Zhang Ling, Li Guang-he, Zhang Xu

Department of Environmental Science & Engineering, Tsinghua University, Beijing 100084, P. R. China

Abstract：It is an important issue that soil is polluted by heavy metals. People pay more attention to the direct effects and neglect the indirect effects caused by heavy metals. The aim of this investigation was to study the indirect effects of Cd on vegetable seeds in laboratory. Wheat were grown in nutrient solutions with different concentration of Cd in water and sand culture. Resolutions of root exudates were collected. The germination of radish seed and cabbage seed planted in the collected wheat root exudates was investigated. The germination of vegetable seeds planted in resolutions of root exudates were lower than contol. The germination of vegetable seeds were decreased by root exudates. The germination was the lowest when the seeds were planted in resolutions of root exudates collected from wheat grown in nutrient solutions with 5mg/L concentration of Cd in water culture and 0.5mg/L concentration of Cd in sand culture. When the concentration of Cd in water culture is lower than 5mg/L, the inhibit of resolutions of root exudates was decrescendo; while the concentration of Cd is higher than 5mg/L it is reverse . When resolutions of root exudates collected from wheat grown in nutrient solutions with 5mg/L concentration of Cd in sand culture , the inhibit of resolutions of root exudates was decrescendo when the concentration of Cd was between 0.5 and15mg/L; while the concentration of Cd was lower than 0.5 or higher than 15mg/L it is reverse. The results showed that resolutions of root exudates inhibted the germination of vegetable to some extend. Its detailed mechanism is refer to allelopathy issue and worthy to be studied further. This paper primarily provides some new information on appropriately culturing in areas polluted by Cd.

keywords: Germination, Root exudates, Stressed by Cd, Vegetable seed, Wheat

1. Intrduction

People pay more and more attention to the issue of pollution in the terrestrial ecosystem caused by heavy metals recently, especially Cd. Though Cd exists in the nature commonly, it doesn’t take part in the structures and mechanism activities of organisms, however, it is fiercely harmful if too much Cd is accumulated in the body of organisms (Leapo, 1987). In many cases, Cd is transferred and accumulated in food chain and then it will be harmful to human beings. Researches were focused on direct effects (Siedleka and Baszynsky, 1993; Wagner, 1993; Kessler and Brand, 1995; Shah and Dubey, 1995) and few research were focused on the indirect effects caused by Cd（Ling and Xue, 2003）. But the indirect effects caused by Cd become more and more important while the issue of pollution caused by Cd is becoming more and more serious and also harmful to the health of human beings. To rotate the crops in soil polluted by Cd is popular in farming. The aim of this investigation was to study the indirect effects of Cd on vegetable seeds germination in laboratory.

This investigation were focused on the indirect effects caused by Cd and supported data for pollution ecology and plant allelopathy. This paper also primarily provides some new information on appropriately culturing in areas polluted by Cd.

2.Materials and Methods

(1)  Materials

Radish (Raphanus sativus) seed and cabbage (Brassica oleracea) seed were bought in Kunming Seeds Station, Yunnan Province of China.

Wheat (Triticum aestivm) seeds without any heavy metal pollution were bought from Kaiyuan City, Yunnan Province of China.

(2)　Methods

(i) Collecting wheat root exudates solution (RES) （Yohan, 1993; Anna et al., 1995; Boeuf-Tremblay et al., 1995; Zhang and Yan, 2003）

Before geminated, wheat seeds had been sterilized by 75% ethanol for 10 minutes and washed by sterilized water for 20minutes, then seedlings were grown in a growth chamber (LRH-250-G, 40 W) with the fluorescent lamps, photo flux density nearly 60 µE m^-2 s^-1 for 12/12 hr photoperiod and control temperature 25/15±1℃(day/night).

After 2 days preculture, seedlings (100 seedlings in each container) were transferred to two series of 1000 mL containers. Half of containers filled with autoclaved Hoagland’s nutrient solution (Hoagland and Aron, 1938)(water-culture, W-culture), the others filled with nutrient solution, vermiculite and sand (vermiculite-culture, V-culture).

The Cd treatment concentrations were 0, 0.5, 5, 15 and 50 mg·L^-1. During the treatment period (9 days), 20 mL nutrient solution contained different concentrations Cd was added to each container under both W- and V-culture everyday. Each treatment was replicated three times.

After 10 days all of the wheat root in each container had been marinated in 100 ml deionized water for 1 h, then been rinsed by another 100 mL deionized water for 10 minutes. The V-cultures were treated as the same (Weerasuriya et al. 1993; Anna et al. 1995; Boeuf-Tremblay et al. 1995). 200 individual was a sample. Ten types of root exudates solution (RES) were collected, then concentrated to 100 mL by a circumrotated distillatory, respectively.

(ii) Measuring rate of vegetable seeds germination

Elite radish seed and cabbage seed had been sterilized by 75% ethanol for 10 minutes and washed by sterilized water for 20minutes. Then 100 seeds geminated in a 1000 mL container filled different ten types of root exudates solution (RES) mentioned above, respectively. 5mL RES were added in every container everyday. Each treatment was replicated three times. Distilled water treatment was control. Tests were carried in a growth chamber (LRH-250-G, 40 W) with the fluorescent lamps, photo flux density nearly 60 µE m^-2 s^-1 for 12/12 hr photoperiod and control temperature 25/15±1℃(day/night). The rate of vegetable seeds germination were measured after one week.

3. Results and Discussion

3.1 Effects of RES under W-culture on vegetable seeds germination

Figure1 Effects of RES under W-culture on vegetable seeds germination

Remark: In figure 1, No.1, No.2, No.3, No.4, No.5 represent the types of RES treatments collected from W-culture in which the Cd treatment concentrations were 0, 0.5, 5, 15 and 50 mg·L^-1, respectively.

CK represents distilled water treatment.

The germination rates of vegetable seeds cultured with RES treatments collected from W-culture were plotted in Figure 1. Each curve represents the average value of three replicates. Figure 1 showed the effects of wheat root exudates under W-culture on germination of vegetable seeds. The germination rates of vegetable seeds cultured with RES were lower than control. The germination rates of vegetable seeds were decreased by root exudates collected from W-culture. The effects of RES on the rate of radish seeds germination were closed to which of cabbage on the whole.

The rate of germination was the lowest when the vegetable seeds were planted in RES collected from wheat grown in nutrient solutions with 5mg/L concentration of Cd in W-culture. When the concentration of Cd in W-culture was lower than 5mg/L, the inhibit of solution of root exudates was decrescendo; while the concentration of Cd was higher than 5mg/L it was reverse.

3.2 Effects of RES under V-culture on vegetable seeds germination

Figure 2 Effects of RES under V-culture on vegetable seeds germination

Remark: In figure 2, ① , ② , ③ , ④ and ⑤ represent the types of RES treatments collected from V-culture in which the Cd treatment concentrations were 0, 0.5, 5, 15 and 50 mg·L^-1, respectively.

CK represents distilled water treatment.

The germination rates of vegetable seeds cultured with RES treatments collected from V-culture were plotted in Figure 2. Each curve represents the average value of three replicates. Figure 2 showed the effects of wheat root exudates under V-culture on germination of vegetable seeds. The germination rates of vegetable seeds cultured with RES were lower than control. The germination rates of vegetable seeds were decreased by root exudates collected from V-culture. The effects of RES on the rate of radish seeds germination were smaller than which of cabbage.

The rate of germination was the lowest when the seeds were planted in solution of root exudates collected from wheat grown in nutrient solutions with 0.5mg/L concentration of Cd in V-culture. In V-culture when the concentration of Cd was lower than 0.5mg/L, the inhibit of solution of root exudates was decrescendo; while the concentration of Cd was between 0.5 and 15mg/L it is reverse.

3.3 Comparing the effects of RES under different cultures on the germination of vegetable seeds

The germination rates of vegetable seeds planted in solution of root exudates were lower than control. The germination rates of vegetable seeds were decreased by root exudates. It was evident that ten types of wheat root exudates solution inhibited vegetable seeds germination.

The germination rates of vegetable seeds in solution of root exudates with W-culture were more than those in solution of root exudates with V-culture when the concentration of Cd was 50mg/L. On the contrary, the concentration of Cd was 0, 0.5, 5, 15mg/L it was reverse.

The effects of RES on the rate of radish seeds germination were closed to which of cabbage on the whole. The effects of RES on the rate of radish seeds germination were smaller than which of cabbage. This indicated that sand medium affected the root secretion.

4. Conclusions

The results showed that lower concentration of Cd had more harm than higher concentration of Cd. Another study suggested that so many kinds of important organic matter, such as sugar, amino acid, organic acid and other SMPs excreted by wheat root were affected by Cd stress (Zhang and Yan, 2003). Those matters would affect the germination rate of vegetable seeds. Also sand played some roles on the root secretion.

The results showed that solution of root exudates inhibited the germination of vegetable seeds to some extend. Its detailed mechanism is referred to allelopathy issue and worthy to be studied further. The mechanisms and rules of roots excreting under the environmental stress need to further investigate, therefore, we will know more about the reciprocity of plant to soil environment and soil micro-organism. This paper primarily provides some new information on appropriately culturing in areas polluted by Cd.

5. References

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