Transgenic doubled haploid barley through microspore culture

A simple and efficient procedure for the production of transgenic doubled haploid barley through microspore culture was developed. Transgenic barley carrying a herbicide resistance gene (bar) as a transformation marker and a gene coding for a fungal thermotolerant endo-1,4-beta-glucanase (eg11) under control of a high-pI alfa-amylase promoter was used as research material. Transgenic mother plants from three different lines were propagated in controlled glasshouse conditions for the production of starting material, spikes for microspore isolation. Spikes were collected when microspores had reached the late uni-nucleate stage. A four week cold pre-treatment at +4°C was needed for the efficient regeneration of green plants. Simple teflon rod maceration was used as microspore isolation procedure without any additional maltose-gradient purification steps. Isolated micropores were plated on solid culture medium at density of 100,000 viable micropores per plate. After four weeks of culture in the dark at 22°C, the developed calli were plated on regeneration medium and grown in the light. The regenerated plantlets were rooted and treated with colchicine for the production of doubled haploids. After colchicine treatment the plants were potted in soil and screened for the presence of the transgene by PCRs. The microspore culture of 16 mother plants (from three different transgenic lines) resulted in 927 green regenerants of which 476 were transferred to soil mix after rooting and colchicine treatment. Of these 476 plants, 380 were transgenic, 358 reached maturity and 350 were fertile with normal seed set. The transgenic DH plants were grown to maturity in glasshouse and the seeds were harvested. A yield of 6.9 kg for the transgenic seed was obtained. These transgenic DH seeds were used in malting, mashing and brewing experiments. Non-transgenic barley (c.v. Golden Promise) was also propagated in glasshouse conditions and used as control.