An EMS mutagenesis protocol for sugar beet and isolation of non-bolting mutantsU.Hohmann1,G.Jacobs1and C.Jung1,21Plant Breeding Institute,Christian-Albrechts-University of Kiel,Olshausenstr.40,D-24098Kiel,Germany;2Corresponding author,E-mail:c.jung@plantbreeding.uni-kiel.deWith3figures and1tableReceived December17,2004/Accepted February23,2005Communicated by F.SalaminiAbstractAn annual sugar beet line homozygous for the dominant gene for early bolting(B)has been mutagenized with different doses of ethylmeth-anesulfonate(EMS).Approximately15000M1seeds were treated with EMS doses between0.5and1%for4,6,8,12and14h.Among 10066M1s,plants with chlorophyll defects and other abnormalities were found.Germination rates ranged between30and100%,whereas the fertility of M1s dropped to36%.A dose of1%EMS applied for 8h was found to yield an acceptable rate of M2sterility(16%). Exactly0.5%of the M2families contained plants with altered bolting behaviour.After selfing these M2plants,five non-bolting M3lines were selected.These plants do not exhibit shoot elongation even after cultivation under long-day conditions.Thus,they are homozygous for new mutagenized,recessive non-bolting alleles.Moreover,four M3 lines showed delayed bolting which was clearly different from the early bolting parent.This demonstrates varying activities of the bolting gene due to different mutational events.Key words:Beta vulgaris—ethylmethanesulfonate—non-bolting—TILLING—vernalizationSugar beet(Beta vulgaris ssp.vulgaris)is a biennial species which forms a storage root in thefirst year and exhibits shoot elongation after a period of cold temperatures in the sec-ond year.Among closely related wild species of the genus B.vulgaris ssp.maritima,early bolting genotypes are abun-dant.These plants start shoot elongation under long-day conditions as early as9–12weeks after germination andfinish their life cycle before winter.A dominant gene B has been found to be the major cause of early bolting in Beta species (Abegg1936)apart from temperature and daylength.The B gene has been mapped with molecular markers to chromosome 2(Boudry et al.1994).Early bolting has an important impact on beet breeding and beet cultivation.Plants must stay in the vegetative phase duringfield production,whereas early bolting can substantially accelerate the breeding process.Conse-quently,there is great interest in this gene which is presently cloned from its position in the genome.High density physical and genetic maps around this gene have been established (El-Mezawy et al.2002,Hohmann et al.2003).For the identification of the bolting gene among candidate sequences from a physical map,mutants with deficient bolting alleles are needed.Numerous techniques for mutagenesis are available which can be categorized by their physical properties and by their mutagenic effects.Gamma irradiation and fast neutron irradi-ation cause chromosome breakage often resulting in transloca-tions while ethylmethanesulfonate(EMS)is highly efficient for inducing transitions by alkylation of guanine bases(N.N.1977).Recently,mutant collections of plants have gained signifi-cance as an important resource for advanced genetic studies. The Targeting-Induced Local Lesions IN Genomes(TILL-ING)strategy relies on large populations of mutagenized plants which are investigated for point mutations within a given sequence(McCallum et al.2000).TILLING projects have been launched with several major crop species such as barley(Caldwell et al.2004),maize(Till et al.2004)and tomato(Menda et al.2004).Crops investigated during the CROPTIL(CROP TILling)initiative are rice,wheat,sor-ghum,pea,medicago,rapeseed,sunflower and melon.In our institute a sugar beet TILLING project has been started recently.As a resource for TILLING and for supporting the identification of the bolting gene a mutant collection of sugar beet has been established.An EMS protocol for efficient mutagenesis which has resulted in the isolation offive mutants deficient in their bolting gene is described here.Materials and MethodsPlant material:The early bolting sugar beet line930190was used for mutagenesis.Under long-day conditions,all plants of this line start shoot elongation9–12weeks after germination.They are homozygous for the dominant bolting allele(BB).M2and M3offspring were germinated in small pots,transferred to multipot plates and planted in afield4–6weeks after germination.Field observation started in June. Between4and12weeks after planting,shoot elongation was recorded. Flowering branches were isolated with plastic bags and seeds were harvested by hand.Between one and30plants were grown from each M2family using the same conditions as for M1cultivation. Mutagenic treatment:Ethylmethanesulfonate mutagenesis of seeds from the early bolting line930190was performed during three growing seasons in the years1999–2001.Batches of100seeds were transferred to50ml Sarstedt tubes that were sealed with a nylon mesh instead of the lid.Several tubes were dipped into200ml distilled water, and the batches of seeds were soaked for8h at room temperature. Seeds were then incubated in200ml of sodium phosphate buffer (0.1M,pH7.4)with gentle shaking(100rpm)and varying EMS concentrations of0.5%(v/v)and1%(v/v),respectively.Incubation periods were for4,6,8,12and14h respectively.In thefirst year,the seeds were treated with1%EMS for12and14h.In the following years,incubation in sodium phosphate buffer was performed for8h with two different EMS concentrations(0.5and1%)and for4,6and 8h.After mutagenesis,the seeds were treated with two washing series in sodium thiosulphate buffer(0.1M,pH7.4)for20min at room temperature with gentle shaking(100rpm).Finally,the seeds were washed in200ml distilled water and then rinsed under running tap water for30–40min.The seeds were dried and kept on wetfilter paperPlant Breeding124,317—321(2005)Ó2005Blackwell Verlag,Berlinwww.blackwell-synergy.com
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