Study on Cadmium Tolerance of Seed Germination and Seedlings Growth of Different Rice Varieties

Guiyuan MENG，Mengyang Ll，Jing ZHOU*，Ting TANG，Shuying ZENG

1.College of Agriculture&Biotechnology，Hunan University of Humanities，Science and Technology，Loudi 417000，China；

2.Collaborative Innovation Center for Farmland weeds control of Hunan Province，Loudi 417000，China

Study on Cadmium Tolerance of Seed Germination and Seedlings Growth of Different Rice Varieties

Guiyuan MENG1，2，Mengyang Ll1，Jing ZHOU1，2*，Ting TANG1，Shuying ZENG1

1.College of Agriculture&Biotechnology，Hunan University of Humanities，Science and Technology，Loudi 417000，China；

2.Collaborative Innovation Center for Farmland weeds control of Hunan Province，Loudi 417000，China

The research used bioassay method to explore seed tolerance of Cd in terms of seed germination and seedling growth.Specifically，12 rice varieties of three types were investigated under Cd stress，covering germination rate，shooting index，vigor index，seedling and shoot lengths，and fresh weights.The results showed that as Cd concentration grew，seed germination and seedling growth kept increasingly inhibited；germination index，vigor index，root length，shoot length and fresh weights went down；inhibition effects on seed germination and seedling growth were conventional rice＞two-line hybrid rice＞three-line hybrid rice.Shenyou 9588，Tianyou 998，Chuanxiang 8，and Y Liangyou 1 performed strongly in Cd tolerance；C Liangyou 651，Nei 5 You 263，Xiangwanxian 16，Huanghuazhan，Xiangwanxian 13，and Zhunliangyou 527 performed poorly in Cd tolerance，and Cd tolerance of rest treatments were moderate.

Variety types；Rice seed；Germination；Seedling growth；Cadmium tolerance

I n Hunan Province，it is a key issue that heavy metal pollution is increasingly deteriorating with industry and development and mineral resources exploitation.For example，Cd，Hg and Pb have become pollutants which are the most dangerous in farmland，posing threats to agricultural production and food safety［1］and more attention is paid to Cd which is easily absorbed by plants and enriches fast in human bodies［2］.Rice is the second most-produced cereal after wheat and the most-consumed cereal in China and rice safety has been closely linked to human health.In Hunan，Cd is a most common heavy metal in farmland soils.Specifically，Cd contents of early-season rice and late-season rice reach as high as 5.33 and 9.36 mg/kg［3-4］in severely contaminated ar eas.In Lengshuijiang，Cd content of contaminated soils tends to be volatile in the range of 2.5-181.6 mg/kg.

Currently，much more attentions have been drawn by remediation of heavy-metal polluted soils by superenrichment plants.For instance，Wang and Liu et al.researched that absorption and accumulation on Cd are different upon rice varieties［5-11］.The re search on Cd-tolerance of rice seeds is basis of selection and breeding of Cd-tolerant varieties，and the test on heavy-metaltolerance during seed germination stage is applied globally［12-14］.Therefore，it is of great importance for selection and cultivating Cd-tolerant rice varieties to explore differences of rice seeds in Cd tolerance.The research investigated effects of Cd stress on seed germination and seedling growth based on 12 rice varieties，providing references for selective breeding of Cd-tolerant rice varieties and for selection of rice varieties with low-absorption capacity of Cd in contaminated farmlands.

Materials and Methods

Test materials

The test materials included local rice，such as Huanghuazhan，Xiangwanxian 13，Xiangwanxian 16，Nongxiang 18；two-line hybrid rice，such as C Liangyou 651，F(xiàn)engliangyou 4，Zhunliangyou 527，and Y Liangyou 1，and three-line hybrid rice such as Shenyou 9588，Tianyou 998，Chuanxiang 8，Nei 5 You 263.

Test design and treatment

The test was conducted in College of Agriculture&Biotechnology agronomy laboratory of Hunan University of Humanities，Science and Technology. At first，fully-grown rice seeds were selected and washed clean，and then sterilized with 0.5%NaClO for 20 min. Subsequently，the seeds were washed repeatedly and sealed with bags.It is notable that a few holes were pricked on bag bottoms.The seeds were merged for 12 h and collected and drain off for 12 h，which were repeated twice.Then，the seeds were collected and placed into culture dishes containing 10 ml 0.5 mmol/L CdCl2with double-layer filter paper.Every culture dish contained 50 seeds.Additionally，the treatment with distilled water was taken as a control and other treatments were repeated three times.Besides，seed culture was conducted at 26℃for 5 d，and the seeds were illuminated for 14 h and in darkness for 10 h every day.The culture solution was supplemented once every day during culture period.

Measured items and methods

During test period，the number of seed shoots was observed and recorded every day and germination standard was determined when seedling grew the length half of the seed length to compute germination rate.Furthermore，root length，shoot length，fresh weights of root and shoot of seedlings were measured on the 6thd.Subsequently，germination index，vigor index，stress index and comprehensive inhibition effect were calculated of different rice varieties［9］.

Germination rate=（The number of germinated seed/The number of test seeds）×100%；

Germination index=Σ（The number of germinated shoots on the specific day/Total germination days）

Vigorindex=The length of seedling length（cm）×Germination index；

Stress index=1-（Treatment value/ Control value）.

The comprehensive inhibition effect refers to averaged stress index of seed of the same rice variety.

Data processing

The test data were processed with Excel 2003 and DPS 3.0.

Table 1 Effects of Cd on seed germination rate and index of different rice varieties

Results and Analysis

Effects of Cd on seed germination，germination index and vigor index of different rice varieties

As shown in Table 1，seeds of different rice varieties kept decreasing upon Cd concentration and the decrease of three-line hybrid rice proved the slowest，followed by two-line hybrid rice and common hybrid rice，reducingby26.49%，63.41%and74.53%，respectively，than that of the control，when Cd concentration reached 1.25 mmol/L.Specifically，when Cd concentration was 0.5 mmol/L，germination rates of most seeds maintained higher than 90.0%，except of Huanghuazhan and Chuanxiang 8（below 85.00%），which indicated that most seeds would not be inhibited in germination by Cd at the concentration of 0.5 mmol/L.However，seed germination rates declined in varying degrees as Cd concentration increased.For example，when Cd concentration grew to 1.0 mmol/L，germination rates of mostseeds dropped considerably，with exceptions of Zhunliangyou 527，Shenyou 9588 and Tianyou 998，which suggested that most rice seeds underwent Cd stress significantly of the concentration.In general，germination rates dropped faster of Xiangwanxian 13，Huanghuazhan，Xiangwanxian 16，showing poor tolerance capacity of Cd；germination rates of Shenyou 9588，Tianyou 998，Chuanxiang 8，and Zhunliangyou 527 decreased slowly under Cd stress at a high concentration，showing higher tolerance capacity；germination rates of rest varieties kept moderate.

As shown in Table 1，relative germination index declined in varying de-grees upon Cd concentrations.Specifically，with Cd concentration in the range of 0-1.0 mmol/L，relative germination rates of different rice varieties dropped slowly，which demonstrated that germination speed is less affected by Cd of the concentrations.When Cd concentration grew to 1.25 mmol/L，rice varieties differed significantly in germination index.For instance，germination indices of local rice and twoline hybrid rice went down by 10.78% and 8.17%than those of Cd at former concentration，and three-line hybrid rice reduced by 4.57%，which incorporated that germination index of threeline hybrid rice is less affected by Cd stress.Of the 12 varieties，Huanghuazhan，Xiangwanxian 13，Y Liangyou 1，Nei 5 You 263，C Liangyou 651，and Xiangwanxian 16 declined the most significantly in terms of germination indices，which proved that the varieties are poorly in Cd-tolerance.In contrast，Shenyou 9588，Tianyou 998，and Chuanxiang 8 dropped dramatically in the germination indices，indicating that the varieties perform better in Cd tolerance.The rest varieties kept moderate.

Table 2 Effects of Cd on seed vigor index of different rice varieties

Table 3 Effects of Cd on root and shoot lengths of different rice varieties

As shown in Table 2，vigor index of seeds declined in varying degrees upon Cd concentration.For example，when Cd concentration was lower than 1.0 mmol/L，vigor indices of the three rice varieties'seeds showed little differences，but seeds of local rice and two-line hybrid rice declined dramatically，which indicated that vigor indices of the varieties in the two classifications underwent Cd stress significantly，showing poor tolerance capacity. When Cd concentration grew to 0.5 mmol/L，vigor indices of the 12 rice varieties declined dramatically and the decreases slowed down when Cd concentration increased to 1.0 mmol/L. When Cd concentration enhanced to 1.25 mmol/L，the vigorindices of Huanghuazhan， Xiangwanxian 13，Nongxiang 18，F(xiàn)engliangyou 4 and Xi-angwanxian 16 dropped considerably，which demonstrated that the varieties performed poorly in Cd tolerance.In contrast，Y Liangyou 1，Shenyou 9588，and Tianyou 998 performed better in Cd tolerance，whose vigor indices reached 5.78%，10.85%and 5.71% with Cd of 1.25 mmol/L，respectively.

Effects of Cd on the length of root and shoot of different rice varieties

As shown in Table 3，both of root and shoot lengths of rice seeds kept decreasing upon Cd concentration，and the higher Cd concentration，the higher inhibition.Specifically，average root lengths differed insignificantly under Cd stresses of different rice varieties，but root length reached maximum of three-line hybrid rice when Cd concentration reached 1.25 mmol/L，which indicated that the varieties performed better in Cd tolerance.In contrast，roots of local rice varieties were shorter，incorporating that the varieties performed poorly in Cd tolerance.On the other hand，shoot lengths from high to low were three-line hybrid rice，two-line hybrid rice and local rice. Generally speaking，with Cd concentration of 1.25 mmol/L，C Liangyou 651 and Shenyou 9588 proved the varieties performed well in Cd tolerance for root lengths reached over 0.4 cm，and Shenyou 9588，Tianyou 998，Nei 5 You 263，Chuanxiang 8，and Y Linagyou 1 also proved well in Cd tolerance，for shoot lengths all reached over 1.3 cm.Besides，root lengths of Huanghuazhan，Xiangwanxiang 13，Nongxiang 18，F(xiàn)engliangyou 4 were below 0.1 cm，and except of the varieties above with better-grown shoots，shoot lengths of the rest rice varieties were all below 0.6 cm，which suggested that the seedlings are significantly influenced by Cd stresses.

Table 4 Effect of Cd stresses on fresh weight root and shoot of different rice varieties

Effects of Cd on seedlings fresh weight of different rice varieties

As shown in Table 4，fresh weights of seedlings and shoots both kept decreasing upon Cd concentra-tion，which incorporated that Cd stress has effects on seeds of different rice varieties.Although changes of root fresh weight differed insignificantly，the weights from high to low were threeline hybrid rice＞two-line hybrid rice＞common hybrid rice，with Cd concentration of 0.75 mmol/L.As for shoot fresh weight，the sequence from low to high was common rice＜two-line hybrid rice＜three-line hybrid rice，and the differences under Cd stress of 0.75 mmol/L were much significant.Specifically，under Cd stress of 1.25 mmol/L，fresh root weights of Shenyou 9588，Chuanxiang 8，and Fengliangyou 4 all kept over 0.3 mg and fresh shoot weights of Shenyou 9588，Tianyou 998，Nei 5 You 263 and Chuanxiang 8 maintained over 3.6 mg.These have demonstrated that seedings of all the rice varieties perform better in tolerance ofCd.In contrast，Huanghuazhan，Xiangwanxian 13，Xiangwanxian 16 and Nongxiang 18 kept below 0.1 mg in terms of both fresh root weight and shoot weight，which incorporated that seedlings of the varieties are significantly influenced by Cd stresses.

Comprehensive evaluations on inhibition effects of Cd on seed germination of different rice varieties

As shown in Table 5，Cd stress on seed germination and seedling growth was the most significant on common rice varieties，followed by two-line hybrid rice and three-line hybrid rice. Specifically，comprehensive inhibition effects were higher （over 0.50）on Huanghuazhan，Xiangwanxian 13，Xiangwanxian 16，C Liangyou 651，Zhunliangyou 527 and Nei 5 You 263，and insignificant （below 0.4）on Y Liangyou 1，Shenyou 9588，Tianyou 998，and Chuanxiang 8.Besides Cd stress on rest seeds kept moderate.

Results and Analysis

Cadmium is a non-essential element for plant growth，and even poses threats to plants if accumulates too much，preventing plant growth［15-16］.The test indicated that under Cd stress，seed germination and seedling growth would be both inhibited；germination index，vigor index，root length，shoot length and fresh weights would go down；differences among rice varieties would increase.These coincide with researches available on rice and alfalfa［17-18］，possibly caused by decline of activities of cell RNA and DNA and reduction of nucleic acid［19］，or inhibited hydrolase vigor by Cd［12，15］.

Nevertheless，Cd tolerance of plants，the comprehensive reflection，is under influence of multiple factors. Therefore，it is necessary for exploring seed tolerance of Cd to conduct a comprehensive evaluation on influential factors［20］.The research investigated germination rate，shooting index，vigor index，root length，shoot length and fresh weightand the results showed that three-line hybrid rice performed better in terms of seed germination or seedling growth in Cd tolerance.Specifically，seed tolerance of Cd tends to be volatile upon rice varieties.Shenyou 9588，Tianyou 998，Chuanxiang 8，and Y Liangyou 1 performed strongly in Cd tolerance；C Liangyou 651，Nei 5 You 263，Xiangwanxian 16，Huanghuazhan，Xiangwanxian 13，and Zhunliangyou 527 performed poorly in Cd tolerance，and Cd tolerance of rest treatments were moderate.It is researched that Cd stress can be detoxicated by plants through fixing，complexing and antioxidase. Hence，tolerance differences in the research of 12 rice varieties require further exploration in the future.

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Responsible editor：Xiaoxue WANG

Responsible proofreader：Xiaoyan WU

不同水稻品種種子萌發(fā)及幼苗生長(zhǎng)耐鎘性研究

孟桂元1，2，李夢(mèng)陽(yáng)1，周靜1，2*，唐婷1，肖瓏1，曾淑英1（1.湖南人文科技學(xué)院農(nóng)業(yè)與生物技術(shù)學(xué)院，湖南婁底 47000；2.湖南省農(nóng)田雜草防控技術(shù)與應(yīng)用協(xié)同創(chuàng)新中心，湖南婁底417000）

為探明不同類型水稻品種種子萌發(fā)及幼苗生長(zhǎng)耐Cd性差異，采用室內(nèi)生物測(cè)定方法，研究了不同鎘（Cd）脅迫處理對(duì)3種不同類型12個(gè)水稻品種種子的萌發(fā)率、發(fā)芽指數(shù)、活力指數(shù)、幼苗根長(zhǎng)、芽長(zhǎng)及其根芽鮮質(zhì)量的影響。結(jié)果表明，隨著鎘離子濃度增加，各類型水稻品種種子萌發(fā)所受抑制作用明顯增強(qiáng)，萌發(fā)率、發(fā)芽指數(shù)、活力指數(shù)、根芽長(zhǎng)及其鮮重均表現(xiàn)呈下降趨勢(shì)；不同類型水稻品種種子萌發(fā)及幼苗生長(zhǎng)受抑制程度均表現(xiàn)以常規(guī)水稻品種＞兩系水稻品種＞三系水稻品種。12個(gè)水稻品種中，深優(yōu)9588、天優(yōu)998、川香8號(hào)、Y兩優(yōu)1號(hào)等4個(gè)品種耐Cd性較強(qiáng)；C兩優(yōu)651、內(nèi)5優(yōu)263、湘晚秈16號(hào)、黃華占、湘13號(hào)、準(zhǔn)兩優(yōu)527等6個(gè)品種耐Cd性相對(duì)較弱，其它為中等耐Cd品種。

品種類型；水稻種子；萌發(fā)；幼苗生長(zhǎng)；耐鎘性

湖南省婁底市科技計(jì)劃項(xiàng)目（2014-1）；雜交水稻國(guó)家重點(diǎn)實(shí)驗(yàn)室資助；湖南省高校農(nóng)藥無(wú)害化應(yīng)用重點(diǎn)實(shí)驗(yàn)室資助。

孟桂元（1977-），男，湖南新邵人，副教授，博士，主要從事作物栽培與生理生態(tài)研究，E-mail：mengguiyuan@126.com。*通訊作者，助理研究員，在讀博士，主要從事作物栽培與生理生態(tài)研究，E-mail：kiki010206@163.com。

2015-08-08

Supported by Loudi City Science and Technology Project（2014-1）；State Key Laboratory of Hybrid Rice；Key Laboratory of Pesticide Harmless Application of Hunan Province.

*Corresponding author.E-mail:kiki010206@163.com

Received:August 8，2015 Accepted:October 10，2015

修回日期 2015-10-10