On the ldentification and Evaluation of Salt Tolerance of Peanut Germplasm

Zhide CHEN，Yonghui LlU，Yi SHEN

Institute of Industrial Crops，Jiangsu Academy of Agricultural Sciences/Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm，Nanjing 210014，China

On the ldentification and Evaluation of Salt Tolerance of Peanut Germplasm

Zhide CHEN*，Yonghui LlU，Yi SHEN

Institute of Industrial Crops，Jiangsu Academy of Agricultural Sciences/Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm，Nanjing 210014，China

For screening out salt tolerance germplasm and providing materials for genetic research of peanut，based on the indexes including relative germination potential，relative germination rate，relative germination index and salt-injury rate，128 peanut germplasms were selected for salt-tolerant identification and estimation under 2.5%NaCl.We found significant but various depressing levels of germination rate under salt stress among different germplasms，and only 5%of 128 germplasms were highly resistant to salinity.We also found that the relative germination index was a useful evaluation index for salt tolerance besides salt damage rate and relative germination rate.Afterall，we comprehensively screened out7 materials（JS011，JS024，JS125，JS491，JS523，JS524 and JS525）as salttolerance germplasms for further research.

Peanut；Germplasm resources；Salt tolerance；Identification and evaluation

Soil salinization is one of the dominant factors restricting agricultural development［1］，there is about 20%culti vated land and 50%irrigated land around theworldharmedbysalt stress［2-3］.In China，there is about 2.7× 107hm2saline-alkali soil［4］，moreover，the area of the salinization and secondary salinization of soil is still increasing with the effects of unreason able crop rotation，blind or excessive application ofchemicalfertilizer［5］. Therefore，excavating the salt-tolerant ability of crops themselves，screening and cultivating salt-tolerant species is the most economic and effective way for developing and utilizing saline-alkali soil at the present stage［6］.

Peanut（Arachis hypogaea L.）is suitable for neutral and meta-acid soil，and is relatively sensitive to salt stress［7］.Salt stress of soil affects the germination of peanut seeds and the growth of plants，reduces the photosynthetic efficiency of plants and the accumulation of photosynthetic products［8-9］；carrying out the salt-tolerance identification of peanut resource，excavating and utilizing excellent salt-tolerance resources is the fundamental work improving the salt tolerance of peanut variety.There have already been some literature reports on salttolerance research， for example，Shandong Institute of Peanuts has already carried out the screening and identification on salt tolerance of 290 kinds ofpeanut resources as early as 1994，and a batch of salt tolerance germplasm was obtained preliminarily［10］.Wang Xiuzhen et al.［11］studied the relationship between main quality characters and salt tolerance of peanut during budding stage，the results showed that the salt tolerance of peanut during budding stage had not significant correlation with linoleic acid，oleic acid，crude protein and crude fat content under non-salt stress.Wu Lanrong et al.［12］adopted pot experi-ment to carry out salt-tolerance identification fortwo-yearwhole growth stage，and the results showed that the salt tolerance of peanut during different growth stages was different，and that during budding stage and seedling stage was the most sensitive. The ability of salt tolerance stress of different peanut varieties was different，and with the increase of salinity，the characters like plant growth rate，plant moisture content，free water content，leafwaterpotential，photosynthetic pigments and total carbohydrate declined obviously［13］.Most studies focused on the effects of salt stress on peanut plant growth，physiology and biochemistry as well as the evaluation index of salt tolerance，etc.，however，the studies on the identification of salt tolerance of peanut resources were fewer，and the identification indexes and evaluation methods of salt tolerance still needed to be perfected［14］.According to the salt stress concentration and identification methods determined at the earlier stage［15-16］，the salt-tolerance identification on 128 peanut resources with different sources and types was carried out，so as to screen out salt tolerance resources，thereby providing a basis material for cultivating salt tolerance varieties and carrying out the genetic research on the salt tolerance of peanut.

Materials and Methods

Research materials

The salt-tolerance identification on 128 peanut resources was carried out，including 40 local resources and 88 improved varieties（strains）.

ldentification methods

0.5%NaCl solution was carried out salt stress treatment，salt stress solution was made of NaCl（analytically pure），and tap water treatment was taken as the control（CK）.30 full and scatheless seeds from each resource were selected for each repetition，then they were placed in the plastic case with cover which was equipped with tap water or 0.5%NaCl stress solution，followed by germinating in the illumination incubator，these were repeated three times.The temperature and light condition of the illumination incubator was dark 24 h/27-28℃.From the 3rdd，the germination number was continuously counted for 5 d taking the germination standard of radicle length≥seed length.To ensure the relatively stable of NaCl stress solution concentration，the stress solution should be changed every 2 days during seed germination period，and the tap water of the CK should be changed.

Character observation

The germination potentialand germination rate of different sources of seeds under 0.5%NaCl stress and CK were surveyed，and the germination index，relative germination potential（germination rate and germination index）and salt-injury rate were calculated according to the following formulas.

Germination potential=（Normal germination number of the 3rdd/Tested seeds）×100%；

Germination potential=（Normal germination number of the 7thd/Tested seeds）×100%；

Germination index=Σ Normal germination seeds every day/The corresponding days；

Relative germination potential（germination rate and germination index）=［Germination potential of treatment groups（germination rate and germination index）/Germination potential of CK（germination rate and germination index）］×100%；

Salt-injury rate=［（Germination rate of CK-Germination rate of treatment groups）/Germination rateofCK］× 100%；

Relative germination potential（germination rate and germination index）and salt-injury rate were taken as the salt tolerance of evaluation index resources.Hereinto，the grading of salt tolerance was carried out as follows taking salt-injury rate as the index［17］:

The classification index of salt tolerance—grade 1 （salt-injury rate≤20%）；grade 2 （20%＜salt-injury rate≤40%）；grade 3 （40%＜salt-injury rate≤60%）；grade 4 （60%＜salt-injury rate≤80%）；grade 5（salt-injury rate＞80%）.

Salt tolerance standard—high tolerance（grade 1）；moderate tolerance（grade 2）；sensitive （grade 3-4）；high sensitive（grade 5）.

Data analysis

All collected data were treated and carried out statistical analysis using Excel and SPSS 19.0.

Results and Analyses

Effect of salt stress on the characters of seed germination

The results of Table 1 showed thataverage germination potential，germination rate and germination index of different germplasms under CK were 31.7%，76.0%and 39.5%，respectively，and these under 0.5%NaCl stress were 8.0%，33.0%and 12.1%，respectively，which were extremely and significantly lower than the corresponding value under CK.The variable coefficients of germination potential，germination rate and germination index of different germplasms under salt stress were 1.79，2.62 and 1.38 times of the corresponding characters under CK.Thus it can be seen that salt stress had prominent inhibiting effect to the germination of peanut seeds，but the inhibiting effects of salt stress were obviously different for different resources.

Under CK and salt stress conditions，the germination index of local varieties was higher than that of improved varieties，moreover，there were extremely significant differences between the two varieties；however，the difference on germination potential and germination rate between the two varieties was not significant（in Table 2），this showed that although there was not significant difference on germination potential and germination rate between the two varieties，the germinating ability of local varieties was higher than that of improved varieties under 0.5%NaCl stress.

The frequency distribution of relative germination potential（germination rate and germination index）

The frequency distribution of relative germination potential（germination rate and germination index）of different germplasms was in Fig.1-Fig.2.The results showed that the relative germination potential of improved varieties was among 0.0-50.0%，which occupied 81.8%of identified resources，in which the interval of 0.0-10.0%occupied 46.6%.The relative germination rate was mainly distributed in the interval of 10.1%-70.0%，which occupied 84.1% ofidentified resources，in which the intervals of 20.1%-30.0% and 40.1%-50.0%respectively occu-pied 18.2%and 19.3%.The relative germination index was mainly distributed in the interval of 0.0-60.0%，which occupied88.6%ofidentifiedresources，in which the interval of 10.1%-20.0% occupied 19.3%.The frequency distribution of relative germination potential（germination rate and germination index）of local varieties was similar to that of improved varieties，and the differences of frequency of different intervals were relatively less.

Comparison on the salt tolerance of different germplasms

The results of salt tolerance identification taking salt-injury rate as the index were in Table 3.Under 0.5% NaCl stress，7 germplasms including 4 improved varieties and 3 local resources from 128 tested germplasms showed high tolerance，which occupied 5.47%of identified germplasms. 28 germplasms including 19 improved varieties and 9 local resources showed moderate tolerance，which occupied 21.88% ofidentified germplasms. There were 67 salt-sensitive germplasms and 26 high sensitive germplasms，which respectively occupied 52.34% and 20.31% ofidentified germplasms.Thus it can be seen that about 75%peanut resource showed sensitivity and high sensitivity to salt stress，and high salt-tolerant resource only occupied about 5%.

Table 1 Germination characteristics of peanut resources under 0.5%NaCl stress and CK %

Table 2Germination characteristics of local varieties and improved varieties under 0.5% NaCl stress and CK %

Comparisononthegermination characteristics of high salt-tolerant and high salt-sensitive germplasms

According to the standard of saltinjury rate，there were 7 high salt-tolerant resources and 26 high salt-sensitive germplasms，in which the salt-injury rates of6 high salt-sensitive sources were higher than 90%，and their relative germination potential，relative germination rate and relative germination index were in Table 4.The results showed that only the calculation method of the salt-injury rate was different from that of relative germination rate，and their results were highly related，the relative germination rates of 7 high salt-tolerant resources were all above 80%，and the relative germination indexes were also higher；the relative germination rates of 6 high salt-sensitive resources were all below 10%，and that of other resources was lower except that of JS359 which was 16.18%.The relative germination potential of 6 high salt-sensitive resources were all lower，in which that of 4 resources was 0，and the other 2 resources were 3.23%and 7.69%，respectively；however，the differences of the relative germination potential of high salt-tolerant resources were obvious.Thus it can be shown that except the character of common salt-injury rate，relative germination index can be used as the salt tolerance index evaluating peanut resource，however，the evaluation using relative germination potential had larger error.

Table 3 The performance of salt tolerance of different types of peanut resources

Conclusions and Discussions

Salt-tolerance identification is the fundamental work of the evaluation of peanutgermplasm resources，the breeding of salt-tolerance varieties and the study of salt-tolerant molecular mechanism.There have already been some literature reports on the identification and evaluation of salt-tolerant peanut varieties and salt-tolerant index study.Wu Lanrong et al.［10］carried out the identification of salt tolerance on 148 peanut resources during the budding stage and seedling stage taking 1.0%NaCl solution as the stress solution，and thought that the salt tolerance of peanut resources was generally weaker.Wang Xiuzhen et al.［11］adopted 32 chemical mutagenesis to treat M2 material including 8708 peanut seeds，and then they were put into 2.0%NaCl solution，finally 81 germinating seeds were screened out，in which only 1 seed emerged and the growth vigor was good.Zhang Zhimeng et al.［18］showed that the salt tolerance of peanut can be divided into 4 types，such as high salt tolerance，salt tolerance，salt sensitivity and high salt sensitivity；with the increase of stress intensity，the quantity of salt-tolerant varieties declined；the suitable salinity identifying the salt tolerance of peanut varieties was among 0.30%-0.45%.Ci Dunwei et al.［19］thought that the salt tolerance of peanut varieties under different salt stress intensities showed differences，so different salt stress concentrations should be selected to evaluate its salt tolerance according to the salt tolerance of the varieties and the requirements of the experiment. This showed that it is of great importance to select appropriate salt stress concentration to identify its salt tolerance to peanut resources.In this study，based on the identification method introduced by Liu Yonghui［16］，NaCl with the concentration of 0.5% was selected to identify the salt tolerance of peanut germplasm.

The identification methods of salt tolerance ofpeanutinclude water planting［20］，saline soil pot［21］and direct planting identification of saline-alkali soil［13］，etc.，and the identification periods include budding stage［22］，seedling stage［15］and whole growth stage［12］，etc. The salt resistance of crops was different in different growth stages，in which they were sensitive to saltstress during budding stage and seedling stage［23］.The salt resistance of peanut during budding stage has the merits of high capacity，short time and strong repeatability，etc.［14］.For the large workload of salt tolerance identification of large amounts of germplasm resources，budding stage was taken as the salt tolerance identification stage of peanut resource in this study，and 7 high salt-tolerant resources including JS011，JS024，JS125，JS491，JS523，JS524 and JS525 were screened out. However，the salt tolerance of peanut during budding stage has not obvious correlation with that of later period［12］，thus based on the identification of budding stage，itis necessary to screen out salt tolerance materials to make further identification evaluation，so as to further verify the stability of its salt tolerance.

Table 4The performance of relative germination characters of several high salt-tolerant and high salt-sensitive resources

The research showed that multiple indexes reflecting the salt tolerance of crops were better than single index，but increasing indexes must increase the workload of identification，and the evaluation results of different indexes had differences.Ci Dunwei et al.［8］set 4 salt stress concentrations and analyzed the salt tolerance of different peanutvarieties from germinationstage to seedling stage through 10 indexes，such as seeding speed，plant morphology and biomass，etc.，the results showed that under the stress of each salt concentration，the salt tolerance indexes had greater variation，and different indexes had certain superposition on salt tolerance evaluation.Salt stress affects the normal germination of wheat［24］，rice［25］and maize［26］seeds，thereby causing the decrease of its germination potential，germination rate and germination index，the germination characteristics of seeds can comprehensivelyreflect their salt tolerance during budding stage，most researchers adopted the indexes like germination rate，germination potential，germination index and vigor index to evaluate the salt tolerance of crops during the budding stage［27-29］.In this study，4 indexes including relative germination potential，relative germination rate and relative germination index and salt-injury rate were selected to carry out salt tolerance evaluation.From the results，salt stress had prominent inhibiting effect to seed germination of peanut，however，the inhibiting effects of salt stress had obvious differences for different resources；under the condition of salt stress，the germination speed of local varieties was higher than that of improved varieties.Based on the analyses of different indexes，the results showed that for high sensitive germplasm，relative germination potential，relative germination rate and relative germination index were lower，and the consistency of the three indexes was better；the relative germination potential of different high-resistance resources had obvious differences，however，the consistency of relative germination rate，relative germination index and salt-injury rate was better.For salt-injury rate and relative germination rate，only the calculation method was different，and their results were highly related，thus in actual analysis，we should choose one. Based on this，to identify the salt tolerance of peanut resource during the budding stage，besides relative germination rate and salt-injury rate，relative germination index can also be used to evaluate.

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Responsible editor：Nanling WANG

Responsible proofreader：Xiaoyan WU

花生资源耐盐性的鉴定与评价

陈志德*，刘永惠，沈一 （江苏省农业科学院经济作物研究所/江苏省农业种质资源保护与利用平台，江苏南京210014）

为筛选耐盐花生种质并为花生耐盐遗传基础研究提供材料，选用相对发芽势、相对发芽率、相对发芽指数和盐害率为指标，以0.5%NaCl为胁迫浓度，对128份花生种质进行芽期耐盐性鉴定。结果表明，盐胁迫对花生种子萌发有显著的抑制作用，但盐胁迫的抑制效应因种质不同而存在明显差异；128份种质中，高耐盐种质仅占5%左右。在耐盐评价指标方面，除盐害率或相对发芽率以外，相对发芽指数可以作为评价指标之一。此外，盐胁迫条件下，地方品种的发芽速度高于育成品种。本研究筛选出JS011、JS024、JS125、JS491、JS523、JS524、JS525等7份高度耐盐种质可用于花生耐盐性基础研究的材料。

花生；种质资源；耐盐；鉴定评价

国家花生产业技术体系“南京综合试验站”（CARS-14）；江苏省农业科技自主创新资金“主要大田作物种质资源的收集、鉴定与共享服务”［CX（14）2001］。

陈志德（1965-），男，江苏宜兴人，研究员，博士，主要从事花生资源与育种研究。E-mail：chen701865@aliyun.com。*通讯作者。

2015-06-13

Supported by National Peanut Industry Technology System “Nanjing Comprehensive Experimental Station”（CARS-14）；the Fund of Independent Innovation of Agricultural Science and Technology in Jiangsu Province “On the Collection，Identification and Shared Service of Germplasm Resources of Main Field Crops”［CX（14）2001］.

*Corresponding author.E-mail:chen701865@aliyun.com.

Received:June 13，2015 Accepted:December 1，2015

修回日期 2015-12-01