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Effects of NaCl Salinity on Growth and Bt Protein Content of Two Transgenic Bacillus thuringiesis Cotton Cultivars

Linjian Jiang, Liusheng Duan, Xiaoli Tian, Baomin Wang, Haifeng Zhang and Zhaohu Li

Center of Crop Chemical Control, Department of Agronomy, State Key Lab of Plant Physiology and Biochemistry,
China Agricultural University, Beijing, 100094, P. R. China www.cau.edu E-mail: lizhaohu@cau.edu.cn

Abstract

The seedling responses of two transgenic Bacillus thuringiesis (Bt) cotton (Gossypium hirsutum L.) cultivars, 99B and SGK9708-41, to NaCl salinity stress were studied under 0, 50, 100 and 200 mM NaCl treatments, respectively. The results indicated that cotton seedling growth of both cultivars was significantly inhibited by NaC1 treatments. The heights, leaf areas, fresh and dry weights of two cultivars were all reduced in a concentration dependent manner. Total N concentrations of seedling roots, stems and leaves of both cultivars were decreased with increasing NaC1 levels, except that of roots of SGK9708-14. 50 and 100 mM NaC1 treatments decreased soluble protein concentrations of roots for both cultivars. 200mM NaC1 increased Bt protein concentrations of stems of both cultivars, but decreased Bt protein concentration of leaves of cultivar 99B. However, the bollworm resistance was not affected by NaC1 treatments.

Media summary

Salinity stress inhibited transgenic Bt cotton growth. Total N and Bt protein concentrations were decreased by 200 mM NaC1 stress. The bollworm resistance was not affected.

Key Words

Transgenic Bt cotton; Salinity stress; Seedling growth; Bt protein

Introduction

Transgenic Bt cotton provides highly effective control of cotton bollworm, reduced reliance on conventional chemical pesticides, and increased yields (Huang et al. 2003). Bt cotton were planted over 6.8 million hectares in the world since produced by Perlak et al. in 1990 (James, 2001). The extremely rapid adoption of Bt cotton demonstrated the outstanding grower satisfaction of the performance and value. In northern China, Bt cotton began to be commercially planted in 1997, and expanded rapidly over 1.0 million ha in 2000, which is nearly 90% of cotton planting area in this region (Huang et al. 2003). However, due to limited rainfall, saline water irrigation, and heavy fertilizer application, soil salinity is one of the most important constraints responsible for low crop yield in this region.

Although extensive work has been carried out on the effects of salinity on conventional cotton (Ashraf, 2002), our understanding of the response of transgenic Bt cotton to salinity stress is still limited, especially for Bt protein expression and resistant to bollworm. Although factors such as parental background had a stronger impact on the expression of Bt gene than environment, environment conditions did affect the expression of Bt gene (Adamczyk et al., 2001). Bruns and Abel (2003) reported that increased N fertilizer likely increased Bt proteins in transgenic Bt maize. Elevated CO2 could cause reduction in Bt protein production (Coviella et al., 2000).

In this experiment, two commercial transgenic Bt cotton cultivars were treated with different NaCl levels in nutrient solution at seedling stage. The objectives were to determine the effects of NaC1 salinity stress on seedling growth, total N, total soluble protein and Bt protein concentrations of two transgenic Bt cotton cultivars.

Materials and Methods

Plant material and culture conditions

The experiment was conducted in a chamber under 20/30 C and 10/14 h day/night condition. Seeds of two transgenic cotton cultivars, 99B (expressing Cry1A toxic protein) and GK9708-41 (expressing proteins of Cry1A and CpTI, cowpea trypsin inhibitor gene), were surface sterilized and germinated. Seven-day-old seedlings were carefully transferred into 5-L pots, filled with half-strength Hoagland solution. Seedlings were allowed to grown in these conditions for 3 days before the initiation of NaCI treatments. The seedlings were subjected to salt stress by adding NaCI to the growth medium in 50 mM increments every 12 h, until the final concentrations of 50, 100, and 200mM were reached. All solutions were changed twice a week and deionized water was added daily to replace the water lost by transpiration. The pH was maintained close to 6.5 by adding concentrated solutions of H2SO4 or KOH.

Seedling Growth

Two-leaf-stage seedlings were separated into roots, stems (including the petiole) and leaves. The fresh weights, leaf areas, and seedling heights were measured for each sample. The dry weights were taken after the samples were oven-dried at 80 C for 48 h.

Total N, Soluble protein and Bt protein concentrations

Oven-dried samples were powdered and digested in 5ml H2SO4 separately. Total nitrogen were quantified according to micro-Kjeldahl method. Frozen samples were homogenized in 5ml distilled water, and the mixtures were centrifuged at 10000g for 10 minutes at 4℃. Total protein of the supernatant was determined spectrophotometrically according to Bradford (1976) using bovine serum albumin as standards. Bt protein contents were determined using ELISA (Wang et al., 1998; Palm et al., 1996).

Insect Bioassay

Fully expanded fresh leaves were placed into petri dishes, individually. And ten just-hatched active bollworm (Lepidoptera: Noctuidae) larvae were placed into each petri dish. Then, the petri dishes were sealed and incubated under 25℃ and 16/8h day/night condition. The survival larvae number in each petri dish was recorded after 3 days incubation.

Experimental Design and Analysis

A completely randomised design with four replications was used for all experiments. The experiments were repeated three times. All data were subject to ANOVA test and means were compared using the appropriate Fisher’s protected LSD (P < 0.05).

Results

Seedling growth

The seedling growth of both cultivars was significantly inhibited by NaC1 treatment. The heights, leaf areas, fresh and dry weights of two cultivars were all reduced in a concentration dependent manner (Table 1). However, the root/shoot ratio of two cultivars intended to increase as the concentration of NaC1 increased, which was also observed by Meloni et al. (2001).

Table 1. Seedling height (HT), leaf area (LA), fresh weight (FW), dry weight (DW) and shoot/root ratio (R) of two transgenic cotton cultivars as affected by NaC1 salinity.

NaC1

99B

 

SGK9708-41

HT

LA

FW

DW

Ratio

 

Height

LA

FW

DW

Ratio

mM

cm

cm2

g

g

   

cm

cm2

g

g

 

0

7.6A

164 A

8.2A

0.66 A

0.25 D

 

8.6A

146 A

7.6 A

0.67 A

0.25B

50

5.9B

123 B

6.5B

0.54 B

0.31 C

 

7.1B

112 B

6.0 B

0.53 B

0.25 B

100

4.9B

104 B

5.9B

0.50 B

0.36 B

 

5.2C

77 C

4.7 C

0.43 BC

0.33 A

200

2.6C

60 C

3.5C

0.33 C

0.39 A

 

2.8D

57 D

3.2 D

0.32C

0.37A

Values in each column followed by the same letter are not significantly different according to Fisher’s protected LSD (P<0.05).

Total N, Ssoluble protein and Bt protein concentrations

The total N concentrations of roots, stems and leaves of both cultivars were effected by NaC1 treatments. There was an obvious tendency that total N concentrations were decreased with increasing NaC1 levels, except that of roots of SGK9708-14 (Table 3).

The soluble protein concentrations of seedling roots of both cultivars were decreased by 50 and 100 mM NaC1 treatments. However, no significant differences were observed between 200 mM NaC1 treatment and control of both cultivars (Table 4). In stems, only 200 mM NaC1treatment decreased soluble protein concentrations significantly for both cultivars. The results also showed that the soluble protein concentrations in fully expanded leaves of both cultivars were significantly increased by 200 mM NaC1 treatment (Table 4).

The Bt protein concentrations of seedling roots of both cultivars were not affected by NaC1 treatments (Table 5). However, 200mM NaC1 treatments increased the stems Bt protein concentrations of both cultivars, and decreased the leaves Bt protein concentration of 99B. There was no difference among 0, 50 and 100mM NaC1 treatments for the Bt protein concentrations of both stems and leaves of the two cultivars (Table 5).

Table 2. Total N concentrations (% of FW) of seedling roots, stems and leaves of two transgenic cotton cultivars as affected by NaC1 salinity.

NaCl
(mM)

99B

SGK9708-41

Roots

Stems

Leaves

Roots

Stems

Leaves

0

3.5 A

3.0 A

4.0 A

3.1 A

2.7 A

4.0 A

50

3.1 AB

2.6 AB

4.0 A

2.8 A

2.1 B

4.0 A

100

2.9 BC

2.2 B

3.5 AB

2.8 A

2.2 B

4.0 A

200

2.5 C

2.1 B

3.3 B

2.3 A

1.8 C

3.4 B

Values followed by the same letter in each column are not significantly different (P<0.05).

Table 3. Soluble protein concentrations (mg/g fw) of seedling roots, stems and leaves of two transgenic cotton cultivars as affected by NaC1 salinity.

NaCl
(mM)

99B

SGK9708-41

Roots

Stems

Leaves

Roots

Stems

Leaves

0

1.34 AB

0.76 A

1.65 B

2.00 A

0.69 A

2.10 B

50

1.15 C

0.78 A

2.03 AB

1.11 B

0.66 A

2.15 B

100

1.28 BC

0.72 A

2.01 AB

1.28 B

0.71 A

2.23 B

200

1.50 A

0.50 B

2.19 A

1.94 A

0.43 B

3.17 A

Values followed by the same letter in each column are not significantly different (P<0.05).

Table 4. Bt protein concentrations (g/g fw) of seedling roots, stems and leaves of two transgenic cotton cultivars as affected by NaC1 salinity.

NaCl
(mM)

99B

SGK9708-41

Roots

Stems

Leaves

Roots

Stems

Leaves

0

1.91 A

1.45 B

0.87 A

2.36 A

1.51 B

1.36 A

50

1.68 A

1.73 B

0.79 A

2.08 A

1.51 B

1.26 A

100

1.61 A

1.45 B

0.98 A

1.73 A

1.79 B

1.41 A

200

2.10 A

3.45 A

0.74 B

2.34 A

2.33 A

1.30 A

Values followed by the same letter in each column are not significantly different (P<0.05).

Insect Bioassay

The bioassay results indicated that NaC1 stress did not affect the bollworm resistance. There was no difference between the two transgenic Bt cotton cultivars (Table 6). Although leaf Bt protein was decreased by 200mM NaC1 treatment for 99B (Table 5), it was still effective to control bollworms efficiently in this experiment.

Table 5. Effect of NaCl on the mortalities of bollworm larvae (%) of two transgenic cultivars.

NaCl (mM)

99B

SGK9708-41

0

94 A

93 A

50

92 A

92 A

100

93 A

93 A

200

94 A

90 A

Values in each column followed by the same letter are not significantly different (P<0.05).

Conclusion

NaC1 salinity stress had adverse effects on transgenic Bt cotton seedling growth, which were observed with conventional cotton (Ashraf, 2002). The heights, leaf areas, fresh and dry weights of two cultivars were all reduced by NaC1 treatments. Varietal differences in total N, soluble protein and Bt protein levels were observed between two transgenic Bt cotton cultivars. However, the bollworm tolerance was not affected in this experiment.

Acknowledgement

The authors thank the National High Technology Research and Development Program of China (grant 2001AA246060) for support, and Dr. Qingwen Zhang for technical assistance.

References

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