Colchicine, trifluralin, and oryzalin promoted development of somatic embryos in Ilex paraguariensis (Aquifoliaceae)

Abstract

A protocol for somatic embryogenesis and plant regeneration of Ilex paraguariensis St. Hil. from embryos cultures was developed. Heart stage zygotic embryos were removed from seeds of immature, light green fruit and treated with antimicrotubule agents (0.1; 0.2, and 0.5% colchicine for 24 and 48 h; 1; 10, and 20 μM of either trifluralin, ααα- trifluoro- 2,6-dinitro-N,N- dipropyl-p-toluidine, or oryzalin, 3,5-dinitro-N4, N-dipropylsulphate during 48 h). The embryos were cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar (1/4MS), and 0.46 μM zeatin. Cultures were incubated in darkness at 27 ± 2 ◦C. All the treatments provoked a diminution of the number of germinated embryos and in some of the treated embryos somatic embryogenesis was induced. Somatic embryo maturation and conversion into whole plants could be achieved by culturing the embryos on 1/4MS lacking hormones and incubated at 27 ± 2 ◦C, 14 h photoperiod (116 μmol m−2 s −1). Most of the plants regenerated from somatic embryos appeared morphologically normal and grew under greenhouse conditions. Only 2 plants out of 152 studied contained the tetraploid number of the chromosomes (2n = 4x = 80), meanwhile the rest of the plants had the normal diploid number of chromosomes (2n = 2x = 40). Somatic embryos with abnormal morphology were also observed.