) [10]. One thing that affects the success of sengon micro cutting is the availability of nutrients and controlled abiotic and biotic environmental conditions. In Table 1 shows the formation rate, callus proliferation and color of callus induced from 3 clones of explant in MS media with a combination of single and double PGRs after 6 weeks of culture. When roots were used as explants, high formation rates of callus (more than 70%) were obtained. Highest formation rates of callus were NAA added in all clones (12 clones), then BAP added (7 clones) and BAP + NAA added (5 clones). The results indicated that BAP and NAA concentrations used in the media were influence the producing callus and affect the amount of callus produced from roots of sengon.
The addition of NAA also gives higher callus proliferation results than the addition of BAP or the addition of a combination of the two hormones. Auxins (NAA) have many effects and can modulate diverse processes and tropic responses (reviewed in [13]. In in vitro culture, exogenous auxins are assumed to orientate developmental pathways and to favour either callogenesis or rhizogenesis according to the concentration [14]. However, there are few comparative detailed studies of the effects of changes in auxin concentrations during callogenesis and rhizogenesis. Although, NAA is rarely used in indirect somatic embryogenesis processes, this hormone has been used to study the switch between distinct organogenic pathways [5]. Table 1 also shows that of the 3 sengon clones tested had no effect on formation rate, callus proliferation and color of callus.
There have been few reports on the regeneration of plants from root cultures of sengon, as these explants have been found to be recalcitrant with respect to the formation of morphogenic callus if their recalcitrant nature could be overcome, roots would be an ideal explant source for obtaining regenerated plants. Roots are plentiful, available at all times, are easy to excise and are also a well-defined source of meristematic tissue. [16] also stated that the unpigmented nature of root protoplasts made them ideal for use as markers in protoplast fusion work. Figure 2 shows that the wall cell has not reached lignification yet because of friable calluses contain much water, and the group of cells can be easily separated from the others. The callus texture from the explant can be distinguished as friable and non-friable. The non-friable callus has compact and tight cells that are difficult to separate. In contrast, a friable callus from an explant has loose cell interaction that is easily detached using tweezers. The resulting color variations might be due to the diverse types of growth regulators, the difference in growth regulator concentration, but not the clones of. Compared to single PGR, a combination of auxin and cytokinin resulted in a callus color that was greener, caused by cytokinin, which tends to promote chlorophyll formation [17] . According to [18], various callus color conditions could be caused by the pigmentation, the influence of light, and the plant parts used as the source explant. Table 1 also shows that of the 3 sengon clones tested had no effect on formation rate, callus proliferation and color of callus.
Callogenesis is the initial response, characterized by the formation of the callus, which starts from the edge of the explant (wounded part) at the top and bottom of the sengon roots that has direct contact with the medium. The callus is formed faster and bigger size on the part that has direct contact with the media (Figure 3). This is probably related to the process of nutrient uptake in the medium by the explant. The appearance of the callus on the wounded part might be caused by the excitement of the tissue on the explant to cover the wound. [19] stated that the cell division that leads to the callus formation occurs from the injuries and both the natural and artificial hormone supply from the outside into the explant. Light is an external factor that influences callus formation. The color change that exists in the callus was because of pigment, nutrients, and environmental factors, such as light. White light could induce callus formation and organogenesis in the plant tissue. A callus that has yellowish green and green color was formed with the addition of cytokinin (BAP).
The green color was because of chlorophyll, mainly because cytokinin has a function in the formation of chlorophyll in the callus and due to environmental factors, such as exposure to light [20]. [21] claimed that the color change in the callus from white to green was due to chlorophyll formation.

Conclusions
In conclusion, this study indicates that 0.5 ml/l BAP; 0.1 ml/l NAA (single PGR); and combination of 0.5 ml/l BAP + 0.1 ml/l NAA (double PGR) can potentially be used to obtain the best callus stimulation from the roots explant source of sengon. The study has also shown that clones had no effect on friable callus formation from root fragments of sengon as explants. Further research needs to be conducted to examine the PGRs for next callus phase of sengon.