Mutagenesis and Identification of Sugarcane Mutants Using Survival on Polyethylene Glycol and Leaf Damage underManaged Water Stress

Drought causes severe damage to sugarcane, reducing its product yield. Given )ailand’s weather conditions and topography, a breeding program to develop new sugarcane genotypes with a high tolerance for water stress is important to the sugarcane industry. )is study created new water stress tolerant sugarcane genotypes using ethyl methanesulfonate (EMS) mutagenesis in the sugarcane cultivar Khon Kaen 3. Using 16mM of EMS for 4 h induced callus mutagenesis (survival rate, 57.5%). )e survival rates of calli treated with 10mM of EMS for 2 and 4 h in selective media with 15% PEG were higher than that of non-EMS-treated calli.)e selected calli survived and grew on selective media with 20% PEG, while non-EMS-treated calli did not grow.)emutant plantlets developed from EMS-treated calli on selective media with 20% PEG for 4 weeks had varying survival rates: 72.25% (10mM of EMS for 2 h), 75.85% (10mM of EMS for 4 h), and 60.61% (16mM of EMS for 4 h). Both healthy mutant sugarcane plants (2,086) and non-mutant plants (234) were cultured on the media with 20% PEG for 16 weeks. Of these, 462 mutant sugarcane plantlets survived and developed on the media, but all the non-mutant sugarcane plantlets died during the selection process. Mutagenesis induced using treatment 4 produced the highest frequency of mutant sugarcane plantlets with water-stress tolerance (45.5%). In total, 136 selected mutant sugarcane plants were transplanted to a greenhouse for evaluation under managed water stress. Fourteen mutant sugarcane plants stayed green after the third cycle of water stress, but the KK3 sugarcane cultivar showed damage on 50% of the leaves. )us, EMS mutagenesis and evaluation using in vitro and greenhouse methods were successful in developing new sugarcane clones with high water-stress tolerance, which is important for sugarcane breeding programs.


Introduction
Sugarcane is an economic crop that has a substantial impact on the economy of ailand, where it is the primary raw material for the production of sugar used in many highvalue products and downstream industries, including bioethanol and electricity production [1]. In 2020, ailand ranked fourth in the world based on the volume of sugarcane produced (74.9 million tons). Drought is adversely affecting many of ailand's economic crops, including sugarcane. Cane production in 2020 was dropped approximately 43%, compared with the previous year, because of the drought [2]. Drought often occurs in the middle of ailand's sugarcane planting season, often having a devastating effect on yield as it reduces the number of shoots per clump and stunts the sugarcane stalks. Sugarcane farmers cannot avoid this annual period of drought because sugarcane grows over a span of 12-15 months. erefore, a systematic breeding program to develop new drought-tolerant sugarcane varieties is essential. Inducing mutation has been shown to be a viable method to create genetic variations, and it was possible to create new sugarcane genotypes for breeding programs.
It is well-known that physical and chemical mutagenesis are efficient methods for inducing genetic variation, with gamma rays, ethyl methanesulfonate (EMS), sodium azide, and 5-azacytidine frequently being used to trigger mutations, thereby creating new plant characteristics [3]. EMS is a chemical mutagen with high mutagenesis efficiency, and in vitro mutagenesis using EMS has been reported as an effective method for creating variations in crop plants [4]. Mutagenesis through EMS has been used to induce imazapyr tolerance, high aluminum resistance, and salt tolerance in sugarcane [5][6][7][8], and it has been used to successfully induce new crop genotypes that are more drought tolerance [9]. Given the high potential of EMS to induce mutagenesis, this should be a realistic method to develop new sugarcane clones that can better tolerate water deficits and drought field conditions. Plant callus cells are an efficient plant material to be used for inducing mutagenesis because target mutant calli can be selected on selective media [10]. Furthermore, selected plant mutant calli can regenerate plants on culture media, and the genetics of cells in a mutant plant regenerated from callus are identical to the mother plant [11]. For sugarcane propagation, cell and tissue cultures are efficient methods for increasing the number of target clones. Calli are usually induced from young leaves and segments of young leaves generally after initiation using different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) [12]. e maximum callus formation (80-85%) was observed in MS media containing 3 and 4 mg/l of 2,4-D [13]. Callus induction medium was reported on Murashige and Skoog (MS) medium supplemented with 2,4-D, with the addition of water enhancing the growth of calli on this medium [14]. e regeneration of sugarcane calli can be controlled using 6-benzylamino purine (BAP), naphthaleneacetic acid (NAA), kinetin, and coconut water. In sugarcane, callus regeneration is highly efficient when plant growth regulators are combined and auxins and cytokinins are often used for supplementation in culture media for callus regeneration [15]. MS medium supplemented with NAA and BAP produced 98.9% regeneration in sugarcane variety CPF-246 [16], while kinetin and BAP could also regenerate plantlets in sugarcane [17].
Water-stress-tolerant plants can be examined and selected based on their capacity to survive on media supplemented with polyethylene glycol (PEG) to create conditions of water stress. PEG is a non-penetrating, inert, osmotic chemical that lowers the osmotic potential of nutrient solutions [18] and can be used to create water stress conditions on synthetic media. It can stimulate water stress in cultured plant cells without damaging the cells, and its high molecular weight reduces the potential of the water in the medium without the water being absorbed by the plant.
In vitro PEG selection for water-stress tolerance has been successful in many plants, such as rice [19] and sugarcane [20]. Selection for water-stress tolerance in sugarcane has been successful under managed water stress using physiological parameters and morphological parameters [21,22]. Common morphological changes due to water stress that occur in sugarcane are leaf rolling, stomatal closure, inhibition of stalk and leaf growth, leaf senescence, and reduced leaf area [23]. However, morphological and physiological changes of sugarcane plants under water stress vary according to genotype, the level of stress, and the type of tissue [24,25]. A leaf damage score under water stress was used for the identification of water-stress tolerance in other crops [26].
Khon Kaen 3 (KK3) is the most popular, commonly planted sugarcane variety in ailand because of its yield performance. However, KK3 is not tolerant to severe water deficit conditions. e presented study applied EMS mutagenesis for the KK3 cultivar to induce new mutant sugarcane genotypes for water-deficit tolerance. e mutant sugarcane genotypes were evaluated in vitro under water deficit conditions at the callus and plantlet stages. After regeneration, the selected mutant sugarcane plants were evaluated in the greenhouse under managed water stress to identify mutant sugarcane plants that thrived despite the stress.

Ethyl Methanesulfonate
Mutagenesis. EMS mutagenesis was adapted from the methods described by Koch et al. [7]. Sugarcane calli were treated by EMS at 10 and 16 mM for 2 h and EMS at 16 mM for 4 h. After EMS mutagenesis, 0.4 g of callus (0.5 mm diameter) was cultured on callus culture medium and incubated in the dark at 25°C ± 2°C at a relative humidity of 40% for 2 weeks. en, calli were subcultured every 2 weeks on the callus culture medium for 18 weeks and checked for calli that survived and grew. e survival rate and growth of EMS-treated calli were recorded.

Selection of Mutant Calli and Plantlets under Selective Media with PEG 15 or 20%.
e optimal PEG concentration for the creation of conditions of water stress in selective media was examined for EMS-treated calli and plantlets. Non-EMS-treated calli and regenerated plantlets of the KK3 sugarcane cultivar were cultured on MS media supplemented with different concentrations of PEG 6000: 0, 5, 10, 15, 20, 25, and 30%. In total, 0.2 g of calli was cultured on selective media for 4 weeks. e survival rate and growth of non-EMS-treated calli were recorded to identify the most efficient PEG concentration for the creation of conditions of water stress.
Calli treated with EMS were cut into pieces (1.5 mm diameter) and placed on semisolid MS media containing 15% and then 20% PEG, each time for 3 weeks. e calli that survived these water stress conditions were selected and transferred to a callus culture media. e selected calli were incubated in the dark at 25°C ± 2°C for 3 weeks; then the calli were subcultured 2 times for 3 weeks each. e fresh weights of the selected calli were recorded.
After in vitro mutagenesis and the selection of waterstressed EMS-treated calli, the surviving calli were regenerated to mutant sugarcane plantlets for evaluation at the plantlet stage. e mutant calli were cultured on MS media, which were supplemented with 10% coconut water, 0.25 mg/l of NAA, and 0.5 mg/l of kinetin for shoot regeneration (Supplementary Table 1). Calli were incubated under cool, white fluorescent light for 16 h a day at 25°C ± 2°C for 4 weeks. Plantlets regenerated from EMStreated calli were transferred to MS media without a plant growth regulator and incubated under cool, white fluorescent light for 16 h a day for 4 weeks.
Regenerated plantlets of the selected calli were cultured on MS media containing 20% PEG, followed by incubation under cool white fluorescent light for 16 h daily at 25°C ± 2°C for 16 weeks. e survival rate, number of shoots, number of roots, and root length were recorded.

Evaluation of Mutant Sugarcane under Managed Water Stress in Greenhouse.
e mutant sugarcane plantlets were transplanted in a greenhouse for evaluation under managed water deficit conditions. e soil moisture was controlled using a wet sensor. After the plantlets had been transplanted for 1 month, the water stress conditions were managed by stopping the application of water that decreased the available soil moisture content to approximately 20% lower than field capacity. e mutant sugarcane plantlets were evaluated under these water stress conditions three times throughout the experimental period. Sugarcane plant leaf damage was scored using a modified method developed by Fen et al. [26]. e experiment was conducted using an augmented randomized complete block design (RCBD) for all three replications. e KK3 sugarcane cultivar was used as a control for the experiment. e mutant sugarcane clones for waterstress tolerance were identified by comparing their leaf damage scores with the leaf damage score of KK3.

Statistical Analysis.
e collected data were statistically analyzed using analysis of variance for the experiments laid out in a completely randomized design and RCBD using the R program software [27]. Means between treatment were compared based on the least significant difference at P ≤ 0.05 (significant) and P ≤ 0.01 (highly significant).

Callus Induction.
Callus formation was discovered on leaf tissue after 7 days of culture on the callus induction media. e MS media supplemented with 3 mg/l of 2,4-D produced the highest rate of callus initiation (96.43%); the second-highest rate was observed in MS media supplemented with 3 mg/l of 2,4-D plus 15% coconut water (74.07%), as shown in Table 1.
e lowest rate of callus induction was produced using the MS media supplemented with 3 mg/l of 2,4-D, and 0.5 mg/l of BAP (9.47%). e calli on the MS media containing 3 mg/L of 2,4-D were yellowish and watery, while the calli on the MS media supplemented with 3 mg/L of 2,4-D and 0.5 mg/L of BAP were brown and watery. Calli induced on MS media supplemented with 3 mg/L of 2,4-D plus 15% of coconut water were mixed, being somewhat yellow and white in color (Supplementary Figure 1). Both friable and compact calli were found on the culture media. e biggest callus was on the MS media containing 3 mg/L of 2,4-D plus 15% coconut water, with the average size of the callus in this treatment being 0.8 cm in diameter.

EMS Mutagenesis.
e survival rates of calli from the EMS mutagenesis treatments and the control were significantly different and varied from 57.5 to 100% (Table 2). e highest survival rates at 100% were for the calli from EMSinduced mutagenesis with 10 mM EMS for 2 h. Friable callus formation was different in each EMS mutagenesis treatment. e lowest average survival rate was in the treatment of 16 mM of EMS for 4 h. e treatment of 16 mM of EMS for 4 h produced less than 20% friable callus formation (Table 2).

In Vitro Evaluation of EMS-Treated Calli under Water
Stress Conditions. Non-EMS-treated sugarcane calli were cultured on MS media containing PEG 6000 concentrations of 0, 5, 10, 15, 20, 25, and 30% for 3 weeks. e results showed that the growth of calli decreased when the PEG concentration increased (Supplementary Figure 2). Concentrations of PEG significantly affected the callus fresh weight compared to the initial callus fresh weight of 0.6 g. e highest callus fresh weight was 1.13 g (188.33% of the initial weight) for callus cultured on the medium without PEG. e calli cultured on MS medium with PEG concentrations of 5 and 10% had slow growth, with fresh weights after culture under the water stress conditions being 0.94 and 0.76 g, respectively.
e growth of calli was inhibited on MS medium with 15% PEG, and the callus fresh weight after the treatment did not differ from the initial weight of 0.6 g. e calli cultured on MS medium with 20% PEG concentration decreased the fresh weight from 0.6 to 0.56 g, with a decline in fresh weight of 6.67%. e callus fresh weights of MS medium supplemented with 25 and 30% of PEG were 0.52 and 0.41 g, respectively, with decreasing rates of 13.33 and 31.67%, respectively. According to this finding, the MS media containing 15 and 20% of PEG were used for screening mutant calli for water-deficit tolerance.
Evaluation of EMS-treated calli on the MS media containing 15% of PEG for 4 weeks showed a progressively decreasing weight (Table 3). e weights of the EMS-treated calli in the three treatments and the control on MS media containing 15% of PEG were significantly different. Evaluation of calli on the MS media containing 15% PEG for 6 weeks revealed that the weight of the EMS-treated calli was International Journal of Agronomy    Means with different letters within a column indicate significant differences (P < 0.05) according to the least significant difference test (LSD).
higher than for non-EMS-treated calli. e weight of the EMS-treated calli ranged from 4.61 to 2.57 g, while the non-EMS-treated callus had the lowest callus weight (0.34 g), as shown in Figure 1. Selected EMS-treated calli from the MS media containing 15% of PEG were cultured on MS media containing 20% PEG for 4 weeks. e weight of EMS-treated calli after culture on the media differed significantly. e weight of EMS-treated calli for each treatment on the MS media with 20% PEG was lower than those on the selective medium with 15% PEG. e selected EMS-treated calli from a selective medium with 15% PEG could survive on selective media with 20% PEG, while the control could not survive on the selective media after only 1 month of culture on the media. e weight of EMS-treated calli ranged from 2.47 to 1.53 g (Figure 2).

Mutant Plantlet Regeneration and Evaluation under Water Stress Conditions.
e number of mutant calli regenerated to plantlets did not differ significantly in the four treatments (Table 4). However, significant differences were found in the average number of regenerated plantlets among the four EMS treatments. Mutant calli treated with 16 mM of EMS for 4 h produced the highest mean number of regenerated plantlets (50.13), while non-EMS-treated calli produced the lowest (18.00).
Mutant sugarcane plantlets regenerated from the EMStreated calli and plantlets of KK3 cultivar were evaluated on MS media with 20% PEG for 4 weeks. e average numbers of roots were significantly different (Table 4). Furthermore, the survival rates of the mutant sugarcane varied noticeably: mutant plantlets from 10 mM of EMS for 2 h (72.25%), 10 mM of EMS for 4 h (75.85%), and 16 mM of EMS for 4 h (60.61%; Table 4). e lowest survival rate of sugarcane plants on selective media was recorded for non-mutant sugarcane plants (39.78%). All the mutant plantlets on the selection media with 20% PEG for 16 weeks survived; however, all the non-mutant sugarcane plantlets died.
In total, 136 sugarcane mutant genotypes were transplanted for evaluation under managed water stress conditions in a greenhouse. Damaged leaf areas were not visible after the first cycle of water stress. However, by the third cycle of water stress, damaged leaf areas were easily visible to the naked eye. Leaf damage scores after the third cycle of water stress were ranked from 0 to 10; the leaf damage score for KK3 (non-mutant) was 5.9. Leaf damage among the samples differed significantly. Out of the 136 sugarcane mutant genotypes, 14 mutant plants were evaluated with leaf damage scores lower than those of KK3. ese 14 mutant  Means with different letters within a column indicate significant differences according to LSD. NS, * , * * non-significant or significant at P < 0.05, 0.01, respectively.
International Journal of Agronomy sugarcane plants had leaf damage scores ranging from 0 to 0.8 ( Figure 3).

Discussion
Compact and friable calli were discovered in equal proportions on the MS media containing 3 mg/L of 2,4-D + 10% (v/v) coconut water, which was consistent with the findings of Patel [28]. Most of the calli induced on MS media containing 3 mg/L of 2,4-D were compact calli, which are difficult to regenerate in plantlets [29]. e MS medium supplemented with 2,4-D plus coconut water has been used effectively in other research studies to induce friable calli in sugarcane [30][31][32]. us, MS medium containing 3 mg/L of 2,4-D + 10% coconut water (v/v) was recommended for callus induction media to prepare calli for EMS mutagenesis.
Concentrations of EMS at 10.00-32.25 mM were used successfully to induce mutations in sugarcane [33]. However, the best conditions for EMS mutagenesis of the sugarcane cultivar KK3 were required to initiate genetic variation. Calli treated with 16 mM of EMS for 4 h resulted in the death of nearly 50% of the plants. e most suitable conditions for EMS mutagenesis of the KK3 sugarcane calli were 16 mM of EMS for 4 h, which was in agreement with Koch et al. [7].
Water stress on selective media using PEG is an efficient method for screening calli for water-stress tolerance [34,35]. If mutant calli can adjust their cell water potential under conditions of water deficit, then the mutant calli will be able to survive on the selective medium containing PEG [36]. Concentrations of PEG at 5 and 10% slowed the growth of calli, while PEG concentrations of 15 or 20% inhibited the growth of calli and the cells died within 8 weeks of culture. PEG concentrations of 25 and 30% decreased the weight of calli after the first week of culture, with most calli cultured on the media not surviving after culture for 4 weeks. PEG concentrations at 25 or 30% were too high to evaluate for water-stress tolerance. erefore, PEG concentrations of 15 or 20% were used as selective media for evaluating the waterstress tolerance of EMS-treated calli.
In the present study, some of the mutant calli could develop on the selective medium supplemented with 15 or 20% of PEG, respectively. Non-EMS-treated calli could not survive on the same selective medium supplemented with 15 or 20% of PEG. e study determined that the selected EMStreated calli were tolerant to water stress. e water stress-tolerant calli were regenerated to plantlets, and the plantlets were evaluated under conditions of water deficit using MS media supplemented with 20% PEG to confirm their tolerance. Mutant sugarcane plantlets regenerated from selected mutant calli survived on selective media containing 20% PEG, while non-mutant plantlets of sugarcane cultivar KK3 died under conditions of water deficit after culture on selective media. During this process of assessing the water-stress tolerance of plantlets, a group of mutant plantlets was identified that could survive on the selective media after culture for 16 weeks, while the non-mutant plantlets all died after culture on the selective media for 16 weeks.
ese results confirmed that the selected mutant plantlets on selective media were tolerant to water stress. Furthermore, the results indicated that in vitro evaluation of water-stress tolerance on MS media supplemented with 20% PEG was a highly effective screening method. e mutant sugarcane clones presented damaged leaf areas in the third cycle of drought stress and scoring the water-stress tolerance of each clone could carry out using visual inspection, while the second cycle of drought produced small dry areas on the sugarcane leaves. is could have been due to the root H 2 O 2 concentration in the third cycle of water stress being higher than in the first and second cycles; consequently, the accumulation of H 2 O 2 in roots in the third cycle resulted in root damage [22]. Reduced leaf area is a common response of plants when plant roots are damaged under water stress conditions [37]. e damaged leaf areas in the third cycle were categorized based on the level of water-stress tolerance of each clone, allowing leaf damage scoring to be applied as an efficient and economical method for the selection of tolerance to water stress in sugarcane clones under managed water stress conditions [38]. e water stress evaluation in the greenhouse confirmed that the in vitro-selected mutant sugarcane clones of the present study were tolerant to serious water stress conditions based on their damaged leaf area. Fourteen mutant sugarcane plants stayed green under serious water stress, but the KK3 cultivar presented approximately 50% leaf damage. Under conditions of water stress on selective media with PEG, the mutant sugarcane plantlets selected for their waterstress tolerance may have had mechanisms to adjust the cell water potential or to protect against cell damage, while the susceptible clones were unable to adapt to water stress and eventually died [39][40][41].
e findings in this study were consistent with other studies that mutant plants presented mechanisms to overcome conditions of dehydration [42,43]. However, only 14 sugarcane mutant plants presented leaf damage scores lower than those of KK3 under serious water stress in a greenhouse. e mutant sugarcane plants evaluated under water stress for 3 cycles displayed high levels of root damage because of the high accumulation of H 2 O 2 in roots, and only sugarcane mutants with defense mechanisms against oxidative damage in root tissue could survive the stress conditions [22,38].
e results of the present study indicated that the 14 mutant sugarcane plants contained mechanisms to overcome conditions of dehydration and had defense mechanisms against oxidative damage in roots. ese 14 mutant sugarcane plants will be utilized as sources of sugarcane germplasm for breeding sugarcane with water-stress tolerance in ailand.

Conclusions
EMS mutagenesis was applied to induce mutation in the KK3 sugarcane cultivar. Using 3 steps of water stress evaluation (at the calli, plantlet, and seedling stages), 14 out of 136 selected mutant sugarcane plants were confidently identified as new sugarcane clones with water-stress tolerance. e 14 sugarcane mutants survived on selective media supplemented with 15% or 20% PEG and stayed green even after the third cycle of water stress conditions in a greenhouse; in contrast, the popular ai KK3 sugarcane cultivar presented 50% damaged leaf areas. During the greenhouse evaluation of the mutant sugarcane plants based on their water-stress tolerance, tolerance was categorized by scoring the damaged leaf area on each plant after the third cycle of water stress.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.