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Effects of activated charcoal, plant growth regulators and ultrasonic pre-treatments on in vitro germination and protocorm formation of Calanthe hybrids

Many Asian orchids are threatened by extinction because of over-collection, habitat destruction and poor germination percentage (2%) in nature. In an attempt to develop a suitable protocol for the asymbiotic germination of Calanthe hybrid seeds, modified Hyponex medium supplemented with different concentrations of activated charcoal (0, 0.01, 0.1 g l-1), α-napthaleneacetic acid (NAA) and 6-benzylaminopurine (BA) (0.1, 0.5, 1.0 mg l-1) were tested. Addition of 0.1 g l-1 activated charcoal without NAA or BA in the modified Hyponex medium significantly accelerated the rate of seed germination. Whereas, the effect of NAA or BA without activated charcoal on the rate of seed germination was shown to be non-effective. However, addition of 0.1 mg l-1 NAA or 0.5 mg l-1 BA in combination with 0.1 g l-1 activated charcoal in the germination medium was proven effective to enhance rate of seed germination compared to other treatments. Meanwhile, the rate of seed germination and number of protocorm formation increased with increasing ultrasonic pre-treatment time (up to 10 min) without destroying embryos, showing significant effect especially for the cultivars characterized as low germination capability in nature.