Microspore-derived embryo formation in radish (Raphanus sativus L.) according to nutritional and environmental conditions

Abstract

Composition of nutrient media, flower bud size, sucrose concentration, heat shock stress, and ethylene inhibitor could have marked effects on microspore embryogenesis. No microspore-derived embryos (MDE) were formed when the microspores were isolated from radish (Raphanus sativus L.) flower buds of 1.0–2.5 mm in size, whereas MDE were formed with microspores isolated from 2.5–4.5 and 4.5–6.5 mm flower buds. The microspores isolated from 2.5–4.5 mm flower buds showed high embryo yields. MDE formation was highest when 150 g·L⁻¹ sucrose was added to the half strength Nitsch & Nitsch (NLN) liquid medium, but at sucrose concentrations less than 100 g·L⁻¹ there was no MDE formation. Microspores cultured on half strength NLN liquid medium containing 0.05 mg·L⁻¹ silver nitrate (AgNO₃) produced the most MDE, showing more than two-fold increase in yield compared to those cultured on medium without AgNO₃. A heat shock pretreatment of microspores at 32°C for 24 h gave high-frequency production of MDE when compare to higher or lower temperatures; no MDE were formed at 42.5°C. The highest yield of MDE was observed when microspores were derived from 2.5–4.5 mm flower buds cultured on half strength NLN medium containing 150 g·L ⁻¹ sucrose, 0.05 mg·L⁻¹ AgNO₃, and precultured with heat shock pretreatment of microspores at 32°C for 24 h, followed by incubation 25°C for 30 days. A polyploidy test indicated that 19.7% of the microspore-derived plants were doubled haploid, other plants were haploid, and chimeras were haploid and diploid.