Improvement of isolated microspore culture of barley (Hordeum vulgare L.): the effect of floret co-culture

Abstract

Barley microspores from five field-grown breeding lines were isolated using an ultra-speed blender and the effect of co-culture with young florets was investigated. Floret co-culture in the induction stage increased the formation of MCS, ELS and green plant regeneration. The florets of teraploid plant were more effective than ones of diploid plant. For line S23, coculture with florets from tetraploid plants gave rise to 2.6 and 7.8 times more MCS and ELS, respectively, than non-co-culture control, whereas co-culture with florets from diploid plants resulted in 1.8 and 6.1 times more MCS and ELS, respectively, than non-co-culture control (Table 2). Florets subjected to cold treatment for 10-20 days induced a greater response than fresh ones, and florets with uninucleate microspores surpassed binucleate microspores. For microspores culture from 15-day cold pre-treated spikes, 93A floret co-culture gave rise to 3.6 and 6.8 times more MCS and ELS, respectively, than the non-co-cultured control, while SD1 floret co-culture resulted in 1.9 and 4.0 times more, respectively. Similarly, for microspore culture from 20-day cold pre-treated spikes, 93A floret co-culture gave rise to 2.6 and 5.1 times more MCS and ELS, respectively, than non-co-cultured control, while SD1 floret co-culture resulted in 1.5 and 3.0 times more, respectively (Table 3). Some microspores formed dense MCS that did not develop further. Compared with the control, floret co-culture resulted in less dense MCS formation, indicating that the isolated florets were beneficial to the normal development of MCS. Floret coculture was only effective when the spikes were cold pre-treated before microspore isolation. Spike cold pretreatment before microspore preparation was crucial for dedifferentiation of cultured isolated microspores, and this could not be replaced by floret co-culture. It is postulated that the florets provided essential substances for in vitro cultured isolated microspores to undergo dedifferentiation and embryogenesis. Both the genotype selection and the physiological status (developmental status and cold treatment) adjustment of the florets for co-culture could improve barley microspore culture. Compared with ovary co-culture, floret co-culture is more efficient. The technique is of simple application in breeding programs and can be a solution for coping with recalcitrant genotypes and or plant donor condition.

Arabinogalactan proteins improve plant regeneration in barley (Hordeum vulgare L.) anther culture

“…This may well be the first observation that the gum arabic accelerates the first androgenic cell division in barley. A similar effect was observed when barley microspores were co-cultured with ovaries (Lu et al 2008). In that study, the authors also reported an increase in the frequency of the MCSs formation.…”

“…One way to achieve this is by making use of a nurse culture which secretes various components to the medium to stimulate microspores to rapid divisions and embryo formation. This approach is often used in wheat (Zheng et al 2002;Cistué et al 2006;Broughton 2008) as well as in barley (Li and Devaux 2001;Lu et al 2008;Lippmann et al 2015). However, nurse culture, such as an ovary-culture, can only be used in liquid media and requires additional labor.…”

Effects of n-butanol on barley microspore embryogenesis

“…Of the different methods for DH production in barley, microspore embryogenesis by anther and/or microspore culture has the greatest potential to produce green DH plants. Even though these methods have been greatly improved in barley (Kasha et al 2001, Cistué et al 1994, Castillo et al 2000, Lazaridou et al 2005, Echavarri et al 2008, Lu et al 2008, Jacquard et al 2006, for review see Ferrie and Caswell 2011, the induction of microspore embryogenesis in cultivars which produce a low number of green plants or none at all, especially in spring genotypes (Muñoz-Amatriaín et al 2008, Asakaviciute 2008, is still a challenge.…”

Expression analysis of three SERK-like genes in barley under abiotic and biotic stresses

“…The microspores were isolated from the pretreated young spikes of barley Hua-30 and cultured in vitro following the procedures described by Lu et al (2008) to induce MDEC (microspore derived embryo callus). After 19-day culture, the induction medium was changed with addition of 8.56 mM·L -1 NaCl as salt stress followed by Liu et al (2016), and the MDECs were collected at 0, 1, 2, 4, 6, 12 and 24 hours after the abiotic treatment for RNA isolation.…”