Screening of safe soybean cultivars for cadmium contaminated fields

The selection and breeding of Cd-safe cultivars (CSCs) has been used to minimize the influx of Cd into the human food chain. The pot-culture experiment combined with the field-culture experiment were conducted to screen out CSCs, i.e. the cultivars accumulating Cd at low enough level for safe consumption in their edible parts when grown in contaminated soils, were screened out and explored among the crop cultivars. We used 25 Chinese soybean cultivars in different Cd contaminated soils to assess the performance of this new method. Variations in uptake, enrichment, and translocation of Cd among these cultivars were studied to screen out soybean CSCs. The accumulation of Cd in the five soybean genotypes was lower than 0.20 mg kg−1 under 1.0 mg Cd kg−1 treatment, and the EF and TF were lower than 1.0. The field studies further identified that cultivar Shennong 10, Tiedou 36 and Liaodou 21 fit the criteria for CSCs, which were suitable to be planted in low-Cd (Cd concentration < 1.22 mg kg−1) contaminated soils. The results can provide scientific methods for screening low-Cd accumulation in soybeans and can provide a path for controlling, treating and remedying Cd-contaminated agricultural soils to make grains safe for human consumption.

www.nature.com/scientificreports/ bone growth. Because of the high economic benefits and suitable natural conditions for planting soybean, farmers are more willing to grow soybean in the world. The amount of Cd that enters human diets from a crop depends on the amount of Cd accumulated in the parts that are consumed, so the translocation of Cd within soybean plants, especially into its seed, is very important for human Cd intake through human diets. Thus, decreasing the content of Cd in soybean seeds is extremely important. The results of this work can provide scientific methods for screening low-Cd accumulation in soybeans and they can provide a path for controlling, treating and remedying Cd-contaminated agricultural soils to make crops safety for human consumption.

Results
Tolerance of 25 soybean cultivars by Cd stress in the pot-culture experiment. In Fig. 1, it describes the plant height of 25 soybean cultivars in different Cd treatments. The plant height of Taiwan 292, Tiefeng 33 and Tiedou 36 under various Cd treatments did not significantly (P > 0.05) differ from CK. In particular, there was a significant (P < 0.05) increase in the plant height of the 17 cultivars (Table 3) under treatments (T 1 and T 2 ) as compared to CK. The result indicated that the above-mentioned Chinese soybeans had tolerance to Cd toxicity. However, the plant height of Tiefeng 30, Tiefeng 35, Kaiyu 13, Kaiyu 11 and Kaijiao 8,157 under T 1 and T 2 decreased significantly (P < 0.05) when compared with CK. It indicated that the five soybean cultivars had poor tolerance to Cd stress. Meanwhile, all the tested cultivars grew normally under Cd stress and had no obvious toxicity symptoms between various treatments. The shoot biomass of soybean plants can also be used to assess plant tolerance to Cd. The seed biomass of 25 cultivars under T 1 and T 2 differed significantly (P < 0.05) from that in the control (Fig. 2). The interactions between Cd treatments and soybean cultivars were highly significant (P < 0.05) for seed weight, indicating that the effects of Cd on the growth and development of various soybean cultivars differed obviously. By their changes in seed weight under T 1 and T 2 , the 25 soybean cultivars can be divided into four groups: (1) the seed weight decreased significantly with an increase in the Cd concentration of Cd in soil, such as Shennong 8, Taiwan Table 1. It showed that there was a significant (P < 0.05) difference in Cd accumulation among 25 cultivar seeds under T 1 and T 2 , ranging from 0.09 to 0.98 and from 0.29 to 2.06, and with the mean Cd accumulation of 0.44 and 0.81 mg kg −1 DW (dry weight) respectively. It demonstrated that Cd concentrations of 80% (20/25) of the samples examined under T 1 exceeded the MPC, and 36% were especially higher than 0.50 mg kg −1 DW. Because soybean seeds are the edible parts for human beings, the concentration of Cd in soybean seeds should be lower than 0.20 mg kg −1 DW and regarded as a rigorous standard for screening out CSCs among 25 soybean cultivars. According to the accumulation of Cd in seeds, the 5 soybean cultivars including Shennong    www.nature.com/scientificreports/ under T 2 , more Cd was accumulated in the seed compared with that under T 1 . The phenomenon can indicate that the accumulation of Cd in seed will increase to some extent with increasing Cd concentration in soils. While no more than 30% of the total Cd uptake was distributed in the seed of the selected cultivars under T 2 except Hefeng 40. Thus, the absolute majority of Cd absorbed by soybean plants was retrained in roots, stems, leaves and pods, and only a very small portion of the Cd accumulation was transferred into soybean seeds.
Enrichment factor and translocation factor of 25 soybean cultivars in the pot-culture experiment. It is a significant (P < 0.05) difference among different soybean cultivars under T 1 and T 2 , ranging from 0.42 to 5.40 and from 0.24 to 3.23, and with the mean of 1.81 and 1.43 respectively (Fig. 4). The EF value in 72% (18/25) of the tested cultivars decreased with an increase in the concentration of Cd in soils. The highest EF value under the two treatments was found in Hefeng 40, up to 5.40 and 3.23 respectively. Under the two Cd treatments, the EF value in the 5 soybean genotypes including Shennong 10, Tiefeng 31, Tiedou 36, Tiefeng 37 and Liaodou 21 was lower than 1.0. In other words, the 5 genotypes had poor ability to uptake Cd from soils. The average TF value of different soybeans under T 1 and T 2 was from 0.09 to 1.99 and from 0.05 to 1.87 respectively (Fig. 5). Under the Cd treatments, the TF value was all lower than 1.0, excepting Hefeng 40. Being similar with the EF value, the highest TF value was also found in Hefeng 40, up to 1.99 and 1.87 respectively. It indicated that Cd uptake in soybean cultivars was limited in their roots, and only a small portion of Cd could be translocated to the edible parts except Hefeng 40. In a word, the EF and TF values in Shennong 10, Tiefeng 31, Tiedou 36, Tiefeng 37 and Liaodou 21 were lower than 1.0.    www.nature.com/scientificreports/ bean cultivars were all higher than 0.20 mg kg −1 . The EF and TF of different soybean cultivars in the field-culture experiment was from 0.41 to 4.2 and from 0.08 to 1.07, respectively, which was relatively lower than that in the pot-culture experiment. The highest Cd concentrations, EF and TF were found in Hefeng 40 in the field-culture experiment, which was similar to the results in the pot-culture experiment. The EF and TF in Tiefeng 31, Shennong 10 and Tiedou 36 were all lower than 1.0.

Accumulation and translocation of Cd in 25 soybean cultivars in the field
Correlation of seed Cd concentrations between the pot-culture and field-culture experiments. Correlation of seed Cd concentrations between the field-culture experiment and the pot-culture www.nature.com/scientificreports/ experiment under T 1 was significantly positive(r = 0.994, n = 25, P < 0.01) (Fig. 8). Meanwhile, correlation of seed Cd concentrations was also found between the field-culture experiment and the pot-culture experiment under T 2 (r = 0.656, n = 25, P < 0.05).

Discussion
In recent years, the pollution of Cd in agricultural soil has attracted worldwide attention 32,33 , mainly because of its strong toxicity to human health. Therefore, breeding crop varieties with high Cd resistance or high tolerance, which can improve crop yield potential and reduce the accumulation of heavy metals in edible parts, is of great significance for safe crop and food production.
In the pot experiment, the results showed that not only four fifths of the 25 tested soybean varieties had no significant decrease in plant height (Fig. 1)    www.nature.com/scientificreports/ decrease in seed biomass in soils treated with two Cd treatments (Fig. 2), and even 7 cultivars showed significant increase in seed biomass in soils treated with two Cd treatments. The results showed that the tested soybean had a certain degree of tolerance to soil Cd toxicity. Similar positive and neutral responses of biomass to heavy metal stress were also observed in crops such as asparagus bean 19 , rice 34 35 . Similar results were observed in this study ( Table 3). The seed Cd concentrations of 25 soybean varieties ranged from 0.09 to 0.98 mg kg −1 DW under 1.0 mg Cd kg −1 treatment, and 80% of the tested soybean varieties had seed Cd concentrations over 0.2 mg kg −1 , which exceeded the MPC of Cd in soybean. This means that even in low cadmium polluted soil, the concentration of Cd in some soybean seeds exceeds the concentration harmful to human body. Therefore, it is necessary to breed soybean varieties with low Cd accumulation. Especially in China, the most populous country in the world, soil Cd pollution has become one of the important obstacles to sustainable agricultural development 5,14 .
Up to now, there is no clear screening criteria for CSCs. Based on previous studies 40,41 , this study used four criteria for screening food safety CSCs: (1) The concentration of Cd in the edible fraction should be lower than that of MPC (0.2 mg kg −1 DW); (2) EF < 1.0; (3) TF < 1.0; (4) As measured by aboveground biomass and height, they can tolerate Cd in polluted soil. In the pot-culture experiment, when the Cd concentration (< 0.2 mg kg −1 ) using edible portion as the standard, five soybean varieties including Shennong 10, Tiefeng 31 and Tiedou 36. Tiefeng 37 and Liaodou 21 in Table 3 could be selected as CSCs under T1 treatment (1.0 mg kg −1 ). In addition, EF and TF values were lower than 1.0 (Figs. 4 and 5). Shennong 10, Tiefeng 31, Tiedou 36, Tiefeng 37 and Liaodou 21 were also used as CSCs (Fig. 2 and Table 2).In the field cultivation experiment, only Tiefeng No. 31 was used, and Shennong 10 and Tiedou 36 were finally determined as CSCs (Fig. 6). However, under treatment T2 the Cd content in the edible part of 25 soybean cultivars was more than 0.2 mg kg −1 DW. In other words, the Cd accumulation in the edible part of 25 soybean cultivars could not be considered as CSCs under T2 treatment with higher Cd content in soil. Therefore, the selected CSCs are only suitable for planting soybean in contaminated soil with low Cd content. In this study, Tiefeng 31, Shennong 10 and Liaodou 21 can be confirmed to be CSCs. In addition, Cd concentration, EFs and TFs of Hefeng 40 were the highest in pot and field experiments, which may pose a high risk to human and animal health through the food chain.
A systematic screening method was used to study the effects of endogenous and environmental factors and their interactions on Cd uptake in soybean cultivars by pot and field screening. Kurz  www.nature.com/scientificreports/ field cultivation experiment 35 . In this study, Tiedou36 and Tiefeng37 were also identified as CSCs in T1 culture. However, in the field culture experiments, because the Cd concentration of seeds is higher than that of MPC, they cannot be considered as CSCs. Obviously, rhizosphere conditions in a closed system are different from those in an open system. In addition, there was a significant positive correlation between seed Cd concentration in field culture experiment and that under low level Cd stress. But not the high level of Cd stress in the pot experiment (Fig. 8). The results showed that the effect of environmental factors on Cd uptake was much greater than that of internal factors. Previous studies have shown that Cd can affect the absorption of other nutrients by crops. Even affect the quality of crops. Liu et al. found that Cd had the most significant effect on the contents of mineral elements in rice roots and leaves, but the results were different for different metal elements, different plant organs and different growth stages 17    www.nature.com/scientificreports/ Liu et al. reported that Cd inhibited the accumulation of K, P, Ca, Mg, Mn and Zn in the aerial part of Chinese cabbage, and the degree of inhibition was different in different varieties 41 . Different reports also give different results about the effect of cadmium on the absorption of microelement [43][44][45] . Therefore, Cd pollution can cause changes in the uptake and transport of other nutrients by crops, which is a very complex problem. However, there are few reports about the correlation between Cd and nutritional elements of soybean. Therefore, the relationship between Cd and other nutrient elements in Chinese soybean should be further studied. Heavy metal pollution rarely occurs on a single metal 18 . For example, Chinese cabbage and cucumber produced in about 10,000 hm 2 of farmland in Shenyang suburb were found to be polluted by Pb and Cd in Liaoning Province, China, and the pollution of Pb, Cd and Hg respectively 46 . Chao et al. reported that much of the land around the Shenyang smelter in northeastern China was found to be heavily contaminated with Cd, Pb and Zn 47 . Bahemuka and Mubov found that vegetables grown along the Sinza and Msimbazi rivers in Dares Salaam, Tanzania, Dares Salaam had relatively high levels of Cd, Pb and Zn in Tanzania. Therefore, our next step is to select CSCs in the case of multi-metal pollution, and verify the feasibility of CSCs strategy 48 .
In this study, clear criteria for screening CSCs were explored. Therefore, only in the polluted soil with low Cd content, the CSCs strategy of soybean varieties is reasonable and feasible. This study provided a scientific basis for selecting soybean varieties with low Cd accumulation, and could be used as a long-term effective and economic means to reduce Cd pollution in crops. Although variety selection will be effective and easy for consumers to improve safer crop and food production, at present, there are still some constraints in the selection process of CSCs. The content of heavy metals in plant tissues varies with the degree of soil pollution, plant genotype and environmental factors 49 . Therefore, further studies to understand the CSCs genotype and environmental effects and their interaction mechanism, as well as the mechanism of pollutant absorption, migration and accumulation in CSCs, is a new direction for the establishment and cultivation of CSCs.

conclusions
A systematic screening methodology, pot-culture combined with field-culture screening, was used to identify CSCs in this study. In the pot-culture experiment, the Cd accumulation in seeds of 25 soybean genotypes had significant (P < 0.05) differences under T 1 and T 2 , with the mean of 0.44 and 0.81 mg kg −1 DW, respectively. The accumulation of Cd in the five soybean genotypes was lower than 0.20 mg kg −1 under T 1 , and the EF and TF were lower than 1.0. There were significant positive correlation of seed Cd between the field-culture experiment and the pot-culture experiment. Therefore, Shennong 10, Tiedou 36 and Liaodou 21 can be regarded as CSCs, which were safe for consumers only when they were cultivated in the low Cd-contaminated soils( Cd concentration should be lower than 1.22 mg kg −1 ).

Experimental site and soil characterization.
A pot-culture experiment was carried out under the open field conditions in the Shenyang Station of Experimental Ecology, Chinese Academy of Sciences (41° 31′ N and 123° 41′ E), which is located at the south of Shenyang City, Liaoning Province, China. Top meadow burozem soil (0-20 cm) was collected from an agricultural area in the station. The average annual temperature in this site was about 6-10 °C and the precipitation was 650-700 mm. The frostless duration was 127-164 days per year. Meanwhile, a field-culture experiment was carried out on a farm (41° 41′ N and 123° 55′ E) in the Dongling District, Shenyang City, Liaoning Province, China. The site meteorology is basically similar to the pot culture experiment site. The basic physical and chemical properties of the pot and field soils were analyzed by the routine analytical methods for agricultural chemicals in soils 50 . In Table 2 it showed the details of the basic properties in soils.
Experimental design. The soil samples were ground to pass through a 4 mm sieve, after mixing with appropriate amount of Cd (in CdCl 2 ·2.5H 2 O solution), then 2.5 kg of soil samples were filled into each plastic pot (Φ = 20 cm, H = 15 cm). There were three treatments including CK (the control, without Cd spiked to soil) and two Cd treatments including T 1 (1.0 mg Cd kg −1 soil) and T 2 (2.5 mg Cd kg −1 soil) were applied. The two levels (1.0 and 2.5 mg Cd kg −1 soil) stood for low and serious contaminations, according to the evaluation methods of single pollution index and the grading standard of polluted soils by heavy metals 18,35,51 . The soil was watered and then left to equilibrate completely outdoors under a waterproof tarpaulin for about 4 weeks. The period is long enough for natural equilibration of the various sorption mechanisms in the soil. These pots were arranged in randomized complete square design with treble replicate in order to minimize experimental errors.
Soybean seeds were obtained from a seed company in Shenyang, China. Twenty-five soybean cultivars of different origins were used in the pot-culture experiment and the field-culture experiment (Table 3). They were sterilized in 2% (v/v) hydrogen peroxide for 10 min and then washed several times with distilled water. Then six seeds per pot were sowed directly into the soil in the pots. In order to imitate field conditions, soybeans in pots were allowed to grow under the open field conditions and no fertilizers were applied. Using tap water (no Cd detected) to maintain 75% of the field water-holding capacity, and a dish was placed under each pot to gather latent leachate during the experiment period. Within 2 weeks after germination, the seedlings were thinned to three strains in per pot. The selected seedlings were about 7 cm in height with 2 leaves. All tested soybeans were harvested at the seed-maturity stage.
And in the field-culture experiment, 25 soybean cultivars were further tested. The soaked seeds were sowed into soil ridges in the field, arranged in a randomized complete block design with six replicates 49 . The farm management was also similar to that of the pot-culture experiment.
Sampling and chemical analysis. Plants were firstly washed thoroughly three times with tap water to remove soil and then carefully washed with de-ionized water for approximately 3 min. The roots, stems, leaves, Scientific RepoRtS | (2020) 10:12965 | https://doi.org/10.1038/s41598-020-69803-4 www.nature.com/scientificreports/ bean-pods and seeds were separated. The samples were dried at 105 °C for 5 min, and then at 70 °C in an oven until completely dried. Every part biomass of plants were weighed and then were ground to power. Soil samples were air-dried and ground using a mortar and pestle, and then passed through a 0.149 mm sieve. The plant and soil samples were digested with a solution containing 87% of concentrated HNO 3 and 13% of concentrated HClO 4 (v/v) 52 . The concentration of Cd was determined by using an atomic absorption spectrophotometer (AAS, Hitachi 180-80 type, made in Japan). The detection limit for the heavy metal analysis is 0.01 mg kg −1 . A certified reference material, soybean material (GBW10013, Qinghai Province, China) was used to monitor the recovery of metals from the plant samples. The geochemistry standard samples were used in this study to validate the soil analyses 33 . The recovery rates for the certified references, material soybean material and geochemistry standard sample, were 98% and 99%, respectively.
Safety standard and statistical methods. According to the National Food Hygienic Standard of China (NFHSC), the maximum permissible concentration (MPC) of Cd (GB2715-2005) in grains for safe consumption is 0.2 mg kg −1 dry weight (DW).The standard was employed to evaluate the safety of consuming the seeds of the tested soybean cultivars grown in heavy metal contaminated soil. For all crops, only the edible portions were sampled as the work was focused on the soil-plant-human pathway of trace metals. Thus, in this study, to evaluate the ability of a plant accumulating heavy metals Cd from soil to seed, the enrichment factor (EF) 35,53 was calculated as follows: where C seed is the average Cd concentration (DW) of the seed of each cultivar, and C soil is the total Cd concentration in corresponding soil.
To evaluate the transfer potential of Cd from root to seed, we calculated the translocation factors (TF) 40,41 as follows: where C seed is the average Cd concentration (DW) of the seed of each cultivar, and C root is the average Cd concentration (DW) of the root of each corresponding cultivar.
Data were analyzed by using the Excel 2003 and SPSS 13.0. All the values are expressed as mean ± standard deviation (S.D.) of the three replicates. Differences were considered significant at P < 0.05. Data were analyzed by one-way ANOVAS with the Duncan's multiple range tests to separate means. All results were expressed on a dry weight basis.