Introduction

The Aloe genus consists of over500 leaf succulent species, ranging from shrubs to large trees, which are extensively distributed in Africa, the Indian subcontinent, Americas, and the Arabian Peninsula1. Aloe saponaria is one of the most important species in the Aloe genus and it is commonly known as the Soap Aloe since sap from its leaves is used as a soap substitute and is able to produce foam in water. This plant has a short stem with green leaves approximately 50 cm long, which are covered with white stripes. This aloe has a less bitter taste compared to other species because it harbors a lower concentration of aloin2. Certain species of Aloe plants are used commercially in food products in Asia and the USA1,3.Aloe plants also have some medicinal properties, such as wound-healing effects, as well as antimicrobial, antitumor and antiseptic activities4-7. The ethanol extract of Aloe saponaria, in particular, has antinociceptive and anti- inflammatory effects8.

Currently, there is an increase in the demand worldwide for this plant due to its medicinal properties. However, the cultivation of  saponariais challenging  since  the  seed germination rate is low, thus it is regenerated in- vitro. The in-vitro plant tissue culture technique (also known as organogenesis) is advantageous as it produces a high yield and protects plant-based resources9,10. Plant regeneration through organogenesis generally involves the induction and development of a shoot from explant tissue, and previous studies have reported the micropropagation of Aloe species11-15.Multiple propagation of Aloe species in-vitrohas been investigated extensively16,17.Additionally, in-vitro shoot organogenesis and plant regeneration of Aloe saponaria have been reported through using meristem explants18 .Efficient in- vitromicropropagation and regeneration of Humuluslupulus plants using a medium containing Gelrite was reported19. Additionally, it has been found that the use of activated charcoal (AC) improvesthe regeneration of banana cultivars20. Previously it had been shown that the best protocol for the micropropagation of A.saponariaexplants included the use of meristems18. Additionally, other studies21,22successfully regenerated plantsin-vitro using MS medium, and found that solid MS media is ideal for the micropropagation of Aloe species23.

Here, we report the development of animproved method for efficient plant regeneration from the meristem explants of A.saponaria by using experimentally defined concentrations of a specific media, gelling agent, and activated charcoal.

Material and Methods

Reagents and chemicals

MS medium, B5 medium, SH medium, Gelrite, activated charcoal,Phytagar,Growth regulators. All the chemicals were purchased from Sigma(St. Louis, MO, USA). All solid media was prepared with Phytagar0.7%(w/v) supplemented with the growth regulator BAP (2mg/L). Before the addition of Phytagar, the pH of each medium was adjusted to 5.7-5.8 using 1N HCL and 1N KOH.

Shoot organogenesis from meristem explants

Two-year-old meristem explants of A.saponaria were collected from the greenhouse at Chungnam National University. The explants were washed thoroughly with running tap water for 30 minutes and then surface sterilized with 70%(v/v) ethanol for 30 sec. The meristem was then disinfected with 1% (v/v) sodium hypochlorite solution for 10 minutes. Next, in a laminar air flow hood, the explants were washed 3 times with distilled water (DI). The explants were then dried with Whatman filter paper. The sterilized meristem explants were cut aseptically at the ends to obtain 4 sections each approximately 0.7 cm in length. Seven explants were then placed on 25 ml of culture medium in Petri dishes (100 × 25 mm).The media used for regeneration included MS24, B5,and SH, as described above.

In order to determine the best conditions for the improvement of shoot regeneration, the concentration of nutrients in the mediumwas optimized by testing the following conditions: ¼ MS, ½ MS,1MSor 2 MS with the growth regulator BAP (2mg/L). Additionally, the optimal concentration and type of gelling agent was determined. The two agents tested were Phytagar(tested at 5,6,7, and 8g/L) and Gelrite(tested at 1,2,3,4 and 5g/L) supplemented with the growth regulator BAP (2mg/l). The ability of the addition of activated charcoal (AC) (tested at 0.1,0.5,1,3, and 5 g/L) in addition to BAP (2 mg/L) to enhance shoot organogenesis was also Cultures were maintained at 25 ± 1°C in a growth chamber with a 16-h photoperiod under standard cool white fluorescent tubes (35 ìmol s- 1·m-2) for 6 weeks. The regenerated shoots were inoculated in rooting medium consisting of MS with 3g/L of Phytagar and 1mg/L of indole-3-butyric acid(IBA). After 6 weeks, the rooted plants were washed with sterile water to remove Phytagar, transferred to pots containing autoclaved vermiculite, and covered with polyethylene bags for 1 week to maintain high levels of humidity. The plants were then transferred to soil, and maintained in a growth chamber with a 16-h photoperiod, and day/night temperatures of 20/18°C, respectively, for 2 weeks. These treated plants were then transferred to a greenhouse.

Statistical Analysis

Data are expressed as the means ± standard deviations of the 30 explants examined.

Results and Discussion

Effect of mediaon shoot regeneration

The effect of different basal media on shoot regeneration of a saponaria was investigated. We observed that shoots began to develop 1 week after explants were cultured. All of the media used in this study had 100% shooting efficiency (Table 1). However, the number of shoots per explant and shoot length were found to be significantly different in MS medium after 2 weeks of culture as compared to the other culture conditions. Indeed, MS medium performed the best, with the highest number of shoots (2.5/ explant) and longest shoot length (1.6 cm), followed by B5 and SH basal media. Therefore, MS medium was selected as the most suitable for the shoot development and growth ofA.saponaria.

Table 1: Effect of different media on the shoot regeneration and growth of A.saponariameristem explants after 6 weeks of culture

*Basal medium supplemented with 2 mg/LBAP.
** From 30 tested explants.
aValues represent the mean ± standard deviation of 50
shoots.

Effect of MS media of different strengths on shoot regeneration

To evaluate the effect of different concentrations of MS media on shoot regeneration of A.saponaria, meristem explants were grown for 4 weeks. The results showed that 1MS medium was optimal for achieving the highest number of shoots (2.5/explant) and longest shoot length (1.6 cm) (Table 2). An increase in the concentration of MSmedium decreased theshoot number and ability for growth. Indeed, shoot number and growth were reduced by almost 2-fold in 2 MS medium compared to ¼ MS medium.

Table 2: Effect of MS media at different concentrations on shoot regeneration and growth of A. saponariameristem explants after 6 weeks of culture

* Basal medium supplemented with 2 mg/LBAP

** From 30 tested explants

aValues represent the mean ± standard deviation of 50 shoots

Table 3: Effect of gelling agents on shoot regeneration and growth of A. saponaria meristem explants after 6 weeks of culture

*MS medium supplementation with 2 mg/LBAP.
** From 30 tested explants.
aValues represent the mean ± standard deviation of
50 shoots.

Table 4: Effect of activated charcoal (AC) on the shoot regeneration and growth of A. saponariameristem explants after 6 weeks of culture

Effect of gelling agentson shoot regeneration

A previous study found that for Rehmanniaglutinosa, shoot organogenesis was more efficient when Gelrite was used as the gelling agent compared to Phytagar, leading to a higher number of shoots and improved shoot growth per leaf explant25. For this reason, we investigated the effect of different gelling agents in MS basal media on the shoot regeneration of A.saponaria. First, shoot explants were grown for 4 weeks in basal medium MSsupplemented with 0.8% Phytagar for initial shoot establishment. Next, in-vitro growing shoots were transferred to MS medium with various concentrations of Phytagar and Gelrite (Table 3). Shoots began to develop 1 week after the explants were cultured on media. Among the Phytagar treatments, an increase in the concentration of Phytagar from 5 to 6 g/L improved the shooting percentage, shoot number, and shoot length. Additional increases in the concentration of Phytagar (i.e., to 7, 8,and 9 g/L) did not improve the shooting response. Utilizing a concentration of Gelriteof 3 g/L as compared to 1 g/L enhanced the shoot number and length; however, further increases inGelrite concentration (4 and 5g/L) did not improve the shoot development.These results were similar to the in vitro rooting response of Syzygiumalternifolium26where increasing the concentration of agar from 0% to 0.8% enhanced rooting, while no improvement was observed from 1.0% to 1.2% of agar.

Effect of activated charcoal on shoot regeneration

Previous studies have revealed that AC improved plant micropropagation27,28, thus we used AC in micropropagation of A.saponaria. To evaluate the effect of various concentrations of AC on shoot regeneration of A.saponaria, meristem explants were grown for 4 weeks on basal MS medium in a controlled environment. Shoot induction was then performed on MSshoot regeneration medium with different levels of activated charcoal. The results demonstrated that 1 g/L of AC provided the optimal conditions for the growth of the greatest number of shoots (3.1 per explant) and longest shoot length (2.7 cm) (Table 4). Furthermore, the shoot length was nearly twice that obtained with the control medium that lacked AC, providing evidence that the addition of AC  improves  the  micropropagation  of  A. saponaria. Further, increasing the amount of activated charcoal(i.e., more than 1 g/L and up to 5 g/L), delayed the initiation of regeneration rate, decreased the shoot number, and stunted shoot growth.

Conclusion

Several studies have reported the in-vitro propagation of Aloe species. However, limited information is available regarding A. saponaria. In this study, we developed an improved, highly efficient micropropagation protocol for the in vitro regenerationof A.saponaria. Although previously established protocols for shoot organogenesis and plant regeneration from meristem explantsof A.saponariahave been described, the global demand for this species required a more efficient production methodology.In order to establish an improved plant regeneration system, we investigatedthe effect of different media, and different concentrations ofgelling agents (Phytagar and Gelrite), and AC on the efficiencyof shoot organogenesis.We found thatbasal MS medium supplemented with 2 mg/L BAP provided the best conditions for shoot induction. Ultimately, this protocol could provide a mechanism for the genetic transformation of Aloe. Additionally, this improved protocol can be implemented in the mass production of plants to meet the ever-increasing global demand for A.saponaria.

Acknowledgements

This study was supported by research funds from Dong-AUniversity, Korea, 2015.

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