Cadmium Tolerance and Bioaccumulation of 18 Hemp ... - Springer Link

Appl Biochem Biotechnol (2012) 168:163–173 DOI 10.1007/s12010-011-9382-0

Cadmium Tolerance and Bioaccumulation of 18 Hemp Accessions Gangrong Shi & Caifeng Liu & Meicheng Cui & Yuhua Ma & Qingsheng Cai

Received: 10 December 2010 / Accepted: 7 September 2011 / Published online: 22 September 2011 # Springer Science+Business Media, LLC 2011

Abstract Hemp (Cannabis sativa L.) is a fast-growing and high biomass producing plant species, which has been traditionally grown as multiple-use crop and recently considered as an energy crop. In order to screen accessions that can be cultivated in cadmium (Cd)contaminated soils for biodiesel production, the ability of Cd tolerance and bioaccumulation of 18 hemp cultivars or ecotypes were evaluated in pot experiment under 25 mg Cd kg−1 (dry weight, DW) soil condition, in terms of plant growth, pigment contents, chlorophyll fluorescence, and Cd accumulation at 45 days after seedling emergence. Results showed that seedlings of all cultivars, except USO-31, Shenyang and Shengmu, could grow quite well under 25 mg Cd kg−1 (DW) soil condition. Among them, Yunma 1, Yunma 2, Yunma 3, Yunma 4, Qujing, Longxi, Lu'an, Xingtai, and Shuyang showed great biomass (>0.5 g plant−1), high tolerance factors (68.6–92.3%), and little reduction of pigment content and chlorophyll fluorescence under 25 mg Cd kg−1 (DW) soil stress, indicating these cultivars had a strong tolerance to Cd stress and could be cultivated in Cd-contaminated soils. Cultivars Longxi, Lu'an, Xingtai, Yunma 2, Yunma 3, Yunma 4, and Qujing exhibited higher Cd concentrations and total Cd in shoots. These cultivars, therefore, are good candidates for the implementation of the new strategy of cultivating biodiesel crops for phytoremediation of Cd-contaminated soils. Keywords Cadmium . Hemp . Tolerance . Accumulation

Introduction Cadmium (Cd) is a highly toxic trace element and has been ranked no. 7 among the top 20 toxins [1, 2]. Large areas of land in many countries have been contaminated by Cd whose G. Shi : C. Liu : M. Cui : Y. Ma College of Life Sciences, Huaibei Normal University, Huaibei 235000, People’s Republic of China Q. Cai (*) College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China e-mail: [email protected]

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Appl Biochem Biotechnol (2012) 168:163–173

presence in the environment is mainly due to industrial processes and phosphate fertilizers [3]. This pollution is especially important when the contaminated lands are used for crop cultivation, since Cd is easily transferred to food chain from the soils, threatening human and animal health [4]. Clean-up of Cd-contaminated soils is difficult. Immobilization or extraction by physicochemical techniques can be expensive and is often incompatible with maintaining soil structure and fertility [5]. Although phytoremediation is considered as a cost-effective, environmentally friendly approach that is applicable to large areas, it also has some disadvantages: (1) The plants that can be used for phytoremediation are herbs with small biomass, which have little economic value [6]. (2) The process of phytoremediation is quite slow and usually takes several years, or even decades, to halve the levels of Cd in the soil [7]. (3) During phytoremediation, the contaminated land cannot be sold or rented, which can cause problems for local economic development [8]. To fully utilize Cd-contaminated soils and to overcome the disadvantage of phytoremediation, we have postulated a new strategy of cultivating energy plants in Cd-contaminated soils for bioenergy production, and this can be combined with phytoremediation [9, 10]. We are interested in the possibilities of hemp (Cannabis sativa L.) as a candidate crop for several reasons. Firstly, hemp is a multiple-use plant, widely employed in many types of non-food industries and providing raw material for the production of natural fiber, insulating board, rope, oil, varnish, and paper [11, 12]. Secondly, hemp is a metal-tolerant organism, and the amount of Cd accumulated in its shoot was not negligible [6, 13–15]. Thirdly, hemp is also a good candidate for soil phytoremediation as it is a high biomass producing plant with about 1-m deep roots that grow fast and easily in dense stands [12, 13]. Finally, it has been recently considered as an energy crop, from which the biomass used as fuel, or the seed oil for biodiesel production [16]. The purpose of this study is to (1) evaluate the Cd tolerance and Cd accumulation capacity of 18 hemp cultivars, (2) screen for cultivars that can be planted in Cdcontaminated areas for bioenergy production, and (3) identify cultivars that can be used for phytoremediation of Cd-contaminated land.

Materials and Methods Experimental Setup After an initial screening, 18 hemp accessions were selected for this study (Table 1). Pot tests were carried out in a greenhouse at the Huaibei Normal University, Huaibei, China (33.57° N, 116.47° E). The average temperature throughout the test period was between 25.9±3.8 °C (daytime) and 21.7±3.6 °C (night), and the relative humidity was 61.5±5.3% (daytime) and 67.4±6.9% (night). The soil type is the gravel blackland (sand/silt/clay; 23.8%:20.3%:55.9%), which is characterized as pH 7.24; N, 68.4 mg kg−1; P2O5, 9.36 mg kg−1; K2O, 75.3 mg kg−1; organic matter, 1.12%; electrical conductivity, 23.6 μs cm−1; Cd, 0.126 mg kg−1. For the Cd treatments (Cd+), 25 mg Cd kg−1 soil (DW) were added into soils as CdCl2·2.5H2O, and for the controls (Cd−), no Cd was added. Each of the treatment had three pots (16×20 cm) filled with 3 kg of soils, and ten uniform seedlings were allowed to grow in each pot at a uniform spacing. All soils were additionally supplemented with 1.0 g KH2PO4 kg−1 soil (DW), 1.5 g NH4NO3 kg−1 soil (DW). The pots were watered daily to 60% of water holding capacity.


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Table 1 Names, abbreviations, origin, and seed providers of hemp cultivars used for pot experiments Cultivar

Abbreviation

Origin

Seed provider

Boyin 5

BY5

Russia

Heilongjiang Academy of Agriculture Sciences

Longxi

LX

Longxi, Gansu

Dingxi Institution of Agriculture Sciences

Zhouqu

ZQ

Zhouqu, Gansu


Xingtai Shuyang

XT SY

Xingtai, Hebei Shuyang, Jiangsu

Heilongjiang Academy of Agriculture Sciences Heilongjiang Academy of Agriculture Sciences

Shenyang

LSY

Shenyang, Liaoning


Shenmu

SM

Shenmu, Shanxi


Yangcheng

YC

Yuncheng, Shanxi


USO-14

U14

Russia


USO-31

U31

Russia


Wuchang 40

WC40

Wuchang, Heilongjiang


Lu'an Huocheng

LA HC

Lu'an, Anhui Huocheng, Xinjiang

Lu'an Institution of Agriculture Sciences Heilongjiang Academy of Agriculture Sciences

Yunma 1

YM1

Kunmin,Yunnan

Yunnan Academy of Agriculture Sciences

Yunma 2

YM2

Kunmin,Yunnan


Yunma 3

YM3

Kunmin,Yunnan


Yunma 4

YM4

Kunmin,Yunnan


Qujing

QJ

Qujing, Yunnan


Evaluation of Plant Growth and Metal Accumulation Seedlings were harvested after 45 days. The plants were washed with running tap water and rinsed with deionized water to remove any soil particles attached to the plant surfaces. The roots and shoots were separated and oven-dried for 30 min at 105 °C, then at 65 °C, until they reached constant weights. The dried tissues were weighed and ground into a powder. Cd concentrations were measured using flame atomic absorbance spectrometry after digestion with mixed acid [HNO3 +HClO4 (3:1, v/v)]. The tolerance index (TI) was expressed on the basis of root and shoot biomass, calculated as the following: TI ¼ 100 ½biomassCd =½biomasscontrol

ð1Þ

The translocation factor (TF), bioconcentration factors (BCFs), and total Cd in plant tissues were calculated as follows [17]: TF ¼ ½Cdshoot =½Cdroot

ð2Þ

BCF ¼ ½Cdshoot or root =½Cdsoil

ð3Þ

Total Cd in plant tissues ¼ ½biomassshoot or root ½Cdshoot or root

ð4Þ

Chlorophyll Concentration Analysis Mature leaves (0.2 g) from plants in each pot were extracted in the dark at 4 °C in a 5-ml mixture of acetone and ethanol (v/v=1:1) until the color had completely disappeared. Total

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chlorophyll and carotenoids were determined with UV-vis spectrophotometers according to the method of Lichtenthaler [18]. Chlorophyll Fluorescence Measurement Before harvesting, four leaves located at the same position of seedlings were used for measuring chlorophyll fluorescence parameters, the ratio of variable to maximal fluorescence (Fv/Fm) and the effective quantum yield of PSII (ΦPS II). This was performed by a pulse of saturating light pulse (5,000 μmol quanta m2 s−1, 1 s), using a Mini PAM (Walz, Effeltrich, Germany). To determine the Fv/Fm values, plants were darkadapted for 30 min, and ΦPS II, under irradiation of 500 μmol quanta m2 s−1. Statistical Analysis Statistical analyses were performed using SPSS Version 13.0 software (SPSS Inc., USA). The effect of the different accessions and the interaction between accessions and Cd treatment on biomass, pigment content, and chlorophyll fluorescence were compared using a two-way ANOVA. The data of tolerance index and Cd accumulation were subjected to one-way ANOVA and Tukey's multiple range test.

Results Biomass and Tolerance Index There were significant differences between cultivars in shoot and root biomass. By contrast, Yunma 1, Yunma 2, Longxi, Qujing, Lu'an, Yunma 3, Yunma 4, Xingtai, and Shuyang showed greatly higher shoot and root biomass for both the control (Cd-) and 25 mg kg−1 Cd (Cd+) treatments (Fig. 1). Treatment with 25 mg kg−1 Cd decreased the shoot and root

Fig. 1 Shoot and root biomass in seedlings of 18 hemp accessions exposed to 25 mg Cd kg−1 soil for 45 days. Data are expressed as means±S.E. of three replicates. ***p