First, the plant species used in this study is Begonia. The specific variety is called President-Carnot.
Second, the materials selected for the experiment include leaves, petioles, and stem sections. These explants were chosen due to their high regeneration potential and ease of handling in tissue culture.
Third, the culture conditions were carefully designed. The basic medium used was MS (Murashige and Skoog) medium. For callus induction, the medium was supplemented with BA (6-Benzylaminopurine) at 3–5 mg/L and NAA (Naphthaleneacetic acid) at 0.1–0.5 mg/L. For cluster bud proliferation, four different media were tested: (1) MS + BA 1.0 + NAA 0.1; (2) MS + BA 0.5 + NAA 0.1; (3) MS + BA 1.0 + NAA 0.1; (4) Strong seedling medium: 1/2 MS liquid. For rooting induction, the medium was 1/2 MS with 0.2 mg/L NAA. All media contained 3.0% sucrose, 0.8% agar, and were adjusted to pH 5.8. Cultivation was carried out at a temperature of 24 ± 2°C under moderate light conditions, approximately 800 lux, with a 10-hour photoperiod per day.
Fourth, the growth and differentiation process involved several key steps. First, sterilization of the explants was performed. Young leaves, petioles, and stem segments were washed thoroughly with tap water and detergent, then rinsed repeatedly. They were surface-sterilized using 75% ethanol for 30 seconds, followed by treatment in a 0.1% HgCl₂ solution for 2 minutes. After several rinses with sterile water, the explants were dried on sterile filter paper and cut into small pieces (0.5 cm² for leaves, 1 cm long for petioles and stems). These were then inoculated onto the callus induction medium.
Next, after one week of incubation, the explants began to swell and thicken, forming a hard texture. Yellow-green callus started to appear. By three weeks, small buds began to differentiate. Over the next five weeks, these buds developed into clusters. The clustered shoots were then transferred to proliferation media (2) and (3), where they continued to produce new buds. The propagation rates for leaves, petioles, and stems were similar across both media, with a multiplication factor reaching 6–9 after about four weeks of subculturing.
After achieving sufficient shoot proliferation, the culture was switched from solid to liquid medium. The shoots were moved to 1/2 MS liquid medium. After three weeks, robust, non-rooted seedlings were observed, which were suitable for future rooting and acclimatization. This step helps improve the plants' adaptability to external environments and enhances their resistance to diseases, making transplanting more successful.
Rooting was induced by transferring 3 cm tall, healthy seedlings to medium (5), which contained 1/2 MS + 0.2 mg/L NAA. Two weeks later, white roots emerged radially from the base of the shoots, with each plant producing 5–7 roots. After three weeks, the roots had elongated to 1.0–1.5 cm in length, and the rooting rate reached 100%.
Finally, the test-tube seedlings were transplanted. The flask lid was opened, and the seedlings were allowed to acclimate in a sterile, ventilated area for three days. They were then removed from the flask, and any remaining culture medium was gently washed off. The roots were briefly soaked in a 0.2% sodium sulfate solution before planting. The seedlings were transplanted into a mixed substrate of sterilized vermiculite, sand, and garden soil (1:1:1). A 0.1% carbendazim solution was sprayed over the foliage to prevent fungal infections. The pots were covered with plastic film, and water was misted twice daily. After one week, the survival rate exceeded 95%, indicating a successful acclimatization process.
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