The propagation of aloe vera is commonly carried out using the ramet method or bud insertion, both of which have low propagation rates and are seasonally restricted. However, tissue culture offers a more efficient and flexible alternative. By inducing callus and shoots from explants and then expanding them through subculturing, it is possible to produce large numbers of aloe plants in a short time, regardless of the season. This technique is especially beneficial for introducing new varieties, as it allows for consistent and controlled production.
**1. Materials and Methods**
- **Plant Species:** Aloe vera
- **Explant Type:** Young stem segments
- **Culture Conditions:** The basic medium used is MS (Murashige and Skoog) medium. Additional growth regulators include:
- 0.5–3 mg/L 1BA (Benzylaminopurine, a cytokinin) + 0.01–0.5 mg/L NAA (Naphthaleneacetic acid)
- 2 mg/L 1BA + 0.5 mg/L NAA
- 0.5–1 mg/L 2BA + 0.01–1 mg/L NAA + 0.2–1 mg/L GA3 (Gibberellic acid)
- 0.2–0.4 mg/L IBA (Indole-3-butyric acid) + 1 mg/L CCC (Chlormequat chloride)
- Culture temperature: 23–27°C
- Light intensity: 1500–2000 lux, with 10 hours of light per day
**2. Experimental Procedure**
Stem segments from actively growing aloe plants are surface-disinfected and cut into 0.5–1 cm pieces. These are then placed on a sterile bench and inoculated onto the first culture medium (Medium 1). After 2–3 weeks, the explants begin to form callus and a few clustered shoots. These are then transferred to Medium 2, where a significant number of shoots develop within one week. Once the shoots reach a height of 1.5–2 cm, they are separated and re-inoculated onto Medium 2. After about three weeks, a complete plantlet forms, with a height of 3–5 cm and root length around 2 cm. For large-scale propagation, the callus can be subcultured on Medium 1 to expand the population.
**3. Clonal Establishment and Storage**
Aloe callus can be stored as a genetic resource at 5°C. Even after five months of storage at this temperature, the callus remains viable and can still be induced to regenerate. Before use, stored callus should be cultured in the lab for three days to check for contamination. If no contamination is observed, the callus can be expanded further. In most experiments, there was minimal contamination, and the media could be kept fresh for extended periods.
**4. Transplanting and Management of Test Tube Seedlings**
Test tube-grown aloe seedlings have limited adaptability to external conditions and require careful acclimatization. Before transplanting, the lid of the culture vessel should be opened gradually, and the seedlings should remain in the growth room for 2–3 days. When removing the seedlings, care must be taken to avoid damaging the roots. The roots should be gently washed in room-temperature water to prevent rotting. A suitable substrate can be a mix of garden soil and vermiculite or pure vermiculite. After planting, the seedlings should be covered with a thin plastic sheet and misted daily for 30 minutes. After four weeks, the seedlings will be ready for transplanting into the field.
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