High frequency callus induction and proliferation of MD2 pineapple (Ananas comosus)

Ananas comosus var. MD2 is currently the most preferred pineapple variety in the international market due to its pleasant aroma and high Brix acidity ratio. In vitro approaches such as callus culture is promising in producing disease-free plantlet. However, there are limited studies reported on callus culture of MD2 variety despite the potential of in vitro regeneration through biotechnological advances. The purpose of the study was to determine the effect of plant growth regulators (PGRs) i.e., 2,4dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and Thidiazuron (TDZ) on callus induction from leaf explant of MD2 pineapple. Leaf base explants were cultured on Murashige and Skoog (MS) media supplemented with varying concentration of 2,4-D (0.5 to 6.0 mg/L) alone and in combination with BAP (1.0 to 3.0 mg/L). The frequency of callus induction was seen significantly highest (91.67±8.33%) with maximum callus fresh weight (0.25±0.07 g) at a combination of 2.0 mg/L 2,4-D and 2.0 mg/L BAP. The shortest duration of callus formation was seen on day 12 with the lowest concentration of 2,4-D, 0.5 mg/L. There is a moderate correlation between the earliness of callus formation and the frequency of callus induction (P<0.01). The most favourable media for callus proliferation was 6.0 mg/L 2,4-D and 2.0 mg/L TDZ as the highest fresh weight of 1.52±0.03 g was recorded. Callus culture has the potential to be a source of plant material and secondary metabolites production. In this study, 2,4-D and BAP have successfully induced callus in MD2 pineapple.


Introduction
Pineapple is one of many commercial fruits available in the global market. Currently, MD2 is the most successful cultivar and listed as a key crop and known by its trade names as "Golden Ripe", "Super Sweet", "Rompine" or "Gold". According to United Nation Conference Trade and Development (UNCTAD, 2016), this variety was farmed in the early 1990s by Del Monte. Due to its deep and lasting transformation, the pineapple industry has been improved. This semi-spiny variety is able to establish as it has high yields and low sensitivity to black spots and soil parasites. Another characteristic of 'MD2' as reported by Thalip et al. (2015) is its mildly sweet in flavour making it very popular among consumers causing it to be one of the outstanding varieties produced for commercial purposes.
Among constraints faced in the production of pineapple is the cultivation of seedlings as the parent plant requires a significant length of time to produce slips and suckers. Biotechnology covers many fields like tissue culture and genetic engineering to the applications like an ideal tool for crop improvement (Bukhori and Khalid, 2013;Yusuf et al., 2016;Hossain and Uddin, 2017). Cytokinin and auxin or auxin alone are normally included in primary culture media for callus induction. Looking at the potential and promises of plant tissue culture technology, efforts have been directed for implementing this technology to improve the productivity of these plants (Wu et al., 2012). Keeping this in view, the present study will focus on callus formation involving PGRs.
The use of PGRs such as auxin with a combination of cytokinin show successful result for callus induction in different cultivars of pineapple (Akbar et al., 2003;De Silva et al., 2008;Hamid et al., 2013;Yusuf et al., 2016;eISSN: 2550eISSN: -2166 © 2020 The Authors. Published by Rynnye Lyan Resources FULL PAPER Hossain and Uddin, 2017;Harahap et al., 2019). Yusuf et al. (2016) reported successful callus induction of MD2 pineapple supplemented with very high cytokinin to auxin ratio, 14 mg/L BAP, and 6 mg/L NAA. Thus, the present study aimed at improving protocol for callus induction of MD2 pineapple and proliferation with the use of a lower concentration of PGRs to become costeffective and affordable for industrial purposes without affecting the growth and potential of callus.

Preparation of sterile mother plant
The leaves of the crown were removed (Figure 1a and 1b), thoroughly washed with detergents, and rinsed. In laminar airflow, the crown was immersed in 90% ethanol for 3 mins and rinsed. Then, it was sterilized in 60% Clorox for 20 mins with 2 drops of Tween 20 followed by 20% Clorox for 10 mins. The crown was again rinsed with sterile distilled water three to five times to ensure the remaining sterilant was washed completely. The crown meristem tip was excised 0.5 to 1.0 cm in size and cultured on solid MS media (Murashige and Skoog, 1962) supplemented with 2.0 mg/L BAP. The cultures were incubated at 25±1°C under a 16/8-h light/dark photoperiod (25-30 μmol photons m -2 s -1 ) using a fluorescent lamp. The leaves obtained from two months old in vitro plantlet ( Figure  1c) were used as the explant for callus induction.

Preparation of Callus Induction (CI) and Callus Proliferation (CP) media
A volume of 1 L MS media was prepared by weighing 4.4 g/L of MS media and 30 g/L sucrose dissolved in 800 mL distilled water in a Schott bottle. After maintaining pH at 5.8, water volume was made up to 1 L and 2 g/L of Gelrite, gelling agent was added to solidify the media. Thereafter, the media was autoclaved at 121°C for 20 mins. The hormone supplements were added as required for different treatments as followed: CI Media: Control (MS medium without hormone) as shown in Table 1 CP Media: Control (MS medium without hormone) as shown in Table 2.

Callus induction
Leaf base segments of two months old plantlet were excised aseptically into pieces (1.0 to 1.5 mm) from the basal site and inoculated on the media fortified by 2,4-D hormones of varying concentration (0.5, 1.0, 2.0, 4.0, and 6.0 mg/L) alone or in combination with BAP (1.0, 2.0 and 3.0 mg/L). All cultures were incubated in the dark at 25±1°C and subcultured every three weeks. -free media) were investigated. After 42 days, the frequency of callus induction (%), fresh weight (FW) of callus (g), and duration of callus formation (days) was recorded before transferring in proliferation media. The cultures were incubated in the dark at 25±1°C and subcultured every three weeks. The morphological changes such as type, texture, and colour of callus were recorded.

Callus proliferation
The callus (0.2 g) obtained from callus induction were inoculated at different 2,4-D concentration (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0 mg/L) combined with constant TDZ (2.0 mg/L) to determine best media for callus proliferation. The cultures were incubated in the dark at 25±1°C and subcultured every three weeks. The FW of callus and morphological changes were documented.

Statistical analysis
The treatments were replicated six times in a Randomized Complete Block Design (RCBD). The frequency of callus induction, FW of callus, and duration of callus formation was analysed through one-way analysis of variance (ANOVA) using Statistical Package for Social Sciences (SPSS) version 25. One-way ANOVA was conducted on the variables with no significance (P>0.05) in terms of the interaction effect of the different PGRs on callus induction and proliferation. Duncan's multiple-range test (P<0.05) was used to determine the significant difference between the means. The correlations between parameters were analysed using the Pearson correlation test (P<0.01).

Sterile mother plant
The most challenging stage in the pineapple micropropagation process was creating healthy and freecontaminant cultures. The pineapple explants were however very prone to the substances of sterilization. Most explants were found to be damaged throughout the sterilization process whether they were unusable by explants contaminated with fungi and bacteria or bleaching. A thorough selection of explants from pathogen-free parent plants together with an effective method of surface sterilization must be identified to achieve high survival. Therefore, preparation of a sterile mother plant part was performed to determine the impact of the concentration of sterilization materials specifically for crown pineapple variety MD2 to obtain viable culture. Notably high survival percent of explants (88.89±0.52%) was observed in 60% Clorox while only 5.56±0.41% of mortality and contamination were recorded. FULL PAPER induction such as frequency of callus induction, FW, and duration of callus formation. The results indicated that control media with the absence of PGRs failed to induce callus even after 42 days of inoculation, thereby affirming that PGRs were essential for callus induction in MD2 pineapple. Auxin 2,4-D used alone and in combination with BAP successfully induced callus. This is supported by Amin et al. (2005) where singly used of 2,4-D and combination with BAP for cultivar Giant Kew pineapple were effective for callus initiation. Conversely, a study conducted by De Silva et al. (2008) which 2,4-D failed to induce callus from meristematic globular bodies (MGB) in Moris and Josapine pineapple.

Effect of plant growth regulators 2,4-D and BAP on callus induction
The auxin 2,4-D alone was capable to induce callus from the explant as early as 12 days of culture in C1 (0.5 mg/L 2,4-D). Meanwhile, C3 (2.0 mg/L 2,4-D) and C4 (4.0 mg/L 2,4-D) both displayed 83.33±10.54% of explant callusing (P<0.05). Among all the concentrations of 2,4-D tested in this study, C3 and C4 also displayed similarly high FW of callus with 0.20±0.09 g and 0.21±0.10 g respectively. The duration for callus formation was delayed as the concentration of 2,4-D were increased. Nevertheless, the frequency of callus induction and callus FW was increased when the concentration of 2,4-D was increased. However, an exceptional decrease for callus induction and callus FW in C5 (6.0 mg/L 2,4-D) were recorded. A positive correlation was seen in callus induction and callus FW as analysed with Pearson's Correlation (P<0.01). The opposite was reported in different plant species, Coffea arabica Code 71 where increased induction rates documented with a lower concentration of 2,4-D (Mwaniki et al., 2019).
In a combination of treatments, the frequency of callus induction was significantly increased with the addition of cytokinin. Compared to all the treatments, C13 (2.0 mg/L 2,4-D + 2.0 mg/L BAP) recorded significantly highest callus induction (91.67±8.33%) and maximum callus FW (0.25±0.07 g) 23 days of culture (P<0.05). The duration for callus formation in combination treatment, C12 (1.0 mg/L 2,4-D + 2.0 mg/L BAP) was the longest with 36.33±0.82 days. The results indicated that there was a moderate correlation (P<0.01) between the frequency of callus induction and the duration of callus formation. It was noted that an additional concentration of 2,4-D beyond 4 mg/L suppressed the rise in frequency of callus induction and FW of callus simultaneously.
The inclusion of auxin in cell culture media has been reported in various plant species including pineapple (Rahayu et al., 2016;Mahadi et al., 2017;Harahap et al., 2019). Auxin plays a significant role in the initiation of callus, but it relies on the type and concentration used in culture media (Wahyuni et al., 2017). Different types of auxin caused different effects on the plants as the responses were highly dependent on plant genotypes and culture conditions (Budisantoso et al., 2017;Mwaniki et al., 2019). For records, auxin 2,4-D has been successfully and widely used in tissue culture due to its strong and significant effect on callus differentiation (Fazeli-Nasab, 2018). The optimum concentration of 2,4-D enhanced the growth and biomass of pineapple as proven by Amin et al. (2005). Results obtained in this experiment were in accordance with previous studies of various pineapple cultivars such as Sipahutar (Harahap et al., 2019), Smooth Cayenne (Istiqomah and Shofi, 2018), and in Tangkit (Zulkarnain and Neliyati, 2017).
Excessive concentrations of 2,4-D in the culture media inhibited the growth and development of MD2 pineapple (Hamid et al., 2013). Such concentrations retarded the rate of protoplasmic streaming and highly toxic for shoot and bud (Thimann, 1939). The common occurrence caused by extremely high auxin were abnormality of shoots and stunted culture (Bukhori and Khalid, 2013). Inadequate supply of oxygen, water, and minerals to the cell obstructed plant growth (Budisantoso et al., 2017). Furthermore, an oversupply of 2,4-D caused a longer duration of callus formation and this result was in agreement with Akbar et al. (2003). An application of a high concentration of exogenous PGRs to accelerate the duration of callus formation was restricted by endogenous PGRs produced by the plant itself. Thus, causing the competition between both exogenous and endogenous PGRs which consequently hindered the normal expression of callus formation (Wahyuni et al., 2017).
Initially, cytokinin promoted organogenesis and cell division where in most cases aided callus induction (Adil et al., 2018;Mwaniki et al., 2019). Combination of PGRs encouraged callus induction as well as shoot multiplication in pineapple (Yusuf et al., 2016;Zulkarnain and Neliyati, 2017;Hossain and Uddin, 2017). According to Indah and Ermavitalini (2013), FW physiologically contains two components, namely water and carbohydrates. The amount of FW generated depends significantly on how rapidly these cells divide, multiply, and then develop the callus (Wahyuni et al., 2017). The result obtained in this study was in agreement with Harahap et al. (2019) where balanced auxin and cytokinin ratio significantly effective in callus initiation of Sipahutar pineapple grown in Indonesia and lead to the highest fresh and dry weight of callus. This suggests that a balanced ratio of 2,4-D and BAP were rather favourable in callus induction of MD2 pineapple. In this sense, current findings were able to improve the protocol proposed by Yusuf  The duration of callus formation indicated the time taken for swelling to explant from the cut to form the callus (Rahayu et al., 2016). The presence of cytokinin BAP was insignificant to accelerate the duration of callus formation. As 2,4-D concentration was increased, the callus formation was further delayed. Conversely, Mahadi et al. (2016) reported addition of cytokinin speed up callus formation as well as when 2,4-D concentration was increased. Nevertheless, application of BAP to the culture media significantly increased (P<0.05) callus induction and callus FW despite a longer duration of callus formation. Time is a critical factor in the determination of effective induction protocols. However, complementary factors together with callus induction frequency, callus biomass, and callus morphology were essential (Mwaniki et al., 2019).
The selection of the best explants for callus induction has been the most critical factor in the micropropagation of many plant species (Bukhori and Khalid, 2013). Callus induction cultured from crown explant took nine weeks of culture for callus formation. Meanwhile, leaf base explant was selected as it took as early as three weeks of culture to produce callus. Moreira et al. (2016) stated that in vitro leaf base explants of Ananas erectifolius were effective for maximum callus induction with a white or creamy coloured base of young leaves. De Silva et al. (2008) acknowledged that the leaf base contains a meristematic region for cell division and enlargement. Figure 2 shows morphological changes of explant at one week, four weeks, and six weeks after culture. The explant was seen swelling after the first week of culture ( Figure 2a) and began to multiply at the wounded site four weeks after culture (Figure 2b). An unorganized clump of white tissue was seen covering the surface of the explant and significantly enlarged after 6 weeks of culture ( Figure 2c). Most of the callus exhibited compact texture. Callus demonstrated from this experiment were heavily lignified and hard in texture (compact), whereas others break easily into small fragments (friable). A mixture of compact and friable callus was recorded in a combination of treatments.
Most pineapple callus produced under supplementation of 2,4-D were compact in texture (Akbar et al., 2003;Amin et al., 2005;Harahap et al., 2019). Recently Harahap et al. (2019) documented compact callus with the inclusion of BAP to the culture media. Compact textured callus occurred due to water potential absorption into the cell which caused it to become rigid . Furthermore, auxin also increased the concentration of intracellular fluid by stimulating the accrual of material structure and chemical component in the cytoplasm (Budisantoso et al., 2019). This was contrary to the experiment by Yusuf et al. (2016) where friable callus was distinguished with a combination of BAP. Despite strong activities to stimulate differentiation, the growth of the callus was sustained proficiently with 2,4-D hormone (Wahyuni et al., 2017).
Since the callus was treated in the dark, the colour of the callus was limited to yellowish white and yellowishbrown after 42 days of completed callus formation. Increased 2,4-D concentration resulted in browning of callus. The addition of BAP to the culture media improved the colour of the callus. After four subcultures, the yellowish-brown callus turned brown completely. Actively dividing callus was usually white, yellow, and green in colour . Auxin in the culture media accumulated ethylene (native plant hormone) often caused browning of culture (Rahayu et al., 2016). The presence of secondary metabolites, long incubation time without subculture and age of callus were factors that caused the browning and necrosis of culture (Budisantoso et al., 2017). As the nutrient in the culture media depleted, an increase in carbon dioxide (CO2) and limited oxygen (O2) resulted in anoxia (Fazeli-Nasab, 2018).
The explant response towards different concentrations of PGRs produced different degrees of callusing; slight callus, moderate callus, and profuse callus ( Table 4). The callus response differed according to concentration and combination of PGRs supplemented in the culture media. Profuse callus occurred in a higher frequency of callus induction. Meanwhile, slight callus was observed in the lower frequency of callus induction. Previous studies have reported contradicting outcomes in where the callus was often varied in colour which is largely contributed by the absorption of light in the cell (Harahap et al., 2019). Similar results testified by Mahadev et al. (2014) profuse and compact callus presented with the use of a higher concentration of 2,4-D. Profuse and compact callus recognized as good callus by Indah and Ermavitalini (2013) as it can accumulate secondary metabolites.  Table 3 shows the effect of PGRs concentration and combination on callus proliferation. The initial weight of callus inoculated on proliferation media was 0.20 g and the FW of callus was collected after 21 days. The maximum FW of callus was observed in P5, 1.52±0.03 g with a mixture of compact and friable texture. The lowest FW of callus was seen in P1, 0.29±0.01 g with a compact texture. The results suggested that FW of callus increased when the concentration of 2,4-D increased up to 6.0 mg/L. Auxin 2,4-D and cytokinin TDZ was shown to give a synergistic effect with an increased FW of callus. Higher FW may be related to an increase in the concentration of cytosolic calcium caused by enhanced media absorption due to the use of higher PGRs. (Ngomuo et al., 2013). Callus growth was frequently associated with callus biomass (Kumlay and Ercisli, 2015) as an increment in callus FW was due to a high number of cells from cell division and enlarged cell size (Wahyuni et al., 2017). However, 8.0 mg/L 2,4-D restricted further increase of callus FW. The results corroborated with Adil et al. (2018) for in vitro grown herbaceous plant Cnidium officinale where biomass was increased from 2.3 to 13 mg/L 2,4-D and any higher concentration of 2,4-D showed low biomass in callus. TDZ unusually induced a 30-fold increase in the growth of callus cultures over other PGRs (Pai and Desai, 2018). Figure 3a PGRs (mg/L)   Auxin and cytokinin may interact through synergistic, antagonistic, and additive mechanisms relying on inoculated tissue culture conditions that promote developmental decision towards callogenesis or shoot formation (Rasud and Bustamam, 2020). CP media fortified with different concentrations of 2,4-D with a combination of TDZ mostly produced compact callus (Table 5). Similar cases were documented by Zulkarnain and Neliyati (2017) whereby a compact structure has good regeneration ability and could be used easily for the shoot and root proliferation. The significance of TDZ has been reported in the callus of various plant cultures, which has also demonstrated a higher rate of cell proliferation and shoot multiplication compared to other PGRs (Budisantoso et al., 2017;Mwaniki et al., 2019).

Effect of combination2,4-D and TDZ on callus proliferation
Colour changes in callus that occurred during the 6week observation indicated a change in the growth phase and the regeneration control of the cells (Amin et al., 2005;De Silva et al., 2008;Ngomuo et al., 2013) The colour of yellowish-white and yellowish-brown showed that cells were still actively dividing (cleavage stage), while the colour of brown showed symptoms of ageing cells (Budisantoso et al., 2017;Nasution and Nasution, 2019). Rasud and Bustamam (2020) state the colour of the callus describes its visual appearance that indicates the activity level of a cell division. Type, concentration, and combination of auxin and cytokinin effects on callus morphology were discussed by several authors in plant tissue culture studies (Hossain and Uddin, 2017;Istiqomah and Shofi, 2018).
Auxin and cytokinin were both essential for in vitro cell proliferation. Moreover, it was discovered that the ratio of these PGRs determines the morphogenetic pathway that the in vitro cultured tissue will follow where high and low ratios of cytokinin to auxin favoured shoot and root regeneration, respectively (Hamid et al., 2013;Zulkarnain and Neliyati, 2017). Meanwhile, balanced concentrations were ideal in the growth of an unorganized cell mass (Akbar et al., 2003;Amin et al., 2005).

Conclusion
PGRs used in this study successfully induced and proliferated callus from MD2 pineapple. The most favourable media for callus induction was MS media supplemented with 2.0 mg/L 2,4-D and 2.0 mg/L BAP. However, in light of cost efficiency, 2.0 mg/L of 2,4-D alone would be sufficient to induce callus. Callus proliferated in this study was 5 times more than the initial inoculated weight in 21 days. Further studies should be established to determine the potential of pineapple callus in secondary metabolite production using biotechnological approach. The results could be used for large-scale commercial production of protease enzyme in pineapple. Values are expressed as mean±SD of 6 replicates for the incubation period of 3 weeks. Values with the same superscript letter within the column are not significantly different at p<0.05. Control = MS alone.