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Aloe Tissue Culture Rapid Propagation
Aloe vera is a perennial, evergreen, succulent herb belonging to the genus Aloe. In recent years, with extensive research into aloe's chemistry and pharmacology, there has been a global surge in interest in aloe-based health products. This has led to a growing aloe cultivation industry in China. However, one of the major challenges is that aloe cannot self-pollinate, making seed propagation difficult. Current breeding methods mainly rely on ramets and tillers, which are not efficient for large-scale production. As a result, aloe seedlings remain expensive. To address this issue, this article introduces an efficient technique for the rapid propagation of aloe seedlings through tissue culture of stem tips. This method allows for the mass production of millions of seedlings in a short time, offering a low-cost and high-efficiency solution.
**Materials and Media**
- Aloe seedlings with a height of 10–15 cm.
- Callus induction medium: MS + (1–6 mg/L) BA + (0.1–0.5 mg/L) NAA.
- Differentiation medium: MS + (2–4 mg/L) BA + (0.1–0.5 mg/L) NAA.
- Rooting medium: MS + (0.1–0.5 mg/L) IBA + (2–5 mg/L) PP33.
**Breeding Method**
1. **Callus Culture**
Healthy aloe seedlings were washed thoroughly with clean water. The leaves and most of the stems were removed, leaving only the shoot tips. These were rinsed again and surface-sterilized using 75% ethanol for 30–60 seconds, followed by mercury treatment for 5–10 minutes. After several rinses with sterile water, the shoot tips were cut into small sections and inoculated onto the callus induction medium. Cultures were maintained under 10–12 hours of light daily at 1000–2000 Lx, with a temperature of 25 ± 2°C. After 15–25 days, callus formation was observed, indicating active cell division.
2. **Subculture**
Once callus was formed, it was carefully transferred to fresh callus induction medium. The process involved removing any residual medium, washing the callus, and cutting it into smaller pieces before re-inoculation. This subculture step can be repeated multiple times, allowing one initial explant to generate a large number of new materials for further differentiation.
3. **Differentiation Culture**
Callus fragments were transferred to the differentiation medium and cultured under similar conditions. After about 30–50 days, buds began to form, followed by the development of small leaves. When the seedlings reached a suitable size, they were moved to rooting medium for further growth.
4. **Rooting Culture**
Seedlings with leaves reaching around 3 cm were transferred to the rooting medium. After approximately two weeks, roots developed, signaling the completion of the rooting phase.
5. **Hardening Off**
Once the roots were sufficiently developed, the seedlings were removed from the test tubes, and the root medium was washed off with clean water. They were then placed in a controlled environment with temperatures between 15–25°C and humidity levels of 60–80% for 12–24 hours to acclimate.
6. **Transplanting**
After hardening, the seedlings were transplanted into vermiculite-based nurseries. They were regularly misted with a nutrient solution and fresh water. Within 15–20 days, the seedlings were ready for field planting.
**Precautions**
1. When preparing the media, all reagents must be measured precisely. Sucrose can be replaced with white sugar to reduce costs. Autoclaving should be done at 120°C, 1.5 kg/cm² for 30 minutes. The culture room must be fumigated with formaldehyde and sterilized with UV light before each use.
2. If contamination occurs, contaminated cultures should be discarded immediately. For minor contamination, aseptically transfer the healthy parts to new media for continued growth.
3. Before transplanting, seedlings can be soaked in a rooting powder solution for 1–2 hours. During the domestication phase, attention must be given to environmental conditions and pest control. Spider mites and aphids can be managed with 40% omethoate EC diluted 1200 times. Black spot disease can be treated with 50% carbendazim WP at 1000 times dilution.