Embryogenesis efficiency and genetic stability of Dianthus caryophyllus embryos in response to different light spectra and plant growth regulators

Aalifar, Mostafa; Arab, Mostafa; Aliniaeifard, Sasan; Dianati, Shirin; Zare Mehrjerdi, Mahboobeh; Limpens, Erik; Serek, Margrethe


Carnation is an important cut flower with industrial and medicinal applications. To establish an efficient protocol without somaclonal variation for micropropagation of Dianthus caryophyllus, direct and indirect somatic embryogenesis (DSE and ISE) were investigated under six different light spectra (white, red, green, blue, red + blue and far red + red) and four combinations of different plant growth regulators (PGRs) never tested so far for carnation. The best results were achieved with 2,4-dichlorophenoxyacetic acid (2,4-D) + N-(2-chloro-4-pyridyl)-N′-phenylurea (4-CPPU) for ISE and picloram + 4-CPPU or naphthoxyacetic acid (NOA) + 6-benzylaminopurine (BAP) for DSE. The DSE method was faster (3 weeks compared to 8 weeks) and easier (no subculturing compared to two rounds of subculture with ISE methods) but the percentage of somatic embryos in the ISE method was higher compared to the DSE method. Our results showed that the highest DSE, formation of embryogenic callus, embryo maturation (generation of globular, heart and torpedo shapes) and ISE rate was observed in carnation explants exposed to blue light (450–495 nm). In contrast, green (495–570 nm), red (610–700) and far red (710–730 nm) lights caused negative effects on embryogenesis compared to white light controls (380–750 nm). For the first time, genetic stability of regenerated carnation plants was estimated using inter-simple sequence repeat (ISSR) markers. The amplified products showed 75 distinct and scorable bands, and regenerants [plants obtained by primary (PSE) and secondary SE (SSE)] were completely identical to the mother plant. Similarly, flow cytometric analysis confirmed that somatic embryo-derived plants had on average 1.53 pg nuclear DNA (2C), and all plants maintained their ploidy. In conclusion, obtained embryos under blue light were big in size and torpedo-shaped and their germination was highest compared to other light spectra. Moreover, blue light was effective for direct and indirect somatic embryogenesis in carnation without induction of somaclonal variation.