Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus

Abstract

Microspore or anther culture has been used to produce desirable meiotic recombinants in numerous species. However, the utilization of these recombinants relies on inefficient genome doubling procedures to obtain fertile doubled haploid plants. This study presents a simple and rapid procedure to generate fertile doubled haploids in Brassica napus cv. Topas using trifluralin (α,α,α‐trifluoro‐2,6‐dinitro‐N,N‐dipropyl‐p‐toluidine), a plant specific microtubule inhibitor. The effects of trifluralin on microtubule depolymerization and chromosome doubling in embryogenic microspore cultures of B. napus were examined and compared with those of colchicine. Indirect immunofluorescence labeling of isolated microspores indicated that microtubules were depolymerized within 30 min of trifluralin treatment and after 3–8 h of colchicine treatment. The direct application of these microtubule inhibitors to microspore cultures resulted in the recovery of fertile doubled haploid plants. Continuous culture in the presence of colchicine, was more effective than 18‐h treatments for fertile plant production but resulted in abnormal embryo formation and recalcitrant plant regeneration. The application of 1 or 10 μM trifluralin during the first 18 h of microspore culture was found to be the superior method for doubled haploid production. The embryos generated after trifluralin treatment developed normally, germinated readily and of the plants produced, close to 60% were fertile. The use of trifluralin to double chromosomes very early in microspore cultures is a simple process requiring minimal manipulation and should be very useful for genetic studies and breeding programs of B. napus and possibly other species.

Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture

“…These results are consistent with Zhao and Simmonds (1995), who demonstrated the efficiency of trifluralin for chromosome doubling in spring oilseed rape, cultivar Topas. The higher efficiency of trifluralin in relation to in vitro chromosome number doubling in comparison with oryzalin was also described by Rudolf et al (1999) in Brassica oleracea.…”

“…in vitro diploidization avoided the occurrence of chimeric plants in comparison with the colchicine treatment of plantlets. According to Zhao and Simmonds (1995), it is so because chromosomes are doubled very early in a microspore culture.…”

“…The higher efficiency of trifluralin in relation to in vitro chromosome number doubling in comparison with oryzalin was also described by Rudolf et al (1999) in Brassica oleracea. According to Zhao and Simmonds (1995), a great advantage of trifluralin use is that embryogenesis is normal and proceeds to direct embryo germination and vigorous plant growth.…”

“…Several microtubule depolymerising herbicides were also proved to be efficient for in vitro chromosome doubling of microspores. Zhao & Simmonds (1995) tested trifluralin and Hansen & Andersen (1996) trifluralin, oryzalin and amiprophos methyl in the spring rapeseed cultivar Topas. They achieved the mean rate of diploidization from 60% to 65%, which is comparable with the application of colchicine.…”

In vitro induction of tetraploid garlic with trifluralin

“…Although considerable progress has been made in the induction and production of Allium plants by antimitotic agents (Campion et al, 1995;Grzebelus and Adamus, 2004), there is no report on tetraploid garlic induction by trifluralin. It was reported that 1 µM trifluralin generated more normal and fertile plants in embryogenic microspore cultures of Brassica napus compared to 25 or 50 µM colchicine (Zhao and Simmonds, 1995) and that trifluralin showed a higher affinity to plant tubulin than did colchicine . Compared to the highest rate of tetraploid induced by trifluralin using root tips from germinated seeds, which was 32.2% in melon (Wei and Jiang, 2008), the garlic tetraploid inducing rate in vitro in this research was much lower (only 3.3%).…”