American Journal of Plant Sciences
Vol.06 No.09(2015), Article ID:56928,11 pages

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

Shiferaw Teshome1,2, Tileye Feyissa2,3

1Department of Biology, College of Natural and Computational Science, Wolaita Sodo University, Sodo, Ethiopia

2Institute of Biotechnology, Addis Ababa University, Addis Ababa, Ethiopia

3Nelson Mandela African Institution of Science and Technology, Arusha, Tanzania


Copyright © 2015 by authors and Scientific Research Publishing Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY).

Received 26 January 2015; accepted 1 June 2015; published 5 June 2015


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-dichloro- phenoxyacetic 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.


Glinus lotoides, Growth Regulator, Kinetin, Medicinal Plant, Regeneration

1. Introduction

Glinus lotoides L. locally known as “Mettere” is a popular medicinal plant. It is herb, whose stems are prostrate or semi-erect, and belongs to Molluginaceae family [1] . This plant is native to Eurasia and Africa and has become widespread in tropical, subtropical and warm-temperate areas worldwide [1] . In Ethiopia it grows at altitudes between 530 - 1650 m.a.s.l. [2] .

G. lotoides has diverse medicinal properties in the folk medicinal systems. In Ethiopia, the seeds of G. lotoides are traditionally used as anti-helminthic for the prevalent tapeworm infestation [3] [4] , to treat Oestrus ovis [5] , Moniezia and Thysaniezia spp. [6] . The seeds of this plant have anticancer activity and nutrition value [7] . In south India, its dried part was used for treatment of purgative, cure for boils, bilious attack, wounds and pains, and tender shoots are cooked and eaten with normal diet twice a day for 5 - 7 days to get relief from urinary disorders [8] [9] . In Pakistan, poultice made up of leaves is applied over wounds and inflammation. The plant is used to treat syphilis and intestinal worms [10] [11] . Its anti-helminthic and taenicidal activity has been attributed to their saponins and flavonoids contents [4] [12] - [15] .

Despite its economic and medicinal importance, only few studies have been carried out on G. lotoides and almost all researches have focused on its medicinal properties. Seed germination (ex vitro and in vitro) was reported [16] . Its seeds contain 10% of crude saponin [17] and 14% of fat [18] [19] . It also serves as source of endophytic microorganism for production of enzymes [20] . The tablet formulations from crude extract of its seeds were reported [21] [22] . The in vivo and in vitro anthelminthic activities on Hymenolopis nana and Taenia saginata worms were reported [4] [14] [17] . Its antitumour activity was evaluated [15] . Toxicological study of its extract was conducted on rats through oral administrations as single and repeat dose on both sexes [23] . The potential effects of this herb on the feeding behavior and survival of nymphs of desert locust grasshopper have been reported [24] . Extraction, isolation, quantification and structural elucidation of five hopane-type saponins (glinusides F, G, H, I, and succulentoside B) and two flavonoids (vicenin-2 andvitexin-2"-O-glucoside) from its seeds were described [21] . Three new hopanesaponins (A-C) and four oleananesaponins (D-G) were also isolated from its root [25] .

Glinus lotoides propagates by seed but poor seed germination percentage is the limiting factor to large-scale cultivation of this species and in these days this plant is threatened [26] . Hence, to overcome all these obstacles, micropropagation is used to propagate plants within short period of time and for in vitro conservation. Callus cultures are used to study cell development, to obtain primary and secondary metabolites through cell suspension cultures. As G. lotoides contains important phytochemicals such as hopane-type saponins, oleananesaponins and flavonoids [21] [25] , for pharmaceutical applications, using callus to extract these chemicals is very important. Moreover, different novel products can be obtained through bio-transformation by using suspension cultures. Developing regeneration protocol through callus has also enormous applications in using this plant to produce novel products through genetic transformation. There is no report on shoot regeneration using through callus in this plant species. Hence, the objective of the present study is to develop in vitro regeneration protocol through callus induction from leaf explants.

2. Materials and Methods

2.1. 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.

2.2. 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.

2.3. 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.

2.4. Rooting

Well developed microshoots were cultured on half strength MS medium containing 15 g/l sucrose and supplemented with different concentrations of IBA (0.0, 0.05, 0.1, 0.5, 1.5, 2.5 mg/l) or NAA (0.0, 0.05, 0.1, 0.5, 1.5, 2.5 mg/l). Number and length of roots were recorded after four weeks of culture.

2.5. 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.

2.6. Culture Conditions

All cultures were maintained in culture room under light intensity of 40 μmol/m2/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.

2.7. 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.

3. Results

3.1. Callus Induction

3.1.1. 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).

Figure 1. Callus induction from leaf explants. (A) 2.0 mg/l 2,4-D; (B) 3.5 mg/l 2,4-D; (C) 1.0 mg/l 2,4-D + 0.5 mg/l BAP. Bars = 2 mm.

Table 1. Effect of 2,4-D on callus induction from leaf of G. lotoides.

3.1.2. 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.

3.1.3. 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).

3.1.4. 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).

3.2. 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 2. Callus induction from leaf explants. (A) 1.0 mg/lNAA + 0.5 mg/lBAP; (B) 2.0 mg/lNAA + 0.5 mg/lBAP; (C) 2.5 mg/lNAA + 0.5 mg/lBAP; (D) 4.0 mg/lBAP + 0.5 mg/lNAA. Bars = 2 mm.

Table 2. Effect of 2,4-D and BAP on callus induction from leaf explants.

Table 3. Effect of NAA and BAP on callus induction from leaf explants.

Table 4. Effect of BAP and NAA on callus induction from leaf explants.

medium containing NAA (1.0, 2.0, 2.5 mg/l) in combination with BAP (0.5 mg/l) were the most responsive in shoot regeneration. The maximum shoot regeneration percentage (20%) with 2.2 ± 0.65 mean number of shoot per callus was achieved on a medium containing 0.5 mg/l BAP (Table 5). Maximum mean number of shoots (2.83 ± 1.22) and mean shoot length (2.16 ± 0.87) were obtained on medium supplemented with the combination of 0.25 mg/l BAP and 0.5 mg/l KIN (Figure 3). Results in Table 5 show that most calli did not result in shoot regeneration.

3.3. 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

Figure 3. Shoot regeneration. (A) 0.25 mg/l BAP + 0.5 mg/l KIN; (B) 0.5 mg/l BAP; (C) 0.5 mg/l BAP + 0.2 mg/l NAA; (D) 1 mg/l BAP + 0.5 mg/l KIN. Bars = 2 cm.

Table 5. Effect of BAP and kinetin in combination with NAA on shoot regeneration from callus.

Means followed by the same letter(s) within column are not significantly different at p < 0.05.

Figure 4. In vitro shoot multiplication on MS medium. (A) 0.5 mg/l BAP; (B) 0.5 mg/l KIN; (C) 1.0 mg/l KIN. Bars = 2 cm.

Table 6. Effect of BAP and KIN on shoot multiplication.

Means followed by the same letter(s) within column are not significantly different at p < 0.05.

on MS medium containing 1.5 mg/l KIN. Maximum shoot length (1.70 ± 0.14 cm) was achieved on growth regulator free medium.

3.4. 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).

Figure 5. In vitro rooting of G. lotoides on 1/2 MS medium. (A) 1.5 mg/l IBA; (B) 0.05 mg/l IBA; (C) 0.1 mg/l NAA; (D) 0.05 mg/l NAA. Bars = 2 cm.

Table 7. Effect of IBA and NAA on rooting of G. lotoides.

Means followed by the same letter(s) within column are not significantly different at p < 0.05.

3.5. Acclimatization

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

4. Discussion

4.1. 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] .

The combination effect of 2,4-D and BAP played a significant role as plant growth regulator and has a noticeable effect on callus induction percentage than NAA in combination with BAP. The interaction effect of 2,4-D and BAP on callus induction of other medicinal plants were reported on Tridax procumbens [34] , Amsonia

Figure 6. Acclimatization of G. lotoides after five weeks. Bar = 2 cm.

orientalis [35] , Swertia chirayita [36] , Strobilanthus foliosus [37] , Stevia rebaudiana [38] , Scrophularia striata [39] .

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.

4.2. 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.

4.3. 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] .

4.4. Rooting

Microshoots were tested on 10 different concentrations of auxins, IBA and NAA, for root induction. IBA was found to be the superior over NAA for root induction in this taxon. Root induction efficiency of IBA was also reported in other medicinal plant species including Psoralea corylifolia [51] , Chlorophytum borivilianum [52] , Stevia rebaudiana [53] , Origanum sipyleum [46] , Solanum nigrum [54] , Cunila galioides [55] , Achyranthes aspera [31] , Majorana hortensis [48] , Operculina turpethum [56] .

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.

5. 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.


The authors are grateful to Institute of Biodiversity Conservation (IBC) for providing seed of Glinus lotoides, Woliata Sodo University and Institute of Biotechnology of Addis Ababa University for unreserved support.


  1. Dequan, L. and Hartmann, H.E.K. (2003) Molluginaceae. Flora of China, 5, 437-439.
  2. Gilbert, M.G. (2000) Magnoliaceae to Flacourtiaceae. In: Edwards, S., Tadesse, M., Demissew, S. and Hedberg, I., Eds., Flora of Ethiopia and Eritrea, Vol. 2, The National Herbarium, Addis Ababa University, Ethiopia, 234.
  3. Kloos, S. (1976) Preliminary Studies of Medicinal Plants and Plant Products in Ethiopian Markets. Journal of Ethiopian Pharmaceutical Association, 2, 18-28.
  4. Mulatu, D. (1978) Taenicidal Activity of Glinus lotoides (Aizoaceae). Journal of Ethiopian Pharmaceutical Association, 3, 9-11.
  5. Gidey, Y., Mekonen, T. and Mezgebe, K. (2011) Survey of Medicinal Plants Used to Treat Human Ailments in Hawzen District, Northern Ethiopia. International Journal of Biodiversity and Conservation, 3, 709-714.
  6. Mesfin, T. and Obsa, T. (1994) Ethiopian Traditional Veterinary Practices and Their Possible Contribution to Animal Production and Management. Revue Scientifique et Technique (International Office of Epizootics), 13, 417-424.
  7. Abebe, E. and Youan, B.C. (2010) Anticancer Activity and Nutritional Value of Extracts of the Seed of Glinus lotoides. Journal of Nutritional Science and Vitaminology, 56, 311-318.
  8. Kumar, G., Banu, G.S., Rajarajan, T. and Sathishkumar, G. (2011) Medicinal Flora of Palayapalayam, Namakkal District, Tamilnadu. Indian Journal of Applied and Pure Biology, 26, 135-158.
  9. Shanmugam, S., Rajendran, K. and Suresh, K. (2012) Traditional Uses of Medicinal Plants among the Rural People in Sivagangai District of Tamil Nadu, Southern India. Asian Pacific Journal of Tropical Biomedicine, 2, 429-434.
  10. Qureshi, R. and Bhatti, G.R. (2008) Ethno Botany of Plants Used by the Thari People of Nara Desert, Pakistan. Fitoterapia, 79, 468-473.
  11. Qureshi, R., Bhatti, G.R. and Memon, R.A. (2010) Ethno Medicinal Uses of Herbs from Northern Part of Nara Desert, Pakistan. Pakistan Journal of Botany, 42, 839-851.
  12. Chopra, R.N., Nayar, S.I. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants. CSIR, New Delhi.
  13. Berhanu, A. and Berhane, T. (1980) A New Triterpenoid Glycoside from the Seeds of Glinus lotoides. Phytochemistry, 19, 1553-1554.
  14. Endale, A., Getachew, M. and Gebre-Mariam, T. (1997) In Vitro Taenicidal Activity of the Seeds of Glinus lotoides on Hymenolepis nana Worms. Ethiopian Pharmaceutical Journal, 15, 46-51.
  15. Kavimani, S., Manisenthilkumar, K.T., Ilango, R. and Krishnamoorthy, G. (1999) Effect of the Methanolic Extract of Glinus lotoides on Dalton’s Ascitic Lymphoma. Biological & Pharmaceutical Bulletin, 22, 1251-1252.
  16. Balcha, A. (2009) Studies on Propagation Biology of Four and Phenology of One Medicinal plant. Ph.D. Dissertation, Addis Ababa University, Addis Ababa, Ethiopia.
  17. Endale, A., Kassa, M. and Gebre-Mariam, T. (1998) In Vivo Anthelminthic Activity of the Extract of the Seeds of Glinus lotoides in Albino Mice Infested with Hymenolepis nana Worms. Ethiopian Pharmaceutical Journal, 16, 34-41.
  18. Biftu, T., Berhanu, A. and Teffera, S. (1979) Fatty Acid Composition of “Mettere” (Glinus lotoides Linne) Seeds by Gas Liquid Chromatography (GLC). SINET: Ethiopian Journal of Science, 2, 19-22.
  19. Endale, A., Schmidt, P.C. and Gebre-Mariam, T. (2004) Standardisation and Physicochemical Characterisation of the Extracts of Seeds of Glinus lotoides. Die Pharmazie, 59, 34-38.
  20. El-Zayat, S.A. (2008) Preliminary Studies on Laccase Production by Chaetomium globosum an Endophytic Fungus in Glinus lotoides. American-Eurasian Journal of Agricultural & Environmental Sciences, 3, 86-90.
  21. Abebe, E. (2005) Isolation, Structural Elucidation, Quantification and Formulation of the Saponins and Flavonoids of the Seeds of Glinus lotoides. PhD Dissertation, Eberhard Karls University, Tubingen, Germany.
  22. Endale, A., Gebre-Mariam, T. and Schmidt, P.C. (2008) Granulation by Roller Compaction and Enteric Coated Tablet Formulation of the Extract of the Seeds of Glinus lotoides Loaded on Aeroperl® 300 Pharma. AAPS PharmSciTech, 9, 31-38.
  23. Demma, J., Gebre-Mariam, T., Asres, K., Ergetie, W. and Engidawork, E. (2007) Toxicological Study on Glinus lotoides: A Traditionally Used Taenicidal Herb in Ethiopia. Journal of Ethno Pharmacology, 111, 451-457.
  24. Ould Ahmedou, M.L., Bouaichi, A. and Idrissi Hassani, L.M. (2001) Evidence of Deterrent and Toxic Effects of Glinus lotoides (Aizoacées) on Nymphs of the Desert Locust Schistocerca gregaria Forskål (Orthoptera, Acrididae). Zoologica Baetica, 12, 109-117.
  25. Hamed, A.I., Piacente, S., Autore, G., Marzocco, S., Pizza, C. and Oleszek, W. (2005) Antiproliferative Hopane and Oleanane Glycosides from Roots of Glinus lotoides. Planta Medica, 71, 554-560.
  26. UNEP (United Nations Environment Programme) (2012) Capacity Building for Access and Benefit Sharing and Conservation and Sustainable Use of Medicinal Plants (Ethiopia ABS CSUMP). Project document.
  27. Murashige, T. and Skoog, F. (1962) A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. Physiologia Plantarum, 15, 473-97.
  28. Priya, G. and Chellaram, C. (2014) In Vitro Derived Callus and Shoot of a Medicinal Herb Solanum trilobatum and Their Effect on Hepatocellular Carcinoma. International Journal of Pharmacy and Pharmaceutical Sciences, 6, 634- 637.
  29. Tanveer, H., Ali, S. and Asi, M.R. (2012) Appraisal of an Important Flavonoid, Quercetin, in Callus Cultures of Citrullus colocynthis. International Journal of Agriculture and Biology, 14, 528-532.
  30. Kaladhar, D.S.V.G.K. (2012) An in Vitro Callus Induction and Isolation, Identification, Virtual Screening and Docking of Drug from Convolvulus alsinoides Linn against Aging Diseases. International Journal of Pharma Medicine and Biological Sciences, 1, 92-103.
  31. Sen, M.K., Nasrin, S., Rahman, S. and Jamal, A.H.M. (2014) In Vitro Callus Induction and Plantlet Regeneration of Achyranthes aspera L., a High Value Medicinal Plant. Asian Pacific Journal of Tropical Biomedicine, 4, 40-46.
  32. Lim, Z.X., Ling, A.P.K. and Hussein, S. (2009) Callus Induction of Ocimum sanctum and Estimation of Its Total Flavonoids Content. Asian Journal of Agricultural Sciences, 1, 55-61.
  33. Arunkumar, B.S. and Jayaraj, M. (2011) Effect of Auxins and Cytokinins on in Vitro Propagation of Ionidium suffruticosum Ging―A Seasonal Multipotent Medicinal Herb. International Journal of Research in Ayurveda and Pharmacy, 2, 198-203.
  34. Wani, M., Pande, S. and More, N. (2010) Callus Induction Studies in Tridax procumbens L. International Journal of Biotechnology Applications, 2, 11-14.
  35. Acemi, A., Ozen, F. and Kiran, R. (2012) Development of an Efficient Callus Production Protocol for Amsonia orientalis: A Critically Endangered Medicinal Plant. EurAsian Journal of BioSciences, 6, 105-112.
  36. Kumar, A., Kaushal, S., Sharma, S. and Sood, H. (2014) In Vitro Callus Induction and Plantlet Regeneration of Swertia chirayita: A Critically Endangered Medicinal Plant. International Journal of Pharmaceutical Sciences Review and Research, 27, 94-96.
  37. Padmavathy, S. (2014) Callus Production Studies in Strobilanthus foliosus and Smilax wightii. International Journal of Innovative Drug Discovery, 4, 41-45.
  38. Guruchandran, V. and Sasikumar, C. (2013) Organogenic Plant Regeneration via Callus Induction in Stevia rebaudiana Bert. International Journal of Current Microbiology and Applied Sciences, 2, 56-61.
  39. Lalabadi, M.A., Jelodar, N.B. and Bagheri, N. (2013) Effect of Plant Hormones on Callus Induction of Explant Types in Endangered Medicinal Herb Tashnedari. International Journal of Biosciences, 3, 39-43.
  40. Sridhar, T.M. and Naidu, C.V. (2011) An Efficient Callus Induction and Plant Regeneration of Solanum nigrum (L.)― An Important Antiulcer Medicinal Plant. Journal of Phytology, 3, 23-28.
  41. Reshi, N.A., Sudarshana, M.S. and Rajashekar, N. (2013) Callus Induction and Plantlet Regeneration in Orthosiphon aristatus (Blume) Miq.―A Potent Medicinal Herb. IOSR Journal of Pharmacy and Biological Sciences, 6, 52-55.
  42. Zare, A.R., Solouki, M., Omidi, M., Irvani, N., Nezad, N.M. and Rezazadeh, Sh. (2010) Callus Induction and Plant Regeneration in Ferula assafoetida L. (Asafetida), an Endangered Medicinal Plant. Trakia Journal of Sciences, 8, 11- 18.
  43. Martin, K.P. (2004) Efficacy of Different Growth Regulators at Different Stages of Somatic Embryogenesis in Eryngium foetidum L.―A Rare Medicinal Plant. In Vitro Cellular & Developmental Biology-Plant, 40, 459-463.
  44. Jain, A.K. and Bashir, M. (2010) In-Vitro Propagation of a Medicinal Plant Portulaca grandiflora. Hook. World Journal of Agricultural Sciences, 6, 327-330.
  45. Aicha, N., Rachida, T.C. and El Abdelmalek, M. (2013) Micropropagation of Thymus satureioides Coss. an Endangered Medicinal Plant of Morocco. Journal of Agricultural Technology, 9, 487-501.
  46. Oluk, E.A. and Çakýr, A. (2009) Micropropagation of Origanum sipyleum L., an Endemic Medicinal Herb of Turkey. African Journal of Biotechnology, 8, 5769-5772.
  47. Echeverrigaray, S., Carrer, R.P. and Andrade, L.B. (2010) Micropropagation of Salvia guaranitica Benth. through Axillary Shoot Proliferation. Brazilian Archives of Biology and Technology, 53, 883-888.
  48. Tejavathi, D.H. and Padma, A.V. (2012) In Vitro Multiplication of Majorana hortensis Moench―An Aromatic Medicinal Herb. Indian Journal of Plant Sciences, 1, 48-56.
  49. Balaraju, K., Agastian, P., Preetamraj, J.P., Arokiyaraj, S. and Ignacimuthu, S. (2008) Micropropagation of Vitex agnus-castus, (Verbenaceae)―A Valuable Medicinal Plant. In Vitro Cellular & Developmental Biology-Plant, 44, 436- 441.
  50. Rasool, R., Kamili, A.N., Ganai, B.A. and Akbar, S. (2009) Effect of BAP and NAA on Shoot Regeneration in Prunella vulgaris. Journal of Natural Sciences and Mathematics, 3, 21-26.
  51. Pandey, P., Mehta, R. and Upadhyay, R. (2013) In-Vitro Propagation of an Endangered Medicinal Plant Psoralea corylifolia Linn. Asian Journal of Pharmaceutical and Clinical Research, 6, 115-118.
  52. Bera, T., Haque, R. and Saha, S. (2009) Micropropagation of an Important Medicinal Plant Chlorophytum borivilianum. International Journal of Physical Sciences, 1, 153-163.
  53. Jitendra, M., Monika, S., Ratan, S.D., Priyanka, G., Priyanka, S. and Kiran, D.J. (2012) Micropropagation of an Anti Diabetic Plant―Stevia rebaudiana Bertoni, (Natural sweetener) in Hadoti Region of South-East Rajasthan, India. ISCA Journal of Biological Sciences, 1, 37-42.
  54. Rathore, V. and Gupta, D. (2013) In Vitro Clonal Propagation of Solanum nigrum. Research Journal of Chemical and Environmental Sciences, 1, 32-33.
  55. Fracaro, F. and Echeverrigaray, S. (2001) Micropropagation of Cunila galioides, a Popular Medicinal Plant of South Brazil. Plant Cell, Tissue and Organ Culture, 64, 1-4.
  56. Sebastinraj, J., Britto, S.J. and Kumar, S.R.S. (2013) Micropropagation of Operculina turpethum (Linn.) Silva Manso. Using Cotyledonary Node Explants. Academic Journal of Plant Sciences, 6, 77-81.
  57. Rout, G.R. (2004) Effect of Cytokinins and Auxins on Micropropagation of Clitoria ternatea L. Biological Letters, 41, 21-26.
  58. Rahman, Md.M., Amin, M.N., Islam, Md.Z. and Sultana, R.S. (2011) Mass Propagation of Solanum surattense Bum. Using Direct Adventitious Shoot Organogenesis from Internode. Acta Agriculturae Slovenica, 97, 11-17.