In Vitro Callus Induction and Shoot Regeneration from Leaf Explants of Glinus lotoides (L.)—An Important Medicinal Plant

G. lotoides L. is a threatened plant that is frequently harvested for medicinal purpose. However, its distribution in the world is limited because of short period of seed viability and poor seed germination. The objective of this study was to develop in vitro propagation protocol for G. lotoides through callus induction. For callus induction, different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid) and BAP (6-benzyl amino purine) were used. Seeds were sown on growth regulator-free MS medium and shoots from the in vitro germinated seedlings were excised and cultured on MS medium containing 0.5 mg/l BAP. Young leaves from these shoots were used as explant for callus induction and shoot regeneration. Maximum callus induction (100%) was observed on medium containing 2,4-D (0.5, 2.0, 3.5 mg/l) or NAA (2.0, 2.5 mg/l) in combination with 0.5 mg/l BAP. However, 2,4-D was the best in overall callus induction. The highest regeneration (20%) frequency was achieved on the medium containing 0.5 mg/l BAP. Highest number of shoot (2.83 ± 1.22) and shoot length (2.16 ± 0.87 cm) per explant were obtained in the presence of 0.25 mg/l BAP + 0.5 mg/l KIN (Kinetin). In shoot multiplication media, maximum mean (6.43 ± 0.87) of shoot was observed on MS medium containing 0.5 mg/l BAP. The best shoot length (1.70 ± 0.14 cm) was recorded on control medium. The highest (95%), maximum root number (14.10 ± 1.47) and root length (1.01 ± 0.10 cm) were obtained on a medium supplemented with 1.5 mg/l Indole-3-butyric acid (IBA). All the plants (100%) were survived after acclimatization in greenhouse. The present study can be useful for callus induction and indirect shoot regeneration form G. lotoides.


Callus Induction
The matured seeds of G. lotoides were purchased from the local market in Addis Ababa and received from Institute of Biodiversity Conservation (IBC), Addis Ababa, Ethiopia. Seeds were surface disinfected with 70% (v/v) alcohol for 3 min, followed by 10% (v/v) sodium hypochlorite solutions containing two drops of Tween-20 for 5 min and subsequently rinsed five times thoroughly with sterile distilled water. Then the seeds were sown on growth regulator-free MS [27] medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. Shoots excised from in vitro germinated seedlings were transferred to MS medium containing 0.5 mg/l BAP for shoot multiplication.
Young leaves from in vitro multiplied shoots were cultured on callus induction MS medium with adaxial (upper) surface in direct contact with the medium. Leaves were wounded perpendicular to its midrib. Culture medium was supplemented with 30 g/l sucrose and different concentrations of 2,4-D (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg/l) and NAA (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg/l) in combination with BAP (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg/l). The pH of the medium was adjusted to 5.8 before addition of 8 g/l agar. The medium was autoclaved at 121˚C for 15 min at 105 Kpa. After autoclaving, 25 ml of the medium was dispensed into Petri dishes (90 mm diameter). A total of 10 leaves per Petri dish with three replications were used. Growth regulator-free MS medium was used as control. The number of shoots that induced callus was recorded after six weeks of culture.

Shoot Regeneration
Six-week-old calli were transferred to shoot regeneration medium containing various concentrations of BAP (0.0, 0.25, 0.5, 1.0, 1.5 mg/l), KIN (0.0, 0.25, 0.5, 1.0, 1.5 mg/l) alone or in combinations with 0.20 mg/l NAA. Culture was maintained under dark conditions at 25˚C ± 2˚C for a month. When shoots emerge, the cultures were placed under dim light for a week followed by transfer to full light of 40 µmol•m -2 •s −1 and 16 h photoperiod and number of calli that regenerated shoots and number of shoots per explant was recorded.

Shoot Multiplication
Regenerated micro-shoots were cultured on shoot multiplication medium containing different concentrations of BAP (0.0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 mg/L) or Kinetin (0.0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 mg/l). After autoclaving at 121˚C for 15 min at a pressure of 105 Kpa, 50 ml of the medium was dispensed into Magenta GA-7 culture vessels. A total of 30 shoots per treatmentwith10 shoots per culture vessel in 3 replications were used. All subsequent subcultures were done at 4 weeks intervals to fresh medium of the same composition. The number of shoots per explant was recorded at each subculture stage.

Acclimatization
Plantlets were carefully removed from culture vessels and gently washed under running tap water followed by transfer to 20 cm diameter pots containing sand, red soil, and compost in 1:3:2 ratio and kept in greenhouse. The potted plantlets were covered with polyethylene bags. The number of survived plants was recorded after five weeks.

Culture Conditions
All cultures were maintained in culture room under light intensity of 40 μmol/m 2 /s and 16 h photoperiod provided by cool-white fluorescent lamp at 25˚C ± 2˚C. Cultures for callus induction were kept in dark room at 25˚C ± 2˚C.

Experimental Design and Data Analysis
In all experiments, Completely Randomized Design (CRD) was used. Data were subjected to one way ANOVA and least significant difference (LSD) test using statistical data analysis software SPSS version 20.0 at 5% probability level.

Effect of 2,4-D
Callus formation was observed after 15 days of culture. Callus formation on the explants occurred at wounding site of major veins, and then continues to growth until it covered the entire leaf section. The best responses (100%) callus induction was observed on the medium containing 0.5, 2.0, 3.5 mg/l 2,4-D ( Table 1). Leaves on each medium containing 2,4-D in the range of 0.5 -4.0 mg/l responded differently. At the highest concentration (4 mg/l) of 2,4-D, callus induction was declined. The formed calli were soft, compact with white, grey-white anddark-grey color (Figure 1).

Synergistic Effect of 2,4-D and BAP
Callus formation primarily occurred at wounding site on leaf veins, and then continued to grow until it covered the entire leaf section. In the medium containing 1.0, 2.5, 4.0 mg/l 2,4-D combined with 0.5 mg/l BAP, the explants showed 96.6% of callus formation ( Table 2). Noticeable callus response variation was observed in their morphology, color and degree of growth with changes in growth regulator combinations. The size of callus in this combination was larger than callus received from 2,4-D alone (Figure 1). Calli were less compact and grey-white color.

Synergistic Effect of NAA and BAP
Among various concentration tested, 2.0, 2.5 mg/l NAA combined with BAP (0.5 mg/l) resulted in a maximum percentage (100%) of fast callus formation ( Table 3). Further increase inNAA concentration reduced callus production. Callus induction was not noticed in presence of 4.0 mg/l NAA and on control medium and the explants dried. In this experiment, calli were dark-grey with soft and less compact texture, fast growing and have well developed roots (Figure 2).

Synergistic Effect of BAP and NAA
After 4 weeks of culture, the most efficient callus induction percentage (93.3%) was observed in a medium supplemented with 3.0 and 4.0 mg/l BAP in combination with 0.5 mg/l NAA ( Table 4). At lower concentration of BAP (0.5, 1.0, 1.5 mg/l) poor callus induction was observed. The calli obtained from various concentrations of BAP combined with 0.5 mg/l NAA were white and grey-white color with soft to compact texture. Under this growth regulators combination, callus has moderate growth rate and roots were developed (Figure 2).

Shoot Regeneration
The calli induced on MS medium containing NAA (1.0, 2.0, 2.5 mg/l) in combination with BAP (0.5 mg/l) and 2,4-D (0.5, 1.0, 2.5 mg/l) showed the highest response in shoot regeneration media. However, calli induced on     (Figure 3). Results in Table 5 show that most calli did not result in shoot regeneration.

Shoot Multiplication
Multiplication of shoot showed the significant difference among the treatments (Table 6, Figure 4). Best number of shoot (6.43 ± 0.87) was observed on medium containing 0.5 mg/l BAP, while 4.60 ± 0.33 was obtained     on MS medium containing 1.5 mg/l KIN. Maximum shoot length (1.70 ± 0.14 cm) was achieved on growth regulator free medium.

Rooting
The highest rooting percentage (95%), root number (14.10 ± 1.47) and root length (1.01 ± 0.10 cm) were obtained on a medium supplemented with 1.5 mg/l IBA ( Table 7). In case of NAA, best mean number of roots (7.75 ± 1.66) and root length (0.76 ± 0.13 cm) were obtained in the presence of 0.1 mg/l. At the base of plantlets, callus was observed in the presence of 2.5 mg/l IBA and 1.5, 2.5 mg/l NAA. All plantlets were healthy and grown vigorously (Figure 5).

Acclimatization
All plants (100%) survived after acclimatization in greenhouse. Plantlets were grown vigorously and any morphological abnormality was not observed (Figure 6).

Callus Induction
Plant tissue culture play a significant role in the conservation of endangered medicinal plant and enhance the production of secondary metabolites. In vitro production of secondary metabolite is used to reduce harvesting of plants from natural habitats. In vitro callus mass propagation and production of their secondary metabolites were reported on Solanum trilobatum [28], Citrullus colocynthis [29], and Convolvulus alsinoides [30]. Sole application of 2,4-Dwas more effective in callus induction than in combination with BAP. Similarly, callus induction frequency was higher in all media containing 2,4-D than the media with NAA and BAP. This indicates the efficiency of 2,4-D in cell division. Effectiveness of 2,4-D in callus formation was also reported in other medicinal herbs such as Achyranthes aspera [31], Ocimum sanctum [32], Solanum trilobatum [28], Ionidium suffruticosum [33].
There was positive interaction result of NAAXBAP relatively in production of fast grown calli than 2,4-D alone or in combination with BAP. Callus production efficiency of NAA and BAP on Solanum nigrum [40] and Orthosiphon aristatus [41] were reported. However, in the present study, spontaneous root formation during callus formation on medium containing NAA and BAP was observed. In Ferula assa-foetida [42] and Eryngium foetidum [43] root formation from callus on medium containing NAA was reported. A significant reduction in the percentage of callus formation with increased NAA concentration while keeping a constant BAP concentration was observed. However, increased BAP concentration at constant NAA concentration led to increased callus induction percentage.

Shoot Regeneration
The plant growth regulator and the explants played a significant role in shoot regeneration. Overall, calli from NAA X BAP were excellent in shoot regeneration as compared to calli from 2,4-D alone or 2,4-D in combination with BAP. Maximum regeneration percentage (20%) was obtained on a medium supplemented with 0.5 mg/l BAP. However, interactive effect of BAP and KIN resulted inmaximum number of shoot per callus. This is in agreement with [38] on Stevia rebaudiana and [31] on Achyranthes aspera.
All concentrations of KIN did not produce any shoots from calli. This is in agreement with [36] in Swertia chirayita. Poor regeneration response was observed in the presence of BAP plus NAA. This is in contrast with the results obtained from [41] Orthosiphon aristatus and [31] Achyranthes aspera.

Shoot Multiplication
The present study revealed that BAP was the most effective cytokinin than KIN for shoot multiplication in this species. Earlier reports showed the efficiency of BAP in production of multiple shoots on Portulaca grandiflora [44], Thymus satureioides [45], Origanum sipyleum [46], and Salvia guaranitica [47]. However, increased concentration of BAP and KIN beyond the optimum level resulted in the reduction of shoot per explants. The earlier reports also showed this phenomenon in Majorana hortensis [48], Vitex agnus-castus [49] and Prunella vulgaris [50].
Shoots developed on MS medium containing various concentrations of KIN were taller than shoots obtained on medium containing BAP. This might be BAP reduced the apical dominance than KIN. Increasing the concentration of cytokinins resulted in the production of stunted shoots in Psoralea corylifolia [51].
Although, root induction was observed on MS medium enriched with different concentrations of NAA, the overall analyzed parameters such as mean root number, root length, shoot number and shoot length were lower than in case of IBA. However, the best root induction competence of NAA was reported in Clitoria ternatea [57], Solanum surattense [58] and Thymus satureioides [45]. At higher concentrations of IBA and NAA, root development was declined.
All plantlets were survived after acclimatization. This might be due to the occurrence of somaclonal variation that different from those of parent plants, which made the plant resistance to environmental difficulty.

Conclusion
An efficient protocol was developed for indirect plant regeneration. MS medium containing 2,4-D (0.5, 2.0, 3.5 mg/l) or NAA (2.0, 2.5 mg/l) in combination with 0.5 mg/l BAP was the most effective for callus induction whereas 0.5 mg/l BAP was optimum for shoot regeneration. Maximum means of shoot and root were observed on MS medium containing 0.5 mg/l BAP and 1.5 mg/l IBA respectively. All the plants were survived after acclimatization in greenhouse. Callus production from rare medical plant is used to harvest secondary metabolites and reduce the over exploitation of plants from their in situ habitats. G. lotoides is the rare medicinal herb with different groups of phytochemicals. Therefore, this protocol is very important for mass propagation of the species and also opens a new way to facilitate secondary metabolites production and isolation of pharmaceuticals from callus rather than harvesting the plant itself.