Effect of Gamma Irradiation on Callus of Handeuleum (Graptophillum pictum L. Griff) Kalimantan and Papua

Handeuleum (Graptophyllum pictum L.Griff) is a medicinal plant that is widely used as a traditional medicine for its healing properties, hence it should be developed as one of the leading Indonesian medicinal plants. The combination of tissue culture and gamma irradiation are two common methods to improve vegetatively-propagated cultivars. This experiment aims to determine the effect of gamma irradiation dose-rate on the callus culture of the two handeuleum accessions. Callus generated from proliferation were irradiated with gamma rays (0 Gy, 15 Gy, 25 Gy


INTRODUCTION
Handeuleum (Graptophyllum pictum L.Griff) is generally used by Indonesian community to cure various diseases since ancient times.The results of phytochemical analysis and active component compound of thirteen accession Graptophyllum pictum L. Griff) conducted by Lestari (2015) have proven that accession number 1 (West Java) and accession number 9 (Papua) to be high in phytochemical content (anthocyanin and carotenoids) that is useful for treatment.According to Juniarti et al. (2021) ethanolic purple leaves extract of handeuleum has antibacterial activity against Lactobacillus acidophilus.Jiangseubchatveera et al. (2015) observed that the essential oils in handeuleum have the ability to poison (cytotoxity) KB, NCI-H187 and Vero.Handeuleum essential oils have antioxidants ability, also have antibacterial against S.areus and E.coli.
Gamma irradiation may affect plant growth and development by altering physiological, biochemical, and genetical processes as well as leading to morphological changes in cells and tissues (Hanafy and Akladious 2018).Gamma irradiation is performed on callus culture because their cells are meristematic, making it more responsive to radioactive compared to adult cells (Hartati et al. 2020).Khalil et al. (2015) found that various gamma irradiation dosage (5 Gy, 10 Gy, 15 Gy, and 20 Gy) on 30-day-old leaf derived callus cultures of stevia (Stevia rebaudiana Bert.).Gamma rays dosage 15 Gy slightly enhanced stevioside content (0.251 mg g -1 ) over the control (0.232 mg g -1 ) along with total phenolics and flavonoids contents.The combination of in-vitro culture technique and gamma ray-induced mutation improve vegetatively-propagated cultivars, such as on gerbera (Ghani et al. 2013) and grapes (Munir et al. 2015).
There has been no information and results of research on the effect of invitro culture and gamma ray irradiation on the variability of handeuleum.Therefore, in vitro and gamma irradiation technique are expected to generate new mutants as a source of variability in handeuleum.This study is aimed to determine the effect of gamma irradiation dose-rate to the variability of callus culture in handeuleum, Kalimantan and Papua accession.

Procedures
The experiment was conducted in 2010 at the Laboratory of Plant Biotechnology and Laboratory of the Department of Agronomy and Horticulture, IPB.The Applications of irradiation was performed at Pusat Aplikasi Isotop dan Radiasi (PAIR) / Center for Isotope and Radiation Applications BATAN, Jakarta.
The experiment used callus obtained from callus proliferation age 3 weeks (eksplant leaves were used to make the callus, and the callus used weighed 0.5 g.), which previously were cultured to the medium WPM 0 for 1 (one) week to eliminate the influence of the previous medium.Callus was then irradiated with gamma rays at some predetermined different dose-rates of treatment.The explants were then grown on the treatment medium for regeneration.The basic medium used consisted of macro-and micronutrients WPM medium, 3% sucrose, 0.2% gelrite, active charcoal, casein hydrolyzate, and growth regulators (BAP and TDZ) in accordance with the treatment.Observations after callus have been irradiated include: 1. RD50, defined using Curve Expert Software1.4.

Data Analysis
The experimental results were analyzed by using test F. If the result of the test real followed by Duncan's Multiple Range Test (DMRT).The distribution of the normality of data was analyzed by the Shapiro-Wilk system.Data was transformed with a scale of normalcy √X + 0.5 to approximate normal distribution.Contrast test was carried out on the callus proliferation experiments to determine the comparisons of treatment.

Reduce Dosage 50 (RD50)
According to Werdhiwati et al. (2020) the use of mutation techniques to induce diversity requires information on the LD50, which is the dose that causes 50% of individuals in the population to die.Based on Sinusoidal Fit Graph (Figure 1) Reduce Dosage 50 of callus culture is 19.25 Gy.The mathematical equation for the graph is y = a + bcos (cx + d).Where a = 9.26113122860E-001, b = 4.61372914461E-002, c = 1.18166181993E-001 and d = -102659525896E+000.
Figures 1 show that the increase in irradiation dose in line with the increase of percentage of dead callus.Irradiation dose of 19.25 Gy causes death in callus above 50%.Increased radiation dose of less than 30 Gy callus death more than 70%.Determination of lethal dose is important factors that must be known to obtain the desired mutant.By theoretical and strengthened by some results research, the LD20-LD50 range can be produce the highest diversity.

Interaction among Accession, Irradiation Dose-rate and Regeneration Media on the Weight of Callus Subculture 1 and Callus Subculture 2
Gamma irradiation very tangibly affected the weight of callus subculture 1 (Figure 2) and callus subculture 2 (Figure 3), as well as the rate of the relative fresh weight growth of callus (Figure 4).Accession, media, and dose-rate interacted to affect the variables.
Figure 2 shows that the highest weight of callus subculture 1 is produced by the Papua accession irradiated by 15 Gy of gamma rays and planted on the medium of 4.44 μM BAP (0.570 g).The highest weight of callus subculture 2 (Figure 3) is produced by Papua accession irradiated by 15 Gy of gamma rays, and grown on medium containing 4:44 μM BAP (0.595 g).
Genotypes that is used determine the success of induced mutations, parts of the plants irradiated and the dose-rate of applied mutagens also play critical role.Nonetheless, the ability to generate high weight of callus was not related to the ability of the respective accession to regenerate after irradiation.The Papua accession handeuleum irradiated with 15 Gy of gamma rays produced the highest weight of callus compared to other treatments, and its weight decreased along with increasing dose-rate of irradiation.Purnamaningsih et al. (2014) states that the irradiated soybean callus is influenced by plant genotype.

Callus Relative Fresh Weight Growth (CRFWG) of Handeuleum Callus Caused by Gamma Ray Irradiation
Gamma ray irradiation has a very significant effect on the relative fresh weight growth of Handeuleum callus on both accessions (Figure 4).The results presented in Figure 4 suggest that The CRFWG is influenced by the interaction between the dose-rate of gamma rays and handeuleum accession.
The highest CRFWG rate of irradiated callus was found in the Kalimantan accession irradiated with dose-rate of 25 Gy (0.448), while the lowest relative growth rate occurred to the Papua accession, irradiated with dose-rate of 35 Gy (0.083).
Gamma ray irradiation affects the relative fresh weight growth randomly; however, there is a general overview of changes in relative fresh weight growth of callus of both accessions.Figure 4 illustrates that the Kalimantan accessions generally have a higher relative fresh weight growth in all irradiation treatment compared to the Papua accession (except treatment of 15 Gy), thus a conclusion can be drawn that the Kalimantan accession is more resistant to gamma ray irradiation treatment compared to the Papua accession.It is in line with the research of Abdelnour-Esquivel et al. (2020) that each ricegenotype generates differing sensitivities for response of several parameters such as survival, regeneration, tissue growth, fresh weight, or dry weight after callus being exposed to gamma rays.Yunita et al. (2020) describe that Batugegi dan Situpatenggang rice variety also show different result in sensitivities in every observation of parameters.

Variability of Relative Fresh Weight Growth of Handeuleum Callus Caused by Gamma Ray Irradiation
This experiment shows that gamma ray irradiation can cause variability in the weight of post-irradiation callus (subculture 1 and 2) and also can be seen from the relative fresh weight growth.Based on the findings, it seemed that the relative fresh weight growth of callus on the control callus population (0 Gy), and the gamma irradiated ones at the dose-rates of 15 Gy, 25 Gy, and 35 Gy resulted in significantly different average values from each other.It shows that the gamma-ray irradiated callus populations generated the highest mean, standard deviation, and variance values, followed by non-irradiated callus population (control).
Table 1 shows that the CV value increases as the dose of gamma ray irradiation increases.The greatest CV value is produced by a 35 Gy gamma ray dose, which means that at this dose the variability is large.According to Dhanavel et al. (2012) morphological diversity in cowpea is caused by the effect of mutations.
Gamma ray irradiations have different effects on the weight of callus, as could be seen from the varying results of the variability analysis.Increasing dose-rate of gamma ray irradiation normally causes a decrease to the regeneration of callus (Nikam et al. 2015) as indicated by the weight loss and discoloration.
A research by Purnamaningsih et al. (2014) states that the regeneration of irradiated callus becomes more restricted than non-irradiated one (control).This is in line with research conducted by Yunita et al. (2020), which describes that irradiation dose of 30 Gy causes death in callus above 50% of Batutegi and Situpatenggang variety rice giving.Increased radiation dose of less than 30 Gy callus death more than 70%.Hong et al. (2018) indicate that treatment with high doses of gamma irradiation has harmful effects on plants growth and development through the increase of free radicals.Oxidative stress caused by highly Reactive Oxygen Species (ROS) that generates by ionizing radiation.ROS can damage nucleic acids, peroxidation and carbohydrates, resulting cellular dam-age (Suzuki et al. 2012).Gamma ray irradiation may affect the primary metabolism, which in turn also affects the level of secondary metabolism in a plant (Jan et al. 2013).

Effect of Gamma Irradiation on the Colors of Two Handeuleum Accessions
The results showed that gamma ray irradiation has a very tangible effect on the color of callus (Figure 5).The colors of callus indicate the state of callus after irradiation treatment.There are four kinds of callus colors; brownish-white, whitish-brown, brown, brownish-black, and black.Black callus indicates dead callus.
Figure 5 and 6 showed that brownish-white callus could be found only in the treatment of 0 Gy (control), amounting to 5%, whereas the 25 Gy irradiation treatment produced brownish-white callus by 2%.The whitish-brown callus existed at all dose-rate of treatment with gamma rays, the dose-rate of 0 Gy (control) made up 24% of whitish-brown callus, dose-rate of 15 Gy and 25 Gy produced whitish-brown callus by 21% and 46% respectively.
The irradiated callus tends to be brown; it even can turn into black on a high-dose irradiation (indicating death of callus).Yunita et al. (2020) report that callus will experience browning after irradiated by gamma rays, because of messed up the biosynthesis of IAA that needed to regenerate somatic cell populations, because of the degradation of the enzyme indolacetol dehydrogenase which plays a role in the biosynthesis.

CONCLUSION
The interaction among the Papua accession, gamma irradiation at the dose-rate of 15 Gy, and regeneration media 4.44 μM BAP produced the highest weight of irradiated callus subculture 1 and 2, 0.570 g and 0.595 g respectively.Irradiation dose-rate of 25 Gy on Kalimantan accession resulted in the highest relative fresh weight growth of irradiated callus (0.448).Dose-rate 25 Gy produced a brownish-white callus of 13%, while other treatments did not.It also produced the highest value of variability in the relative fresh weight growth of irradiated callus (0.0047).

Figure 1 .
Figure 1.Callus weigt responses curve in different level of gamma ray irradiation

Figure 2 .Figure 3 .
Figure 2. Callus weight from different level of gamma ray irradiation at subculture 1 after treatment

Table 1 .
Based on Table 1, of the irradiated callus The variability of the relative growth rate of irradiatedcallus of handeuleum, Kalimantan and Papua accession.