An EMS mutagenesis protocol for sugar beet and isolation of non-bolting mutants

Abstract

An annual sugar beet line homozygous for the dominant gene for early bolting (B) has been mutagenized with different doses of ethylmethanesulfonate (EMS). Approximately 15 000 M1 seeds were treated with EMS doses between 0.5 and 1% for 4, 6, 8, 12 and 14 h. Among 10 066 M1s, plants with chlorophyll defects and other abnormalities were found. Germination rates ranged between 30 and 100%, whereas the fertility of M1s dropped to 36%. A dose of 1% EMS applied for 8 h was found to yield an acceptable rate of M2 sterility (16%). Exactly 0.5% of the M2 families contained plants with altered bolting behaviour. After selfing these M2 plants, five non-bolting M3 lines 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.

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

“…This approach is supported by the finding that many disease-resistance genes are derived from DNA base substitutions or deletions (Tian et al, 2004). The bolting gene B was mutated at a frequency of 0.3% by seed treatment with 1% ethylmethanesulphonate mutagen and further characterised by TILLING (Hohmann et al, 2005). Mutagenesis using transposable elements is an effective method to generate a large number of mutants for further phenotyping and functional gene analysis.…”

Evolutionary Conservation of the FLOWERING LOCUS C-Mediated Vernalization Response: Evidence From the Sugar Beet (Beta vulgaris)

“…During evolution, any number of mechanisms, both within and outside the vernalization pathway, could be recruited to produce a vernalization requirement. Second, by mutagenizing an annual line (BB) with ethyl methylsulfonate, Hohmann et al (2005) produced beets that performed as biennials: they had lost the ability to flower under long days alone, but retained an obligate vernalization requirement, flowering only after cold treatment. Hohmann et al's (2005) results demonstrate that a vernalization requirement may be imposed in beet via loss-of-function mutations with no obvious impact on the vernalization response.…”

Induced Mutations and Crop Improvement

“…To maximise recovery of mutants, it is possible to adjust the dose of the mutagen, for example, the germinating seeds of tetraploid cotton ( Gossypium hirsutum ) were exposed to three to fi ve times the LD 50 rate of EMS (3 % v/v) to ensure recovery of mutants (Auld et al 1998 ). In contrast, EMS treatment of germinating sugar beet seeds required only 0.5 % (v/v) EMS to elicit a suffi ciently mutagenic response (Hohmann et al 2005 ). Over 3,200 M 2 families were derived from an early bolting line treated with EMS, of these only 9 families exhibited the desired non-bolting trait and eventually gave rise to 5 lines with the non-bolting phenotype (Hohmann et al 2005 ).…”

“…In contrast, EMS treatment of germinating sugar beet seeds required only 0.5 % (v/v) EMS to elicit a suffi ciently mutagenic response (Hohmann et al 2005 ). Over 3,200 M 2 families were derived from an early bolting line treated with EMS, of these only 9 families exhibited the desired non-bolting trait and eventually gave rise to 5 lines with the non-bolting phenotype (Hohmann et al 2005 ).…”