Jianxia LIU

College of Agronomy and Life Sciences, Shanxi Datong University, Datong 037009, China

Buckwheat breeding is always important. Both traditional and modern breeding techniques have played a very important role in buckwheat breeding[1]. Reproduction problem is an important factor limiting the yield of common buckwheat.Therefore, how to overcome the adverse features is the key to breakthroughs in buckwheat breeding. EMS is a commonly-used inducer for improving breeding traits. For the induction by EMS, the selection of parent materials and induced sites is an important part. The easily-handled and induction-sensitive organs are generally selected as the induced sites.Seed is the earliest and most commonly used material for EMS induction treatment. In this study, common buckwheat seeds were selected as the material for EMS induction treatment.Although there are many factors limiting the seed germination induced by EMS, seeds have following advantages compared with other materials[2].First, seed is an independent organ of plants. It can not only maintain its vitality but also preserve mutant genes throughout the induction process[3].Second, seed germination induction is unnecessary to be carried out under sterile conditions, thereby greatly simplifying the operation. Third, although the mutagenic effect is not immediately shown, and some new traits will be even expressed in the second or third generation, the obtained new traits can always be passed on to offspring,thereby omitting follow-up identification work.

Material and Methods

Material

Plant material In this study, the used plant material was seeds of Jinqiaomai No.3,a local cultivar in Shanxi Province. Jinqiaomai No.3 was bred by the Crop Research Institute of Shanxi Academy of Agricultural Sciences.

Reagents The EMS (ethylmethane sulfonate)liquor with purity higher than 99% was purchased from the Beijing Solarbio Science & Technology Co.,Ltd.

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) according to volume ratio of 61:39.

A certain amount (24.8 g) of sodium thiosulfate(NaS2O3·5H2O)was dissolved in a certain volume (500 ml)of cooled boiling water. Subsequently, a certain amount (0.2 g) of sodium carbonate (Na2CO3) and a certain volume(500 ml) of cooled boiling water were added. Thus the sodium thiosulfate(0.1 mol/L)solution was prepared. Different concentrations of EMS solutions were prepared as described in Table 1.

Methods Treatment of buckwheat seeds by EMS

Induced concentration and time of EMS The induced concentrations of EMS were arranged as 0.3%, 0.5%,0.7%, 1.0%, 0.5% and 1.7% (W/V).The different concentrations of EMS solutions were prepared by mixing EMS inducer and phosphate buffer(pH 7.0). The induced times were arranged as 4, 8 and 12 h. In the CK groups, EMS was not added. Total 21 treatments were designed.

Seed selection and soaking The common buckwheat seeds were soaked in a breaker filled with water.They were stirred appropriately. The empty or broken seeds, weed seeds and impurities floating on the water surface were all removed.Finally,total 5 000 large and plump common buckwheat seeds with uniform size were selected. The selected buckwheat seeds were soaked in clear water at 25 ℃for 14 h.

Treatment of seeds by EMS The soaked buckwheat seeds were spread evenly into 24 labeled petri dishes(including 21 treatment groups and 3 control groups).In each petri dish,total 100 common buckwheat seeds were placed. In the petri dishes of the 3 control groups,certain volumes (13 ml for each) of clear water were added.Similarly, the test was repeated once.All the common buckwheat seeds were incubated at 25 ℃.

Ending of induction treatment After treated by EMS, the common buckwheat seeds were rinsed with sodium thiosulfate(0.1 mol/L,10 ml for each petri dish) three times. Subsequently, they were rinsed with clear water three times.And then,the seeds were spread uniformly into petri dishes laid with two layers of qualitative filter paper. Appropriate amounts of water were added to the petri dishes to keep the seeds completely wet. The petri dishes were placed in an incubator at 25 ℃.

Transplanting of seedlings The common buckwheat seedlings were transplanted into soil with transplanting depth of 2-3 cm.

Table 1 Preparation of different-concentration EMS solutions

Table 2 Multiple comparisons of germination vigor, germination rate, relative germination vigor and relative germination rate of common buckwheat seeds among different concentrations of EMS solutions

Index measurement and methods

The germination rate referred to the percentage of germinated seeds within 7 d. The relative germination rate referred to the ratio of germinated seeds between certain treatment group and certain control group within 7 d. The germination vigor referred to the percentage of germinated seeds within 3 d. The relative germination vigor referred to the ratio of germinated seeds between certain treatment group and certain control group within 3 d.

2.1 蘋果虎皮病 蘋果虎皮病發(fā)病初期，果皮呈淡黃褐色，表面平或略有起伏，或呈不規(guī)則塊狀，以后顏色逐漸變深，呈褐色至暗褐色，稍凹陷。病部果皮可成片撕下，皮下變?yōu)楹稚?。病果肉綿，略帶酒味。病變多發(fā)生于果實(shí)陰面未著色部分，嚴(yán)重時(shí)才延及陽面著色部分?；⑵げ‰m然不影響果實(shí)風(fēng)味，但嚴(yán)重影響果實(shí)外觀，降低商品價(jià)值。

The obtained data were analyzed using Duncan’s new multiple range test.The multiple comparisons for various factors were performed using SPSS.

Results and Analysis

As shown in Table 2, with the increased induced concentration of EMS,the germination vigor and germination rate of common buckwheat seeds were all trended to be decreased. However, when the concentration of EMS ranged from 0.3% to 1.0%, there were no significant differences between the treatment and control groups, indicating that EMS solutions with concentrations of 0.3%-1.0%had no significant effects on germination vigor and germination rate of common buckwheat seeds. When the concentrations of EMS were 1.5%and 1.7%, the germination vigors of common buckwheat seeds were decreased to 1.7% and 0, respectively from 15.3% in the control group (P＜0.05), and the germination rates were decreased from 4.2% and 0.2%, respectively from 33% in the control group (P ＜0.05). It indicated that the EMS solutions with concentrations of 1.5%-1.7% inhibited greatly germination vigor and germination rate ofcommon buckwheat seeds.

At the same time, with the increased concentration of EMS, the relative germination rate and relative germination vigor of common buckwheat seeds were also trended to be decreased. Similarly, when EMS concentration ranged from 0.3% to 0.7%,there were no significant differences in relative germination rate or relative germination vigor between treatment and control groups. However, when the EMS concentration was increased to 1.0%, the relative germination vigor of common buckwheat seeds was reduced from 1.0 (CK)to 0.5 (P＜0.05),and the relative germination rate was reduced from 1.0 (CK) to 0.56 (P ＜0.05). Generally, when the relative germination vigor and relative germination rate are around 0.5, the treatment concentration is considered to be the half lethal concentration, which is also considered to be the optimal induced concentration. Therefore, 1%was the optimal induced concentration of EMS for common buckwheat seeds.When the EMS concentrations were increased to 1.5%and 1.7%,the relative germination vigors of common buckwheat seeds were reduced to 0.1 and 0, respectively from 1 in the control group (P ＜0.05), and the relative germination rates were reduced to 0.14 and 0.01, respectively from 1.00 in the control group (P＜0.05). It suggested that under concentrations of 1.5%-1.7%, the germination of common buckwheat seeds was greatly inhibited by EMS. So, 1.5% was the lethal concentration of EMS for common buckwheat seeds.

Table 3 showed that no significant differences were observed in germination rate, relative germination rate,germination vigor or relative germination vigor between the induced times of 4 and 8 h. However, when the induced time was increased to 12 h,significant differences were shown, and the relative germination vigor and relative germination rate were reduced to 0.22 and 0.23,respectively.

Table 3 Multiple comparisons of germination vigor, germination rate, relative germination vigor and relative germination rate of common buckwheat seeds among different induced times of EMS

Conclusions and Discussion

The determination of appropriate dose range of mutagen is the key to the success of tests. Certain mutagen has different dose ranges for different plants. Only in the appropriate dose range,species can maintain their good traits and generate more new mutations[4].

Considering the induced time,this study concluded that the induced time of EMS should be more than 8 h for inducing germination of common buckwheat seeds.

In the test, another important aspect was to control bacteria. It has been found that common buckwheat seeds are easy to be contaminated during their germination.A suitable environment facilitates the growth and propagation of bacteria, inhibiting the normal germination of seeds through infection. Therefore, before the test,the petri dishes must be disinfected completely. In addition, excessive infestation of bacteria must be prevented during the observation and statistics of test data.

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