Mature dry seeds of six elite Malaysian Indica rice varieties (MRQ50, MR269, MR276, MR219, MRQ74 and MR84) were used. All seeds were supplied by the Malaysia Agriculture Research Development Institute (MARDI). In addition, the experiment was designed based on Randomized complete block design (RCBD).

Hull from all mature seeds containing scutellar region of embryo was removed. Dehulled seeds were sterilized with 70% alcohol for 1 min, followed by shaking in a 40% sodium hypochlorite solution containing three drops of polyoxyethylene sorbitan monooleate in an orbital shaker, at 120 rpm for 20 min. Finally, the explants (seeds) were rinsed with sterile distilled water five times.

Five sterile seeds were placed in each Petri dish (100 mm × 15 mm) containing 20 mL of MS media supplemented with 2,4-D (MS salts and vitamins, 300 mg/L casamino acid, 2 mg/L 2,4-D, and 2.8 g/L gelrite, pH 5.8). Each four Petri dishes were measured as a replicate and totally 12 Petri dishes, three replicates, were analyzed for every one variety. Then, the cultured seed were incubated in the dark at 25 °C. After three weeks, yellowish white embryogenic calli had developed on the scutellar surface.

The produced calli were transmitted to the MS media supplemented with 2, 4-D containing MS salts and vitamins, 300 mg/L casamino acid, 2 mg/L 2,4-D, and 2.8 g/L gelrite, pH 5.8 for proliferation of the calli. The calli were kept in the dark at 25 °C for 10 days.

The healthy surviving MR219 embryogenic calli were transferred to the MSKN regeneration medium (MS salts, MS vitamins, 2 mg/L kinetin, 1 mg/L NAA, 300 mg/L casamino acid, 30 g/L sucrose, 0.1 g/L myo-inositol, 2.8 g/L gelrite, pH 5.8). The cultures were kept in the dark for 20 days. The emerging shoots were harvested and then transferred to the fresh regeneration media (MSKN). The cultures were placed in the light at 27 °C with a 16-h photo period (110 μmol/m²/s) for 20 days.

Finally, the healthy shoots were transferred into the rooting medium (MSO) containing MS salts, MS vitamins, 300 mg/L casamino acid, 30 g/L sucrose, 0.1 g/L myo-inositol, 2.8 g/L gelrite, pH 5.8. The cultures were kept exposed to light at 27 °C with a 16-h photo period (110 μmol/m²/s) for 14 days.

After 14 days, when a well-developed root system was observed, the culture medium was removed gently from the roots of plantlets using water. After that, the plantlets were transferred individually into the Yoshida culture solution in a greenhouse with a 14-h photo period (160 μmol/m²/s), 95% relative humidity, and 29 °C day/light temperature and kept for 21 days. Subsequently, the plants with vigorous root systems were transferred into the pots containing paddy soil, water (1 L per pot and 500 mL each day until maturity, and sufficient initial fertilizer – a mixture of fertilizers, 2.5 g of (NH₄) SO₂, 1.25 g of P₂O₅, 0.75 g of K₂O per pot). About 2.5 g of (NH₄) SO₂ were added into each pot at the initial stage of flowering. Panicles were harvested when 85% of the color of the grains turned straw gold.

Various important traits were measured during the experiment to explore the responses of the different varieties to the regeneration procedure. The considered traits included callus induction day, number of contaminated seeds, number of dead seeds, percentage of dead seeds, number and percentage of callus induction frequency, number of explants producing embryogenic callus, EF (Embryogenic Frequency), browning rate (%), plant regeneration frequency, fresh weight and dry weight of four- to nine-week-old calli.

The collected data were statistically analyzed by SAS version 9.3. The level of significance was evaluated from analysis of variance (ANOVA). The mean of different traits among varieties were investigated using Duncan's multiple range.

There were no significant differences among varieties in term of number of contaminated seeds. The lowest number of contaminated seed (3.33) was observed in MR269 and MR276 varieties. While, the highest number of contaminated seed (4.66) was recorded for MR219 and MRQ50 (Tables 1–4).

Calli were variably developed from the scutellar region of seeds and were visible within 6–20 days. The varieties were significantly differentiated in terms of callus-induction day (51.06^**), callus-induction frequency (149.16^**), number of calli induced (5.96^**), number of dead seeds (3.03^**) and percentage of dead seeds (75.83^**) (Tables 1 and 2). The comparison of the varieties as regards the callus-induction day showed that the minimum number of days needed for callus induction corresponds to MR219 and the maximum one to MR84. The highest and lowest callus induction frequency (70% and 51.66%) and number of induced calli from seeds (14 and 10.33) were observed in MR269 and MRQ74, respectively. After the callus induction step, the number (5.33 and 2.66) and the percentage of seeds dead (26.66 and 13.33) were counted and showed that the highest number of dead seeds corresponded to MRQ74 and the lowest one to MR269, respectively (Tables 3 and 4 and Fig. 1).

The rice varieties used in this study were significantly differentiated based on the number of explants producing embryogenic calli (5.83^*), embryogenic frequency (145.83^*) in 5% of probability level and browning rate of calli (681.25^**) at the 1% of probability level (Tables 1–4). The maximum and minimum numbers (7.66 and 4) and frequencies of embryogenic calli (38.33% and 20%) were recorded in MR219 and MRQ74, respectively. However, the highest browning rate was observed in MR84 (87%) and the lowest rate in MRQ50 (46%) (Tables 3 and 4). The maximal fresh and dry weight of calli during four up to nine weeks of culture was observed in the sixth and seventh weeks in all varieties (Figs. 2 and 3).

The variance of rice varieties was significantly variable based on the plant regeneration frequency (681.25^**) at the 1% level of probability. The majority of plants regenerated from embryogenic calli were obtained from MRQ50 (54%) and the minimum number of plants from MR84 (Fig. 4).

Assessment of simple correlation coefficients of traits among six rice varieties illustrated that obtaining embryogenic calli was affected positively by the callus induction day (r = 0.596^**). In addition, the frequency of callus induced from the seeds was affected negatively by the number of dead seeds (r = –0.781^**). However, the plant regeneration frequency was significantly negatively correlated to the browning rate (r = –1^**). The negative but non-significant correlations were founded among different traits, including callus induction day (r = –0.370), number of callus induction (r = –0.262) and embryogenic frequency (r = –0.366) with browning rate and number of dead seeds with embryogenic frequency (r = –0.405) (see Table 5).

The six Malaysian rice varieties were clustered to two categories based on the measured characters and responses to specific media. Cluster I was composed of the varieties that weakly responded to the media in different stages (MRQ74 and MR84), while cluster II was comprised of four varieties that satisfactorily reacted to the media (MRQ50, MR219, MR269 and MR276) (Fig. 5).