Forage crops are important as animal feed and have other benefits, such as cover crops, preventing erosion, and producing nitrogen in the soil, which can improve soil nutrients (Hasan et al. 2019; McGourty et al. 2005). Hence, forage crops can be regarded as a fundamental component of sustainable agriculture (Allen et al. 2011). Plant breeding is one of the earliest sustainable agricultural technologies to increase production, quality, and resistance against biotic and abiotic stresses (Al-Yassin et al. 2010; Dormatey et al. 2020). The development of science in plant breeding has recently grown massively. The current method to improve plant genetic modification has resulted in tremendous success, especially for food crops. The chemical and physical procedures to enhance plant genetics are commonly employed in forage restoration. Species like Indigofera0 (Khaerani et al. 2021; Musa et al. 2021; Musa et al. 2023), leucaena tarramba (Khaerani et al. 2023), alfalfa (Harianja et al. 2021), Stylosanthes (Tan et al. 2009), clitoria (Sajimin et al. 2015), panicum grass (Fanindi et al. 2016), purple grass (Fanindi et al. 2019; Pongtongkam et al. 2005), Dwarf Napier grass (Pongtongkam et al. 2006; Tan et al. 2009) and Wilman lovegrass (Álvarez-Holguín et al. 2022) have been upgraded through plant breeding. One of the high production and quality has been grown, called Pakchong Napier grass, also known as Super Napier grass. It is a new hybrid produced by crossing Pennisetum purpureum and Pennisetum glaucum (Somjai et al. 2020). This plant has unique characteristics of high nutritional value compared to other cultivars. The highest nutrient content is obtained when harvested 45 days after planting (Bangprasit et al. 2017; Mohamad et al. 2022). However, there is a lack of genetic variability in this species since it is a new hybrid. In the present study, the authors consider using gamma irradiation sourced by Cobalt 60 (⁶⁰Co) as the mutagenic agent. The advantages of gamma irradiation include high penetration waves, the ability to release or capture electrons, and direct disruption of chemical bonds of molecules when passing through the target (Irfaq et al. 2003; Lagoda 2012).

Efficient induction of mutagenesis by gamma irradiation requires identifying the optimal irradiation dose. Radiosensitivity tests can estimate the optimal dose, which indicates the varying effect of radiation exposure and the dose reducing 50% of the population (Ke et al. 2019; Masuda et al. 2006; Matova et al. 2021). However, very few studies have ventured into the radiosensitivity responses of forages with different sources and radiation dose activity rates. This study focused on radiosensitivity based on activity rate. This study aims to determine the optimum dose after exposure to varying gamma activity rates of Super Napier grass, where the low activity rates are generated by multipurpose panoramic irradiator 70 Ci and high activity rates are generated by Gamma cell 3 kCi. The Gamma cell 3 kCi emits a high activity rate of radiation, leading to acute radiation effects. In contrast, multipurpose panoramic irradiator 70 Ci emits a low dosage rate of radiation, resulting in chronic radiation effects. Acute irradiation is exposure to high doses of radiation over a shortened period, while chronic irradiation is exposure to low doses of radiation continuously over an extended period.

Plant material and method

The Super Napier grasses (Pennesetum purpureum×Pennisetum glaucum) originated from Livestock Breeding and Forage Crops Center of Mataram Livestock and Animal Health Service, West Nusa Tenggara, Indonesia (8°35'20.2"S 116°05'58.3"E) with site temperature of 25℃-30℃, relative humidity level of 73%, and precipitation around 10-19 mm. Stem segments selected the targeted material plants in vegetative propagation.

Gamma irradiation and preparation

Irradiation treatments were performed at the Center for Isotope and Radiation Application, National Nuclear Energy Agency (CIRA-NNEA) sourced from Cobalt-60 (⁶⁰Co) of gamma irradiation and two activity rates, i.e., low activity rate (Multipurpose panoramic irradiator 70 Ci) and high activity rate (Gamma Cell irradiator 3 kCi). For each gamma activity rate, as many as 140 stems were prepared into 7 irradiation dose treatment including the doses were (Ctrl) without applying irradiation, 10 Gy, 20 Gy, 30 Gy, 40 Gy, 50 Gy, and 60 Gy.

Plant growth condition

The stem segments were planted under greenhouse conditions at 26℃-27℃ with an optimal relative humidity level of 75%-80% in the 40 x 60 cm polybags at the Center for Isotope and Radiation Application, National Nuclear Energy Agency's Experimental Farm Indonesia (CIRA-NNEA) (6°17'40.8"S 106°46'27.0" E) for 21 days. A total of 56 polybags were filled with soil and compost at a ratio of 1:2. The stem segments were well-watered every two days. Completely randomized design at a specific irradiation dose with four replicates for each low and high activity rate. The implementation of the experiment is described in Fig. 1.

Figure 1. Graphical abstract of pre-irradiation, irradiation and post-irradiation of Super Napier grass.

Data collection

To identify the effect of low activity rate (Multipurpose panoramic irradiator 70 Ci) and high activity rate (Gamma Cell irradiator 3 kCi) gamma irradiation on Super Napier grass, it can be determined based on the agromorphological characteristics of the plant. The assessment of radiosensitivity is based on the observed survival rate (%), growth rate (%), plant height (cm), and number of leaves (unit) after the stems were exposed to gamma irradiation. Plant height (cm) and number of leaves (unit) were recorded every week, and the changes were observed at the end of the observation. The collected data survival rate (%) and growth rate (%) for each gamma dose were calculated using the following formula:

Statistical analysis

The experiment employed a Data analysis performed using the Kruskal Wallis non-parametric test on survival percentage (%) and growth rate (%) and analysis of variance on plant height (cm) and number of leaves (unit) with the Statistical Tool for Agricultural Research (STAR) 2.0.1. Lethal Dose (LD₅₀) was identified and analyzed using Curve Expert 1.3. The highest variability of mutants occurred in gamma-ray irradiated mutants with LD₅₀ and GR₅₀. The least significant difference test (LSD) was employed to compare meansStandard Deviation (SD).

Agromorphological parameters of Super Napier grass post-gamma irradiation

Agromorphological characterization is one of the steps that are considered important in plant breeding. Mainly in determining lethal dose and dose reduction after gamma irradiation. Primarily involved in assessing the lethal dose and growth reduction strategies following exposure to gamma radiation. The agromorphological parameters of post-gamma irradiated Super Napier grass are presented in Table 1 and Fig. 2.

Table 1 . Agromorphological parameters of Super Napier grass post-gamma irradiation.

Gamma source	Doses	Number of leaves (unit)	Survival rate (%)	Growth rate (%)	Plant height (cm)
Low activity rate (Multipurpose panoramic irradiator 70 Ci)	Ctrl	2.68±2.74a	42.5±0.00	42.5±7.50	12.30±16.53a
	10Gy	1.24±3.53b	38.75±8.26	38.75±8.26	5.30±19.33b
	20Gy	0.51±3.82c	33.75±5.54	35.00±6.12	2.24±18.19bc
	30Gy	0.31±3.59c	33.75±7.18	33.75±7.18	1.11±14.16c
	40Gy	0.29±2.80c	21.25±9.87	21.25±11.25	0.74±8.69c
	50Gy	0.03±2.02c	22.5±5.20	26.25±8.15	0.08±6.64c
	60Gy	0.00±0.06c	11.25±3.14	21.25±6.25	0.01±1.40c
LSD		**	ns	ns	**
High activity rate (Gamma Cell irradiator 3 kCi)	Ctrl	9.95±0.98a	75.00±0.00a	75.00±7.50c	67.42±9.97a
	10Gy	1.17±3.94b	55.00±3.53b	55.00±3.53b	34.30±24.61b
	20Gy	0.32±3.73c	33.75±7.18c	33.75±7.18c	11.96±16.31c
	30Gy	0.08±2.82c	31.25±8.50c	31.25±8.51c	5.17±9.80c
	40Gy	0.03±2.08c	33.75±11.25c	33.75±11.25c	1.59±3.44c
	50Gy	0.00±0.44c	16.25±1.25cd	16.25±1.25cd	0.30±1.31c
	60Gy	0.00±0.44c	2.50±2.50d	2.50±2.50d	0.01±1.32c
LSD		**	**	**	**

Means with a similar superscript letter in the same column are not significantly different. Least Significant Difference (LSD) Test level of significance = 1%. ** Significant; ns non-significant.

Figure 2. Agromorphological parameters of Super Napier grass post-gamma irradiation.

Table 1 shows the differences in agromorphological parameters (p<0.01) resulting from exposure to different irradiation doses in Super Napier grass. The high activity rate (Gamma Cell irradiator 3 kCi) showed differences in the number of leaves, survival rate, growth rate, and plant height. However, no significant effects were observed (p>0.01) on survival rate and growth rate at the low activity rate (Multipurpose panoramic irradiator 70 Ci). In multipurpose panoramic irradiator 70 Ci and gamma cell 30k Ci, the control (ctrl) shows significantly different effects to10Gy for both gamma sources. Meanwhile, the 20Gy dose showed no significant difference from 30Gy to 60Gy in the number of leaves and plant height parameters. Nevertheless, gamma cell 3 kCi shows a better effect on the morphology of Super Napier grass compared to a low activity rate of multipurpose panoramic irradiator 70 Ci where gamma cell 3 kCi indicates significant difference among control (ctrl), 10Gy, and 50Gy.

Based on Table 1, the number of leaves and plant height (Fig. 2), Gamma cell 3 kCi generally illustrate better results for those agromorphological characteristics than Multipurpose panoramic irradiator 70 Ci. Additionally, for number of leaves, ctrl (Multipurpose panoramic irradiator 70 Ci 2.68±2.74^a; Gamma cell 3 kCi 9.95±0.98^a) shows a higher average than the other doses treatment such as 10Gy (Multipurpose panoramic irradiator 70 Ci 1.24±3.53^b; Gamma cell 3 kCi 1.17±3.94^b), 20Gy (Multipurpose panoramic irradiator 70 Ci 0.51±3.82^c; Gamma cell 3 kCi 0.32±3.73^c), this trend is also similar to plant height where ctrl shows the highest value (Multipurpose panoramic irradiator 70 Ci 12.30±16.53^a; Gamma cell 3 kCi (67.42±9.97^a). The average of survival and growth rate with a high activity rate (Gamma cell 3 kCi) showed better results compared to the low activity rate Multipurpose panoramic irradiator 70 Ci in both ctrl (75.00±0.00^a; 42.50±0.00) and 10Gy (55.00±3.530^b; 38.75±8.26), respectively. In plant height, ctrl showed higher results on all doses generated by a high dose rate than doses at a low dose rate. The lowest were found at 60Gy (Multipurpose panoramic irradiator 70 Ci 0.001±1.40^c; Gamma cell 3 kCi 0.01±1.32^c), and the highest were found in ctrl (Multipurpose panoramic irradiation 70 Ci 12.30±16.53^a; Gamma cell 3 kCi 67.42±9.97^a). The growth value of the number of leaves and plant height drastically decreases along with a higher irradiation dose.

The LD₅₀ of post-gamma irradiation of Super Napier grass

The LD₅₀, or lethal dose 50, refers to the radiation dose estimated to result in the death of 50% of an exposed population by post-gamma irradiation. The LD₅₀ can also determine the optimal dose of gamma radiation for promoting genetic diversity in plants. The LD₅₀ of post-gamma irradiation of Super Napier grass with different gamma sources is presented in Table 2.

Table 2 . The LD₅₀ of post-gamma irradiation of Super Napier grass.

Gamma Source	Parameter	Model	Equation	R2	LD50
Low activity rate (Multipurpose panoramic irradiator 70 Ci)	Number of leaves (unit)	Linear	y=15.03-0.20x	0.67	55.71
	Survival rate (%)	Linear	y=93.97-1.23x	0.92	35.82
High activity rate (Gamma Cell irradiator 3 kCi)	Number of leaves (unit)	Linear	y=18.17+0.28x	0.77	28.98
	Survival rate (%)	Linear	y=90.89-1.41x	0.88	28.99

Based on Table 2, lethal dose (LD₅₀) in low and high activity rates had different results. At the low activity rate (Multipurpose panoramic irradiator 70 Ci), the lethal dose (LD₅₀) ranges from 35.82Gy to 55.71Gy, and it is dramatically higher compared to the high activity rate (Gamma Cell irradiator 3 kCi), ranging from 28.98Gy tp 28.99Gy. A similar trend is also observed in the growth reduction (GR₅₀) (Table 3), in which the low activity rate (Multipurpose panoramic irradiator 70 Ci) is sharply higher than the high activity rate (Gamma Cell irradiator 3 kCi). The values are about 38.33Gy and 20.76Gy, respectively.

Table 3 . The GR₅₀ of post-gamma irradiation of Super Napier grass.

Gamma Source	Parameter	Model	Equation	R2	GR50
Low activity rate (Multipurpose panoramic irradiator 70 Ci)	Plant height (cm)	Linear	y=31.59-0.49x	0.72	38.33
High activity rate (Gamma Cell irradiator 3 kCi)	Plant height (cm)	Linear	y=57.12-1.12x	0.81	20.76

The GR₅₀ of post-gamma irradiation of Super Napier grass

The growth reduction post-gamma irradiation on plants refers to the specific dose of gamma radiation that causes a decline in plant growth or survival rate. The growth reduction can be determined by measuring the values of the lethal dose (LD₅₀) or growth growth reduction (GR₅₀). The impact of gamma irradiation on plant growth can vary depending on the radiation dose. In this study, the GR₅₀ of post-gamma irradiation of Super Napier grass was measured according to the plant height parameter in Table 3 below.

As can be seen from Table 2, lethal dose (LD₅₀) and growth reduction (GR₅₀) in low and high activity rates are significantly different. At a high activity rate of Gamma Cell irradiator 3 kCi, irradiation dose for lethal dose (LD₅₀) and growth reduction (GR₅₀) were found on 28.99Gy and 20.76Gy. The results are considered relatively high compared to the low activity rate of multipurpose panoramic irradiator 70 Ci with LD₅₀ and GR₅₀ were obtained at 35.82Gy and 38.33Gy. Each morphological parameter, including survival rate, plant height, and the number of leaves, also indicates a positive correlation R<1 at low and high activity rates.

Graphic models post-gamma irradiation of Super Napier grass

The graph in Fig. 3 illustrates various graphic models, such as linear graphic models in which (R) correlation is closer to 1.

Figure 3. Lethal dose and growth reduction of Super Napier grass in different activity rates.

As can be seen from Table 2, lethal dose (LD₅₀) and growth reduction (GR₅₀) in low and high activity rates are significantly different. At a high activity rate of Gamma Cell irradiator 3 kCi, the lethal dose (LD₅₀) was identified at 28.99Gy and growth reduction (GR₅₀) was identified at 20.76Gy. The results are relatively high compared to the low activity rate of multipurpose panoramic irradiator 70 Ci with LD₅₀ of 35.82Gy and GR₅₀ of 38.33Gy. Each morphological parameter, including survival rate, plant height, and the number of leaves, also indicates a positive correlation R<1 at low and high activity rates.

This study induced the Super Napier grass by gamma rays, Cobalt-60. Gamma rays with various energy forms from electromagnetic radiation have higher energy levels with greater penetrating power than other irradiation rays (Anne 2020). Gamma-ray will enhance plant characteristics and productivity (Pandit et al. 2021; Yamaguchi 2018). A comparative study clarified how the observed effects after exposure were highly affected by many factors. Some are related to the plant characteristics (species, cultivars, growth stage, cell architecture, and genome organization), and others are related to the quality, dosage, and irradiation exposure duration (Jan et al. 2012). Activity rates focus on the rate at which radioactive decay or nuclear transformations occur. They provide information about the intensity of a radioactive source. On the other hand, irradiation doses measured in grays focus on the amount of energy deposited by ionizing radiation in a material, allowing us to understand its potential effects on living organisms or materials. Most gamma-ray irradiators in mutation breeding employ a high activity rate (Gamma Cell irradiator 3 kCi).

Multipurpose panoramic irradiator 70 Ci known as IRPASENA 70 Ci is a low activity rate irradiator that causes chronic irradiation, while gamma cell 3 kCi is a high activity rate that causes acute irradiation. Gamma radiation at high concentrations inhibits physiological plant processes, affecting growth and development. Many crops, including Bambara groundnut (Muhammad et al. 2021), mustard (Malek et al. 2012), and black gram (Yasmin et al. 2020), have shown that higher doses of low and high activity rates of gamma radiation can substantially damage plant growth and physiological development in seeds, seedlings, or propagates.

The average survival rate and growth rate of Super Napier grass (Fig. 2) exhibit a similarity in the high dosage rate and low activity rate. Survival rates can be moderately increased at a dose of 60Gy. There is a noticeable difference when comparing the low dosage rate of Multipurpose panoramic irradiation at 70 Ci to the high activity rate of Gamma cell at 3 kCi. In contrast, according to Hong et al. (2018), low activity rate, chronic gamma irradiation, causes severe damage and inhibition of plant growth in wheat much more than high activity rate, acute irradiation. Choi et al. (2021) stated that high activity rate, acute irradiation produces rice seeds with drastically reduced fertility, while at low activity rate, and chronic irradiation fails to produce fertile seeds. High activity rate causes immediate and more significant damage to plant physiology, whereas a low activity rate causes long-term damage, leading to reproductive failure. Compared to acute radiation, chronic radiation can result in a significantly increased frequency of homologous recombination (Kovalchuk et al. 2000). Chronic irradiation results in a much greater occurrence of homologous recombination (HR) in comparison to acute irradiation. Homologous recombination caused by persistent radiation can result in a variety of genetic resources in plants (Hong et al. 2018).

Furthermore, plants grown under low activity rates and chronic radiation exposure exhibit complex biological effects and altered plant metabolism and gene expression patterns (Voronezhskaya et al. 2023). Gamma cells are specifically used in research and applications requiring low doses and modest throughputs (International Atomic Energy 2006). It is commonly used to subject biological and non-biologic materials to radiation to evaluate how the targets will react. Meanwhile, multipurpose panoramic irradiator 70 Ci, is widely used when many samples need to be irradiated simultaneously, especially for culture cells (Kim et al. 2008).

The response of germination and plant growth post-gamma irradiation treatment generally depends on species and plant genotype, heritability material, and irradiation characteristics such as quality, the quantity of radiation dose, exposure time, and intensity of exposure (Jan et al. 2012; Saha et al. 2019; Ulukapi et al. 2018). The probability of plant mutations through physical mutagenesis, gamma irradiation, can be described at a dose of 50% (LD₅₀) where the irradiated sample dies or when 50% of plant growth decreases (GR₅₀) (Álvarez-Holguín et al. 2019; Muhammad et al. 2021; Thole et al. 2012). The mutagen dose induces 50% mortality (LD₅₀) in the first generation of the mutant population (M1) due to its ability to produce a higher range of mutational phenotypes (Ke et al. 2019). In this study, the effect of irradiation doses from 10Gy to 40Gy still demonstrated relative growth, but at doses of 50Gy and 60Gy, the plants died. Such a phenomenon is supported by many studies stating that lower radiation doses lead to improved germination and plant growth. While the higher doses result in growth abnormalities, delayed germination, or even the death of exposed plants (Kovalchuk et al. 2003; Majeed et al. 2017; Singh et al. 2010).

The first step in identifying the mutation efficiency effect of gamma irradiation is to determine the optimal radiosensitivity of irradiation dose or reducing 50% of the population (lethal dose median, LD₅₀). Parameters commonly shown in LD₅₀ include the survival rate (Talebi et al. 2012). Radiosensitivity test enables the identification of accurate irradiation doses to induce optimal mutation with the most ignored effect in gene complex (Matova et al. 2021; Riviello-Flores et al. 2022). Concerning the mutation involving gamma-ray irradiation, generally, the mutation causes genetic variability within the LD₅₀ dose interval and below. The identified optimal dosage will be used as the standard to achieve genetic variability in M2 plants (Hanafiah et al. 2010).

Table 3 shows that the correlation coefficient indicates a strong positive correlation between survival rate, number of leaves, and plant height. Fig. 3 also describes linearly the plant's growth reduction damage and mortality in 50Gy and 60Gy of gamma irradiation dose. As can be seen, the higher dose of gamma irradiation implies more damage affecting the plants. Gamma rays are electromagnetic irradiation that affects plant growth and development by changing morphology, physiology, biochemistry, and genetics in cells and tissues. At certain doses, the effect of gamma irradiation causes oxidative stress (Irfaq et al. 2003; Kiani et al. 2022; Marcu et al. 2013).

After identifying LD₅₀, the most accurate doses can be selected as treatments. LD₅₀ is commonly selected as the appropriate dose for irradiated plants by comparing the irradiation outcome in similar species and families of plants. For further evaluation, the following generation will require more verification concerning the expected traits to produce new germplasm (Álvarez-Holguín et al. 2022). Generally, grasses and vegetatively propagated plants have lower doses than the other species and seed-propagated plants. Thus far, many improved studies have been related to lethal and reduced doses in grasses. The studies explained that LD₅₀ with low dose (below 100Gy ), such as 93Gy as LD₅₀ for Augustine grass (Çakir et al. 2017), 85Gy for Bermuda grass (Mutlu et al. 2015), and 68.85Gy for swamp rice grass(Sukmasari et al. 2021). However, the findings only explained that the irradiation was sourced from Cobalt-60 (⁶⁰Co). Further study has not been found regarding the gamma activity rate, such as Multipurpose panoramic irradiator 70 Ci and Gamma Cell irradiator 3 kCi. Therefore, this study becomes the latest research in forage plant breeding, specifically in gamma irradiation mutation.

Super Napier grass radiosensitivity test has been optimized with a low activity rate, Multipurpose panoramic irradiator 70 Ci, and a high activity rate, Gamma chamber 3 kCi. In low activity rate (Multipurpose panoramic irradiator 70 Ci), the lethal dose (LD₅₀) ranges from 35.82Gy to 55.71Gy, and it is higher compared to the high activity rate (Gamma Cell irradiator 3 kCi), ranging from 28.98Gy tp 28.99Gy. In reduction dose (RD₅₀), the low activity rate (Multipurpose panoramic irradiator 70 Ci) was identified at 38.33Gy, which is higher than high activity rate (Gamma Cell irradiator 3 kCi), which was identified at 20.76Gy. The correlation coefficient indicates a strong positive correlation between survival rate, number of leaves, and plant height. However, the higher irradiation dose resulted in a decrease and subsequent inhibition in plant growth and the survival rate and growth rate, growth reduction damage and mortality in 50Gy and 60Gy of gamma irradiation dose. This study becomes a preliminary study for mutagenesis breeding for forage crops. Further study concerning the mutant traits and vegetative mutant generation will be necessary.

The authors would like to express their gratitude to The Ministry of Education and Culture of Indonesia through the Directorate General of Higher Education of Indonesia under Program Magister Doktor Sarjana Unggul (PMDSU) for funding this research, appreciation to Hasanuddin University, the Center for Isotope and Radiation Application, National Nuclear Energy Agency of Indonesia (CIRA-NNEA) and Livestock Breeding and Forage Crops Center of Mataram Livestock and Animal Health Service of West Nusa Tenggara, Indonesia for significant contribution.