Yuan-Ling Chen,Hui-Lin Liang,Xing-Liang Ma,Su-Lin Lou,Yong-Yao Xie,Zhen-Lan Liu,Le-Tian Chen and Yao-Guang Liu

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,College of Life Sciences,South China Agricultural University,Guangzhou 510642,China

Introduction

Plant mutants are important bio-resources for crop breeding practice and gene functional studies.Several methods have been used to create rice(Oryza sativa)mutant libraries,including physical and chemical mutagenesis(Gu et al.2005;Ye et al.2006;Bhat et al.2007),DNA tagging with TDNA(Jeon et al.2000;Jeong et al.2002;Sha et al.2004),transposons and retrotransposons(Miyao et al.2003;Kolesnik et al.2004;Kumar et al.2005;Jiang et al.2007),and enhancer trapping(Wu et al.2003;Sallaud et al.2004).So far,eleven rice mutant libraries created by T-DNA or transposon tagging carried out in different institutions have been integrated into the Rice Functional Genomic Express Database(http://signal.salk.edu/cgi-bin/RiceGE5).Although rice mutant libraries based on T-DNA and transposon tagging have played great roles in gene cloning and functional studies,generation of more mutants by physical or chemical mutagenesis is still needed in these research areas,especially for screening mutants for agronomically-important traits.Physical or chemical mutagens induce not only loss-of-function or enhanced function mutations,but also neomorph mutations that confer novel functions to the mutated genes.More importantly,desirable mutants created by physical or chemical mutagens can be directly used for breeding,without concern of the bio-safety effects of the marker genes from T-DNA tagging.For physical mutagenesis,electromagnetic radiation such as X-rays and gamma rays,and particle radiation such as neutrons,and β and α particles are wildly used(Kodym and Afza 2003).Although physical mutagens induce a high mutation rate,they frequently result in large chromosomal segment aberrations,which are unfair to gene mapping or may cause the undesirable deletion of linked genes.

Ethyl methanesulfonate(EMS;CH3SO2OC2H5)is an alkylating agent most commonly used for plant mutagenesis.EMS frequently alkylates guanine at the N-7 position followed by adenine at the N-3 and cytosine at the N-1,resulting in the transition of these bases.In addition,the alkalyted guanine can be separated from the deoxyribose,leaving it depurinated,which finally results in small base deletions or insertions during DNA replication(Kodym and Afza 2003).EMS treatment is easily handled and has a high mutagenesis efficiency.In fact,mutagenic treatment of seeds has become the most common method in seed-propagated crops(Kodym and Afza 2003).However,this method is not ideal for rice,because EMS has difficulty permeating through the rigid husks into the embryos,and also can not easily enter the apical meristem of germinated seeds.This results in a low mutagenesis efficiency,and usually produces chimeric mutants.

Chemical mutagenesis of plant suspension-cultured cells dates back to the 1970s.Sung(1976)studied the effectiveness of EMS and nitrosoguanidine(NG)on cell growth using cell suspensions of soybean(Glycine max Merrill.)and wild carrot(Daucus carota L.).Treatment with 0.4%EMS for 2 h resulted in a survival rate of about 50%in soybean cell suspension culture.The compounds tested usually increased mutation frequency by one order of magnitude over the naturally-occurring rate.In 1980,Weber and Lark reported that chemical mutagens such as hycanthone,NG and EMS induced inheritable changes in the ability of maltose utilization in suspension cell cultures of soybean(Weber and Lark 1980).Hofmann et al.(2004)treated embryogenic suspension cultures of soybean with different concentrations of EMS and found that the mean survival rate of embryogenic cultures decreased from 74%(1 mM EMS)to 43%after 30 mM EMS treatment.More recently,Jain(2010)reported a procedure for EMS mutagenesis using banana suspension cell cultures with clump sizes of 3–4 cells.However,these reports did not provide information on the mutant plants regenerating from the mutagenized cell clones.

Large-scale screening of mutations of target genes in rice is laborious and time-consuming.Increasing mutation rate and at the same time decreasing the harmful effects of the mutagens to mutant plants are big challenges.Considering that somatic embryogenesis is the main regeneration method in the rice in vitro culture system,and that somatic embryos arise from single cells(Vasil and Vasil 1981;Ling et al.1988;Jones and Rost 1989),rice suspension cultures with tiny cell clusters are ideal for chemical mutagenesis.Since tiny cell clusters consist of dozens to hundreds of cells,usually hundreds of micrometers in diameter,mutagen solution easily permeates into the cells,thus increasing mutation rate.The mutated single cell can develop into a somatic embryo to regenerate a mutant plant,and chimera mutations are thus avoided.

In this paper,we describe the development of an EMS mutagenesis system based on suspension cell cultures in rice,to increase the mutagenesis efficiency and obtain mutants regenerated from somatic embryogenesis without mosaic plants.We show that treatment with 0.4%EMS for 18–22 h is an optimal condition for this mutagenesis system.We obtained various types of mutants with dominant,semi-dominant,or recessive mutant traits.Several typical mutants involved in important agronomic traits are presented as successful examples of this mutagenesis system.

Results

Establishment of an efficient EMS mutagenesis system in rice based on suspension cell culture

Theoretically,single cells or protoplasts are the most efficient materials for EMS mutagenesis,but the culture procedures for rice single cells or protoplasts(Kao 1982)are much more complicated than for cell clusters.Furthermore,it is difficult to regenerate plantlets from single cells or protoplasts,while suspension cells can be regenerated more easily.Therefore,we chose suspension cultures with tiny cell clusters consisting of dozens to hundreds of cells for EMS treatment.Using dried mature seeds or immature embryos of japonica rice(variety Nipponbare)for the callus induction,this system took about 2.5 months to establish tiny suspension cultures suitable for EMS treatment and another 1.5 months to obtain regenerated plantlets.A typical procedure is shown in Figure 1.In order to remove large cell clusters before pre-differentiation culture,the suspension cells treated with EMS were passed through a 20-mesh-number cell sieve(with a pore size of 830µm).Most obtained cell clusters ranged from 200 to 700µm in diameter,with an average of 467.2 µm(Figure 2A–C).

In order to establish optimal conditions for the mutagenesis,two concentrations of EMS(0.4%and 0.6%)with different treatment times(18 h and 22 h)were tested.As shown in Figure 3,the survival and proliferated calli with 0.4%EMS treatment were much more than those with 0.6%after being cultured in pre-differentiation medium for 15 d.In fact,most cell clusters treated with 0.6%EMS for 22 h died(Figure 3D).Next,the alive calli were transferred to differentiation medium and cultured for 20 d.The calli from the cell clusters treated with 0.4%EMS(Figure 3E)for 18 h and 22 h proliferated rapidly,and efficiently regenerated many plantlets,whereas those treated with 0.6%EMS proliferated poorly and plantlets were rarely regenerated(Figure 3F,G).Usually,a 2-mL volume of cell clusters treated with 0.4%EMS produced more than 400 independent regeneration lines.Thus,considering the survival rate,a 0.4%EMS treatment for 18–22 h is more optimal than a 0.6%treatment.

Figure 1.A flowchart for rice(Oryza sativa)ethyl methanesulfonate(EMS)mutagenesis based on cell suspension-culture cells.This mutagenesis system takes about 2.5 months to establish tiny suspension cultures suitable for EMS treatment,and another 1.5 months to obtain regenerated plantlets.

Figure 2.Suspension-cultured cells used for ethyl methanesulfonate(EMS)mutagenesis.(A)Suspension cells ready for EMS-treatment.(B)Appearance of the cell clusters sieved through a 20-meshnumber cell sieve.Bar=1,000µm.(C)Frequency distribution of diameters of cell clusters after sieving.

Construction of a mutant library with a high frequency of inheritable phenotypic mutations

In total,more than 3,000 M1plantlets were obtained from three EMS mutagenesis experiments.We planted 304 M2lines in the field,with about 100 plants per line,for phenotyping during the entire growth period.

At the seedling stage,12.2%and 12.1%of the M2lines in two sub-libraries descending from the treatments with 0.4%and 0.6%EMS,respectively,had leaf color variations(Table 1).Albino mutants appeared most frequently,and other variations were yellow seedlings or striped leaves.Various other types of mutants were obtained,including large grain,large panicle,multi-ovaries and stamenless,male sterility,rolling leaf,variations in plant height,tiller angle or number,heading date,grain or panicle shape(Table 1 and Figure 4).Male sterile mutants were produced in high frequencies in both sub-libraries.Dominant,semi-dominant,or recessive mutants were observed according to the segregation phenotypes and their ratios,and the mutant traits were inherited steadily to the next generation.The two sub-libraries produced similar frequencies(29.4%and 33.6%)of mutation rates(Table 1).No mosaic mutants were observed in the mutant lines tested;however,some mutants showed multiple phenotypic variations(Figure 4).

A large-grain mutant was screened out from this mutant library.When compared with the wild-type,the 10-grain-length,10-grain-width,and 100-grain-weight of this mutant(M2)were increased by 10.6%,7.7%,and 24.9%,respectively(Figure 5).

Identification of a new eui allelic mutant caused by point mutations

A recessive mutant with an abnormally-elongated uppermost internode was obtained,which showed a segregation ratio fitting 1:3(9 mutants:39 wild-type)in the M2generation.The plant height of the mutant was similar to that of the wildtype before heading,but during heading the uppermost internode of the mutant elongated rapidly(Figure 6A).The uppermost internode of the main panicles of the mutant reached 38.8 cm,an increase of 40%compared with the wild-type(27.5 cm)(Figure 6B).The phenotype of this mutant was similar to the elongated uppermost internode(eui)mutant of the EUI gene,which encodes a cytochrome P450 monooxygenase that functions to deactivate gibberellins(Zhu et al.2006).Therefore,we sequenced EUI in the mutant and detected an A308-to-C308base substitution and a deletion of G311residue in the coding region of the gene,and named it eui-4(Figure 6C).The base deletion of eui-4 leads to a frame-shift.Transformation of the wild-type EUI gene into this mutant recovered the wild-type trait(Figure 6D),indicating that this new eui-4 allele is the cause of loss-of-function in the mutant.This allele may be useful for developing new male-sterile lines for hybrid rice breeding to overcome the wrapround panicle phenomenon,a common problem in male-sterile lines.

陪同毛德君妻子来的还有市中心医院副院长周大国，一个风度翩翩的学者模样，他从车上一下来就发现了出门迎候的卢局长一行人，老远就伸出手喊卢局长。原来他们是认识的。卢成功也亲热地握着他的手说：“想不到也惊动了周院长。”

Figure 3.Two-step differentiation of calli from the ethyl methanesulfonate(EMS)-treated rice(Oryza sativa)suspension-cultured cells.(A–D)Comparison of callus proliferation of cell clusters cultured on pre-differentiation medium for 15 d after treatment with different concentrations of EMS and times as indicated.The same amount of EMS-treated cell clusters were placed on each plate.(E–G)The second step culture of the calli from the EMS-treatments on differentiation medium for 20 d.Few or no calli proliferated from the treatments with 0.6%EMS for 18h or 22 h(F,G).

Discussion

Neomorph functional mutations and various allelic mutations that produce different degrees of functional variations of genes of interest may be obtained by point mutations,which are very useful for plant breeding and functional genomics research.Compared to other mutagens such as electromagnetic radiation and T-DNA or transposon tags,EMS is an ideal mutagen for creating such mutations,since EMS mutagenic treatment results mainly in base substitutions or small base deletions or insertions,rather than large DNA fragment changes.

Table 1.Various mutant types obtained in M2 populations derived from different ethyl methanesulfonate(EMS)treatments.

Currently,EMS mutagenic treatment of seeds is the most widely used method in seed-propagated plants,but this method usually results in a low mutation efficiency in rice,and some varied phenotypes may be lost in the next generation due to chimeric mutations.For example,Ye et al.(2006)treated seeds of an indica rice variety with 0.4%EMS and obtained a mutation rate of 4.64%for visible phenotypes in M2lines,but the mutation rate decreased to 3.78%in the M3generation.Gu et al.(2005)achieved a higher mutation rate of 12.4%for visible phenotypes using a strategy of successive mutagenesis in two generations of a japonica rice variety,with 0.5%EMS treatment of seeds in the first generation(M1)and 0.5%or 0.7%EMS treatments of the M2seeds.In this study,we have established an efficient mutagenesis method based on EMS treatment of suspension-cultured cells in rice,which can produce much higher mutation rates than the traditional methods by mutagenesis of seeds.Using this method,we obtained more than 3,000 M1plantlets,and from 304 investigated M2lines we found 94 mutants with various visible traits(an average of 30.9%mutation rate),including several important agronomic traits such as large grain,large panicle,eui mutant,and male sterility.This mutation efficiency is much higher than those reported from the rice mutant libraries.If suitable methods are used to screen other traits such as biotic and abiotic resistance,grain quality,etc.,the total mutation rate would be higher.In order to increase mutation rate and types,a combinational use of other chemical mutagens could be tested,such as acridines,which mainly induce frame-shift mutations.This EMS mutagenesis system could also be applied to other plant species,such as soybean,wheat,and maize,in which suspension cell culture systems are already well-established,and somatic embryogenesis is the main means of regeneration(Vasil and Vasil 1986;Finer and Nagasava 1988;Vasil et al.1990).

Figure 4.Several mutants involved agronomic-important traits obtained from the ethyl methanesulfonate(EMS)mutagenesis library.(A,B)A large-grain mutant showing 10-grain length(A)and 10-grain width(B).N,Nipponbare;m,mutant;w,wild-type segregated from heterozygous mutant.(C–F)A mutant with large panicle(C,at the heading stage)and yellow color in spikelet(D),culm(E),and uppermost internode(F).(G)A large panicle mutant.(H)A mutant with multi-ovaries and lacking stamens and lodicules.(I)A male-sterile mutant without pollen.(J)A short anther mutant,of which the width(diameter)of the anthers was normal but the length(ca.1 mm)was much shorter than that of the wild-type(ca.2 mm).(K,L)A mutant with less-tiller(K)and without spikelet(L).(M,N)A rolling-leaf mutant.(O)A less-tiller mutant.

Figure 5.Comparison of the large-grain mutant shown in Figure 4 A-B with its wild-type.The 10-grain-length,10-grain-width,and 100-grain-weight of the M2 mutant increased by 10.6%(n=13),7.7%(n=13),and 24.9%(n=10),respectively.

Mutated genes that are not from DNA-tagging are usually isolated by map-based(positional)cloning.This strategy requires the construction of mapping populations by crossing the mutants with other parental lines that genetically diverge from the mutant donor parents,so as to establish reasonable amounts of polymorphic DNA markers covering the whole genome for the mapping of target genes.Recent advances in high-throughput sequencing make it possible to efficiently map a gene conferring the mutant phenotype through a wholegenome resequencing strategy,using pooled genomic DNA of homozygous mutant plants selected from an iso-genic mapping population that is created by crossing the mutant with its donor parent(Abe et al.2012).Therefore,once a mutant of interest is identified from the mutant library,it can first be checked for any sequence differences between the mutant and the wildtype in the reported genes with the similar phenotype.If the mutant involves an unreported gene,the mutant can be used to construct an iso-genic segregation population for wholegenome re-sequencing to map and isolate the target gene.

On the other hand,for the screening of allelic point mutations in genes of interest in EMS-mutagenized populations,Targeting Induced Local Lesions in Genomes(TILLING)is an effective strategy(Till et al.2003,2007).Although it is a high-throughput technique per se,in terms of its PCR system,the efficiency of this technology largely depends on the mutation rate of the mutant library.Therefore,a combination of our mutagenesis system with TILLING will facilitate the high-throughput screening of allelic mutations of target genes.

Materials and Methods

Hormones and EMS

Phytohormones for culture media and ethyl methanesulfonate(EMS)were purchased from Sigma-Aldrich.

Medium composition

Medium for Callus induction:N6(Chu 1978)macro nutrients,B5(Gamborg et al.1968)micro nutrients and vitamins,2,4-D 2.5 mg/L,sucrose 30 g/L and agar 8 g/L(pH 5.8);Medium for callus subculture:N6 macro nutrients,B5 micro nutrients and vitamins,2,4-D 2.5 mg/L,casein hydrolysate 300 mg/L,L-proline 500 mg/L,L-glutamine 500 mg/L,sucrose 30 g/L and agar 8 g/L(pH 5.8);Medium for suspension culture:N6 basal nutrients,2,4-D 4 mg/L and sucrose 30 g/L(pH 5.8);Medium for pre-differentiation(Yang et al.1999):N6 macro nutrients,MS micro nutrients(Murashige and Skoog 1962),B5 vitamins,CuSO4·5H2O 1 mg/L,ABA 5 mg/L,6-BA 6 mg/L,NAA 2 mg/L,casein hydrolysate 500 mg/L,L-proline 500 mg/L,sucrose 30 g/L and agar 8 g/L(pH 5.8);Medium for differentiation:N6 macro nutrients,MS micro nutrients,B5 vitamins,6-BA 3 mg/L,NAA 1 mg/L,sorbitol 18 g/L,sucrose 30 g/L and agar 8 g/L(pH 5.8);Medium for rooting:N6 macro nutrients,MS micro nutrients,B5 vitamins,NAA 1 mg/L,sucrose 30 g/L and agar 8 g/L(pH 5.8).

Callus induction

Suspension culture

Fresh granular calli were crumbed into small pieces with tweezers and grown in suspension culture medium.The suspension cell clusters were cultured at 26°C in the dark on a shaker at 100 rpm and sub-cultured at a 7-d interval.Two mL packed volume cell clusters(i.e.without liquid)were transferred into 20 mL fresh medium with a graduated pipette,the mouth of which was 2 mm in diameter,to select the cell clusters smaller than 2 mm.

Figure 6.Identification of an elongated uppermost internode mutant with new allelic variations in the EUI gene.(A,B)After heading,the uppermost internodes(arrowed)of the mutant elongated to an average of 38.8 cm,40%longer than those(arrowed)of the wild-type(27.5 cm).(C)Sequencing of EUI and eui-4(from the reverse direction of the gene).Arrows indicate the positions of the base substitution and deletion in the eui-4 allele.The base positions of the EUI sequence are based on the coding region sequence of EUI(Accession No.AY987039.1).(D)Complementation of eui-4 with EUI recovered the wild-type phenotype in a transgenic plant.

EMS treatment

A volume of 2 mL cell suspension was transferred into 20 mL fresh suspension culture medium and cultured for 4 d under the same conditions.After discarding the medium,30 mL fresh suspension culture medium was added into the cell clusters,and sterile EMS solution(sterilized by filtration)was added at a final concentration of 0.4%or 0.6%(w/v).EMS treatment was sustained at 26°C for 18 or 22 h on a shaker at 100 rpm.

Measurement of cell clusters

Diameters of cell clusters that passed through a 20-meshnumber sieve(with a pore size of 830µm)were measured with Image-pro Express 6.3 software under a SZX10 stereo microscope(Olympus,Japan).In total,300 cell clusters were measured in three replications.

Two-step differentiation regeneration

After EMS treatment,the liquid was discarded and the cell clusters were rinsed 4 times with 30 mL fresh suspension culture medium and shaken at 100 rpm for 10 min each time.At the last rinsing,the cell clusters were poured through a 20-mesh-number cell sieve,and the tiny cell clusters that were obtained,smaller than 0.9 mm in diameter,were blotted on filter paper.Note that the sieving step is necessary for a suspension established in fewer than 2 months,because the sizes of many cell clusters were larger than 1 mm.The cell clusters from each 2 mL suspension culture were divided into 6 aliquots and placed on 6 plates of pre-differentiation medium.After preculture at 26°C for 15 d in the light under a regimen of 12/12 h photoperiod,the proliferated calli were separately transferred to the differentiation medium piece-by-piece in order to obtain independent mutant plantlets.Small shoots of about 1 cm in height were transferred on rooting medium for regeneration.Plantlets regenerated from the same callus probably arose from a single cell,and were thus treated as the same line.

Planting and phenotyping

The regenerated M1plantlets with well-developed roots were transplanted in pots.M2and M3lines(～100 individuals for each line)were planted in the field to investigate phenotypic variations and segregation ratios at the whole growth period.

Photomicrography

Mutant spikelets were observed under a SZX10 stereo microscope,and images were acquired with Image-pro Express 6.3 software.

Complementation test of eui-4 mutant

The native EUI gene was PCR-amplified and sub-cloned into the binary vector pCAMBIA1300.The construct was transferred into the eui-4 mutant by Agrobacterium-mediated transformation.

This work was supported by grants from the Ministry of Science and Technology of China(2012AA10A303)and the Ministry of Agriculture of China(2011ZX08010-001).

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