An Effective Protocol for Callus Induction and Plant Regeneration in an Indica Rice Cultivar RD43

Abstract

Rice (Oryza sativa L.) is a staple food for billions of people globally. Rice cultivar RD43 has been recognized for its health benefits but has faced declining productivity due to climate change. Plant tissue culture serves as a powerful tool for studying and improving rice cultivars, yet a standardized protocol for rice cv. RD43 is lacking. This study aims to establish an efficient plant tissue culture protocol for rice cv. RD43 by evaluating concentrations of plant growth regulators for callus induction, proliferation, and regeneration. Callus induction was most effective with 4.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), while callus proliferation was effective with 2.0 mg/L of 2,4-D. Furthermore, 2.0 mg/L of 6-benzyladenine (BA) yielded the highest plant regeneration, achieving a 50% regeneration rate and producing 9.60 shoots per callus. These findings lay the groundwork for a robust tissue culture protocol for rice cv. RD43 as a means for advanced breeding studies and contributing to global food security amid climate change challenges.

1. Introduction

Rice (Oryza sativa L.) is one of the most important carbohydrate sources worldwide, serving as a staple food for over three billion people. It is projected that global rice demand will increase by approximately 40% by the year 2030. In Thailand, the Rice Department has registered the indica rice cultivar RD43 (SPR99007-22-1-2-2-1), notable for its medium-to-low glycemic index (GI) and low amylose content. These characteristics contribute to slower digestion and absorption, helping maintain lower postprandial blood glucose levels. Rice cv. RD43 also reduces the number of pregnant women with diabetes who need insulin [1]. However, its productivity is often hindered in drought conditions. Under drought stress, rice cv. RD43 shows marked decreases in key agronomic traits, including shoot height, leaf number, tiller number, flag leaf area, panicle weight, panicle length, seed fertility, and grain weight [2].

Plant tissue culture techniques have become vital tools in rice breeding, particularly for screening stress tolerance and elucidating the physiological and biochemical responses under stress [3]. The three fundamental steps of plant tissue culture, callus induction, callus proliferation, and plant regeneration, are highly dependent on the genotype and the plant growth regulators employed. Commonly, callus formation from mature rice seeds relies on 2,4-dichlorophenoxyacetic acid (2,4-D), used alone or in combination with other plant growth regulators. Different rice genotypes may require different types or concentrations of growth regulators [4]. After callus induction, callus proliferation involves expanding, multiplying, and maintaining undifferentiated cells in vitro, typically on a medium supplemented with 2,4-D and/or a cytokinin such as benzyladenine (BA) or kinetin [5]. Subsequent plant regeneration often employs media containing both auxins and cytokinins in balanced ratios, with lower auxin-to-cytokinin ratios favoring shoot formation and higher ratios promoting root development [6].

Despite rice cv. RD43’s valuable nutritional traits, an effective in vitro tissue culture protocol for this cultivar is lacking, especially in identifying the effective concentrations of plant growth regulators at each stage. Additionally, many indica rice genotypes exhibit suboptimal callus induction, proliferation, and regeneration efficiencies in vitro [7]. This study aims to identify the levels of auxins and cytokinins to improve callus induction, callus proliferation, and shoot regeneration in rice cv. RD43, thereby establishing a reliable tissue culture protocol that can support future breeding and improvement programs for this important cultivar.

2. Materials and Methods

2.1. Explant Preparation and Sterilization

Mature seeds of rice cultivar RD43 were manually dehusked and soaked in 70% ethanol for 1 min. Next, the seeds were surface-sterilized in 15% (v/v) Clorox^® solution (6% sodium hypochlorite) (The Clorox Company, California, USA) with 2–3 drops of Tween-20 for 10 min. Following sterilization, the seeds were rinsed three times with autoclaved distilled water (5 min per rinse) to remove residual disinfectant.

2.2. Callus Induction

Sterilized seeds were placed on a Murashige and Skoog (MS) medium pH 5.8 [8] supplemented with 30 g/L sucrose, 8 g/L agar, and 2,4-D at concentrations of 0 (control), 1.0, 2.0, 3.0, 4.0, or 5.0 mg/L. One seed was cultured per bottle (120 mL glass bottle containing 20 mL of the medium). Cultures were maintained in the dark at 25 ± 2 °C for 2 weeks. The following parameters were recorded to determine a suitable 2,4-D concentration for callus induction in rice cv. RD43: callus induction frequency, callus diameter, fresh weight, and dry weight.

2.3. Callus Proliferation

Calli (approximately 2 mm in diameter and 5.2 mg in fresh weight), induced from rice seeds on the MS medium containing 4.0 mg/L 2,4-D for 2 weeks, were used. These calli were transferred to the MS medium supplemented with 2,4-D at 0, 2.0, or 4.0 mg/L in combination with 0 or 1.0 mg/L BA. Cultures were maintained in the dark at 25 ± 2 °C for 10 weeks. Data on callus diameter, fresh weight, callus characteristics, and callus color were recorded to determine the most suitable conditions for callus proliferation in rice cv. RD43.

2.4. Plant Regeneration from Callus

Calli induced from rice cv. RD43 mature seeds were transferred to the MS medium containing 0 or 1.0 mg/L 1-naphthaleneacetic acid (NAA) combined with 0, 1.0, 2.0, 3.0, 4.0, or 5.0 mg/L BA. Cultures were maintained under a 16 h photoperiod at 25 ± 2 °C for 8 weeks. The parameters recorded included shoot regeneration frequency, number of shoots per callus, and number of roots per callus.

2.5. Statistical Analysis

The data were analyzed using R Statistical Programming Version 4.3.3 [9] with RStudio software (version 2024.12.1+563). Normality was tested using the Shapiro–Wilk test. As the data were not normally distributed, the Kruskal–Wallis test was used to assess differences among the treatments, followed by Fisher’s least significant difference (LSD) test with Holm’s method for p-value adjustment in post hoc comparisons. A main effects model was applied, focusing on primary factors without considering interactions. Outliers were identified using the interquartile range (IQR) method.

3. Results

3.1. Callus Induction of Rice Cv. RD43 Using 2,4-D

Mature seeds of rice cv. RD43 was cultured on an MS medium containing various concentrations of 2,4-D for 2 weeks. Each treatment consisted of 20 bottles, with one seed per bottle. Callus formation occurred in all treatments supplemented with 2,4-D, with no callus formed in the control (0 mg/L 2,4-D). The callus induction frequency was 75.00% on the MS medium containing 1.0 mg/L 2,4-D, whereas media with 2.0, 3.0, 4.0, and 5.0 mg/L 2,4-D achieved 100.00% (

Table 1. Effects of different concentrations of 2,4-D on callus induction after 2 weeks of culture. Twenty seeds were used for each treatment. Values represent the mean ± standard error (SE). Data were analyzed using the Kruskal–Wallis test followed by Fisher’s least significant difference (LSD) test with Holm’s method at p ≤ 0.05. Means followed by the same letter within the same column are not significantly different. Asterisks indicate significant differences.

MS Medium Supplemented with 2,4-D (mg/L)	Callus Induction Frequency (%)	Callus Diameter (mm)	Fresh Weight (mg)	Dry Weight (mg)
0 (control)	00.00 ± 0.00 c	-	-	-
1.0	75.00 ± 9.93 b	2.72 ± 0.20 c	3.80 ± 0.75 c	2.21 ± 0.45 ab
2.0	100.00 ± 0.00 a	3.45 ± 0.17 ab	7.51 ± 0.94 ab	2.33 ± 0.24 a
3.0	100.00 ± 0.00 a	3.66 ± 0.15 a	8.13 ± 0.80 ab	2.48 ± 0.21 a
4.0	100.00 ± 0.00 a	3.77 ± 0.18 a	9.98 ± 1.25 a	2.85 ± 0.31 a
5.0	100.00 ± 0.00 a	3.04 ± 0.18 bc	5.48 ± 0.73 bc	1.33 ± 0.16 b
Kruskal–Wallis test	<0.001 *	<0.001 *	<0.001 *	<0.001 *

). Although these four higher concentrations showed no statistically significant differences among themselves, they were significantly different from the control and 1.0 mg/L treatment (p ≤ 0.05).

Callus diameter generally increased with higher 2,4-D concentrations. The largest diameter (3.77 mm) was observed with 4.0 mg/L 2,4-D, followed by diameters of 3.66, 3.45, and 3.04 mm at 3.0, 2.0, and 5.0 mg/L 2,4-D, respectively, and 2.72 mm at 1.0 mg/L 2,4-D (Figure 1). Fresh weight of the callus increased as 2,4-D concentration increase, reaching 9.98 mg at 4.0 mg/L 2,4-D (highest). The fresh weights of calli induced at 2.0 and 3.0 mg/L 2,4-D were 7.51 mg and 8.13 mg, respectively, with no significant difference among these three concentrations. However, calli cultured with 5.0 mg/L 2,4-D showed a minor decrease in fresh weight (5.48 mg) (Table 1). Dry weight followed a similar trend. In media with 2,4-D at 1.0, 2.0, 3.0, 4.0, and 5.0 mg/L, callus dry weights were 2.21, 2.33, 2.48, 2.85, and 1.33 mg, respectively. Although 5.0 mg/L 2,4-D resulted in a minor drop (1.33 mg), there were no significant differences among 1.0–4.0 mg/L 2,4-D.

3.2. Callus Proliferation of Rice Cv. RD43 Callus

Calli induced with 4.0 mg/L 2,4-D (2 weeks old) were subcultured onto MS media containing combinations of 2,4-D (0, 2.0, 4.0 mg/L) alone or with BA (0, 1.0 mg/L) for 10 weeks (

Table 2. Callus proliferation of rice cv. RD43 after 10 weeks of culture. Each treatment was performed using 15 calli. Values represent the mean ± standard error (SE). Data were analyzed using the Kruskal–Wallis test followed by Fisher’s least significant difference (LSD) test with Holm’s method at p ≤ 0.05. Means followed by the same letter within the same column are not significantly different. Asterisks indicate significant differences.

MS Medium with		Callus Diameter (mm)		Fresh Weight (mg)		Characteristics	Color of Callus
2,4-D (mg/L)	BA (mg/L)
0	0	9.65 ± 0.26	abc	156.98 ± 8.80	b	friable callus with numerous roots	Dark brown–Black
2	0	10.70 ± 0.23	a	250.95 ± 9.66	a	friable callus	Yellow–Pale yellow
4	0	10.41 ±0.14	ab	231.68 ± 6.91	a	friable callus	Yellow–Pale yellow Some parts brown
0	1	9.08 ± 0.06	abc	160.45 ± 3.54	b	friable callus with roots	Dark brown–Black
2	1	8.76 ± 0.02	bc	124.95 ± 2.10	b	friable callus	Yellow–Dark brown
4	1	8.18 ± 0.26	c	133.68 ± 6.10	b	friable callus	Yellow–Dark brown
Kruskal–Wallis test		<0.001 *		<0.001 *

, Figure 2). For the MS medium without plant growth regulators, calli reached a diameter of 9.65 mm and a fresh weight of 156.98 mg. They were friable and rooted but turned dark brown, indicating partial tissue death. For the MS medium supplemented with 2,4-D (2.0 or 4.0 mg/L) alone, calli were friable, yellow–light brown, and generally larger. For instance, calli on 2.0 mg/L 2,4-D attained a fresh weight of 250.95 mg and a diameter of 10.7 mm. For the MS medium with BA only (1.0 mg/L), calli were friable with dark brown coloration and root formation (diameter 9.08 mm, fresh weight 160.45 mg). Finally, for the MS medium supplemented with 2,4-D (2.0 or 4.0 mg/L) and 1.0 mg/L BA, calli were smaller (diameters 8.76–8.81 mm) and fresh weights (124.95–133.68 mg) but remained friable with a yellow-to-dark brown hue.

Comparing treatments, calli cultured on the MS medium containing 2.0 mg/L 2,4-D alone showed the highest diameter and fresh weight. Although 4.0 mg/L 2,4-D also supported substantial proliferation, there was a slight reduction in callus size and weight. MS media lacking 2,4-D produced many roots but also led to significant callus browning and partial cell death. Taken together, the MS medium supplemented with 2.0 mg/L 2,4-D provided viable calli, exhibiting both the highest diameter and fresh weight. It appears to be a suitable proliferation medium for rice cv. RD43 calli.

3.3. Plant Regeneration of Rice Cv. RD43 Callus

Calli obtained from an MS medium supplemented with 4.0 mg/L 2,4-D after 2 weeks of induction were transferred onto regeneration media, where green spots indicative of shoot initiation appeared at around 2 weeks. Both shoots and roots formed on the same medium by 4 weeks, and the shoots elongated by 8 weeks (Figure 3). Regeneration rates ranged from 0.00% to 50.00% (

Table 3. Plant regeneration of rice cv. RD43 callus after 8 weeks of culture. The number of regenerated shoots and roots is expressed in relation to the number of explants that produced regenerants. Data were analyzed using the Kruskal–Wallis test followed by Fisher’s least significant difference (LSD) test with Holm’s method at p ≤ 0.05. Values represent the mean ± standard error (SE). Means followed by the same letter within the same column are not significantly different. Asterisks indicate significant differences (NS: not significant).

MS Medium Supplemented with		Plant Regeneration (%)	The Number of Shoots Per Callus	The Number of Roots Per Callus	Number of Calli Tested
NAA (mg/L)	BA (mg/L)
0	0	18.75 ± 10.08 ab	3.67 ± 0.14 b	11.33 ± 0.14	16
0	1.0	30.00 ± 15.28 ab	3.00 ± 0.84 b	7.67 ± 0.48	10
0	2.0	50.00 ± 16.67 a	9.60 ± 0.28 a	10.00 ± 0.50	10
0	3.0	0.00 ± 0.00 b	-	-	16
0	4.0	44.44 ± 17.57 ab	5.25 ± 1.10 ab	8.00 ± 1.19	9
0	5.0	12.5 ± 12.50 ab	1.00 ± 0.00 b	4.00 ± 0.00	8
1.0	0	0.00 ± 0.00 b	-	-	16
1.0	1.0	14.29 ± 9.71 ab	1.00 ± 0.00 b	5.50 ± 0.57	14
1.0	2.0	6.67 ± 6.67 ab	1.00 ± 0.00 b	5.00 ± 0.00	15
1.0	3.0	18.18 ± 12.2 ab	7.50 ± 0.21 ab	4.00 ± 0.43	11
1.0	4.0	28.57 ± 18.44 ab	1.00 ± 0.00 b	4.50 ± 0.27	7
1.0	5.0	0.00 ± 0.00 b	-	-	12
Kruskal–Wallis test		<0.05 *	<0.05 *	NS

). The number of tested calli per treatment was varied due to contamination. However, no statistically significant differences (p ≤ 0.05) were observed for the number of roots per callus.

For the MS medium with BA only, BA concentrations of 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/L provided regeneration frequencies of 18.75%, 30.00%, 50.00%, 0.00%, 44.44%, and 12.50%, respectively. The number of shoots per callus ranged from 1.0 (5.0 mg/L BA) to 9.60 (2.0 mg/L BA), and the number of roots per callus ranged from 4.0 (5.0 mg/L BA) to 11.33 (0 mg/L BA). The 0% regeneration rate at 3.0 mg/L BA was unexpected, given the general trend observed with other BA concentrations. To confirm this result, we conducted an additional experiment using more calli under the same 3.0 mg/L BA treatment, but no regeneration was observed in this treatment. For the MS medium with 1.0 mg/L NAA and BA, BA concentrations of 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mg/L produced regeneration frequencies of 0%, 14.29%, 6.67%, 18.18%, 28.57%, and 0%, respectively. The number of shoots per callus ranged from 1.00 (1.0 mg/L BA) to 7.50 (3.0 mg/L BA), while the number of roots per callus ranged from 4.00 (3.0 mg/L BA) to 5.50 (1.0 mg/L BA). Overall, the highest regeneration frequency (50.00%) and maximum shoots/roots were observed on an MS medium containing 2.0 mg/L BA alone. However, overall regeneration was markedly lower in all NAA-containing treatments compared to BA alone, suggesting an inhibitory effect of NAA on regeneration in rice cv. RD43 calli.

4. Discussion

In this study, rice cv. RD43 callus formation was first observed at approximately one week of culture, consistent with Thadavong et al. (2002) [10], who noted that callus generally emerges around the base of the shoot arising from the seed. Specifically, callus development occurs via cell division in the scutellum and mesocotyl regions of the embryo, stimulated by 2,4-D. An MS medium without plant growth regulators did not induce any callus formation, whereas the callus induction frequency rose from 0% (control) to 100% on media supplemented with 2,4-D at concentrations ranging from 2.0 to 4.0 mg/L. These concentrations differed significantly (p ≤ 0.05) from the control, underlining the effectiveness of 2,4-D for rice cv. RD43 callus induction.

Callus diameter and fresh weight peaked at 4.0 mg/L 2,4-D, then slightly declined at 5.0 mg/L. This is likely due to the inhibitory effect of excessive 2,4-D on cell division, corroborating previous findings [11]. Mondal et al. (2013) [12] similarly reported that 2,4-D levels ≥ 4.0 mg/L can be toxic for certain rice genotypes. Although the dry weights did not differ significantly among 1.0, 2.0, 3.0, and 4.0 mg/L 2,4-D, they did decrease under 5.0 mg/L, reinforcing the notion that overly high auxin concentrations can harm callus growth.

Here, MS media supplemented with 4.0 mg/L 2,4-D was selected as the most suitable concentration for callus induction of rice cv. RD43 because it consistently produced the largest and healthy calli. Although the differences were not statistically significant, the calli induced at 4.0 mg/L were more actively proliferating, which we considered important for supporting subsequent callus proliferation and regeneration stages. While this concentration is somewhat higher than the range reported in other varieties (2.0–3.0 mg/L) [4,13], it suggests that rice cv. RD43 may require a unique auxin dosage. Genetic variability between rice cultivars likely explains differences in 2,4-D responsiveness.

For callus proliferation, culturing on 2.0 or 4.0 mg/L 2,4-D produced calli that remained yellow-to-pale yellow and viable. However, 4.0 mg/L 2,4-D occasionally showed toxic effects, potentially causing localized browning and a decrease in diameter and fresh weight. Media without 2,4-D, meanwhile, induced rooting and caused partial callus death, implying that minimal residual 2,4-D from the induction stage might be insufficient for prolonged callus proliferation, though sufficient for root formation. This aligns with observations by Morini et al. (2000) [14], who reported that high 2,4-D levels reduce root formation but promote cell expansion without differentiation. Furthermore, higher concentrations of 2,4-D stimulated cell wall elongation, resulting in cell enlargement without differentiation into organs [15].

Adding BA to the proliferation medium diminished callus size somewhat compared to the medium supplemented with only 2,4-D, presumably due to an imbalanced auxin: cytokinin ratio [16]. Adjusting BA concentrations could enhance callus proliferation, as noted by Dhamotharan et al. (2021) [17] and Sawant et al. (2021) [18], where moderate 2,4-D combined with lower levels of kinetin or BA led to improved proliferation in various rice cultivars.

Auxin–cytokinin combinations frequently promote shoot regeneration in rice. For example, Binte Mostafiz and Wagiran (2018) [4] observed regeneration frequencies of 40–82% in certain rice varieties when the MS medium was supplemented with both auxin and cytokinin. In this study, an MS medium containing 2.0 mg/L BA yielded the highest regeneration rate of 50% for rice cv. RD43. Such variability in regeneration is driven by multiple factors, including genotype, explant developmental stage, and the concentration and ratio of plant growth regulators [19]. Here, callus viability appeared to decline during the regeneration phase, likely due to suboptimal growth regulator concentrations or callus sizes/ages that were not ideal for regeneration. Therefore, adjusting these factors could potentially enhance the efficiency of plant regeneration.

5. Conclusions

Callus induction in rice cv. RD43 reached 100% on MS media supplemented with 2.0, 3.0, 4.0, or 5.0 mg/L 2,4-D. The effective medium for callus induction was MS containing 4.0 mg/L 2,4-D, producing the greatest callus diameter, fresh weight, and dry weight. Meanwhile, an MS medium with 2.0 or 4.0 mg/L 2,4-D supported callus proliferation effectively, with the largest callus diameter and fresh weight achieved at 2.0 mg/L 2,4-D. For plant regeneration, the highest rate (50%) and maximum shoots per callus (9.60) were obtained with an MS medium supplemented with 2.0 mg/L BA. The effective protocol for callus induction, proliferation, and plant regeneration described here provides a robust framework for rice cv. RD43 improvement programs, especially under climate-change challenges.