Utilization of zygotic embryos of an economic rattan palm Calamus thwaitesii Becc. (Arecaceae) for somaplant regeneration and cryobanking

Zygotic embryos excised from immature green fruits of the rattan palm, Calamus thwaitesii and cultured for 16 weeks under optimum culture conditions in Murashige and Skoog (MS) medium supplemented with 31.67 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 35.23 μM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) produced mixed (compact and friable) calli at 70 and 92 % rates. The semi-friable part of the callus (~500 mg) separated and subcultured in medium containing 2.22 μM 6-benzyladenine and 1.07 μM α-naphthalene acetic acid produced groups of 10.37 ± 0.60-21.52 ± 0.48 discrete globular embryoids of varied size in 6-8 weeks. Calli raised in presence of 2,4,5-T were relatively more prolific, friable and embryogenic than those induced by 2,4-D. Embryoids (2.0-3.0 mm) isolated and cultured in basal medium germinated into plantlets at 65 % efficiency while the immature (0.5-2.0 mm) ones produced calloid structures. Approximately 15 % of the in vitro plantlets raised from the 2,4-D-induced embryogenic calli produced secondary immature embryoids on the sheath and lamina parts of leaves which were isolated and cultured in basal medium developed into rooted plantlets at 62 % rate in 12-16 weeks. The continued growth of the embryo-derived callus through successive subcultures together with differentiation of embryoids into plantlets, and the formation of immature embryoids on in vitro plantlets in MS basal nutrient medium reports for the first time a reliable method of producing at least 116 plants from a single embryo in a year. Rooted plantlets treated with 50 % glycerin survived at 78 % rate after hardening and 82.7 % of the hardened plants reintroduced into forest segments showed uniform growth free of morphological abnormalities after 3 years of observation. In addition to embryogenesis, cryopreservation of the zygotic embryos through simple drying and encapsulation-dehydration methods resulting 60-70 % recovery rates also offers another option for long-term conservation and sustainable utilization of this plant genetic resource.