High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree (Neolamarkia Cadamba)

In this works, a simple, efficient and repeatable protocol was developed for in vitro regeneration via callus-mediated organogenesis of Neolamarkia Cadamba using cotyledonary petioles and hypocotyls. Effects of basal medium, plant growth regulators, the types and age of explant on the formation of adventitious buds/shoots were studied. Meanwhile, histological analysis for early ontogenic stages and genetic stability assessment by flow cytometry were investigated. Our investigation demonstrated that, compared with 6-benzyladenine (BA), N(6)-(2-isopentenyl) adenine (2-ip), Thidiazuron (TDZ) was the optimal cytokinin for buds/shoots induction on cotyledon and hypocotyl explants. Douglas-fir and sugar pine medium (DCR) supplemented with 22.7 μM TDZ and 0.27 μM α-naphthalene acetic acid (NAA) was most effective on bud induction, with the highest bud-induction rate and numbers of buds on cotyledon and hypocotyl explants. The available shoot per explant hit 35.2 when the induced callus sub-cultured to a medium without TDZ. It was found that TDZ could promote induction of the callus and the buds, however, continuous exposure beyond 4 weeks of supplemented high concentration (exceed 11.35 μM), TDZ was harmful to the proliferation and growth of buds/shoots. DCR appeared more efficiency than Murashige and Skoog medium (MS), Woody Plant medium (WPM), anther culture of cereal crops medium (N(6)) on bud induction. Age of cotyledon and hypocotyl explants in 20-day to 25-day was most beneficial to adventitious buds/shoots formation. Histological investigation confirmed that the buds originated from the wounded incisions of cotyledonary petiole and hypocotyl fragments, with callus formation. The regeneration plantlets were successfully acclimatized in greenhouse, yielded above 95% survival rate in field, exhibited normal morphology and growth characteristics. The analysis of flow cytometry on N. cadamba indicated no variation in the ploidy levels between the regenerated plantlets and the donor trees. The developed procedure can be used for mass production, germplasm exchange and transgenic studies to improve the resistance of the species via Agrobacterium-mediated.