Detailed Explanation of Key Points of Aseptic Seeding Techniques for Orchid Seeds

I. Capsule Treatment

1. Selection and Pretreatment

Select capsules at a certain number of days after pollination, generally choosing capsules with a slightly yellow outer skin and lusterless pericarp.

For the collected capsules, first rinse off obvious dirt such as mud and dust on the surface under running tap water, then use cotton dipped in diluted washing powder solution to wipe the surface and the grooves, then rinse under running tap water for 15-30 minutes, and use a scalpel to cut off the front section (be careful not to cut too much, to prevent the capsule from splitting).

2. Surface Disinfection

After the rinsed capsules are air-dried, proceed on the ultra-clean workbench depending on the situation.

a. When the capsule is relatively large and the pericarp is thick and hard, place it in 75% alcohol to soak for several seconds to several minutes, then take it out and burn it for sterilization; surface disinfection is complete once it is burned dry.

b. When the capsule is relatively small, first soak it in 75% alcohol for 5-10s, then place it in 0.1% mercuric chloride with added Tween for disinfection for 15-20min, then rinse the capsule with sterile water 4-5 times, 1-2min each time, take it out and air-dry it on sterilized kraft paper.

II. Sowing

On the ultra-clean workbench, place the already disinfected capsule on a sterile pad, hold the capsule with sterilized tweezers, use a scalpel to cut off both ends of the disinfected capsule (until powdery seeds are visible), then split the capsule open from the suture, scrape out the seeds inside the capsule, and evenly spread them on the surface of the culture medium.

III. Induction Culture

1. Culture Medium

For the induction medium, Hyponex No. 1 and MS medium are often used, with added activated charcoal, sucrose, agar, and 6-BA, NAA, etc. in proportions according to the different species, with the pH adjusted to 5.4-6.0.

2. Culture Conditions

The culture temperature is about 26°C. In the early stage, dark culture or scattered light is sufficient. After the protocorms have noticeably grown larger, provide light for 12h/d, with a light intensity of about 2000 Lx.

After a certain number of days from sowing, the seeds begin to germinate after being cultured on the induction medium for a certain number of days, but no root elongation occurs; instead, the embryo gradually swells. When one end of the seed coat ruptures, the swollen embryo is yellow, similar to callus tissue. With continued culture, the embryo surface becomes covered with many small conical protocorm-like bodies. As the culture time is extended, the small conical bodies gradually grow larger, forming green spherical protocorms that can be used for proliferation, subculture, and differentiation.

3. Subculture

After a certain period from sowing (about 50-60d), after the seeds have germinated into sufficiently large protocorms, the space in the original induction culture flask is almost completely filled. To increase the growth space, flask division can begin. When dividing into flasks, try to separate the small plantlets as much as possible and distribute them evenly in the culture medium. Generally, divide into flasks once every (60-80d).

IV. Seedling Strengthening and Rooting Culture

After several rounds of subculture, some protocorms gradually grow into small plantlets with 1-2 small leaves, and some small plantlets also have 1-2 thick, short green roots. At this time, these small plantlets are not yet ready for direct hardening and transplanting, so seedling strengthening culture can be carried out. Depending on the orchid variety and plant size, add banana puree, coconut water, etc. to the seedling-strengthening and rooting medium, and continue culturing until they grow into small plantlets with 3-5 leaves, 3-4 roots, and a height of 5cm, after which they are transplanted and acclimatized.

The culture temperature is about 26°C. In the early stage, the light intensity is about 2000 Lx, and the light duration is 12h/d. In the later stage, scattered sunlight can be used, with a light intensity of (800-1000) Lx.