Simultaneous production of triploid and hexaploid plants by endosperm culture with colchicine treatment in diploid Haemanthus albiflos

Abstract

Haemanthus albiflos (Amaryllidaceae) is a monocotyledonous plant with high ornamental value. To increase the ornamental value of this species, polyploidy breeding was programmed in this study by employing endosperm culture for producing triploid plants. Further, we tried to produce hexaploid plant by colchicine treatment to endosperm-derived callus. As an initial step, endosperm explants harvested 8 weeks after flowering were cultured on Murashige and Skoog (MS) medium containing 5 mg L−1 4-amino-3,5,6-trichloropicolinic acid (picloram) and 5 mg L−1 6-benzylaminopurine (BAP) for callus induction. Induced calli were maintained on the same medium. Somatic embryos and adventitious shoots were observed after transfer the callus segments onto 1/2MS medium without plant growth regulators (PGRs). Further development of somatic embryos and adventitious shoots were achieved by transferring the callus segments onto 0.5 mg L−1 1-naphthaleneacetic acid (NAA) and 1 mg L−1 BAP. Complete plantlets were established after placing the regenerated shoots and developing embryos on 1/2MS medium without PGRs. Flow cytometric analysis revealed that diploid, triploid, and tetraploid plantlets were involved in these regenerants. When the endosperm-derived callus segments were treated with 0.2% (w/v) colchicine for 24, 48, and 72 h, 4 hexaploid plants were regenerated through the same regeneration procedures as mentioned above. Ploidy levels of the representative diploid, triploid, and hexaploid plants were also confirmed by counting chromosome numbers. Availability of endosperm culture in combination with colchicine treatment was discussed as the means for producing polyploid plants. Triploid plants were regenerated from endosperm-derived callus via somatic embryogenesis and adventitious shoot formation in diploid Haemanthus albiflos. Hexaploid plants were induced by colchicine treatment to the endosperm-derived callus.