Healthy cuttings of A. paniculata were collected from Moulvibazar and Sunamganj district of Bangladesh. Collected cuttings were planted in the research field and rooftop of the Department of Genetics and Plant Breeding for explant preparation.

For explant (stem, leaf and midrib) preparation, at first a shoot was excised from healthy plant of A. paniculata and swabbed with 70% alcohol-soaked cotton and the shoot was cut into small pieces. After that the small pieces were washed in running tap water for 20 min followed by washing with household detergent for 5 to 7 min. They were brought to laminar air flow after washing with distilled water. Then the shoot pieces were treated with 70% ethyl alcohol (MERCK, Germany) for 2 min for surface sterilization and rinsed in sterile double distilled water 3 to 4 times. Again, the shoot bits were surface sterilized with 10% Chlorox (Sodium hypochlorite, The Clorox Company, Oakland, USA) for 8 minutes, and were rinsed with sterile double distilled water 3 to 4 times to remove the effect of surface sterilizing agents. The explants were then split into little pieces and used as explants after being surface sterilized. Finally, the segment of stem, leaf and midrib (5-7 mm in length) from excised shoot were used as explant for the callus induction.

Stem, leaf and midrib explants (5-7 mm in length) were inoculated in MS (Murashige and Skoog, 1962) media complemented with several concentrations of BA (99%, Duchefa Biochemie, the Netherlands) (0.5, 1.0, and 2.0 mg/L), NAA (98%, Duchefa Biochemie, the Netherlands) (0.1, 0.5 and 1.0 mg/L) and 2,4-D (96%, Duchefa Biochemie, the Netherlands) (0.1, 0.5 and 1.0 mg/L) to find the best medium for callus initiation. The process was carried out entirely in a laminar airflow cabinet. In each culture jar containing 50 ml callus induction media, five explants were put. On the surface of the medium, the stem, leaf, and midrib segments were inoculated horizontally (Fig. 1a, b & c) and implanted in culture room with high light intensity (16/8 hours light/dark) produced by 144 W white fluorescent lamp, a temperature of 25±2°C and a relative humidity of 70%.

After attaining a convenient size, the calli were re- cultured on freshly prepared shoot induction media (Fig. 1 d & e) containing MS salts and vitamins, 3% sucrose, 1% agar and different strengths of 2,4-D (0.5 and 1.0 mg/L), NAA (0.1, 0.5 and 1.0 mg/L) and BA (1.0, 2.0 and 3.0 mg/L). When in vitro raised shoots grew about 2-3 cm in height was aseptically transferred into full strength and half strength MS media separately supplemented with different strengths of NAA (0.5, 1.0 and 2.0 mg/L) and IBA (0.5, 1.0 and 2.0 mg/L). A permanent marker was used to mark the cultivated vessels after being sealed with parafilm to denote each treatment. They were then placed in a culture room.

After development of adequate root system, 4-6 cm plantlets were removed cautiously from the cultivated vessels without damaging roots and rinsed smoothly in running tap water to eliminate agar medium and sucrose trace elements to avoid fungal infection. Then the complete rooted plantlets were transferred into plastic pots containing wet soil and provide a glassware cover (beaker) to prevent dehydration (Fig. 1k). When the plantlets were successfully adapted to the natural environment then they were transferred to field condition (Fig. 1l).

The experiment was designed following completely randomized design (CRD) with three replications. The recorded data on the percentage of callus initiation, shoot regeneration and root regeneration and statistically analyzed to ascertain the significance of the experimental results. MS Excel 2010 was used for calculating the mean and standard deviation for all treatments. Dunkan’s Multiple Range Test (DMRT) was used for evaluating the significance and difference between means by utilizing R software (version Rx64 3.4.3).

Callus initiation was started within a week from thestem, leaf and midrib explants of A. paniculata afterincubation of explants on MS medium containingseveral combinations of BA (0.5, 1.0 and 2.0 mg/L),2, 4-D (0.1, 0.5 and 1.0 mg/L) and NAA (0.1, 0.5and 1.0 mg/L). Among the combinations tested, MSmedium supplemented with 0.5 mg/L NAA and 2.0mg/L BA showed the highest callus initiationfrequency for all three types of explants withsignificant heterogeneity among the explants (Fig. 2 and Fig. 3). The highest callus formation was 100% 93.33% and 73.33% for stem, leaf and midrib explantsrespectively in 0.5 mg/L NAA and 2.0 mg/L BAcombination, while the lowest recorded callusformation for stem (26.67%), leaf (20%) and midribexplant (13.33%) found in MS medium supplementedwith 0.1 mg/L 2,4-D and 0.5 mg/L BA combination (Fig. 2). However, the callus initiation frequency ofstem, leaf and midrib explants on rise with the increaseof NAA and 2,4-D up to 0.5 mg/L individually in acombination with BA up to 2mg/L. Afterword, withthe increase of NAA and 2,4-D the callus initiationfrequency has decreased. The phytohormone 2,4-D ina combination with BA produced 80%, 66.67% and 53.33% callus for stem, leaf and midrib explantsrespectively, whereas, NAA along with BA induced100%, 93.33% and 73.33% for stem, leaf and midribexplants respectively. So, it is clear that callusinitiation frequency is mostly regulated by NAA and 2, 4-D and NAA was more responsive compared to 2, 4-D (Fig. 2). On the other hand, higherconcentration of NAA induced excessive rooting in thecallus (Fig. 6a) and eventually reduced the shootforming capacity of the callus. However, in hormonefree MS media, none of the explants produced callus.

In this study, stem and leaf explants showed better response for callus induction than midrib explants are in conformity of previous finding of War et al., 2018. Jindal et al., 2016 reported that best callus induction was obtained as MS medium containing 1 mg/L 2, 4- D and 1 mg/L NAA by using leaf explant. War et al., 2018 suggested that optimal callus induction medium for leaf explant was MS medium containing 2, 4-D (1.0 mg/L) and Kinetin (0.5 mg/L) followed by 2,4- D (1.5 mg/L) and Kinetin (1.0 mg/L) and optimal callus induction medium for stem explant is MS medium supplemented with 2,4-D + Kinetin (1.5 + 1.0 mg/L). Sharma and Jha (2012) reported that, highest callus initiation was achieved on MS media consist of 1 mg/L 2,4-D and 1 mg/L NAA as well as combinations of 1 mg/L 2,4-D and 0.5 mg/L Kin and 1 mg/L BAP and 1 mg/L NAA.

The physical appearance of a callus is influenced by the type of explants and the composition of the culture media (Sharma and Nautiyal, 2009). In this study, several types of distinguishable callus in terms of consistency and colour were generated from different types of explants (Fig. 3) in different auxin (NAA and 2, 4-D) and cytokinin (BA) combinations (Fig. 4). When the concentration of auxin (NAA and 2, 4-D) and cytokinin (BA) were increased (above 1 mg/L) very compact, brownish, brownish white or white calli were found. Whereas, at low NAA, 2,4-D and BA (below 0.5 mg/L) concentrations fragile, brownish or whitish colour calli were produced (Fig. 4). Again, in the presence of NAA and BA, leaf explants generate compact, green-colored callus. On the other hand, 2,4- D and BA combinations generated friable white callus from leaf explant.Therefore, compact, fragile, greenish, whitish and brownish calli derived from various explants in various combinations and concentrations of growth regulators. However, these types of callus morphology were also found in some other plants like Ipomoea obscura L. (Mungole et al., 2009), Gynura procumbens (Nurokhman et al., 2019). A green compact callus was found in Orthosiphonsta mineus from a balanced concentration of NAA and BA (Elangomathavan et al., 2017). In contrast, higher concentration of NAA and BA provided yellowish white compact callus in Dianthus caryophyllus (Arif et al., 2014).

However, it is revealed that, varied concentration and combination of plant growth regulators also influenced the callus morphological responses. Differences in plant growth regulator concentrations and combinations have a significant impact on morphogenic responses of callus and the morphogenic response is affiliated with the mineral nutrition that is applied (Avilés et al., 2009). Additionally, color of callus can change due to changes in chlorophyll as a result of reaction among different plant growth regulators; explant types and environmental culture factors like light exposure and temperature (Elias et al., 2015).

In this experiment, stem and leaf explants had a stronger callus induction response and produced robust, vigorous calli than the midrib explants. Hence, calli derived from stem and leaf explants were employed in shoot regeneration.Two types of auxins (2,4-D and NAA) and cytokinin (BA) were used to find out the best medium for shoot regeneration. 21 days old healthy calli were cultured on half strength MS medium containing a variety of BA, 2,4-D and NAA combinations and concentrations (Fig. 5). After three weeks of callus culture, shoot bud formation started from the calli. Both the calli and calli with the shoot buds was sub cultured on shoot regeneration

Fig. 1:
In vitro regeneration of Kalmegh (a) Stem explants on callus induction medium (MS + 0.5NAA + 2.0BA) at 1st day of culture, (b) Leaf explants on callus induction medium (MS + 0.5NAA + 2.0BA) at 1stday of culture, (c) Midrib explants on callus induction medium (MS + 0.5NAA + 2.0BA) at 1st day of culture, (d) 15 days old stem callus on callus induction media, (e) 15 days old leaf callus on callus induction media, (f) 15 days old midrib callus on callus induction media, g) shoot bud initiation from stem callus in shoot induction medium (½MS + 0.1NAA + 3.0BA) on 27 days of culture, (h) shoot bud initiation from leaf callus in shoot induction medium (½MS + 0.1NAA + 3.0BA) on 27 days of culture, (i) multiplication of shoot in shoot induction medium (MS + 0.1NAA + 3.0BA) on 6 weeks of culture, (j) initiation of root in MS + 2 IBA medium, (k) acclimatized plant in soil, (l) plants that survived in natural condition.Scale bars represent 0.5 cm (a, b & c), 1 cm (d, e, f, g & h), 2.5 cm (i, j & k), 1.0 cm (l).

Fig. 2:
Callus initiation frequency from In vivo stem, leaf and midrib explants of A. paniculata on MS media fortified with various concentrations of BA, NAA and 2,4-D. Data consist of three replications and 5 explants were utilized in each replication. Bars reflect the standard deviation of means. Values with different letters are significantly different at P value = .05 (DMRT).

Fig. 3:
Morphology of callus at 3rd week of culture periodbased on explant type

Fig. 4:
Morphology of callus at 3rd week of culture period based on PGR concentration and combination

media to obtain complete shoot buds. The percentage of shoot regeneration was varied with the difference of concentrations and combinations of growth hormones. Out of different combinations, ½ MS + 0.1 mg/L NAA + 3.0 mg/L BA was showed the highest 73.33% and 60% shoot regeneration frequency for leaf as well as stem explants respectively (Fig. 5). This result has proven that, ½ MS + 0.1 mg/L NAA + 3.0 mg/L BA is the best media combination for shoot initiation. Other findings of in vitro shoot regeneration of A. paniculatas (Bansi and Rout, 2013) also suggest that ½ MS in a combination with 3.0 mg/L BA produce higher number of shoots. However, the better performance of stem explant in shoot regeneration is also supported by the findings of Roy (2014).

The strength of MS medium also influenced the shoot regeneration from stem and leaf callus. Full MS medium showed browning of the callus (Fig. 6c) and reduced shoot elongation process (Fig. 6b). These observations might be influenced by the high mineral concentrations, present in full strength MS media, excessive in amount for shoot morphogenesis (Purkayastha et al., 2008; Kataky and Handique, 2011).

From the above data, it was found that the frequency of shoot regeneration from stem explant was higher than that of leaf explant (Fig. 5). To investigate the reason behind more shoot regeneration frequency of stem explants compared with leaf explant, anatomical study of stem and leaf segment of A. paniculata was carried out. Transverse section (T.S.) of stem possesses epidermis, collenchyma, chlorenchyma, sclerenchyma, phloem, xylem, pith cells (Fig. 7) whereas T.S. of leaf contains layer of epidermis, palisade parenchyma, spongy parenchyma (Fig. 7). Among them chlorenchyma, pith, palisade parenchyma, spongy parenchyma is parenchymatous cell. In microscopic observation, number of active parenchymatous cells was found more in stem than that of leaf (Fig. 7). And collenchyma cell was found in the stem which is absent in the leaf (Fig. 7).

Possibility of the highest shoot regeneration from the stem explant was due to presence of more active parenchymatous cells than leaf explant (Fig. 7). On the other hand, collenchyma cells serve the growing part of the plant like leaf and shoot which is absent in the leaf (Fig. 7). So, the highest shoot regeneration in the stem explant may be due to the presence of more active parenchymatous cells and collenchyma cells in the stem compared to the leaf explants (Abhilasha and Arpita, 2017).

For root initiation, well grown shoots were rescued and sub-cultured into MS media supplemented with different concentration of NAA and IBA (0, 0.5, 1.0 and 2.0 mg/L). Among the fourteen tested media, the

Fig. 5:
Shoot regeneration frequency at 6 weeks of culture of stem and leaf explant of A. paniculata on half strength MS media strengthen with various concentrations of BA, NAA and 2,4-D. Data consist of three replications and 5 explants were utilizedin each replication. Bars reflect the standard deviation of means. Values with different letters are significantly different at P value = .05 (DMRT).

best result was obtained in half strength MS media containing 2.0 mg/L IBA. The maximum percentage of rooting (93.33%) was noticed in half MS medium fortified with 2.0 mg/L IBA whereas the minimum root formation frequency (33.33%) was found in full strength MS medium reinforced with 0.5 mg/L NAA (Table 1).Root development was noted within 7 days of transplanting, and plantlets established a well- developed root system by 15 days (Fig. 1j). After that the plants were transferred to pot soil of acclimatization room for acclimatization (Fig. 1k). The acclimatized plantlets were grown successfully in field condition.

Fig. 6:
(a) Excessive rooting in the callus-on-callus induction medium contained higher concentration of NAA and 2,4-D (more than 1 mg/L), (b) no elongation of shoot in full strength MS medium, (c) browning of callus on full strength MS medium.Scale bars represent 1 cm (a, b, c).

Fig. 7.
(a) T.S. of Stem of A. paniculata Nees (× 40) where 1. Epidermis, 2. Collenchyma, 3.Chlorenchyma, 4. Sclerenchyma, 5. Phloem, 6. Xylem and 7. Pith cells; (b), T.S. of Leaf of A. paniculata(× 40) where 1. Cuticle, 2. Lower epidermis, 3. Palisade parenchyma, 4. Upper epidermis and 5. Spongy parenchyma.

Table 1:
Root initiation frequency of A. paniculata on different strength of MS media reinforced with various concentrations of NAA& IBA.

Data consist of three replications and 5 explants were used for each replication. The mean values were compared by DMRT. Mean ± SD followed by same letters are not significantly different at P = 0.05.

Growth hormone, NAA or IBA alone or in a combination provide positive effects on rooting in vitro for A. paniculatas (Bansi and Rout, 2013; Roy, 2014; Kataky and Handique, 2011). In this study, two auxins (NAA & IBA) were used for root initiation. Six weeks old shoots,obtained from the subsequent culture in callus initiation and shoot formation media, were used for root formation. However, the regenerated shoot did not produce any roots in control (full and half strength MS medium without hormones) even after six weeks of culture. This type of in vitro rooting behavior of A. paniculatas also reported by Kataky and Handique (2011);Bansi and Rout (2013); Jindal et al. (2015) and Chandran et al, (2017). Root initiation was observed in full and half strength MS media supplemented with NAA or IBA after 7 days of inoculation. The highest (93.33%) rooting was found in half strength MS medium supplemented with 2.0 mg/L IBA whereas the lowest (33.33%) rooting was occurred in MS media with 0.5 mg/L NAA (Table 1). Similarly, Jindal et al. (2015); Chandran et al. (2017); Deshmukh et al. (2017); found maximum root formation in 2.0 mg/L IBA.

In this study, half strength MS media responded better to root initiation than full strength MS media which is in accordance with Chandran et al. (2017); Deshmukh et al, (2017); Roy (2014); Bansi and Rout (2013); Kataky and Handique (2011). This could be owing to half-strength MS media having lower osmotic strength and nutritional content than full-strength MS media. Half-strength MS media provided low osmotic potential for ease acclimation of plants and also created partial stress, causing plants to develop more roots and stimulate rhizogenesis.

Any medium with lower concentrations of auxin showed a poor response in root formation as compared to medium with higher concentrations of auxin and IBA was proved more responsive than NAA which is in accordance with Roy (2014) and Purkayastha et al. (2008) findings.