CALLUS INDUCTION IN LEUCAENA ( Leucaena leucocephala (Lam.) de Wit) AS AN EFFORT TO PROVIDE TARGET TRANSFORMATION THROUGH Agrobacterium tumefaciens

Leucaena is a plant that produces biomass productivity in the form of hardwood for fuel. However, Leucaena is also classified as an invasive plant which can cause the urgency of native plant species and ecosystems in Indonesia. Therefore, the formation of sterile Leucaena needs to be done, one of which is through genetic transformation using Agrobacterium tumefaciens . Callus is used as a target for transformants in the genetic transformation process, so it is necessary to use appropriate media and PGR. This study aimed to determine the type of media and the concentration of 2.4-D on callus induction. This research is an experimental study with a completely randomized design (CRD) method with two factors. The first factor is the type of media (MS and WPM) and the second factor is the concentration of 2.4-D (0; 0.25; 0.50; 0.75; 1.00; 1.25 and 1.50 mgL -1 ). Parameters observed were callus initiation, callus fresh weight (gram), callus texture and color. Quantitative data is analyzed by analysis of variance (ANOVA). The results showed that the use of media had a significant effect (P<0.05) on callus fresh weight. The use of 2,4-D concentration had a significant effect (P>0.05) on callus texture. The use of WPM media resulted in the fastest callus emergence time (6.67±0.57), the best callus texture (crumb callus type 2) and the best callus color (green). Meanwhile, the highest fresh weight (2.48±0.83) was in the use of MS media. The fastest callus emergence time occurred in the control (without the addition of 2.4-D) (7.33±0.57 and 6.67±0.57), the highest average fresh callus weight (2.48±0.83 and 2.35±0.32) occurred in the treatment with the addition of 1.00 mgL -1 2.4-D with a crumb callus texture of type 2 and 0.25 mgL -1 2.4-D produced callus with green color.


INTRODUCTION
Leucaena leucocephala (Lam.) de Wit (Fabaceae), commonly known as 'Lamtoro' in Indonesia and is the finest known species of the genus Leucaena.leguminous tree that develops in a wide extend of soil sorts in numerous tropical and sub-tropical nations (Saafi and Borthakur, 2002;Jube and Borthakur, 2008).It is considered an indispensable tree species in agroforestry because of its disease resistance, efficient development and its branches can be used as food for feeding organisms.Leucaena leaves are high in protein, but they also contain a toxic called mimosine (Saafi and Borthakur, 2002).
Leucaena has a high biomass, but this plant is classified as an invasive plant.
Invasive plants originating from within the country and abroad can cause the urgency of native species and ecosystems (Radiansyah et al., 2015).Leucaena was declared as an alien invasive plant in Indonesia with a higher density of tiller growth (11.78%) in uninhabited houses and 4.42% in inhabited houses (Iqbar et al., 2017).It is also declared as invasive plants along the hiking trail of Panderman Mount, East Java.This plant can grow at an altitude of more than 1400 meters above sea level (masl) and is able to withstand rainfall of 500-3500 mm (Septiadi et al., 2018).One of the reasons is the easy spread of Leucaena seeds, either through biotic or abiotic aids.
However, even without apparent fruit or seed morphological adaptation, endozoochory can be an effective mechanism for an alien species to spread to a new environment, even if it did not spread in this way within its home range (Gallego-Fernandez et al., 2020).
Prevention needs to be done on the  (Sorensson and Brewbaker, 1994).
Another method that can be done to produce sterile Leucaena is by genetic transformation.

Hereditary change utilizing
Agrobacterium tumefaciens or the biolistic strategy requires a productive tissue culture recovery convention.Tissue culture work of this tree has been started in a number of research facilities with constrained victory (Saafi and Borthakur, 2002).The initial stage of the genetic transformation process is callus production in vitro (Dwiyani et al., 2016).Callus is a transformation target which will be inoculated with A.
tumefaciens suspension in the genetic transformation process.The use of callus as a target for transformation has been carried out on various plants.Correia (2014) used callus as a transformation target in the manufacture of Leucaena plants with low phenolic exudate, low cell necrosis and a stronger root system mediated by A. tumefaciens.

Materials
The materials used were Leucaena seeds cv.Tarramba (brown color), MS

Seed Sterilization
The sterilization of Leucaena seeds followed the method of Sapsuha et al.
(2011) with modifications.Leucaena seeds were washed with 0.5 g of detergent for 15 minutes then rinsed with sterile water for 10 minutes.Seeds were then soaked in 1% fungicide solution and 1% bactericide solution for 20 minutes each.Then, the seeds were rinsed again with sterile water.
The seeds were soaked in a 10% bleach and 5% bleach solution for 10 minutes each and rinsed again with sterile water 3 times for 5 minutes each.Finally, to break dormancy, the seeds were soaked in warm water for 30 minutes in LAF.The sterile seeds were cultured under aseptic conditions in LAF.
Seeds were cultured in preconditioned media and kept in a culture room at 25 0 C under 12 watt LED lighting (18 hours light and 6 hours dark) for 14 days to induce shoots growth.

Callus Initiation
The young stem explants were cut into 0.5-1.0cm pieces and cultured on MS and WPM medium supplemented with 30 gL -1 sucrose and 8 gL -1 agar.The  Kadir (2006).

Data Analyses
Statistical

Callus fresh weight
Growth is a permanent increase in the size of an organism or part of a plant which is the result of an increase in the number and size of cells.Growth is characterized by an irreversible increase in weight, so the measurement of callus fresh weight can represent the callus growth variable (Indah and Ermavitalini, 2013).
Fresh weight of callus in this study was determined by looking for the difference between callus weight at week 4 after induction and the first day of culture.The callus fresh weight yield data are presented in Table 2.  (Sari et al., 2018).The fresh weight produced is very dependent on the speed at which these cells divide, multiply, and then continue to grow (Idris and Paserang, 2019).According to Marisa et al. (2021), physiologically fresh weight consists of water and carbohydrates.Indah and Ermavitalini (2013) described that the large fresh weight of callus was due to the high water content.Rahayu et al. (2016) showed that the fresh weight of callus tended to increase at a concentration of 2 to 4 mgL -1 2.4-D but decreased at a concentration of 6 mgL -1 2.4-D in Centella asiatica plants.

Callus texture and color
Explant growth in vitro culture could be observed from its visual appearance in the form of callus texture and color.Callus texture is one of the markers used to assess callus quality (Indah and Ermavitalini, 2013).Observation of callus texture was carried out at 4 week after culture which resulted in varied callus textures (Figure 1).The results of the ANOVA test on the texture of the Leucaena callus are presented in Table 3.   Callus texture is a marker used to determine the quality of a callus so that it can be seen that cells are still actively dividing or have stagnated in cell division.
A good callus is assumed to have a friable texture because it facilitates separation into single cells and increases oxygen aeration between cells (Sari et al., 2014).Compact callus is more difficult to separate because of its strong texture.However, compact callus usually produces high secondary metabolites which are commonly used in the health sector (Arieswari, 2018).
Callus color is one of the indicators of explant growth on in vitro culture.Callus tissue produced from an explant usually brings out different colors (Indah and Ermavitalini, 2013).Callus color observations were carried out at 4 week after culture (Figure 2).The results of the ANOVA test on the color of the Leucaena callus are presented in Table 4.

Based on
experiment employed Randomized Completely Design with two factors.First factor was basal medium used (MS and WPM).Second factor was 2.4-D concentration (0; 0.25; 0.50; 0.75; 1.0; 1.25 and 1.50 mgL -1 ).Each treatment was repeated three times and one explant was planted in one Petri dish.Based on that, there were 42 experimental units, namely 2 types of media × 7 concentration of 2.4-D × 3 replications.Cultures were incubated at 25 0 C under white fluorescent tubes at 18 hour light and 6 hour dark.Variables observed were time of callus initiation, callus fresh weight, callus texture and color were measured following callus scoring of Gultom et al. (2012) and

Note:
The mean value ± SD followed by different letters in the same column indicate significant difference according to Duncan's Multiple Range Test (DMRT) at a significance level α = 0.05.The results showed that WPM treatment with a concentration of 2.4-D 1.0 mgL -1 gave a significantly different effect on callus texture compared to other treatments.Meanwhile, MS treatment without 2.4-D and WPM without 2.4-D did not show a significantly different effect with MS treatment with a concentration of 2.4-D 1.50 mgL -1 , WPM with a concentration of 2.4-D 0.25 mgL -1 , WPM with a concentration of 2.4-D 0.50 mgL -1 , WPM with a concentration of 2.4-D 0.75 mgL -1 , WPM with a concentration of 2.4-D 1.25 mgL -1 and WPM with a concentration of 2.4-D 1.50 mgL -1 .
of 2.4-D 1.25 mgL -1 , ie brownish yellow.Meanwhile, yellowish white color was only produced in MS treatment with a concentration of 2.4-D 0.50 mgL -1 .Callus color indicates cell division activity that occurs in callus (Damanik et al., 2018).Green, white, yellow and brown colors indicate that cells are still actively dividing, while brown, black or blackish brown colors indicate signs of cell aging.Callus discoloration is caused by the synthesis of phenolic substances in cells (calus) (Nasution and Nasution, 2019).The brown color of the callus is caused by the metabolism of toxic phenolic compounds, and often occurs as a result of the explant sterilization process.Phenol compounds generally inhibit growth or even cause tissue death (Nasution and Nasution, 2019).I'anatushshoimah, et al. (2020) stated that the browning event is actually a natural event and a process of adaptive change of plant parts due to physical influences such as stripping and cutting.

Table 1 .
ANOVA test results of Leucaena callus initiation time Note: The mean value ± SD followed by different letters in the same column indicate significant difference according to Duncan's Multiple Range Test (DMRT) at a significance level α = 0.05.

Table 2 .
ANOVA test results of Leucaena callus fresh weight

Table 3 .
ANOVA test results of Leucaena callus texture

Table 4 .
ANOVA test results of Leucaena callus color Note: The mean value ± SD followed by different letters in the same column indicate significant difference according to Duncan's Multiple Range Test (DMRT) at a significance level α = 0.05.