In vitro physio and biochemical characterization of salt tolerance in rice (Oryza sativa L.) genotypes

doi:10.21203/rs.3.rs-2593583/v1

Salt stress induces oxidative damage to the cell by synthesizing reactive oxygen species. Salt-tolerant plants are potentially equipped with some defense mechanisms, such as enzymatic and non-enzymatic anti-oxidant properties. We attempted to characterize the physiochemical properties of the callus to select promising lines of rice at the cellular level under artificial salt stress induced by NaCl in vitro. In this study, we report the highest (19%) embryogenic callus induction frequency in CO 46 at higher NaCl stress (150 mM NaCl). Concerning the relative growth rate of the callus, we observed the highest RGE in BPT5204 and TRY1 suggesting that these could maintain a higher water potential and a fresh mass of the callus to survive salt stress. The genotype TRY 1 had the highest regeneration frequency (35%) in control (0 mM NaCl), but a significant reduction in RF (66%) was observed at 150 mM salt stress. The genotypes CO 50, CR 1009, and BPT-5204 registered the lowest regeneration frequency (6.7%) and produced a moderate number of shoots. Concerning the proline content high proline content in both the tolerant (BPT-5204) and sensitive (CO-46) cultivars was observed. Our result provided unique insight into the anti-oxidant properties of callus culture in rice. The anti-oxidative enzyme activities had increased progressively with increasing NaCl concentration in the medium. Genotypes BPT-5204 and TRY1 had a significant level of enzyme activities even at the highest NaCl treatments. Among the six genotypes, BPT-5204 and TRY1 were better in their performance with respect to the above parameters, which showed the physiological and biochemical homeostasis of the genotypes to salt stress.

In vitro

antioxidants

ROX

salt stress

homeostasis

For the development of salt tolerant lines, the elite plants selected from BPT 5204 and TRY regenerated in different NaCl concentrations were maintained at the greenhouse and can be uses for the downstream process.

Soil salinity is one of the prime stresses on the plant community, affecting the production of a vast range of agricultural crop plants (Qureshi et al. 2019). Approximately 20 to 50 percent of fully fertile irrigated soils on all continents have become saline, posing significant challenges for the over 1.5 billion people attempting to grow their food (FAO 2021; Hazman, 2014). Around the world, there are over 833 million hectares (8.7 percent of the planet) of salt-affected soil (Munns and Tester 2008), of which 6.73 million hectares is in India (Amaravel et al. 2019). In Tamil Nadu the area affected by salinity is 0.0427 million hectares, out of which the maximum extent of saline soil in the state occurs in Ramanathapuram (16,200 ha) and Trichy (11,165 ha) (Pandian and Vijayakumar 2017; Tamaraiselvi and Arulmozhiselvan 2020) Human activities such as industrialization, inappropriate agricultural practices, excessive or inappropriate use of fertilizers, deforestation, seawater inundation, or intrusion into groundwater are the prime cause of soil salinity which also aggravated by scarce availability of good-quality irrigation water in the country (Ahmad et al. 2019). Mounting pressure to produce more food per unit of arable land forced extensive use of brackish groundwater for irrigation, causing an excess salt accumulation in the soil. Rice (Oryza sativa L.) is the prime food crop, feeding approximately half of the world's population (Khokhar et al. 2021; Lou et al. 2012), and over 75 percent of the Asian population rely on rice as their primary food source (Hoang et al. 2016). Out of 148 million hectares of total arable land, rice accounts for 42.2 million hectares in India (Agricultural Statistics at a glance, Custom & DGCIS 2015 & information collected from trade Division). In the fiscal year 2021, India's rice production volume was 124 million metric tonnes (Statistical Research Department, October 5, 2022, in Production volume rice India FY 2010–2022). In the future, increase of per-unit area grain production could be a challenge because every unit increase in salinity above a certain threshold level will undoubtedly reduce rice production significantly. The threshold salinity level of rice is 3.0 dS m^-1, and above this threshold level, there is a reduction in yield of 12 percent per unit increase in dS m^-1 (Chandrashekhar 2020). Rice is a semi-aquatic cereal and moderately tolerant to salt (Kalaiyarasi et al. 2019). Soil salinity affects the plant at every stage, from germination to flowering, resulting in a crop with a low yield. Salt stress affects an array of the morphological, physiological, and biochemical functions in plants ranging from membrane instability, loss of ion- homeostasis, loss of turgor, and higher accumulation of reactive oxygen species (ROS) that affect the growth and development of the plant (Chinnusamy et al. 2005; Asada 2006). Salt-tolerant plants are potentially equipped with some defence mechanisms, such as enzymatic and non-enzymatic antioxidant properties, to ward off the deleterious effects of salt (Mamik and Sharma 2017.). Several of these antioxidants scavenged the ROS and avoided the potential damage caused to the cell. Among the known ROS scavengers, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) are the important components of the plant antioxidant defense system. The primary role of individual concern, SOD produces H₂O₂ upon reacting with superoxide radicals (Xing et al. 2015), mostly catalase (CAT) is present in plant peroxisomes where it chiefly removes H₂O₂ (Dat et al. 2000; Mittler 2002), and ascorbate peroxidase (APX) is involved in the detoxification of H₂O₂, maintaining membrane stability, and CO2 fixation (Ozyigit et al. 2016). Most of the evidence, by several authors, suggests that the salt-tolerant lines have improved activity of these biochemical properties compared to the susceptible lines. Conventional field screening techniques are extensive and tedious, alternatively biotechnological approaches specifically through in vitro screening have great potential in selection of promising elite line (Aazami 2010). Considering the above fact, the present investigation aimed to study the extent of morpho, physio, and biochemical properties involved in the potential salt stress response in the rice genotypes based on the screening work under in vitro conditions was carried out.

The present in vitro salinity screening work in rice was performed in the tissue culture unit, Department of Biotechnology, Vanavarayar Institute of Agriculture, Pollachi, during 2020–21.

Explant preparation

Six rice genotypes, viz., BPT–5204, CR 1009, TRY1, CO 50, Jodimattai, and CO 46, were collected from the Tamil Nadu Agricultural University, Coimbatore, and the Anbil Dharmalingam Agricultural College and Research Institute, Trichy. The technique of mature embryo culture was used for in vitro screening. The endosperm with an intact embryo was manually extracted from healthy, well-matured seeds and surface sterilized with 0.1% (w/v) HgCl₂ for 15 minutes followed by 70% ethanol for 30 seconds under aseptic conditions under laminar airflow chamber. The sterilized wet seeds were then thoroughly rinsed three times with sterile distilled water and blotted in 90-mm Petri dishes lined with filter paper.

Growth media and culture condition

Murashige and Skoog (1962) salts and vitamins supplemented with 2 mg L^-1 2, 4-D + 0.5 mg L^-1 Kinetin used for callus induction. The callus induction media consisted of various concentrations of NaCl (0, 20, 40, 60, 80, and 100 mM). Each treatment was maintained with three replications. The sterilized blotted-dried seeds were transferred to control as well as NaCl media and kept at 23 ± 2ºC under dark conditions. Callus induction was initiated 8-10 days after inoculations. Embryogenic calluses were then subcultured on fresh media after every two weeks for callus proliferation.

Morpho, Physio and biochemical analysis

Callus induction frequency (CIF)

At 15 days after incubation, the callus induction frequency (CIF) was calculated by dividing the number of seeds with callus by the total number of seeds inoculated and is expressed as a percentage as follows:

Embryogenic callus induction percentage (ECIP)

Calluses with creamy white, shiny, and friable would differentiate into plants when transferred to regeneration media are called embryogenic calluses. One-month-old calli isolated from explants were cultured on the same medium for another four weeks. The ECIP was calculated using the following formula:

Relative growth rate (RGR)

The relative growth rate of the callus was measured 60 days after inoculation according to the method of Amin et al. 2013. To determine the initial and final weight of the calli, the Petri plates along with media were weighed before and after inoculation with calli. The RGR was calculated according to the following formula:

Regeneration percentage (RP)

Embryogenic calli selected from each treatment were transferred to regeneration media containing 4 mg/L^-1 BAP and 0.1 mg/L^-1 Naphthalene acetic acid (NAA). Callus was incubated at 25 ± 2°C with a 16/8 h light/dark cycle and a light intensity of 40 μmol m⁻² S⁻¹. Each treatment comprised three replications, with six calli cultivated in each replicate. The RP was calculated using the following formula:

Enzymes extraction and assay

Proline content

The proline content of the callus was measured according to the standard procedure of Bates et al. 1973. The 0.5 g of fresh callus tissue was homogenized in 3% sulfosalicylic acid and centrifuged for 15 min at 5000 rpm. About 2 ml of supernatant was collected and incubated in acid ninhydrin and glacial acetic acid reaction mixture for 1 hr at 100^⸰ C. After incubation, the reaction mixture was kept at room temperature for cooling and extracted with toluene. The absorbance value was read at 520 nm in a spectrophotometer, where toluene was used as blank.

CAT activity

The CAT activity was measured according to the standard procedure given by Islam et al. 2009. In liquid nitrogen (N2), 0.5 g callus tissue had homogenized into fine powder. Powdered callus was then incubated in a reaction mixture containing 50 mM Tris-HCl buffer, 0.25 mM EDTA, and 20 mM H2O2. The suspension was centrifuged, and the supernatant was collected to measure the CAT activity. The absorbance had read at 290 nm in a spectrophotometer.

APX activity

The APX activity was measured according to the method of Hoque et al. (2007). The 0.5 g of the sample was homogenized in liquid nitrogen (N₂), and the fine powder was incubated in a reaction mixture containing 50 mM KH₂PO₄ buffer, 0.1 mM EDTA, 0.1 mM H₂O₂, and 0.5 mM AsA. The homogenate was then centrifuged, and the supernatant was collected to measure the APX activity.

SOD activity

SOD was assayed according to Cakmak and Marschner (1992). The 0.5 g of frozen callus homogenate had incubated in an assay mixture consisting of distilled water, 0.05 M pyrophosphate buffer, 186 M Phenazine Meta Sulfate (PMS), and 300 M Nitroblue Tetrazolium (NBT). In the end, 0.1 mM NADH was added to start the reaction, and the reaction mixture was incubated at 30^oC for 90 sec. Then the reaction was terminated by adding 1 ml of glacial acetic acid. The optical density (OD) was read at 560 nm.

Statistical analysis

The experiment was laid- out in a completely randomized design with three replications and six treatments [control (0 mM) and 30 mM, 60 mM, 90 mM, 120 mM, and 150 mM NaCl treatments]. Replicated data of various traits were analyzed using SPSS 16.0 software package. The mean difference had adjusted by Duncan’s multiple-range tests, and the results were furnished as mean ±SD (P ≤ 0.05) to compare the genotypes.

Callus culture is an indirect pathway for plant regeneration that could be an effective way to create new variations (Kadhimi et al. 2014; Al-Khateeb et al. 2020). The variations could be generated naturally and maintain the plant's nature. In vitro screening could be a potential alternative to field screening to identify promising genotypes by screening at the cellular level (Wani et al. 2010). The drastic effect of salts is convincingly sensed more readily at the cellular level than in the whole plant. The cell lines subjected to the stress could have gained the potential by regenerating into more tolerant genotypes (Rattana and Bunnag, 2014). To select promising lines at the cellular level, the implications of physiological and biochemical properties could be a handy tool. By considering the importance of various selection criteria, the aim of the present study was to elucidate the effect of NaCl on different selection parameters of callus.

Effect of NaCl on growth characteristics

Rice is a salt-sensitive crop compared to other cereals, and growth is highly disturbed when the salinity level exceeds 3.5 dS m⁻¹ (Zeng and Shannon 2000). To improve the rice salt tolerance it is reasonable to know about morpho, physio and biochemical changes in callus derived plants under salt stress (Kibria 2017; Bhowmik et. 2021). Our study shows the effect of salt on growth characteristics such as callus induction frequency (CIF), embryogenic callus induction frequency (ECIF), relative growth rate, and regeneration percentage of six genotypes subjected to various NaCl treatments. The results observed on CIP (Table 1, Fig. 1 & 2) showed a significant decrease in CIP at the highest salt stress (150 mM NaCl) in all the genotypes. Among the six genotypes, JODIMATTAI (traditional variety) registered with the lowest reduction in CIP (66.3%) when compared to the control (84.3%). Genotypes TRY 1 (salt-tolerant control) and BPT-5204 (moderately tolerant) were observed with 65.0% and 60.7% of CIP, respectively, at the highest salt treatment, on par with JODIMATTAI. CO 46 (salt-sensitive) had the lowest CIP at both control (63.67%) and 150 mM NaCl (43.3%). The rest of the genotypes, CO 50 and CR 1009, had moderate levels of CIP. Our result on CIP was in conformation with report of Alhasnawi et al. 2017, Rosimah Nulit et al. 2018, and Bhowmik et al. 2021 in rice. Many published studies adequately stated that salt inhibits callus induction by affecting ion homeostasis and causing membrane instability (Ashraf 1994). In contrast to salt-tolerant genotypes, salt-sensitive genotypes have a rapid loss of turgor (water relation) and reduced nutrient uptake, reducing a genotype's buffering capacity to salt stress (Ashraf 2009; Errabii et al. 2007). Embryogenic calluses, characterized by loose friable, and translucent nature, can differentiate into plants in regeneration media (Hoque et al. 2004; Ming et al. 2019).

Table 1. Callus induction frequency (CIF) of genotypes at various NaCl concentrations

Genotypes	Callus induction frequency (%)
Genotypes	Control	30 mM	60 mM	90 mM	120 mM	150 mM
BPT-5204	83.7±1.5^b	82.0±1.0^a	79.3±1.2^a	74.0±1.0^a	67.3±1.5^b	60.7±1.5^b
CR1009	73.3±2.9^c	71.3±3.2^b	65.0±1.0^b	59.3±1.5^c	55.0±1.0^d	46.7±1.5^d
TRY1	87.7±0.6^a	81.7±1.5^a	80.0±1.0^a	74.3±1.5^a	73.0±1.0^a	65.0±1.0^a
CO 50	82.7±1.5	71.7±2.9^b	67.0±2.6^b	62.7±2.5^b	59.0±1.0^c	55.3±1.5^c
JODIMATTAI	84.3±0.6^ab	83.3±0.6^a	79.7±2.5^a	75.3±1.5^a	70.7±2.5^a	66.3±1.5^a
CO 46	63.67±1.5^d	60.3±1.5^c	57.3±1.5^c	49.7±1.5^d	47.3±1.5^e	43.3±1.2^e

Values are mean ±SD (n=3) followed by different letter in the same column indicated significantly different at the P = 0.05probability level, based on Duncan’s multiple range test

The embryogenic nature of the genotypes presented in this study demonstrated the differential responses of six cultivars to the frequency of embryogenic callus induction. Callus which was 14-days-old was subcultured on fresh media to induce embryogenic callus. Results on ECIP presented in Table 2 showed that the genotype BPT 5204 was registered the highest (64%) ECIP at control followed by CO 46 (54.3%) and TRY 1 (54%). Compared to the control, the percentage reduction of embryogenic callus induction was very high (61%) at 150 mM NaCl treatment, as registered in CO 50. The ECIP was reduced with an increase in concentration of NaCl in the medium (Fig. 3 & 4). The genotype CO 46 had the highest ECIP (19%) at 150 mM NaCl. While the genotypes CO 50 and BPT 5204 were on par with CO 46, the genotype CR1009 showed low embryogenic callus. It was observed, in many reports that the callus exposed to the higher salt concentration appeared to have brown necrotic tissue that became compact and non-embryogenic (Reddy and Vaidyanath 1986; Rosimah Nulit et al. 2018).

Table 2. Embryogenic callus induction frequency (ECIF) of genotypes at various NaCl concentrations

Genotypes	Embryogenic callus induction frequency (%)
Genotypes	Control	30 mM	60 mM	90 mM	120 mM	150 mM
BPT-5204	64.0± 1.0^a	51.3±1.5^a	36.0±1.0^b	28.3±2.1^c	24.0±1.0^b	16.7±1.5^a
CR1009	48.7±2.1^c	38.0±1.0^c	27.0±1.0^d	18.0±1.0^e	12.3±1.5^c	8.0±1.0^c
TRY1	54.0±1.0^b	46.7±1.5^b	43.0±1.0^a	34.7±1.5^b	24.3±1.5^b	16.3±1.5^a
CO 50	45.0±1.5^c	40.3±1.5^c	35.7±1.5^b	28.3±1.5^c	22.3±2.1^b	17.7±1.5^a
JODIMATTAI	47.0±2.6^c	34.3±2.3^d	30.0±2.6^c	20.3±1.5^d	14.7±1.5^c	11.7±3.1^b
CO 46	54.3±1.5^b	50.3±1.5^a	44.7±1.5^a	37.7±1.5^a	29.3±1.5^a	19.0±1.0^a

Values are mean ±SD (n=3) followed by different letter in the same column indicated significantly different at the P = 0.05probability level, based on Duncan’s multiple range test

The RGR of rice callus was significantly affected by the application of salt stress in the medium. The salt-affected callus showed a significantly reduced fresh mass and water content of the tissues. The result on callus RGR presented in Table 3 showed that BPT–5204 had the RGR (1.5) at the highest salt treatment (150 mM NaCl) as against the control (2.4). The lowest reduction in RGR (28%) had observed at the highest salt concentration. While the highest reduction was observed in JODIMATTAI (52%) and TRY 1 (50%), the RGR (0.8) had recorded in CO 50 and CO 46. The genotypes BPT 5204 and TRY1 appeared to have the ability to maintain a balanced water potential and a fresh mass of the callus. The survival of the callus was significantly affected by the RGR of the callus. The results demonstrated that genotypes CO 46 and CO 50 had low RGR and were more susceptible to salt stress. Hahmed et al. (2007) reported the RGR rate of callus in rice genotypes decreases under NaCl stress. Similarly, Gul et al. (2022) in sugarcane reported a drastic change in the relative growth rate in response to stress, which was in agreement with our result. Salt stress reduced the callus induction and thus dry matter production (Alharby et al. 2016).

Table 3. Relative growth rate (RGR) of different rice cultivars in response to different level of salinity

Genotypes	Relative growth rate/ 8 week
Genotypes	Control	30 mM	60 mM	90 mM	120 mM	150 mM
BPT-5204	2.4±0.1^a	2.2±0.1^ab	2.1±0.1^a	1.9±0.1^a	1.7±0.2^a	1.5±0.3^a
CR1009	2.00±0.2^bc	1.9±0.3^c	1.7±0.2^b	1.6±0.3^bc	1.3±0.2^c	1.1±0.1^bc
TRY1	2.4±0.2^a	2.2±0.1^a	2.0±0.1^a	1.8±0.1^ab	1.5±0.2^ab	1.2±0.1^b
CO 50	1.5±0.1^d	1.4±0.2^d	1.2±0.1^c	1.0±0.1^d	0.9±0.1^d	0.8±0.1^c
JODIMATTAI	2.1±0.2^ab	1.9±0.2^bc	1.8±0.2^b	1.5±0.1^c	1.3±0.0^bc	1.0±0.1^bc
CO 46	1.7±0.3^cd	1.4±0.2^d	1.2±0.1^c	1.1±0.1^d	1.0±0.1^d	0.9±0.1^c

Values are mean ±SD (n=3) followed by different letter in the same column indicated significantly different at the P = 0.05probability level, based on Duncan’s multiple range test

Callus obtained in high salt concentration showed physiological adaptation and homeostasis, which was clear by from their ability to develop shoots. Embryogenic calluses (0.5 g in each treatment) were transferred to regeneration media. Table 4 presents the frequency of regeneration, which shows that the number of shoots regenerated decreased significantly as the concentration of NaCl increased. The current study found that genotypes exposed to salt stress had pronounced changes in RP. TRY 1 yielded the greatest number of plantlets (35%) in the control treatment. But there was a significant reduction (66%) in the number of regenerates when subjected to 150 mM NaCl. CO 50 and CR 1009 had the lowest RP (6.7%). The genotype CO-46 was on par with JODIMATTAI, while BPT 5204 showed an intermediate number of shoots. The salt-tolerant genotypes were observed to produce the most shoots under stress levels comparable to those at which the others failed to regenerate. Our findings are consistent with the report of Hannachi et al. (2021); Siddeswar and KaviKishor (1989) in rice in vitro screening.

Table 4. Regeneration frequency (RF) of different rice cultivars in response to different level of salinity

Genotypes	Regeneration frequency (%)
Genotypes	Control	30 mM	60 mM	90 mM	120 mM	150 mM
BPT-5204	26.7±1.5^c	22.7±0.6^c	19.0±1.0^b	15.7±1.5^b	12.0±1.5^b	8.3±1.5^bc
CR1009	21.3±1.5^d	20.0±1.0^d	17.0±2.0^c	13.0±1.0^c	10.3±2.1^c	8.0±1.0^c
TRY1	35.0±2.0^a	31.0±1.0^a	24.7±1.2^a	20.7±2.1^a	17.3±1.5^a	12.0±1.0^a
CO 50	20.3± 1.5^d	17.3±1.5^d	14.0±1.0^c	12.0±1.0^c	9.3±1.5^c	6.7±0.6^c
JODIMATTAI	30.7±1.5^b	26.3±0.6^b	22.7±1.5^a	18.7±1.5^a	14.3±1.5^b	10.0±1.0^ab
CO 46	28.7±1.5^bc	27.0±1.0^b	23.0±1.0^a	17.7±1.5^a	12.3±1.5^b	9.7±2.1^ab

Values are mean ±SD (n=3) followed by different letter in the same column indicated significantly different at the P = 0.05probability level, based on Duncan’s multiple range test

Proline activity in salt stress

Plants avoid osmotic stress caused by drought and salt by accumulating some intercellular solutes and proteins. Proline is one of the vital stress proteins and acts as an osmoprotectant in plant cells when subjected to stress (Handa et al. 1986; Summart et al. 2010; Aazami et al. 2021). Salt stress exhibited drastic changes in intercellular proline content (Fig. 1). The proline content was low at the control and progressively increased from 30 mM to 90 mM, but higher salt in the media drastically reduced the proline content. The result showed that CO 46 registered highest proline content (3.20 µmol/g^-1) at 90 mM and reduced (1.71 µmol/g^-1) at 120 mM and 1.33 µmol/g^-1) at 150 mM NaCl. The lowest proline activity was registered in TRY 1. The results suggested that the genotypes CO 46 and BPT 5204 had the highest intercellular proline activity, in contradiction to the previous report that the highest proline content has been attributed only to the salt-tolerant genotype. This report conclusively showed that proline supports survival rather than growth. However, a higher proline level at the highest NaCl could have drastic effects on callus tissue, leading to necrosis and the death of the cells. This supplements the report of Ahmad et al. (2007); Shah et al. (2012); Rosimah Nulit et al. (2018); and Alhasnawi et al. (2016) discussed the same in rice.

Antioxidants activity in salt stress

Salt stress induces oxidative damage to the cell by synthesizing reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide, and hydroxyl radicals (OH), which are responsible for membrane instability and cellular damage (Noctor and Foyer 1998; Munns et al. 2006; Gupta and Huang 2014). Several reports demonstrated that plants are naturally equipped with ROS scavengers to avoid lipid peroxidation, pre-radical formation, and protein destabilization (Cui et al. 2010; Tsai et al, 2004; Swapna 2003; Banu et al. 2009; Hasanuzzaman et al. 2014). The important ROS scavengers are antioxidants such as CAT, SOD, and APX. Fig. 8 displays the CAT activity, which shows that CAT activity increased with increasing NaCl concentration of the media. The highest CAT activity (115.3 µMol/Min^-1/g^-1) was registered in BPT-5204 at 150 mM, NaCl, which was significantly higher than the control (73 µMol/Min^-1/g^-1). The lowest (98.3 µMol/Min-¹/g^-1) CAT activity was noticed in CR1009 at 150 mM NaCl. The genotype JODIMATTAI was on par with BPT 5204, while others showed significantly varied CAT activity in response to different NaCl treatments. The result of SOD enzymatic activity is presented in Fig. 9. A significant difference in SOD activity was observed between the control and NaCl treatments. SOD activity exhibited a progressive increase in response to the increased NaCl concentration in the media. The highest SOD activity (280 µMol/min-¹/g^-1) had registered in BPT 5204 at 150 mM NaCl than the control (202 µMol/min^-1/g^-1). The genotype CO 50 had the lowest SOD activity (238.3 µMol /min^-1/g^-1) than the control and other genotypes. TRY 1 was observed to be on par with BPT 5204. The effect of NaCl on ascorbate peroxidase (APX) activity is presented in Fig. 10. The result showed that TRY 1 registered the highest APX activity (93.0 µMol/Min^-1/g^-1) at 150 mM NaCl. The APX activity of the callus grown on NaCl media displayed a several-fold increase and a significant difference among six cultivars at different NaCl treatments than the control. The genotype CR1009 showed the lowest (73 µMol/Min-1/g^-1) APX activity at the highest NaCl treatment. The results indicated that antioxidative enzyme activities increased progressively with increased NaCl concentration in the medium. Significant differences were observed in all cultivars for APX activity. Among the six genotypes, BPT 5204 and TRY1 showed significantly higher antioxidative enzyme activities at the highest NaCl treatment than the control. The present results are consistent with previous reports by Vaidyanathan et al. 2003; Alhasnawi et al. 2016, and Bhowmik et al. 2021, in rice.

Thus the result indicated that a progressive increase in salt concentration could drastically change the callus growth characteristics such as CIF, ECIP, RGR, and RF. increased activity of antioxidants in proportion to salt concentration in the media was observed in all the genotypes. Among the six genotypes, BPT 5204 and TRY1 were superior in their performance on the above parameters, which showed the physiological and biochemical homeostasis of the genotypes to salt stress. The results are to be validated in different salinity regime in soil.

SOD, Superoxide dismutase; CAT, Catalase; APX, Ascorbate peroxidase; CIP, Callus induction frequency; ECIF, Embryogenic callus induction frequency; RF, Regeneration frequency; RGR, Relative growth rate; ROS, Reactive oxygen species; H₂O₂, Hydrogen peroxide.

Acknowledgments

The authors would like to express their gratitude to the Professor and Head of the Department of Crop Improvement, Vanavarayar Institute of Agriculture, for the valuable support and appreciation for the conduct of the experiment.

Author contributions

G. Thamodharan: supervision, conceptualization, methodology, validation, resources, review and editing. All authors read and approved the final manuscript. P. Mathankumar: data analysis, curation and editing. T. Veeramani: conducting in vitro culture, methodology and biochemical analysis.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

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In vitro physio and biochemical characterization of salt tolerance in rice (Oryza sativa L.) genotypes

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Version 1

Abstract

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Key Message

Introduction

Material And Methods

Results And Discussion

Conclusions

Abbreviations

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References

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Version 1