Reduced ascorbate and reduced glutathione improve embryogenesis in broccoli microspore culture

Abstract

Isolated microspore culture (IMC) is an effective alternative technique for the production of doubled haploid (DH) homozygous lines in plant breeding. However, death of most of the cultured cells during culture is a critical obstacle that affects embryo production in the practical application of IMC technology. In this present paper, we evaluated the effects of two antioxidants of both reduced ascorbate (ASC) and reduced glutathione (GSH) on microspore viability and the induction of embryogenesis in the IMC of broccoli (Brassica oleracea L. var. italica). The results showed that cell death occurred soon after stress treatment (at 32.5°C for 24h), and the number of dead cells reached about 80% after 2d. The addition of 10mgl−1 ASC and 20mgl−1 GSH significantly decreased microspore mortality, and had a strong effect on the number of embryos produced. In the genotype ‘B415’, embryo yields increased approximately 1.2-fold and 2.5-fold after the addition of the 10mgl−1 ASC and 20mgl−1 GSH, respectively. Moreover, the addition of ASC and GSH also influenced positively the rate of plant regeneration, which respectively increased by 4.2% and 9.7% for ‘B415’. Taken together, our results show that the induction of embryogenesis in broccoli is strongly affected by the use of ASC or GSH. The effects on embryogenesis induction could confirm the importance of the redox environmental regulation in IMC, specifically at the early phase of IMC. The obtained knowledge may accelerate and guide the application of microspore-derived DH plants as a breeding tool in broccoli.