Enhancement of microspore culture efficiency of recalcitrant barley genotypes

The culture response of isolated microspores of seven recalcitrant cultivar s of barley has been largely improved by identifying an appropriate pretrea tment and utilizing ovary co-cultivation. After comparison of three pretrea tment media, medium B was shown to be most efficient for inducing microspor e embryogenesis, while 0.3 M mannitol frequently used for the responsive cv . Igri was found to be ineffective for recalcitrant genotypes. A further si gnificant improvement of embryogenesis was achieved by using ovary co-cultu re, which resulted in an overall 2.1-fold increase in embryo formation and 2.4-fold increase in green plant regeneration from all cultivars compared w ith the control. Optimal co-culture conditions were identified as 5 ovaries /ml medium kept over 20 days in induction culture. Microspore plating densi ties in cultures with and without co-culture were found to be optimal at 4x 10(4)/ml and 8-12x10(4)/ml, respectively. The most effective and reproducib le method for culturing microspores of recalcitrant genotypes appeared to b e the combination of medium B pretreatment with ovary co-culture. By using this procedure, the genotypic difference in microspore embryogenesis could be reduced. It was found that medium B mainly enhanced percent live embryog enic microspores, and ovary co-culture subsequently improved cell division and embryogenic development. The method described here is important for the application of the microspore culture technique to barley breeding and bio technology.