Rhizome Buds Disinfection for Preparation of Red Ginger (Zingiber officinale Roxb. var. rubrum Rosc.) In Vitro Culture

The success of culture initiation depends on explant surface sterilization techniques. Sui­ table concentration, combinations, and duration of exposure of sterilizing agents are important to raise in vitro culture successfully. The aim of this work is to obtain the suitable sterilization method for ex­ plant buds of red ginger rhizome to get the axenic culture. Four sterilizing agents, fungicide, bacteri­ cide, Cefotaxime antibiotic, and NaOCl were tested for sterilization by various concentration and dura­ tion of exposure. The results showed that sterilizing agents 200 mg/L Cefotaxime and 100 mg/L Beno­ myl combined with NaOCl decreased the contamination of explants, and achieved 20% axenic culture.


Introduction
Red ginger (Zingiber officinale Roxb. var. ru brum Rosc.) belonging to the family Zingibera ceae is one of the most important components of Indonesian herbal medicine, locally called as jamu. Red ginger is also one of the most impor tant export commodities. High fluctuation price of Indonesian red ginger is caused by the lack of the market stock and serious damage due to pests and diseases [1]. The use of healthy planting ma terial is necessary to be considered to increase red ginger productivity [2].
Plant tissue culture technique has been used for the purpose of in vitro propagation on vari ous species. In vitro propagation aims to achieve healthy plants that will be used as seeds [3]. Therefore, we need the suitable sterilization pro cess to eliminate microorganism contaminant in explants so as not to interfere plant growth.
The explants sterilization is the process of making explants contamination free before es tablishment of cultures. It is important that the explants be free of any contaminants including endophytic prior to tissue culture without losing their biological activity [4]. These factors such as source of explants, plant species, age and other climatic changes affect the success of steriliza tion methods [5]. The problem at the beginning of culture is contamination of microorganisms and browning in explants. Therefore, suitable sterilization techniques are needed to eliminate all microorganisms on the surface and inside ex plants so that they do not interfere and reduce the growth of explant cells.
Internal contaminants are very difficult to overcome because sterilization is only limited to the surface of explants. In plant material that con tains inside contaminants, systemic antibiotics or fungicides must be used [6][7][8][9].
Various sterilizing agents are used to decon taminate plant tissues for in vitro cultures. The sterilizing agents are also toxic to the explants, so it is necessary for suitable concentration of ster ilant, exposure duration of the explants to vari ous ste rilant, the sequences stage of using these sterilant have to be standardized to minimize explants injury and increase survival rate while ensuring effective sterilization [10].
According to Srivastava et al. [11], concen tration and exposure duration of the sterilizing agents differ for different plant and plant parts. Tiwari et al. [12] reported that the use of only one sterilizing agent is not successful to sterilize the sugarcane leaf sheath. Sodium hypochlorite (NaOCl) combined with antibiotics is an effec tive steri lizing agent to eliminate bacterial con taminants in red durian leaf explants [13], shoot tip and leaf sheath explant of Ensete ventricosum [14]. Patel et al. [9] reported that the submersion of nodal explants of pomegranate cv. Bhagva in antibiotic solution effectively produce axenic cul ture that are able to grow. The focus of this study is to develop a standard protocol for the steriliza tion of rhizome bud from red ginger rhizomes to obtain axenic culture.

Chemicals
Murashige-Skoog (MS) medium was used as a culture medium, gelled with 2.5 g/L Phytagel, and 3% sucrose as carbon source. HCl or NaOH adjusted pH of medium to 5.8. Bayclin (NaOCl 5.25%) and Cefotaxime antibiotic were used as sterilizing agent to eliminate the surface conta minant of explants. Fungicide (Dithane M-45) and bactericide (Benlate), Benomyl fungicide, Cefotaxime antibiotic were used as systemic sterilizing agent to remove the contaminant inside tissue of explant. Plant growth regulators were added to medium, i.e. benzyl aminopurine (BAP), naphthalene acetic acid (NAA), and gibberellic acid. Polyvinylpyrrolidone (PVP-40) was used as antioxidant to prevent browning at explants, and Tween-20 was used as surfactant to help the sterilizing agent penetrate in to tissue of explant.

Plant materials
The explants used were part of the bud meristem of the red ginger rhizome from Yogyakarta. The red ginger rhizomes were washed thoroughly in running water for 30 minutes before being sterilized with fungicide and bactericide.

Method development 2.3.1. Explant surface sterilization
Rhizome bud explants were washed for 30 min with running tap water with liquid detergent. All thin leaves were removed from rhizome. Rhizome buds were then soaked in fungicide (2 g/L) for 120 min, washed three times by sterile distilled water, then treated with bactericide (2 g/L) (for 180, 360 min). Thereafter, the rhizome bud explants were treated by applying 6 sterilization methods (M1-M6) as presented in Table 1. In contrast to the sterilization methods (M7) without soaked in fungicide and bactericide, the rhizome bud explants were immediately soaked in antibiotic (Cefotaxime) and fungicide (Benomyl).
Shoot apex meristem (± 2.0 mm) were excised from buds after sterilizing treatment, and planted on each treatment media containing gibberellic acid or without gibberellic acid. The cultures were incubated with a photoperiod of 16 h/8 h light/dark, temperature 25±2 °C and 60 to 70% relative humidity.

Culture media
There were 2 treatments of plant growth regulator (0.5 mg/L gibberellic acid or without gibberellic acid) added to MS medium. MS medium supplemented with 3.0 mg/L BAP + 0.5 mg/L NAA + 200 mg/L PVP.

Data analysis
Experiments were organized according to completely randomized design with 20 explants per treatment. The cultures were maintained for 8 weeks. Culture had also been documented photographically with the use of stereoscopic Table 1. Explants surface sterilization procedure Treatment code Sterilization procedure M1 2 g/L fungicide (120 min), washed, 2 g/L bactericide (360 min), washed, NaOCl/sterile distilled water (1:1) plus 2 drops of Tween-20 for 5 min, washed, NaOCl/sterile distilled water (1:2) for 10 min, followed by five times washing using sterile distilled water.
microscope. The percentage of contaminated explants and also the survived free contaminated were counted.

Results and discussion 3.1. Explant surface sterilization
After observing for one week of planting all surface sterilization treatment were almost contaminated. As shown in Figure 1A and B, there were no browning at the explants. The addition of 200 mg/L PVP to MS medium improved the recovery of isolated meristem and also prevented the browning. Similar results with PVP (300 mg/L) was observed in overcoming explant browning of young stem segments of Hongyang kiwifruit by Chai et al. [15]. Table 2 revealed the percentage of survived or contaminated explants. Bacterial and fungi contaminants caused mortality of explants (treatment code M1, M2, M3) which were also caused by too long exposure duration to sterilizing agents, so the tissue of explant became damage. High concentration and long exposure duration to sterilizing agents resulted contaminant free explants but loss of explants viability (treatment code M4, M5, M6). The contaminant within the explants is not easily controlled. Antibiotic with broad spectrum activity is prerequisite to obtain better results. Cefotaxime antibiotic is mainly used against bacteria attack in humans. However, it is also used to eliminate bacteria in plant genetic transformation and low toxicity to plants [16]. Higher concentration of 300 -400 mg/L Cefotaxime could inhibit the growth and differentiation of plant tissue. Based on Table  2, the explants exposed to 100 mg/L Benomyl for 60 min, 200 mg/L Cefotaxime for 60 min, followed by NaOCl with lower concentration and shorter duration of treatment (M7 code) gave 20% axenic cultures and survived. Oo et al. [8] explained that strawberry explants which were treated with reduction time of exposure (20% NaOCl for 5 min, 70% ethanol for 5 min, 0.1% mercuric chloride HgCl2 for 4 min) and higher concentration of antibiotic (500 mg/L Ciprofloxacin for 60 min) gave the best results with higher survival percentages 25%, 10%, 55%, 20% respectively on the shoot tips, runner tips, nodal segments, and leaf segments. Patel et al. [9] stated that exposing nodal segments of Punica granatum L. to antibiotics 300 mg/L Bavistine for 18 min, 200 mg/L each of Streptocycline and Cefotaxime for 18 min and 8 min followed by 1000 mg/L HgCl2 for 5 min were achieved to be most the effective treatment to obtain 93.33% axenic culture. Mercuric chloride is often effectively used to overcome microbial contamination on underground rhizomes. The use of mercuric chloride in this work was avoided because it is

rhizomes.
Main contaminant at explants were bacteria that appeared after 3 days of culture. It is well known and most of the literature report NaOCl has broad spectrum of antimicrobial activity to rapidly kill bacteria, fungi, protozoa, and viruses.  For that reason, all treatment of sterilization methods in the present work uses NaOCl.

Growth of shoot apex meristem explant on MS medium
There were different developments of the culture on MS media containing gibberellic acid ( Figure 2) or without gibberellic acid. Elongation of shoot apex meristem showed that the addition of gibberellic acid improved shoot elongation (Figure 3), nevertheless shoot multiplication had not yet been achieved, even though cultured in MS medium with the addition of BAP 3.0 mg/L. Gibbrellic acid has relatively broad spectrum in the form of cell enlargement. On the contrary, Zuraida et al. [17] reported that BAP concentration between 3.0-5.0 mg/L was very effective to promote ginger microshoots after 35 days resulted 2 microshoots per explant. BAP is commonly used to produce ginger multiple shoots [18,19].

Conclusions
The best results with 20% axenic culture and alive were obtained using combinations of 200 mg/L Cefotaxime for 60 min, 100 mg/L Benomyl for 60 min, and NaOCl for 5 min.