Improving the efficiency of isolated microspore culture in six-row spring barley: II-exploring novel growth regulators to maximize embryogenesis and reduce albinism

Abstract

Two alternative cytokinins, thidiazuron and meta-topoline, were tested in isolated microspore culture on recalcitrant barley genotypes (six-row, spring), and green plant regeneration was improved substantially. Doubled-haploid (DH) plants are coveted in plant breeding and in genetic studies, since they are rapidly obtained and perfectly homozygous. In barley, DHs are produced mainly via androgenesis, and isolated microspore culture (IMC) constitutes the method offering the greatest potential efficiency. However, IMC can often be challenging in some genotypes because of low yield of microspores, low regeneration and high incidence of albinism. Six-row spring-type barleys, the predominant type grown in Eastern Canada, are considered recalcitrant in this regard. Our general objective was to optimize an IMC protocol for DH production in six-row spring barley. In particular, we explored the use of alternative hormones in the induction medium (thidiazuron and dicamba), and in the regeneration medium (meta-topoline). This optimization was performed on two typical six-row spring (ACCA and Léger), a two-row spring (Gobernadora) and a two-row winter (Igri) barley cultivar. When 6-benzyl-aminopurine (BAP) was replaced by a combination of thidiazuron and dicamba in the induction medium, a 5.1-fold increase (P < 0.01) in the production of green plants resulted. This increase was mainly achieved by a reduction of albinism. Moreover, a 2.9-fold increase (P < 0.01) in embryo differentiation into green plants was obtained using meta-topoline instead of BAP in the regeneration medium. Together, these innovations allowed us to achieve a substantial improvement in the efficiency of IMC in this recalcitrant type of barley. These results were later successfully validated using sets of F1s from a six-row spring barley breeding program.

The effect of copper on plant regeneration in barley microspore culture

“…The applicability of the isolated microspore system is dependent on the proportion of obtained embryos converted into green plants. For this reason much effort is dedicated to establishing efficient protocols of the microspore culture (Esteves et al 2014). It is well documented that in barley anther culture additional Cu 2+ supplementation during both the pretreatment and the induction phase of androgenesis improves GP regeneration (Jacquard et al 2009).…”

Early embryo achievement through isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. ‘Monreal Rosso’ and ‘Nules’

“…To our knowledge, this alternative cytokinin has not been used to induce microspore embryogenesis by anther or isolated microspore cultures. Esteves et al (2014) recently tested meta-Topolin in the regeneration medium of isolated microspore culture of recalcitrant barley genotypes. It increased embryo differentiation into green plants by 2.9-fold.…”

Effect of thidiazuron on microspore embryogenesis and plantlet regeneration in Chinese flowering cabbage (Brassica rapa. var. parachinenis)

“…TDZ improved green plant regeneration in recalcitrant six-row, spring barley genotypes (Esteves, Clermont, Marchand, & Belzile, 2014).…”

“…A previous study suggested that adding TDZ to the inducing medium is more efficient and can even replace the cytokinin benzylaminopurine (BAP) in isolated microspore culture; TDZ also affected peanut somatic embryogenesis presumably (Murch, Victor, Krishnaraj, & Saxena, ). TDZ improved green plant regeneration in recalcitrant six‐row, spring barley genotypes (Esteves, Clermont, Marchand, & Belzile, ). TDZ could be used to enhance the gynogenesis of unfertilized ovule cultures of Chinese long cucumber (Li, Si, Cheng, Li, & Liu, ); high induction frequencies (7.85%–12.14%) were found at the time of anthesis with concentrations of 0.03–0.07 mg/dm 3 .…”