Volume 13, number 1
 Views: (Visited 312 times, 1 visits today)    PDF Downloads: 1524

Lee S. Y, Baskar T. B, Kim J. K, Park S. U. Enhanced shoot organogenesis in Aloe saponaria following treatment with ethylene inhibitors and polyamines. Biosci Biotech Res Asia 2016;13(1)
Manuscript received on : 20 December 2015
Manuscript accepted on : 15 January 2016
Published online on:  29-02-2016
How to Cite    |   Publication History    |   PlumX Article Matrix

Enhanced shoot organogenesis in Aloe saponaria following treatment with ethylene inhibitors and polyamines

Sook Young Lee1, Thanislas Bastin Baskar2, Jae Kwang Kim3 and Sang Un Park2*

1Regional Innovation Center for Dental Science and Engineering, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju, 501-759, Korea 2 Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-Gu, Daejeon, 305-764, Korea 3Division of Life Sciencesand Bio-Resource and Environmental Center, Incheon National University, Incheon 406-772, Korea

DOI : http://dx.doi.org/10.13005/bbra/1997

ABSTRACT: Plants belonging to the genus Aloe are wide spread in Africa and are important in the pharmaceutical industry. Here, we investigated the role of ethylene inhibitors and polyamines in the enhancement of shoot regeneration from meristem explants of Aloe saponaria. A rapid, highly effective shoot regeneration procedure was designed using meristem explants. First, we found that Murashige and Skoog (MS)with 2mg/L6-benzyl amino purine (BAP) was the best for shoot initiation. Shoot regeneration was assessed using different concentrations of AgNO3,aminoethoxyvinylglycine (AVG), and CoCl2 (1, 5, 10, and 20mg/L).While AgNOinduced shoots (3.5/explant) and increased shoot length by 2.5cm at concentrations of 10mg/L, an additional increase in the concentration of AgNO3 decreased shoot production and shoot length. We also analyzed the effects of three different polyamines(putrescine,spermidine, and spermine) at various concentrations (10, 30, 70, and 100mg/L),in addition toMS with 2mg/L BAP, on shoot regeneration and length. Among these compounds, putrescine best augmented shoot regeneration and length, with the largest increase in the number of shoots (3.7/explant) and a moderate augmentation in shoot length 2.5cm at 70mg/LusingMS with 2mg/L BAP, followed by spermidine and spermine. This study revealed that the addition of ethylene inhibitors and polyamines couldimprove shoot regeneration and length in Aloe species. The procedures established in the present study could be used to produce a larger number of shoots, as well as enhance plant growth over a short period, and could be used as an important tool in future gene transfer studies.

KEYWORDS: Aloe saponaria; Meristem explants; Micropropagation;  Ethylene inhibitors; Polyamines

Download this article as: 
Copy the following to cite this article:

Lee S. Y, Baskar T. B, Kim J. K, Park S. U. Enhanced shoot organogenesis in Aloe saponaria following treatment with ethylene inhibitors and polyamines. Biosci Biotech Res Asia 2016;13(1)

Copy the following to cite this URL:

Lee S. Y, Yan Y. Z, Arasu M. V, Al-Dhabi N. A, Park S. U. Seasonal variation of saponin contents in Platycodon grandiflorum). Biosci Biotech Res Asia 2016;13(1). Available from: https://www.biotech-asia.org/?p=6825

Introduction

The genus Aloe, which belongs to the Xanthorrhoeaceae family,includes over 500 known species, of which,Aloe saponaria,is one of the most important species in Africa. This speciesis commonly known as soap aloe or zebra aloe and is a salt tolerant plant widely occurring in South Africa, Zimbabwe, and America. Recently,Aloe species have been extensively studied and are also being used in the pharmaceutical industry for their medicinal valueand mainly in the manufacture of soaps for foam production. Additionally, A.saponariahas a less bitter taste compared to other Aloe species and has recently become the most favored plant in California for its ornamental value. However, A.saponariais difficult to identify as it hybridizes easily with other Aloe species; its leaves are red orgreen and are characterized by anH shaped spot in the leaves. Furthermore, A. saponariapopulations are sensitive to changes in temperature and water.

Currently,the study of human diseases has been increasing worldwide, and new medicines for controlling disease have been developed. Aloe species have medicinal properties; however, owing to their low population, the global demand for these species is high. The gel extracted from A.saponaria leaves contains polysaccharides, proteins, fats, aloin, and resins2. Polysaccharides of A.saponariahave immunomodulatory activities, as has been reported in both in-vivo and in-vitro experiments3. Furthermore, previous studies on A.saponaria demonstrate that this plant can have haemagglutinating properties and can prevent the multiplication of tumor cells while also improving the normal human cell growth cell. In particular, the compound mannan from A.saponariahas been shown to inhibit tumor cell activation and proliferation4. Aloe species have both wound healing and anti-inflammatory properties5-7, as well asantinociceptive and anti-inflammatory properties8.

In recent years, the demand for Aloe species has increased due to its medicinal importance. It is very difficult to induce plant proliferation due to its low growth rate in-vivo conditions. Thus, tissue culture plays an important role in plant regeneration and micro propagation. The in-vitro plant micro propagation and regeneration of aloe species has been previously reported9-11, with the invitro regeneration of A.saponaria using meristem explants previously explored12.Ethylene is a volatile compound produced in excess by plants and affects plant growth. Ethylene inhibitors have been used invitro in order to improve cell growth and regeneration rates. In particular, ethylene inhibitors increase shoot regeneration and cell growth in Aloe arborescens13, as well as in the hypocotyl explants of the Chinese radish14. Both aminoethoxyvinylglycine (AVG) and AgNo3 enhanced shoot regeneration in mustard15. In Chinese cabbage, polyamines improved the regeneration frequency16 and the use of these compounds to induce embryo genesis was investigated17.

The currently available protocol for the in-vitro regeneration of A.saponaria using meristem explants is inefficient and leads to a very low frequency of shoot regeneration, with no improvement in cell growth. In this study, we report the development of an improved method for enhanced shoot regeneration and shoot length from meristem explants of A.saponaria using different concentrations of ethylene inhibitors and polyamines.

Materials and Methods

Shoot organogenesis from meristem explants

One-year-oldA.saponaria meristem explants were collected from the green house of Chungnam National University, washed in running tap water for 30 min,surface-sterilized with 70% (v/v) ethanol for 30 s and 1% (v/v) sodium hypochlorite solution for 10 min, then rinsed three times in sterilized water to remove the sodium hypochlorite solution. The sterilized meristem explants were cut aseptically at the ends, into four sections approximately 0.7 cm in size. Meristem explants were placed on a Petri dish (100 × 25 mm) with approximately 25 mL of culture medium. Seven explants were cultured in each Petri dish. The basal medium consisted of MS18medium and was solidified with 0.7% (w/v) Phytagar. The pH was adjusted to 5.8 before adding Phytagar. The media were sterilized by autoclaving at 1.1 kg cm-2 (121 °C) for 20 min. For shoot regeneration from meristem explants, the MS medium consisted of 0, 0.1, 0.5, 1, 2, or 4 mg/L kinet in and BAP. In order to improve shoot regeneration, the medium was optimized by testing the effect of various concentrations of anti-ethylene agents (0, 1, 5, 10 or 20 mg/L of AgNO3, AVG, and CoCl2) on shoot formation and growth. Enhancement of shoot organogenesis was tested at different concentrations of polyamines (10, 30, 70, and 100 mg/L of putrescine,spermidine, and spermine). Inoculated cultures were maintained at 25 °C in a growth chamber with a 16-h photoperiod under standard cool white fluorescent tubes (35 mmol s-1 m-2) for 6 weeks. The shoots were transferred to a new MS medium every 15 days.

Rooting of regenerated shoots

Regenerated shoots (approximately 1cm long) were placed in MS medium, which was solidified with 3g/LPhytagar and dispensed at 30 mL per Magenta box, with four shoots cultured in each box. Regenerated shoots were incubated at 25°C in a growth chamber with a 16h photoperiod under standard cool white fluorescent tubes (35 mmol s-1 m-2) for 5 weeks.After six weeks, the rooted plants were washed with sterile water to remove Phytagar, transferred to pots containing autoclaved vermiculite, and covered with polyethylene bags for one week to maintain conditions with 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 20/18 °Cfor 2 weeks. These plants were then transferred to the greenhouse.

Results And Discussion

Effect of different ethylene inhibitors on shoot regeneration and shoot length

Overall, the addition of ethylene inhibitors improved shoot regeneration as well as shoot length. In this study, the effect of three different ethylene inhibitors and different concentrations was assessed. A.saponaria cultured on MS medium with 2mg/L BAP produced significant changes in shoot regeneration. After 15 days, the inoculated culture began to regenerate from the meristem explants. A significant effect was observed with the addition of 10 mg/L of AgNO3 and AVG; however, further increase in the concentration of AgNO3 and AVG decreased shoot regeneration rates and shoot length. The maximum shoot regeneration and shoot length was obtained at 1/2 MS with 2mg/Lof BAP and 10mg/L of AgNO3,with slightly inferior results observed with the addition of AVG, and no improvement under treatments with different concentrations of CoCl2 showed in Table 1.

Effect of different polyamines on shoot regeneration and shoot length

To evaluate the effect of polyamines at different concentrations on shoot regeneration of A.saponaria, meristem explants were grown for 4 weeks. The addition of polyamines increased shoot number and shoot length, with a significant improvement using putrescineat 70mg/L in shoot regeneration and shoot length and no further improvement observed with a further increase in concentration. The highest shoot number and shoot length was induced byputrescineat 70mg/L, followed by spermidine and spermine at the same concentration (Table 2).

Plant tissue culture is the main tool for micropropagation and regeneration of various plants. In order to establish a plant regeneration system, we investigated the effects of different ethylene inhibitors and various concentrations of anti-ethylene agents (AgNO3,AVG, and CoCl2) as well as polyamines(putrescine, spermidine, and spermine) on the efficiency of shoot organogenesis in A.saponaria.Previously, a protocol was established for shoot organogenesis and plant regeneration from meristem explantsofA.saponaria12, in which the regeneration of aloe species using MS medium with auxins and cytokininsat different concentration19 was assessed. More recent studies reported that ethylene induces regeneration and shoot elongation in selected plants20. Ethylene inhibitors have been shown to improve regeneration and shoot length21 in A.arborescens and AgNOenhances micropropagation and regeneration frequency22. Since in many cases plant regeneration is affected by the increased production of ethylene23, we investigated the use of ethylene inhibitors in the regeneration medium and found that the accumulation of ethylene reduced the regeneration frequency. We established a regeneration method for A.saponaria using different ethylene inhibitors at various concentrations. We found that AgNOat 10mg/L induced the highest number of shoots (3.5 per explant [Table 1]). AVG and CoCl2 produced fewer shoots. In addition, we studied the ability of polyamines to regenerate A.saponaria. It has been previously reported that polyamines improve shoot regeneration in Cucumissativus and With ania somnifera24,25. Furthermore26 investigated the addition of spermidine, which significantly improved shoot proliferation in apricot leaves, and 27demonstrated that putrecine induced root development in Hedera helix.In this study, we used three different polyamines (putrescine, spermidine, and spermine), and found that putrecine produces the greatest number of shoots. Both spermidine and spermine were less efficient compared to putrecine.We also devised a highly efficient system involving the use of meristem explants, ethylene inhibitors, and polyamines for the in-vitro micropropagation of A. saponaria. To our knowledge, this is the first study that uses polyamines for the regeneration of Aloe species.

Table 1. Effect of different ethylene inhibitors on shoot formation and growth ofAloe saponaria on MS media supplemented with BAP 2 mg/l

Ethylene inhibitors(mg/L) No. of shoots/explant Shoot length(cm)
Control0 2.3 ± 0.2 1.5 ± 0.1
AgNO3 1 2.5 ± 0.3 1.6 ± 0.1
5 3.1 ± 0.3 1.9 ± 0.2
10 3.5 ± 0.3 2.3 ± 0.2
20 2.1 ± 0.2 1.3 ± 0.1
AVG 1 2.4 ± 0.2 1.5 ± 0.2
5 2.9 ± 0.3 1.7 ± 0.2
10 3.3 ± 0.4 2.1 ± 0.2
20 2.2 ± 0.3 1.4 ± 0.1
CoCl2 1 2.3 ± 0.2 1.4 ± 0.1
5 2.6 ± 0.3 1.6 ± 0.2
10 2.0 ± 0.2 1.4 ± 0.1
20 1.7 ± 0.2 1.1 ± 0.1

The values are the means ± SD

Table 2. Effect of different poly amines on shoot formation and growth of Aloe saponaria on MS media supplemented with BAP 2 mg/L

Ethylene inhibitors(mg/L) No. of shoots/explant Shoot length(cm)
Control            0 2.3 ± 0.2 1.5 ± 0.1
Putrescine 10 2.6 ± 0.2 1.7 ± 0.2
30 3.2 ± 0.3 2.0 ± 0.2
70 3.7 ± 0.4 2.5 ± 0.3
100 3.5 ± 0.3 2.1 ± 0.2
Spermidine 10 2.3 ± 0.2 1.4 ± 0.1
30 2.5 ± 0.3 1.6 ± 0.2
70 2.6 ± 0.3 1.8 ± 0.2
100 2.1 ± 0.2 1.5 ± 0.2
Spermine 10 2.2 ± 0.2 1.5 ± 0.2
30 2.3 ± 0.2 1.4 ± 0.2
70 2.5 ± 0.3 1.5 ± 0.2
100 1.9 ± 0.2 1.3 ± 0.1

The values are the means ± SD

Conclusion

In conclusion, plant regeneration protocols are essential for inducing genetic transformation and accelerating plant growth. Currently, shoot organogenesis is extensively used for in-vitro plant regeneration and transformation. The protocol established in the present study is an effective method for regenerating A.saponaria as well as producing additional plants within a short period of time. Remarkably, the ethylene inhibitors AgNOand AVG promoted the frequency of shoot organogenesis in this species. In addition, polyamines enhanced the shoot number and length. Together, our findings potentially form the basis for the improvement of genetic tools for Aloe species.

Acknowledgements

This study was supported by research fund of Chungnam National University in 2015.

References

  1. Chaudhary, A.K., Ray, A.K., Jha, S., Mishra, I.N. Callus formation, shoot initiation and in       vitro culture of Aloe vera. BiotechnolBioinformaBioeng.,2011;1(4):551–553
  2. Robson, M.C., Heggers, J.P., Hagstrom, W.J. Myth, magic,witchcraft or fact? Aloevera are visited. J Biol Chem., 1982;3:157–63.
  3. Reynolds, T., Dweck, A.A. Aloe vera gel leaf: a review update.J Ethnopharmacol 1999;68:3–37.
  4. Sampedro, M.C.,Artol, R.L.,Murature, M., Murature, D.,Ditamo, Y., Roth, G.A.,Kivatinitz, S. Mannan from Aloe saponaria inhibits tumoral cell activation and proliferation, International Immunopharmacology.,2004;4: 411– 418
  5. Capasso, F., Borrelli, F., Capasso, R., Di Carlo, G., Izzo, A.A., Pinto, L., Mascolo, N., Castaldo, S., Longo, R. Aloe and its therapeutic use. Phytotherapy Research.,1998; 12: 124–127.
  6. Barros, F.M.C.,Pereira,K.N.,Zanetti,G.D.,Heinzmann, B.M. PlantasdeUso Medicinal no Municı´piodeS~aoLuizGonzaga,RS,Brasil.Latin American Journal of Pharmacy.,2007; 26: 652– 662.
  7. Loots, D.T.,VanDer Westhuizen,F.,Botes, L. Aloe ferox leaf gel phytochemical content,antioxidant capacity,and possible health benefits.Journal of Agricultural and Food Chemistry.,2007; 55: 6891–6896.
  8. Silva, M.A., Trevisan, G., Klafke, J.Z., Rossato, M.F., Walker, C.B., Oliveira, S.M., Silva,C.R., Boligon,A.A., Flores, F.C., Silva, C.B., Athayde, M.L., Ferreira, J. Antinociceptiveandanti-inflammatoryeffectsof Aloe saponaria Haw on the rmalinjuryinrats .Journal of Ethanopharmacology.,2013; 146: 393-401.
  9. Velcheva, M., Faltin, Z., Vardi, A., Eshdat, Y., Perl, A. Regeneration of Aloe arborescens via so matic organogenesis from young inflorescences. Plant Cell Tissue Organ Cult.,2005;83:293–301.
  10. Bairu, M.W., Stirk, W.A., Dolezˇal, K., Van Staden, J. Optimizing the micropropagation protocol for the endangered Aloe polyphylla:can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin?.Plant Cell Tissue Organ Cult.,2007; 90:15–23.
  11. Bedini, C., Caccia, R., Triggiani, D., Mazzucato, A., Soressi, G.P., Tiezzi, A.Micropropagation of Aloearborescens Mill: a step towards efficient production of its valuable leaf extracts showing anti proliferative activity on murine myeloma cells. Plant Biosyst.,2009; 143:233–240.
  12. Chae, S.C., Kim, H.H., Park, S.U. Shoot organogenesis and plant regeneration of Aloe saponaria. Life science journal.,2013;10(3): 575-578.
  13. Suh ,S.Y., Baskar, T.B., Kim, H.H., Al-Dhabi, N.A., Park, S.U.Ethylene inhibitors promote shoot organogenesis of Aloe arborescens Miller. South Indian Journal of Biological Sciences., 2015; 1(1): 43-46.
  14. Pua, E.C.,Sim, G.E., Chi, G.L., Kong, L.F.Synergistic effect of ethylene inhibitors and putrescineon shoot regeneration from hypocotyl explants of Chinese radish(RaphanussativusL. var. longipinnatusBailey) in vitro. Plant Cell Reports.,1996;15: 685-690.
  15. Chi, G.L, Barfield, D.G., Sire, G.E., Pua, E.C.Effect of AgNO3 and Aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypesPlant Cell Rep., 1990;9:195-198.
  16. Chi, G.L., Lin, W.S., Lee, J.E.E., Pua, E.C. Role of polyamines on de novo shoot morphogenesis from cotyledons of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro.Plant Cell Rep., 1994;13:323-329.
  17. Venkatachalam, L., Bhagyalakshmi, N.Spermine-induced morphogenesisand effect of partial immersion system on the shootcultures of banana. ApplBiochemBiotechnol., 2008;151(2):502–511.
  18. Murashige, T.,Skoog, F. A revised medium for rapid growth and bioassays with Physiol Plant.,1962;15:473-497.
  19. Hashemabadi, D., Kaviani, B. Rapid micropropagation of Aloe veraL. via shoot multiplication. Afr J Biotechnol.,2008;7(12):1899–1902.
  20. Trujillo-Moya, C., Gisbert, C. The influence of ethylene andethylene modulators on shoot organogenesis in tomato. PlantCell Tissue Organ Cult., 2012;111:41–48.
  21. Kumar, P.P., Rao, C.D.,Goh, C.J. Influence of petiole andlamina on adventitious shoot initiation from lamina explants ofPaulownia fortunei. Plant Cell Reports.,1998;17: 886–890.
  22. Pius, J., George, L.,Eapen, S.,Rao, P.S.Enhanced plant regeneration in pearl millet (Pennisetumamericanum) byethylene inhibitors and cefotaxime. Plant Cell, Tissue and Organ Culture.,1993;32: 91-96.
  23. Kevers, C., Gal, N.L.,Monteiro, M., Dommes, J., Gaspar, T., Gal, N. Somatic embryogenesis of Panax ginseng in liquidcultures: a role for polyamines and their metabolic pathways.Plant Growth Regul.,2000; 31: 209–214.
  24. Vasudevan, A., Selvaraj, N., Ganapathi, A., Kasthurirengan, S., RameshAnbazhagan, V., Manickavasagam, M., Choi, C.Leucine andspermidine enhance shoot differentiation in cucumber (Cucumissativus L.). In Vitro Cell DevBiol Plant.,2008;44(4): 300–306.
  25. Sivanandhan, G., Mariashibu, T.S., Arun, M., Rajesh, M., Kasthurirengan, S., Selvaraj, N., Ganapathi, A. The effect of polyamines onthe efficiency of multiplication and rooting of Withaniasomnifera(L.) Dunal and content of some with anolides in obtainedplants. ActaPhysiol Plant.,2011;33:2279–2288.
  26. Petri, C., Alburquerque, N., Tornero O.P., Burgos, L.Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve adventitious regeneration from apricot leaves and Agrobacterium-mediated transformation of leaf tissues. Plant Cell Tissue Organ Cult.,2005;82(1):105–111.
  27. Geneve, R.L., Hackett, W.P. Ethylene evolution and endogenous polyamine levels during adventitious root formation in Englishivy. Curr Topics Plant Physiol.,1990;5:332–334.
(Visited 312 times, 1 visits today)

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.