Riyu WEN,Yaodong GUO,Jianxia LIU,Lianshou ZHENG,Lisheng FAN,Xiaoping WU

1.Maize Research Institute,Shanxi Academy of Agricultural Sciences,Xinzhou 034000,China;

2.College of Agriculture and Life Sciences,Datong University,Datong 037009,China

Responsible editor:Tingting XU Responsible proofreader:Xiaoyan WU

At current,the studies on tarary buckwheat mainly focus on genetic diversity,taxonomy and other basic aspects of biology[1].The studies on breeding of tarary buckwheat are extremely important.Among them,both the traditional and modern breeding techniques all play very important roles.Reproductive problem is one of the important limiting factors for improvement of tarary buckwheat yield.Therefore,how to overcome these adverse features,such as breeding excellent traits using EMS mutagenesis,will be the key to make outstanding progresses in breeding of tarary buckwheat.

The selection of mutagenic parent material and position is an important part of EMS mutation breeding.Generally,the organs that are easy to be treated and sensitive to mutagenesis are selected as mutagenic positions.Seed is the earliest and most commonly used material for EMS mutagenesis.Although the seed mutagenesis by EMS is restricted by many factors,it still has huge advantages that other materials do not have[2].Seed is an independent organ of plant,and it can maintain its vitality and preserve the mutagenized gene for a long time across the mutagenesis treatment[3].Seed mutagenesis is not needed to be performed under aseptic conditions,which largely reducing the processing steps.Although the effect of seed mutagenesis can not be shown immediately and some of the traits will even be expressed in the second and third generation,the new traits obtained from mutagenesis can still be passed on to offspring,omitting the subsequent identification works.To this end,the seeds of tarary buckwheat were used as the material for EMS mutagenesis in this study so as to investigate the effects of different-concentration EMS on germination of tarary buckwheat seeds.

Material and Methods

Material

Plant materialThe seeds of Jinqiaomai No.4,a local tarary buckwheat variety in Shanxi Province,were provided by the Crop Research Institute of Shanxi Academy of Agricultural Sciences.

Reagents(1) EMS mother liquor.Ethyl methane sulfonate (EMS),with purity＞99%,was purchased from the Beijing Solarbio Science&Technology Co.,Ltd.

(2) Phosphate buffer.The phosphate buffer (0.2 mol/L,pH 7.0) was prepared by mixing disodium hydrogen phosphate (Na2HPO4,0.2 mol/L)and sodium dihydrogen phosphate(NaH2PO4,0.2 mol/L)(v/v,61/39).

(3) Sodium thiosulfate solution.Certain amount (24.8 g) of sodium thiosulfate (NaS2O3·5H2O,0.1 mol/L)was weighed and dissolved in 500 ml of cooled boiling water.Subsequently,certain amount (0.2 g)of Na2CO3and 500 ml of cooled boiling water were added.

(4) Different-concentration EMS solutions.The different-concentration EMS solutions were prepared as shown in Table1.

Methods

Treatment of tarary buckwheat by EMS(1) EMS concentrations and mutagenesis times.The concentrations of EMS were arranged as 0.3%,0.5%,0.7%,1.0%,1.5% and 1.7%.The seeds of Jinqiaomai No.4 were treated for 4,8 and 12 h,respectively.Thus a total of 21 treatments were arranged.The seeds of Jinqiaomai No.4 that were not treated with EMS were treated as control(CK).

(2) Seed selection and soaking.The seeds were soaked in a breaker with water and stirred moderately.The empty seeds,broken seeds,grass seeds and impurities floating on the water surface were removed.Then a total of 5 000 large,plump and sizeuniform tarary buckwheat seeds were selected so that the germination rate and germination vigor would be improved.The selected seeds were soaked in clear water at 25 ℃for 14 h.

(3) Treatment of seeds by EMS.The selected seeds were distributed evenly in 24 labeled dishes,including 21 treatments and 3 controls.A total of 100 tarary buckwheat seeds were placed in each of the 24 dishes.Subsequently,13 ml of EMS solution at a certain concentration was added to each of the 21 dishes.Correspondingly,13 ml of clear water was added to each of the three controls.Similarly,another 24 dishes were arranged as above.All the dishes were placed in a constant temperature incubator(25 ℃).

(4) Termination of mutagenesis treatment.After the treatment,the remaining EMS in the dishes was drained.Subsequently,10 ml of Na2S2O3(0.1 mol/L) was added to each of the dishes to rinse the seeds.The rinsing was repeated three times.After the three times of washing with Na2S2O3,the seeds will be rinsed with clean water.The seeds were then transferred in a new dish laid with twolayer filer paper.The seeds were evenly distributed in the new dishes.After adding certain amount of water,all the dishes were placed in a constant temperature incubator(25 ℃).

(5) Transplanting of germinated seeds.After the seeds germinated,they were all transplanted in soil.

Indicator measurement and methodsGermination rate = Number of germinated seeds within 7 days/Tested seeds×100%;

Relative germination rate = Number of germinated seeds in treatment groups within 7 days/Number of germinated seeds in control groups within 7 days×100%;

Germination vigor = Number of germinated seeds within 3 days/Tested seeds×100%;

Relative germination vigor =Number of germinated seeds in treatment groups within 3 days/Number of germinated seeds in control groups within 3 days×100%.

Data statistics and analysis

Multiple comparisons of various factors were performed with Duncan’s new multiple range method using SPSS software.

Table1 Preparation of different-concentration EMS solutions ml

Table2 Effects of different-concentration EMS on germination of Jinqiaomai No.4

Table3 Effects of different treatment times on germination of Jinqiaomai No.4

Results and Analysis

Effects of EMS on germination of Jinqiaomai No.4 seeds

Effects of different-concentration EMS on germination of Jinqiaomai No.4 seedsTable1 showed that with the increase of EMS mutagenic agent concentration,the germination vigor and germination rate of Jinqiaomai No.4 all trended to be decreased.When the concentrations of EMS ranged from 0.3%to 1.0%,there were no significant differences between treatment and control groups; but when the concentration of EMS reached 1.5%and 1.7%,the germination vigors of Jinqiaomai No.4 were decreased from 85.0% (CK) to 46.7%(P＜0.01)and 27.3%(P＜0.01),and the germination rates were reduced from 96.0% (CK) to 68.0% (P＜0.01) and 45.3% (P＜0.01).It was indicated that 1.5%and 1.7%of EMS had significant inhibiting effects on germination vigor and germination rate of Jinqiaomai No.4.

At the same time,with the increase of EMS concentration,the relative germination rate and relative germination vigor of Jinqiaomai No.4 were all trended to be decreased.When the EMS concentrations ranged from 0.3% to 1.0%,no significant difference was found in relative germination vigor between treatment and control groups; but when the EMS concentration was increased to 1.5%,the relative germination vigor of Jinqiaomai No.4 seeds was decreased from 1.00 (CK) to 0.55 (P＜0.01).When the EMS concentrations ranged from 0.3%to 1.0%,there were no significant differences in relative germination rate of Jinqiaomai No.4 seeds between treatment and control groups,but when the EMS concentrations reached 1.5%and 1.7%,the relative germination rates of Jinqiaomai No.4 seeds were decreased from 1,00 (CK) to 0.71 and 0.47.Therefore,1.7% was considered to be the optimum concentration of EMS for mutagenesis of tarary buckwheat.

Effects of different treatment times on germination of Jinqiaomai No.4 seedsAs shown in Table2,when the treatment times of EMS were 4 h and 8 h,there were no significant differences in germination rate and relative germination rate of Jinqiaomai No.4 seeds between treatment and control groups; but when the treatment time was increased to 12 h,the differences in germination rate and relative germination rate reached the significant levels.However,there were no significant differences in germination vigor and relative germination vigor between treatment and control groups among the three treatment times.

Conclusions and Discussion

The determination of optimum concentration of mutagenic agent is the key for success of mutation breeding.Different plants require different concentration ranges of certain mutagenic agent.Only within the optimum concentration range of mutagenic agent can species maintain their good traits and generate more new mutation[4-5].

In this study,the seeds of Jinqiaomai No.4,a local tarary buckwheat variety in Shanxi Province,were used as the material.They were mutagenized with different -concentration EMS for different time.The results showed that EMS had significant effects on germination rate of Jinqiaomai No.4.When the concentration of EMS was increased to 1.7%,the relative germination rate was decreased to 0.46,close to 0.50.Therefore,1.7%was considered to be the median lethal concentration,and was also considered to the optimum concentration for mutagenesis[4].In addition,the mutagenic time should be best more than 8 h.

The control of bacterial infection was also another important aspect of this study.During the test,the germination of tarary buckwheat is easily infected with microorganisms.The incubating environment is suitable for growth and propagation of microorganisms,affecting the normal germination of tarary buckwheat seeds.So before the starting of the test,the dishes must be disinfected thoroughly.In addition,the process of data observation and statistics also needs to prevent bacterial infection.

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