Asymbiotic Germination and Leaf Explant-Based Regeneration of the Endangered Medicinal Orchid Hemipilia cucullata from Mature Seeds

My research focuses on optimizing tissue culture protocols for endangered orchids to enhance conservation, large-scale propagation, and sustainable utilization of these valuable medicinal plants. A major challenge is achieving consistent germination and regeneration rates across orchid species as each requires species-specific media optimization and the precise control of growth conditions. We have developed efficient tissue culture protocols for multiple endangered plants, enabling species recovery and supporting reconstruction into natural habitats.

No efficient reproducible tissue culture system exists for Hemipilia cucullata handling propagation, restoration, and conservation. Terrestrial orchids often need specific symbiotic fungi whose isolation and identification are complex. A symbiotic method simplify propagation and bypass these steps.

Our practical enables high germination and the rapid plant regeneration from mature states, eliminating fungal dependency and offering a faster, more reliable approach for Hemipilia cucullata propagation. To begin, place the capsule in a beaker. Add one to two drops of detergent into the beaker for rinsing.

Wash the capsule under running tap water for five minutes. Then using sterile absorbent paper, remove the remaining surface water from the capsule. Now, immerse the capsule in 75%ethanol for 30 seconds.

Sterilize the capsule by immersing it in 50 milliliters of 20%sodium hypochlorite solution containing approximately 5%available chlorine. Then add two drops of undiluted household dishwashing detergent to the solution and incubate the capsule for 10 to 12 minutes. Rinse the capsule five times with sterile distilled water to remove any residual disinfectant.

Then using a sterile scalpel, remove approximately one millimeter from both the apex and the pedicel ends of the capsule. Make a small incision at the upper end of the capsule to expose the seeds and collect them in a sterile Petri dish. Using sterile forceps, evenly disperse approximately 500 to 600 seeds into each culture bottle containing the prepared medium.

Allow the seeds to absorb water and settle at the bottom of the liquid medium. Maintain the cultures at 25 degrees Celsius with a 12-hour light and dark cycle and a light intensity of 36 micromoles per square meter per second. Observe seed germination weekly.

Record any visible morphological changes and photograph the cultures for documentation. Select healthy plantlets one to two centimeters in size without visible blackening or complete yellowing for shoot elongation. Using sterilized and cooled forceps, aseptically transfer 8-12 plantlets into each bottle containing freshly prepared shoot elongation medium.

Evenly distribute the plantlets within each bottle. After five weeks of shoot elongation, select plantlets with fully developed shoots and healthy leaves for leaf explant sampling. Excise fully expanded sterile leaves from a healthy plantlet using the sterile scalpel.

Remove the apical region and leaf margins. Then cut each leaf into approximately 0.5 by 0.5 centimeter segments using a sterile scalpel under aseptic conditions. Place 10 leaf segments onto the PLB induction medium with the adaxial surface facing upward.

Make sure each segment touches the medium firmly. Then culture them for five weeks. Finally, assess the number of successfully induced PLBs and calculate the induction rate as a percentage.

Record the average bud height in centimeters and count the number of buds formed per explant. Seed germination in liquid medium was visibly characterized by the emergence of green embryos from the seed coat at around 60 days, which subsequently formed spherical protocorms by 80 to 85 days. The highest germination rate of 72%was achieved in liquid medium supplemented with 0.5 milligrams per liter NAA.

B5 medium containing 0.5 milligrams per liter benzyladenine and 0.2 milligrams per liter NAA yielded the highest protocorm proliferation rate averaging 5.3 newly formed protocorms per explant. Protocorms cultured in B5 medium with the optimized growth regulator combination displayed vigorous growth and healthy green pigmentation, outperforming those grown on MS medium under the same conditions. The highest number of branches per plantlet averaging four was observed with one milligram per liter of each benzyladenine and NAA, followed closely by NAA alone at 0.5 milligram per liter.

Shoot elongation was most enhanced with the combination of 0.5 milligrams per liter benzyladenine and one milligram per liter NAA, resulting in an average plantlet height of 4.8 centimeters. The highest induction rate of protocorm-like bodies 44.3%was achieved using three milligrams per liter benzyladenine with 0.2 milligrams per liter NAA.