First, the plant species: Begonia. The specific variety is not mentioned, but it refers to a type of begonia commonly used in tissue culture experiments.
Second, the material category includes leaf, petiole, and stem sections, which are the explants used for the tissue culture process.
Third, the culture conditions: The basic medium used is MS (Murashige and Skoog). For callus induction, the medium is MS supplemented with BA (Benzylaminopurine) at 3–5 mg/L and NAA (Naphthaleneacetic acid) at 0.1–0.5 mg/L. For cluster bud proliferation, there are four different media: (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 is 1/2 MS in liquid form. Rooting medium is 1/2 MS + NAA 0.2. All media include 3.0% sucrose, 0.8% agar, and pH adjusted to 5.8. The cultivation temperature is maintained at around 24°C, with moderate light intensity (about 800 lux) and a 10-hour light cycle per day.
Fourth, growth and differentiation:
(1) Obtaining sterile materials: Young leaves, petioles, and stem segments were first washed with water and detergent, then rinsed thoroughly under running tap water. Under aseptic conditions, they were wiped with 75% alcohol and immersed in a saturated bleach solution for 10 minutes, followed by 0.1% HgCl₂ treatment for 2 minutes. After several rinses with sterile water, the explants were dried on sterilized filter paper and cut into small pieces—0.5 cm² for leaves, and about 1 cm long for petioles and stems. These were then inoculated onto the callus induction medium (1).
(2) Induction and propagation of cluster buds: After one week of incubation, the explants began to swell and harden, forming yellow-green callus. By the third week, small buds started to appear. After five weeks, these buds developed into clusters. The clustered shoots were then transferred to media (2) and (3) for further proliferation. Within four weeks, the proliferation rate reached 6–9 times. This indicates that both leaf and stem explants showed similar efficiency in producing new shoots.
(3) After reaching a large number of proliferated shoots, the culture was switched from solid to liquid medium. The shoots were transferred to 1/2 MS liquid medium. After three weeks, robust, non-rooted seedlings were observed. This step helps strengthen the plants, improve their adaptability to external conditions, and increase disease resistance, making them more suitable for transplanting later.
(4) Root induction: Seedlings approximately 3 cm tall were placed on rooting medium (5). Two weeks later, radial white roots began to form, with each shoot developing 5–7 roots. After three weeks, the roots had elongated to 1.0–1.5 cm, and the rooting rate reached 100%.
(5) Transplanting test-tube seedlings: The flask lid was opened, and the seedlings were acclimatized in a ventilated area for three days. They were then carefully removed, and any remaining culture medium was gently washed off. Before planting, the roots were briefly soaked in a 0.2% Naâ‚‚SOâ‚„ solution. The seedlings were then transplanted into a mix of sterilized vermiculite, sand, and garden soil (1:1:1). A 0.1% carbendazim solution was sprayed over the foliage, and the pots were covered with plastic film. Water was misted twice daily. After one week, the survival rate exceeded 95%, indicating successful acclimatization.
Gelatin Size 1 Empty Capsule,Hpmc Empty Gelatin Capsules,Empty Capsules Size 1,Mixed Empty Pill Capsules
Ningbo Jiangnan Capsule Co., Ltd. , https://www.jncapsule.com