Establishment for a successful
in vitro culture can be identified as the sufficient surface sterilization of explants while percentage of fungal and bacterial contaminations and bleaching of explants decides the efficiency of culturing procedure. In most cases, the primary reason for failure of commercial tissue culture laboratories is the inability to control contaminations (
Leifert and Woodward, 1997). Hence, when developing a callus production protocol for
A. marmelos for the secondary metabolite extraction
, a proper surface sterilization procedure should be identified as the first step. Sodium hypochlorite (NaOCl) has been identified as a highly effective sterilant against most of the bacteria and fungi which cause contaminations in plant tissue culture (
Yildiz, 2012). It is a practical selection as it can be easily diluted to the required concentration and readily available
(Tiwari et al., 2012). Clorox
® bleach is a common sterilizing agent containing NaOCl as the main ingredient which has been used to sterilize many plant types
(Daud et al., 2012). In the current study explants were surface sterilized with 10% (v/v) Clorox ® for 10 min as the main sterilization procedure.
Immature leaves were bleached after surface sterilization and could not use for further observations (Table 1). Fungal contaminations were significantly higher in mature leaves (80%) and shoot tips (90%) than seeds (10%). Bacterial contaminations were occurred rarely in mature leaf and shoot tip explants.
Immature leaf, mature leaf and shoot tip explants have to be considered as inferior to seed explants due to contaminations and bleaching in this study. Hence seed explants were selected as the most viable and practical option for callus induction from
A. marmelos. Seeds and cotyledon explants were reported to be used even in previous callus induction studies of this plant
(Hossain et al., 1993); (
Hazeena and Sulekha, 2008);
(Joyner et al., 2014); (Devi, et al., 2014). Other than seeds and cotyledons, some other explants of the plant as nodal segments
(Islam et al., 2007) Leaf discs
(Thangavel et al., 2008); (Arumugam et al., 2008) and stem segments
(Bhardwaj et al., 1995) have also been reported to develop good quality callus
.
Though contaminations occurred rarely, seeds established with seed coat did not produce callus even after five weeks from culture initiation (Table 2). Hundred per cent callus production was observed in the seeds established after removing seed coat followed by split into two halves (Fig 1). Response for callus induction was considerably low in the seeds established without split in to two halves though seed coat was removed. Therefore, seed cultures should be established after removing seed coat followed by splitting for a better callus induction.
Plant growth regulators play an important role in production of higher amount of high-quality callus. Auxins induce callus formation and proliferation while 2,4 D can be identified as the most efficient plant growth regulator related to callus culture
(Luciani et al., 2006). Different concentrations and combinations of 2, 4 D and BAP were tested for the callus induction from seed explants of
A. marmelos in the current study.
According to Kruskal-Wallis one-way ANOVA test no significant differences were observed in quality of callus and growth of callus among treatments. Therefore, MS medium supplemented with combinations BAP ranging from of 0.5 to 1.5 mgL
-1 and 2,4 D ranging from 1.0 to 2.0 mgL can be used to induce callus from seed explants of
A. marmelos. For commercial purposes the combination with lowest concentrations (MS medium supplemented with 0.5 mgL
-1 BAP and 1.0 mgL
-1 2, 4 D) can be used.
In previous studies 2, 4 D alone or together with another plant growth regulator has been used for the callus induction from different explants of
A. marmelos. According to
Das et al., (2009) creamish friable callus was obtained from nodal segments of
A. marmelos on MS medium supplied with 4.0 mgL-1 2, 4 D within two weeks of inoculation. At the same time, nodal segments of
A.marmelos were reported to produce maximum amount of callus in MS medium supplemented with 0.3 mgL
-1 BA and 2 mgL
-1 2,4 D
(Islam et al., 2007). Cotyledon explants of
A. marmelos showed highest response for the callus induction in MS medium supplemented with 2.2 µM BA and 2.26 µM 2,4 D (
Hazeena and Sulekha, 2008). In another study,
A. marmelos callus cultures were initiated from leaf explants on B5 medium supplemented with 0.5 mgL
-1 2,4 D and 0.2 mgL
-1 BA
(Arumugam et al., 2008). Current study has proved that MS medium supplemented with combinations BAP ranging from of 0.5 mgL
-1 to 1.5 mgL
-1 and 2,4 D ranging from 1.0 mgL
-1 to 2.0 mgL
-1 could also be used to induce superior callus from seed explants of
A. marmelos as a new addition to the literature. Not only that, a combination of 0.5 mgL
-1 2, 4 D and 1.0 mgL
-1 BAP resulted in highest percentage of callus production in black turmeric (
Curcuma caesia) (
Abubakar and Pudake, 2019).
Once initiated, callus should be multiplied to obtain a sufficient amount for the secondary metabolite extraction. Auxins alone or together with a cytokinin can be used for the process according to the preference of the plant species. MS medium supplemented with combinations of 2, 4 D (0.5, 1.0 (mgL
-1) either with BAP 0.5, 1.0 (mgL
-1) or Kinetin 0.5, 1.0 (mgL
-1) was tested for the callus multiplication of
A. marmelos in this study.
The highest biomass (132.58 gL
-1 fresh weight) was recorded in the callus in MS medium supplemented with 0.5 mgL
-1 2, 4 D and 1.0 mgL
-1 Kinetin when introduced to the callus multiplication media. The result was not significantly different from the treatment with 1.0 mgL
-1 2, 4 D and 0.5 mgL
-1 Kinetin (123.58 gL
-1 fresh weight). The lowest fresh weight was recorded in the MS medium supplemented with 1.0 mgL
-1 2, 4 D and 1.0 mgL
-1 BAP, which was not significantly different from the treatment with 1.0 mgL
-1 2, 4 D and 0.5 mgL
-1 BAP. Significant differences were observed among treatments at p ≤ 0.05.
In previous callus culture studies, use of 2, 4 D and Kinetin in combination, alone or together with some other plant growth regulators has been recorded in many occasions. In most cases callus has been proliferated in the same medium used for callus induction.
Thangavel et al., (2008) have been obtained actively proliferating callus tissues from leaf disc explants of
A. marmelos by culturing the young leaf discs in MS medium supplemented with 2.0 µM Kinetin and 6.0 µM2, 4D. Callus has been induced and multiplied successfully from cotyledon explants of
A. marmelos in MS medium supplemented with Kinetin and NAA (
Bindhu, 2015). According to
(Hossain et al., 1994) slow-growing calli were induced and proliferated from nucellar explants of
A. marmelos in MS medium supplemented with 5.0 mgL
-1 NAA and 1.0 mgL
-1 kinetin. Current study revealed that, MS medium supplemented with 0.5 mgL
-1 2, 4 D and 1.0 mgL
-1 Kinetin and MS medium supplemented with 1.0 mgL
-1 2, 4 D and 0.5 mgL
-1 Kinetin can be successfully used for the callus multiplication of
A. marmelos.
When consider about the extraction of secondary metabolites,
in vitro callus cultures proved that
A. marmelos has as much potential in diabetes management as the original leaf extract
(Arumugam et al., 2008) showing that no distortion happens in secondary metabolites extracted from the callus cultures. Than, maintaining many trees of
A. marmelos, having callus cultures within a small space is highly beneficial in extracting metabolites for medicinal purposes.
