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 value¹ and 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 an^“H^“ 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 properties^5-7, 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 reported^9-11, 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 AgNO_3,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 (AgNO_3,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 AgNO₃ enhances 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 AgNO₃ at 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 somnifera^24,25. 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

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

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 AgNO₃ and 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.

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