In Vitro Mutagenesis of Etlingera elatior by Gamma Ray Intermittent Irradiation

Etlingera elatior is tropical ornamental plant commonly called “torch ginger” from Zingiberaceae family. Conventional breeding of E. elatior is limited by cross incompatibility, poor fruit set and low seed production. In this study irradiation of E. elatior with gamma ray performed to induce mutation. This study was aimed to increase morphological diversity and to obtain unique morphological characters to increase the aesthetic value of E. elatior as ornamental plants and cut fl ower. Two genotypes of E. elatior, red and white fl owers, were tested. The LD20, LD35, and LD50 were determined following intermittent (split dose) gamma irradiation with a two-hour gap between each gamma ray shot. Red genotype E. elatior explants were irradiated with dose of 3 + 3 Gy (LD20); 4 + 4 Gy (LD35) and 5 + 5 Gy (LD50); white genotype were irradiated with a dose of 2 + 2 Gy (LD20); 2.8 + 2.8 Gy (LD35) and 3.7 + 3.7 Gy (LD50). Non-irradiated explants were set as control. The results of this study indicated that the increase in dose of gamma ray irradiation changed shoot length and number of leaves in the red genotype per explant as compared to control. Morphological changes occurred in leaf shape at 5 + 5 Gy and 3.7 + 3.7 Gy and formation of variegated leaves at 2.8 + 2.8 Gy and 5 + 5 Gy.


Introduction
Zingiberaceae is the largest family of the order Zingiberales. Many species from Zingiberaceae family are economically important as source of food, spices, medicine and ornamentals. Etlingera elatior (Jack) R.M.Sm is known as kecombrang or honje in Indonesian. E. elatior fl owers, fruits and seeds have been used as food ingredients in Asia, including Indonesia. E. elatior fl owers have an attractive colour with varying shades of pink. The red shade bracts and fl owers make this species very attractive plant for ornamental purposes (Figure 1) such as for decoration in hotels and offi ces.
Etlingera fl owers have a short vase life; most fl owers only last for six to seven days (Sabu et al., 2013). Cut fl owers from a tropical country get less attention than fl owers originated from sub-tropical countries like roses, carnations and gerberas. Research and breeding of tropical fl owers including E. elatior is very limited and most studies focus on the phytochemical content of rhizomes for medicinal rather than for ornamental purposes. Chan et al. (2011) reported that phytochemicals from E. elatior has antioxidant, antibacterial, antifungal, cytotoxic, hepatoprotective and tyrosinase inhibitor activity. white (Anderson, 1994) Despite having attractive shape and colours E. elatior fl owers are large and heavy, making, the cost of air transportation expensive (Dwiatmini et al., 2009). E. elatior can be a potential cut fl ower but it requires continuous improvement in fl ower colour, morphology, longevity, size, odour and reduced time to fl ower. Unfortunately, conventional breeding of E. elatior is limited by cross-incompatibility, poor fruit set and low seed production, therefore gamma ray induction mutation is performed to increase morphological diversity.
Mutation is one of sources of genetic variation. Natural mutations are rarely occurring so to increase the frequency of occurrence of natural mutations induction mutations has been performed using mutagens (Aisyah et al., 2009). Among the physical mutagens, χ -and γ (gamma) rays are the most commonly used mutagens in mutation breeding. Gamma rays pass through a tissue by ionizing process (Mba and Shu, 2011). Gamma ray irradiation can damage DNA molecules such as the double strand damage, loss of nitrogen bases and changes in chemical base structure that can cause gene mutations. The existence of gene mutations in plant can cause changes in phenotypes which inherited to their offspring called mutant (Van Harten, 2002). Putative mutants are plants which are considered as mutants that expected in next generation to be genetically proven. In vitro culture methods have facilitated the use of mutation-assisted breeding technique for improvement vegetative propagated crops (Bala and Kanwar, 2015).
The success of irradiation to increase population diversity is largely determined by irradiated radio sensitivity of the genotype. Radio sensitivity can be measured based on the value of LD 50 (Lethal Dose 50) which is the dose that causes 50% mortality of the plant population. The optimum dose that can produce the most mutants usually occurs around LD 50 . LD 25 -LD 50 is the dose that can produce the highest mutation rate. Research conducted by Yunus et al. (2013) reported that LD 50 in shoots in vitro is 10 Gy. Based on LD 50 , LD 35 and LD 20 have been obtained in the preliminary study using dose range of 5-25 Gy. In this study, gamma ray intermittent irradiation treatment used in red and white genotype E. elatior. Induction of gamma ray intermittent irradiation mutation is expected to increase genetic diversity and can be obtained unique properties that can increase the aesthetic value of ornamental plants and fl owers.

Plant Materials
This preparation and propagation of plant materials was conducted in the Tissue Culture Laboratory 3, Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University in February to August 2017. The explants were collected from fourteen-month-old E. elatior plants with red and white fl owers from Mandiri Jaya Flora Nursery, Megamendung, West Java, Indonesia. Individual shoots were excised and cultured on MS medium (Murashige and Skoog, 1962) containing 3 % sucrose (w/v), 0.7 % gel, 31.08 μM BAP. pH medium was adjusted to 5.8 before autoclaving at 121 o C for 15 min. The cultures were incubated for six weeks at 23 ± 1 o C under 24 hours light with intensity 1200-1500 lux prior to irradiation treatment.

Gamma Ray Irradiation
The irradiation process was carried out at PAIR BATAN, South Jakarta. E. elatior plantlets (in tissue culture fl asks) of 15 to 30 mm shoot length were irradiated with the 60 Co radioisotope source (Gamma Chamber 4000A; dose rate of 288.55 Gy/ hr). Irradiation was conducted intermittently with a split dose of LD 20 , LD 35 , and LD 50 with a gap of two hours between irradiation. The values of LD 20 , LD 35 and LD 50 had been determined for both E. elatior genotypes in our previous study (unpublished) i.e. 6 Gy, 8 Gy and 10 Gy, respectively, for the red fl ower genotype, and 4 Gy, 5.6 Gy and 7.4 Gy, respectively, for the white fl ower genotype. In the current study, red genotype plantlets were irradiated at 0 Gy (control); 3 + 3 Gy (P1); 4 + 4 Gy (P2) and 5 + 5 Gy (P3) whereas the white genotype plantlets were irradiated at 0 Gy (control); 2 + 2 Gy (P1); 2.8 + 2.8 Gy (P2) and 3.7 + 3.7 Gy (P3). There were three replications per dose, and each replication consisted of 10 explants.

Post Irradiation Handling of the Explants
The irradiated explants are designated as MV0. After irradiation treatment, MV0 explants were transferred to a MS medium supplemented with 31.08 μM BAP for shoot multiplication; the new shoots grew from MV0 were designated as MV1. When the MV1 shoots were 15 to 30 mm in length they were excised from the irradiated explants (MV0) and sub-cultured to a new medium with the same hormone composition (MS + 31.08 μM BAP).
Sub-culture was conducted at fi ve weeks intervals for shoot multiplication up to the second regeneration cycle (MV2). Cultures were maintained on the same type of medium by regular sub-culturing every fi ve weeks over a period of 10 weeks. Plant cultures were incubated at 23 ± 1 o C with 24-h light photoperiod with light intensity 1200-1500 lux. A radio sensitivity test was determined by measuring the percentage of plantlet survival at fi ve and ten weeks after irradiation. Quantitative characters were measured on the number of leaves per explant, number of shoots per explant and shoot maximum height. Colour and shape of leaves were recorded visually after ten weeks of culture.

Data Analysis
The data from MV1 and MV2 were analysed using boxplot with Microsoft Excel Professional Plus 2016 to show the data characteristics and distribution. Leaf colours were characterized using the Royal Horticultural Society Mini Colour Chart as a reference.

Result and Discussion
The increasing dosage of irradiation decreased the percentage of surviving plantlets at fi ve and ten weeks after irradiation (Table 1).
The number of shoot, shoot length and number of leaves per explant in E. elatior MV1 and MV2 generation are presented in Table 2. The irradiation treatment did not affect number of shoots, shoot length and number of leaves because gamma ray mutations occurred on individuals from each treatment randomly. Boxplot (Figure 2 to 7) was used to determine data distribution in each treatment for the analysis of quantitative character so that changes in one individual can be detected on the graph. The existence of different individuals is indicated by the existence of outliers or extreme values on the graph (Amri et al., 2009). The number of shoots in both genotypes has abnormal data distribution in both generation (Figures 2 and 3). White genotype produces fewer shoots than red genotype but, white genotype have individual explants which have the most shoots with P3 treatment dose (LD 50 ). Irradiation treatment in MV2 has extreme values in P1 (LD 20 ) and P3 (LD 35 ) in red genotypes and P1 (LD 20 ) in white genotypes. The existence of extreme values in explant growth following irradiation treatment indicated potential mutants.
Shoot length in MV1 and MV2 generation of both genotypes have asymmetrical data distribution (Figures 4 and 5). The shortest shoots in red genotype were with P2 or LD 35 (Table 1). The phenotypes of MV2 shoots are likely to be unstable, so changes might still occur in the following generation. Similar to shoot length, number of leaf per explant of both genotypes has an asymmetrical data distribution on    Leaf colour and shape was recorded when the plants were 10 WAI (weeks after irradiation). Irradiation treatment produced putative mutant plants in MV1 and MV2 generation. Untreated red genotype ( Figure  8 A) had two shoots which can clearly be distinguished from the parent, whereas plantlets irradiated with 5 + 5 Gy produced plantlets with clustered shoots (Figure 8 B) and were diffi cult to distinguish from their parent, branched or variegated plantlets ( Figure  8C). Untreated white genotype has more leaves than   irradiated plantlet (Figure 9 A). Irradiation treatment at 2.8 + 2.8 Gy resulted in variegated leaves (Figure 9 B), whereas treatment 3.7 + 3.7 Gy resulted in branched (Figure 9 C) and asymmetrical leaves (Figure 9 D). The leaf colour from all treatments in both genotypes (Table 3) did not show any differences, except in the treatment dose of 5 + 5 Gy in red genotype which had several plantlets with yellow green leave, or RHS 149D. All changes that occurred in these plantlets have the potential to become mutants.
Determination the radio sensitivity of in vitro explants to gamma rays is the initial step of an early screening of variants with possible altered genetic patterns. The critical level of gamma irradiation where mutations were induced is usually within the range of tolerance for regeneration. LD 50 is recommended irradiation dose which slightly inhibits plant development (Zhou et al., 2006). Based on the results of this study, red genotypes are more sensitive to gamma rays than white genotypes. The percentage of survived plantlets that are irradiated intermittently at LD 50 (5 + 5 Gy and 3.7 + 3.7) was not show 50% dead plantlets, so as at LD 35 (4 + 4 Gy and 2.8 + 2.8 Gy) and LD 20 (3 + 3 Gy and 2 + 2 Gy) ( Table 1). It shows that the effects of the LD 50 acute irradiation was different from LD 50 intermittent irradiation. Another method to determine the plant's radio sensitivity is based on the value of Reduction Dose (RD 50 ), i.e. a dose that causes 50% of the plant population to experience growth inhibition. The lower the RD 50 value, the higher the radio sensitivity level (Herison et al., 2008).
The higher dose of irradiation increased damage to the plant, so it can inhibit plant growth including the number and length of shoots. Gamma ray irradiation has high penetration rate so it can reach multicellular layers (Yunus et al., 2013). However, gamma ray can also cause random mutations that can result in physiological damage in cell metabolism, resulting in faster or slower plant growth (Anshori, 2014). Increasing the dose of gamma ray irradiation to a certain level increased the frequency of mutations in a linear manner, but will likely reduce the ability to survive linearly. Putative mutants are generally formed in LD 50 range (Aisyah et al., 2009).
In this study, gamma ray irradiation treatment at LD 50 had produced mutants. Additionally, Sianpar et al. (2013) reported that irradiation of T. fl agelliforme at LD 50 on 25 Gy produces mutants with taller plants with more shoots than its parents. Anshori et al. (2014) reported that gamma ray irradiation on LD 50 range in Curcuma domestica obtained plants with faster growth than control plants. Dwimahyani and Widiarsih (2010) reported in vitro mutagenesis of chrysanthemum (Dendranthema grandifl ora) at 10 Gy increased the multiplication of shoots by 10% compared to the control plants.
Genetic alteration in fi rst generation after irradiation treatment can easily be seen through morphological changes, so that the plants can be called putative mutants (Anshori et al., 2014). Changes in leaf shape due to gamma ray irradiation are thought to be due to abnormal mutant cells that develop into different tissues or organs compared to those from the parent cells (Cahyo and Dinarti, 2015), while leaf colour changes are caused by disruption of chlorophyll synthesis (Datta, 2012). Gamma irradiation to Coleus blumei purple/green resulted in fi ve putative mutants based on changes in leaf colour and pattern (Togatorop et al., 2016), whereas Romeida (2012) reported nine mutants of S. plicata orchids. Gamma irradiation at 50 and 70 Gy produced changes in leaf colour and shape of Curcuma domestica (Anshori et al., 2014).

Conclusion
Explants of E. elatior red irradiated with gamma ray at 5 + 5 Gy and 3.7 + 3.7 Gy had changes in leaf shape; formation of variegated leaves were obtained with gamma ray irradiation at 2.8 + 2.8 Gy and 5 + 5 Gy. Irradiation at 5 + 5 Gy to the red genotype and 3.7 + 3.7 Gy to the white genotype have the potentials to produce putative mutants.