Manuscript accepted on : 20 April 2016
Published online on: 14-06-2016
Effect of Carbon Sources and Sucrose Concentrations on Shoot Organogenesis of Aloe Saponaria
Jae Kwang Kim1, Thanislas Bastin Baskar2 and Sang Un Park2*
1Division of Life Sciencesand Bio-Resource and Environmental Center, Incheon National University, Incheon 406772, Korea.
2Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-Gu, Daejeon, 305-764, Korea.
Corresponding Author E-mail: supark@cnu.ac.kr
DOI : http://dx.doi.org/10.13005/bbra/2116
ABSTRACT: In the present study, the effect of various carbon sources and sucrose concentrations on in vitro organogenesis of Aloe saponaria was investigated and a rapid micropropagation protocol was developed from in vitro-derived meristem explants. Meristem explants were cultured in initial shoot regeneration media with five different carbon sources (fructose, glucose, lactose, maltose, and sucrose), and sucrose as the best carbon sources for shoot regeneration and shoot elongation was investigated at five different concentrations (10, 20, 30, 40, 50 mg L-1). The treatment with sucrose resulted in the highest number of shoots (2.7 ± 0.2) per explant and produced the longest shoots (16.4 ± 1.3 mm), whereas the treatment with maltose was the least efficient in promoting shoot number (1.5 ± 0.1) and shoot elongation (10.4 ± 0.9 mm). The highest shoot regeneration (3.3 ± 0.3) and the longest shoots (19.1 ± 1.5 mm) were observed in treatments with 40 g L-1 sucrose. Further increase in sucrose concentration delayed shoot induction, resulting in stout shoots stunted in their growth. Our results suggest that carbon sources, particularly sucrose, could be used for micropropagation and in plant transformation protocols for regeneration of Aloe species.
KEYWORDS: Aloe saponaria; Shoot organogenesis; Fructose; Glucose; Lactose; Maltose; Sucrose
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Introduction
The monocotyledonous leaf succulent genus Aloe, a member of the Liliaceae family, presently comprises over 500 species, ranging from small shrubs to large tree-like forms1. Of those, five species, A. vera, A. arborescens, A. perryi, A. ferox, and A. saponaria are used primarily for therapeutic medicinal purposes, with A. vera being the most known for commercial and therapeutic uses2. Aloe saponaria, commonly known as the soap aloe, is one of the most popular species in the Aloe genus. Its leaves are used as a soap substitute as they are able to produce foam in water. This plant has a short stem with green-white striped leaves approximately 50 cm long. This aloe has a less bitter taste compared to other species because of lower concentration of aloin3. Aloe species are well characterized on the African Continent, Arabian Peninsula, Madagascar, and eastern Indian Ocean Islands4. Aloe, native to South Africa, is now spread worldwide. The genus has been used as a multipurpose folk remedy, and the products of A. vera have been used in the medicinal and cosmetic industries. The species has been an essential component in the traditional medicine of several contemporary cultures, such as China, India, the West Indies, and Japan5. Aloe vera plants have high water content, ranging from 99–99.5%6. The remaining 0.5–1.0% is the solid material comprised of over 75 various biologically active compounds including fat-soluble vitamins, minerals, enzymes, simple/complex polysaccharides, phenolic compounds, and organic acids.
In recent years, many researchers have studied this species because of its medicinal value. The gel extracted from aloe has been used as a natural ingredient of many products in cosmetics, topical preparation, and foods. Aloe vera gel has been reported to exhibit wound healing, anti-bacterial, anti-fungal, immune stimulating, and anti-inflammatory activities7-9. Furthermore, it has been used as an agent in treatment of peptic ulcers, diabetes mellitus, and cancer8,10. The ethanol extract of A. saponaria has pharmacological properties, including anti-oxidant, antinociceptive, and anti-inflammatory activity11. Research on medicinal active compounds of Aloe has developed in scope during the last three decades and the production or transformation of value-added compounds, which are medicinally important, still needs to be investigated.
In last decades, the demand for this aloe plant species has increased due to its medicinal properties. The commercial cultivation of A. saponaria is challenging because of the low seed viability, low germination rate, limited availability of raw material with high quality, and slow vegetative growth. Tissue culture technique is an alternate method for preservation of those beneficial medicinal plants12-14. Large scale propagation and conservation of plants by tissue culture is recognized as one of the key areas in biotechnology techniques.
Several studies investigated shoot regeneration and proliferation of different species of the Aloe genus15,16. Most of the studies used shoot tips and axillary buds as explants for regeneration of A. vera17. Moreover, the presence of the plant growth promoter is essential for regeneration17-19 reported on axillary shoot formation using indole-3-butyric acid (IBA), whereas Roy and Sarkar20 and Natali et al.21 regenerated shoots on a medium containing 2,4-D and kinetin. Richwine et al.22 examined the initiation of shoots by zeatin, and Debiasi et al.,23 and Liao et al.19 investigated the effects of benzyladenine, indole-3-acetic acid, and naphthaleneacetic acid on bud initiation. In plants, carbohydrates are a crucial source of carbon in biosynthesis processes. In vitro cell, tissue, and organ cultures of plants are not fully autotrophic, requiring a source of carbohydrates to maintain the osmotic potential of the culture media and to provide energy and carbon for developmental processes with high energy requirement, such as organogenesis, root induction, embryogenesis, emission, and shoot multiplication24. Hence, sugars have a promising effect on the growth, differentiation of cells, and physiology25. The objective of the present investigation is to determine the influence of carbon source such as glucose, fructose, lactose, maltose, and sucrose on shoot organogenesis using meristem explants and to evaluate different concentrations of sucrose on rapid in vitro propagation of A. saponaria.
Materials and Methods
Plant material and culture medium
Aloe saponaria seeds were purchased from Richters Herbs (Goodwood, ON, Canada) and stored at 4°C. The seeds were surface-sterilized with 70% (v/v) ethanol for 30 s, rinsed with 2% (v/v) sodium hypochlorite solution for 10 min, and washed with sterilized distilled water three times in a laminar air flow hood. Ten seeds were inoculated on 25 mL of agar-solidified culture medium in Petri dishes (100 × 15 mm). The basal Murashige and Skoog (MS) medium consisted of salts and vitamins (Murashige and Skoog, 1962)26, and it was solidified with 0.7% (w/v) agar. The pH of the medium was adjusted to 5.7 to 5.8 using 1 N hydro chloric acid (HCL) and 1 N potassium hydroxide (KOH) before adding agar, and then sterilized by autoclaving at 121°C for 20 min. The seeds were germinated in a growth chamber at 25°C, illumination of 35 µmol s-1 m-2 provided by standard cool-white fluorescent tubes, and a 16-h photoperiod.
In vitro shoot organogenesis
Meristem explants of A. saponaria were taken from plants grown in vitro and cut aseptically at the ends into sections approximately 0.7 cm long. Explants were placed on the medium in Petri dishes (100 × 25 mm). Each Petri dish contained approximately 25 mL of basal medium supplemented with 30 g L-1 sucrose, 7 g L-1 Phytagar, and 2 mg L-1 6-benzylaminopurine (BAP). The pH of the medium was adjusted and the medium was sterilized using the same procedures described for the germination medium, and seven explants were inoculated in each plate. For enhancement of shoot regeneration, the medium was optimized by testing the effect of 30 g L-1 of each carbon source (fructose, glucose, lactose, maltose, and sucrose) and different concentration (10, 20, 30, 40, and 50 g L-1) of sucrose. Inoculated plates were incubated at 25 ± 1°C in a growth chamber with a 16-h photoperiod and illumination provided by standard cool-white fluorescent tubes (35 µmol s-1 m-2) for 6 weeks.
Rooting of regenerated shoots
Regenerated A. saponaria shoots (~1.5 cm in length) were transferred to 1/2 MS medium in a Magenta box (Magenta LLC, Chicago, IL, USA). After 3 to 4 weeks, the regenerated shoots were transferred into the rooting medium, consisting of MS medium with 8 g L-1 of Phytagar and 1 mg L-1 of IBA. Four shoots were cultured in each culture vessel. Regenerated shoots were incubated at 25 ± 1°C in a growth chamber with a 16-h photoperiod and illumination of 35 μmol·s-1·m-2 provided by standard cool-white fluorescent tubes for 5 weeks. After 5 weeks, the rooted plants were washed with water to remove agar, transferred to pots containing autoclaved vermiculite, and covered with polyethylene bags for 1 week to maintain high humidity. The plants were then transferred to soil and maintained in a growth chamber with a 16-h photoperiod and a day/night temperature of 18/20°C for 2 weeks. These hardened plants were then transferred to the greenhouse.
Statistical analysis
Data were expressed as means ± standard deviation of 50 leaf explants tested.
Results and Discussion
A protocol was enhanced for in vitro shoot organogenesis of A. saponaria27, but shoot development efficiency using this protocol was not sufficient. We used various carbon sources, i.e., fructose, glucose, lactose, maltose, and sucrose to study the effectiveness of shoot organogenesis in A. saponaria. Different types of carbohydrates have been found to play substantial roles in micropropagation of A. saponaria. Among the five different carbon sources used in this study, addition of sucrose resulted in the highest number of shoots and longest shoots. The highest shoot number (2.7 ± 0.2 per explant) and the longest shoots (16.4 ± 1.3 mm) were observed in explants cultured on carbon media (MS media with 2 mg L-1 BAP) supplemented with 30 g L-1 sucrose, followed by glucose (Table 1). The treatment with maltose showed the lowest efficiency in shoot regeneration (1.5 ± 0.1) and shoot elongation (10.4 ± 0.9 mm) in A. saponaria. Sucrose produced 1.6-fold greater number of shoots per explant and 1.3-fold longer shoots than the lowest producing carbon sources maltose.
Table 1: Effect of carbon sources on shoot regeneration and growth of Aloe saponaria explants after 6 weeks of culture on regeneration medium (Murashige and Skoog medium with 2.0 mg L-1 6-benzylaminopurine).
Carbon sources (30 g L-1) | No. of shoots per explant* | Shoot length* (mm) |
Fructose | 2.0 ± 0.2 | 12.5 ± 1.2 |
Glucose | 2.5 ± 0.3 | 14.8 ± 1.4 |
Lactose | 1.7 ± 0.2 | 11.2 ± 1.1 |
Maltose | 1.5 ± 0.1 | 10.4 ± 0.9 |
Sucrose | 2.7 ± 0.2 | 16.4 ± 1.3 |
* Values represent the mean ± standard deviation of 50 shoots
To determine the optimum concentration for increased regeneration in A. saponaria, the generation medium (MS medium with BAP at 2 mg L-1) was supplemented with different concentrations of sucrose (10, 20, 30, 40, and 50 g L-1). Increasing the concentration of sucrose increased the shoot number and shoot length; the highest shoot number (3.3 ± 0.4) and shoot length (19.1 ± 1.5 mm) were observed in treatments with 40 g L-1 sucrose. Thereafter, further increase of sucrose concentration reduced the shoot number and shoot length (Table 2).
Table 2: Effect of sucrose concentration on shoot regeneration and growth of Aloe saponaria explants after 6 weeks of culture on regeneration medium (Murashige and Skoog medium with 2.0 mg L-1 6-benzylaminopurine).
Sucrose (g L-1) | No. of shoots per explant* | Shoot length* (mm) |
10 | 1.8 ± 0.2 | 12.9 ± 0.9 |
20 | 2.2 ± 0.2 | 14.5 ± 1.1 |
30 | 2.7 ± 0.2 | 16.4 ± 1.3 |
40 | 3.3 ± 0.3 | 19.1 ± 1.5 |
50 | 3.2 ± 0.4 | 18.7 ± 1.2 |
* Values represent the mean ± standard deviation of 50 shoots
The results suggested that the inclusion of carbon sources (carbohydrates) is important for enhanced shoot development; shoot regeneration of A. saponaria is affected by both the type and concentration of sugar in the culture medium (Tables 1 and 2). The highest shoot regeneration and shoot elongation were obtained in media supplemented with sucrose at 40 g L-1. Similar results were reported for Echinacea angustifolia28. A few studies reported that sucrose at 3% level was the optimal concentration to achieve the highest level of shoot regeneration and shoot elongation29,30. Sucrose enhanced shoot regeneration of A. saponaria compared to fructose and glucose31 and stimulated shoot organogenesis of Prunus domestica and increased shoot number compared to glucose32. Previous report suggested that glucose, fructose, and maltose elicit very low levels of shoot regeneration and shoot elongation33, and similarly, the effect of different monosaccharides used as carbon source on regeneration was relatively low compared to sucrose34. However, in vitro beech cultures showed high adventitious shoot regeneration and axillary branching in treatments with glucose35. In other plants, including Alnus glutinosa 36, A. cremastogyne37, Corylus avellana38, Prunus mume39, Juglans regia40, and Rosa41, the best shoot regeneration was obtained in treatments with glucose and fructose compared to sucrose. Many studies reported that 3% sucrose enhanced the most the micropropagation of plants, whereas in the present study, we showed that the highest level of shoot regeneration and elongation of meristem explants of A. saponaria were achieved in treatments with 4% sucrose.
Conclusion
Previously established protocols for shoot organogenesis and plant regeneration from meristem explants were not successful in the regeneration of A. saponaria. In the present study the effect of carbon source and sucrose concentrations on in vitro regeneration of A. saponaria was studied. It was observed that sucrose was the best carbon source and that 4% sucrose was the optimum concentration for healthy shoot regeneration and elongation. This protocol can be helpful for large scale proliferation of Aloe plant species and thus contribute to effective conservation of the species. The results presented herein will facilitate research on genetic enhancement of A. saponaria.
Acknowledgements
This study was supported by research fund of Chungnam National University in 2015.
References
- Grace, O.M. Current perspectives on the economic botany of the genus Aloe L. (Xanthorrhoeaceae). S Afr J Bot., 2011; 77:980– 98.
- Park, Y.I., Lee, S.K. New perspectives on Aloe.In Kim,Y.S. (ed.) Carbohydrates. Springer, New York, USA., 2006.
- Li, J., Wang, T., Shen, Z., Hu, Z. Relationship between leaf structure and aloin content in six species of Aloe L. Acta Botanica Sinica., 2003; 45(5):594–600.
- Cousins, S.R., Witkowski, E.T.F. African Aloe ecology: a review. Arid. Environ, 2012; 85:1–17.
- Foster, M., Hunter, D., Samman, S. Evaluation of the nutritional and metaboliceffects of Aloe vera. In: Benzie IFF, Wachtel-Galor S, eds. Herbal Medicine: Biomolecular and Clinical Aspects. 2nd ed. Boca Raton: CRC; 2011.
- Hamman, J.H. Composition and applications of Aloe vera leaf gel. 2008; 13:1599-1616.
- Reynolds, T., Dweck, A.C. Aloe vera leaf gel: a review update. Ethnopharmacol., 1999; 68: 3-37.
- Harlev, E., Nevo, E., Lansky, E.P., Ofir, R., Bishayee, A. Anticancer Potential of Aloes: Antioxidant, Antiproliferative, and Immunostimulating Attributes. Planta Med., 2012; 78: 843-852.
- Ibrari, M., Muhammad, N., Shah, W., Barakatullah. Evaluation of trace and toxic heavy metals in selected crude drugs used in Khyber Pukhtonkhawa, Pakistan. J. Bot., 2013; 45(1): 141-144.
- Langmead, L., Feakins, R.M., Goldthorpe, S., Holt, H., Tsironi, E., Desilva, A., Jewell, D.P., Rampton, D.S. Randomized, double-blind, placebo-controlled trial of oral Aloe vera gel for active ulcerative colitis. Pharmacol. Theo., 2004; 19: 739-747.
- Yoo, E.A., Kim, S.D., Lee, W.M., Park, H.J., Kim, S.K., Cho, J.Y., Min, W., Rhee, M.H. Evaluation of antioxidant, antinociceptive, and anti-inflammatory activities of ethanol extracts from Aloe saponaria Phytother Res 2008; 22:1389-95.
- Rout, G.R., Mahato, A., Senapati, S.K. In vitro clonal propagation of Nyctanthes arbortristis. Biol Plant, 2008; 52:521–524.
- Bantawa, P., Saha-Roy, O., Kumar, S., Ghosh, R., Mondal, K.T. In vitro regeneration of an endangered medicinal plant Picrorhiz scrophulariiflora. Biol Plant, 2011; 55:169–172.
- Swarna, J., Ravindhran, R. In vitro propagation and assessment of genetic integrity of Talinum triangulare (Jacq.) Wild: a valuable medicinal herb. Acta Physiol Plant, 2012; 10:1007–1017.
- Hosseini, R., Parsa, M. Micropropagation of Aloe vera grown in South Iran. Pak. J. Biol. Sci., 2007; 10(7): 1134-1137.
- Albanyl, N.J., Vilchez, S., Lion, M.M., Chacin, P. A methodology for the propagation in edge Aloe vera L. Rev. Fac. Agron., 2006; 23: 213-222.
- Meyer, H.J., Staden, J.V. Rapid in vitro propagation of Aloe barbadensis Plant cell, Tiss. Org. Cult., 1991; 26: 167-171.
- Aggarwal, D., Barna, K.S. Tissue culture propagation of elite plant of Aloe vera Linn. J. Biochem. Biotech., 2004; 13: 77-79.
- Liao, Z., Chen, M., Tan, F., Sun, X., Tang, K. Micropropagation of endangered chinese Aloe. Plant cell Tiss. Org. Cult., 2004; 76: 83-86.
- Roy, S.C., Sarkar, A. In vitro regeneration and micropropagation of Aloe vera. Scientia Hort., 1991; 47: 107-114.
- Natali, I., Sanchez, I.C., Cavallini, A. In vitro culture of Aloe barbadensis Micropropagation from vegetative meristem. Plant Cell, Tiss. Org. Cult., 1990; 20: 41-47.
- Richwine, A.M., Tipton, J.L., Thompson, A. Establishment of Aloe gasteria, and Haworthia shoot cultures from inflorescence explants. Hort Sci. 1995; 30: 1443-1444.
- Debiasi, C., Silvia, C., Pescadore, R. Micropropagation of Aloe vera L. Bras. Pl. Med., Botucatu., 2007; 9: 36-43.
- Yaseen, M., Ahmad, T., Sablok, G., Standardi, A., Hafiz, I. Role of carbon sources for in vitro. Mol Biol Rep., 2013; 40(4): 2837- 2849.
- Gibson, S.I. Plant sugar-response pathways. Part of a complex regulatory web. Plant Physiol., 2000; 124:1532–1539.
- Murashige, T., Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 1962; 15:473-497.
- Chae, S.C., Kim, H.H., Park, S.U. Shoot organogenesis and plant regeneration of Aloe saponaria. Life Sci.., 2013; 10(3): 575- 578.
- Chae, S.C. Influence of carbon sources on shoot organogenesis in Echinacea angustifolia DC. Life Sci., 2013; 10:1300-1303.
- Reddy , M.C., Bramhachari, P.V., Sri Rama Murthy, K. Optimized plant tissue culture protocol for in vitro morphogenesis of an endangered medicinal herb Ceropegia. Tropical and Subtropical Agroecosystems, 2015; 18: 95 – 101.
- Gauchan, D.P. Effect of different sugars on shoot regeneration of maize (Zea mays L.). Kathmandu University Journal of Science, Engineering and Technology, 2012; 8: 119-124.
- Baskaran, P., Jayabalan, N. Role of basal media, carbon sources and growth regulators in micropropagation of Eclipta alba – a valuable medicinal herb. Kmitl Sci J., 2005; 5(2):469- 482.
- Amutha, S., Ganapathi, A., Muruganantham, M. In vitro organogenesis and plant formation in Vigna radiata (L.) Wilczek. Plant Cell, Tiss. Org. Cult., 2003; 72:203-207.
- Trejgell, A., Jarkiewicz, M., Tretyn, A. The effect of carbon sources on callus induction and regeneration ability in Pharbitis nil . Acta Physiol Plant., 2006; 28(6): 619-626.
- Cuenca, B., Vieitez, A.M. Influence of carbon source on shoot multiplication and adventitious bud regeneration in in vitro beech cultures. Plant Growth Regul., 2000; 32: 1-12.
- Nowak, B., Miczy´nski, K., Hudy, L. Sugar uptake and utilization during adventitious bud differentiation on in vitro leaf explants of ‘W¸egierka Zwykła’ plum (Prunus domestica). Plant Cell, Tiss. Org. Cult., 2004; 76: 255- 260.
- Welander, M., Welander, N.T., Brackman, A.S. Regulation of in vitro shoot multiplication in Syringa, Alnus and Malus by different carbon sources. J Hort Sci., 1989; 64: 361–366.
- Yu, X., Reed, B.M. Improved shoot multiplication of mature hazelnut (Corylus avellana L.) in vitro using glucose as a carbon source. Plant Cell Rep., 1993; 12: 256–259.
- Tang, D., Ishii, K., Ohba, K. In vitro regeneration of Alnus cremastogyne Burk from epicotyl explants. Plant Cell Rep., 1996; 15: 658–661.
- Harada, H., Murai, Y. Micropropagation of Prunus mume. Plant Cell, Tiss. Org. Cult., 1996; 46: 265–267.
- Gruselle, R., Nicaise, C., Boxus, P. Regulation of in vitro shoot multiplication in Persian walnut by different carbon sources and by ammonium phosphate. Bull Rech Agron Gembloux., 1995; 30: 47–53.
- Hsia, C.N., Korban, S.S. Factors affecting in vitro establishment and shoot proliferation of Rosa hybrida L. and Rosa chinensis minima. In Vitro Cell Dev Biol-Plant., 1996; 32: 217– 222
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