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Studies of differential sensitivities between conventional and transgenic Bt cotton cultivars to potassium deficiency

Zhiyong Zhang, Xiaoli Tian, Zhaohu Li, Liusheng Duan, Baomin Wang and Zhongpei He

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: tian_xiaoli@163.com and lizhaohu@cau.edu.cn

Abstract

It was observed that transgenic Bt cottons (Gossypium hirsutum L.) showed premature senescence and K+ deficiency symptoms more often than the conventional cultivars in the fields. To verify if the transgenic cottons were more susceptible to K+ deficit, the effects of K+ levels (High, 0.5 mM and Low 0.02 mM of KC1) on seedling growth and K+ absorption were compared between transgenic cultivars and conventional cultivars. The results indicated that seedling fresh weights of both transgenic DP99B and conventional Zhong 35 were obviously affected by K+ levels. Seedlings fresh weights were higher when grown at 0.5 mM K+ than 0.02 mM K+ for both cultivars. Under low K+ level, the fresh weights of roots, stems and leaves of transgenic cultivar DP99B were significantly lower than those of the conventional Zhong 35. At low K+ level, the dry weights of two transgenic cultivars, DP99B and Zhong 41, were also significantly lower than those of two conventional cultivars, Zhong 35 and Zhong 36. However, K+ absorption results indicated that transgenic DP99B showed a better K+ absorption ability over Zhong 35. K+ concentrations in roots and leaves of DP99B were all higher than those of Zhong 35 under both high and low K+ levels, and DP99B had a higher K+ absorption rate than Zhong 35 under low K+ conditions. This experiment indicated that the differential responses between transgenic and conventional cottons could be the result of differences in response to available K+.

Media summary

Seedling growth of transgenic cottons was reduced more under low K+ than conventional cultivars. K+ absorption abilities did not account for the differences.

Key Words

Transgenic cotton; potassium; efficiency; deficit

Introduction

Potassium plays an important role in ensuring pH stabilization, osmoregulation, membrane transport processes and enzymes activation (Marschner 1995). The ability of plants to uptake and efficiently use potassium varies inter-species and intro-species within a wide range (Glass and Perley 1980). Different species have specific K+ absorbing and utilization mechanisms to support high vegetable and reproductive growth (Chen and Gabelman 1995, Chen and Gabelman 1999).

Cotton requires K+ at the late growing stage. K+ deficiency can reduce cotton yield and quality (Gormus and Yucel 2002) and result in premature senescence (Zhu et al. 2000). Recently, transgenic Bt cottons are increasing rapidly due to their resistance to bollworm and other benefits (Huang et al. 2003). Therefore, the aim of this study was to evaluate the effects of K+ deficiency on the seedling growth and K+ absorption of transgenic and conventional cottons to determine whether transgenic cottons were more sensitive to K+ deficiency.

Materials and Methods

Plant material and culture conditions

The experiments were conducted in a chamber under 20/30 ℃ and 10/14 h day/night under 450μmol m-2 s-1 conditions. Two transgenic cotton cultivars, DP99B (expressing Cry1A toxic protein) and Zhong 41 (expressing proteins of Cry1A and CpTI, cowpea trypsin inhibitor gene), and two conventional cultivars, Zhong 35 and Zhong 36, were used. The seeds were surface sterilized and germinated. Seven-day-old seedlings were carefully transferred into 35x27x12 cm pots. The seedlings were placed into the holes of foamy lids. The pots were filled with a modified Hoagland solution, which included 2.5mM Ca(NO3)2, 1mM MgSO4, 0.5mM (NH4)H2PO4, 210-4mM CuSO4, 110-3mM ZnSO4, 0.1mM EDTA Fe Na, 210-2mM H3BO3, 510-6mM (NH4)6Mo7O24, 110-3mM MnSO4. KC1 was used as the K+ source, at low (0.02) K+ and high (0.5mM) K+ concentrations. All solutions were changed twice a week and deionized water was added daily to replace the water lost by evapotranspiration. The pH was maintained close to 6.5 by adding concentrated solutions of H2SO4 or NaOH. Air was bubbled into the nutrient solution by an air pump to provide O2 and give a mixing action to ensure nutrient partitioning homogeneously.

After 21 days, at the stage of five leaves, six uniform seedlings were equally separated into two groups for each cultivar and each treatment. Seedlings from a group were harvested for biomass and determining K+ concentration. The remaining plants were used to determine absorption.

Plant Biomass and K+ content

The seedlings were separated into roots, stems and leaves, and fresh weights recorded. Samples were then oven-dried at 75C for 48 h, and weighed. Root and leaf samples were ground to pass a 0.5mm screen, then soaked in 1 M ammonium acetate (pH 7.0) solution for 5h, vibrated for 30 minutes and filtered. Extraction solution was analysed for K+ by atomic absorption spectroscopy (Christain and Feldman 1970).

K+ absorption

Three uniform seedlings of each variety from different K+ treatments were selected, and put into 500-mL bakers. Each baker was filled with 300mL of nutrient solution, except for the nil K+ treatment. After the seedlings were starved of K+ for 48h, the seedlings were removed from the solution. The roots were washed with 0.5mM CaSO4 three times. Seedlings were then put into 300 mL containers containing a solution of 0.2 mM KCl and 0.5mM CaSO4, where Ca2+ can guarantee integrity of membranes and minimize ions efflux (Jensen et al., 1987). After 70 min, uptake was stopped by removing seedlings and disposed of as described below. K+ in solution was measured by using atomic absorption spectroscopy.

Statistical analysis

Each pot was referred as a replicate. A completely randomized design with five replications was used for all experiments. All data were subject to ANOVA test and means were compared using the appropriate Fisher’s protected LSD (P ≤ 0.05).

Results

K+ levels on seedling growth

The results indicated that seedling fresh weights of both cultivars, DP99B and Zhong 35, were affected by K+ levels (Table 1). The fresh weights of roots, stems and leaves of the high K+ treatment were significantly higher than those of the low K+ treatment for both cultivars. However, there was a difference between varieties in the low K+ treatment. The fresh weights of roots, stems and leaves of transgenic cultivar DP99B were significantly lower than those of the conventional cultivar Zhong 35 under low K+ (Table 1). While there were no such differences observed at high K+ between the two cultivars. Further more, the ratio of roots to (stems + leaves) of DP99B was also decreased by low K+ (Table 1).

Table 1. Effects of K+ levels on seedling roots (R), stems (S) and leaves (L) fresh weights and ratio of R / (S+L) of cultivars DP99B and Zhong 35.

Cultivar

R

S

L

R / (S+L)

Low K+

High K+

Low K+

High K+

Low K+

High K+

Low K+

High K+

 

- g -

- g -

- g -

   

DP99B

0.36 bB

3.02 aA

1.47 bB

5.83 aA

1.81 bB

10.0 aA

0.11 bB

0.19 aA

Zhong35

1.04 aB

2.72 aA

2.33 aB

7.20 aA

2.72 aB

10.4 aA

0.20 aA

0.15 bA

Values in each column followed by the lower-case letters for comparison between cultivars and upper-case letters for comparison between K+ levels within a cultivar at P<0.05.

Further experiments with four cultivars, including two transgenic cottons, DP99B and Zhong 41, and two conventional cottons, Zhong 35 and Zhong 36, also showed the same trend. Two transgenic cultivars were more significantly affected than the conventional cultivars under low K+ than were the conventional varieties. The dry weights of roots, stems and leaves of transgenic cultivars DP99B and Zhong 41 were significantly lower than those of conventional cultivars Zhong 35 and Zhong 36 (Table 2). The dry weight ratios of roots to (stems + leaves) of two transgenic cultivars were also lower than those of the two conventional cultivars. These results indicated that transgenic cultivars were more sensitive to K+ deficit than the conventional cultivars. This could be why the transgenic cottons were more subjected to premature senescence disorder than the conventional cottons under the field condition.

Table 2. Effects of K+ deficiency on seedling roots (R), stems and Leaves (S+L) dry weights (g) and ratio of R/(S+L) of transgenic cultivars DP99B and Zhong 41 and conventional cultivars Zhong 36 and Zhong 35.

Cultivar

R

S+L

R / (S+L)

DP99B

0.027 b

0.329 b

0.083 b

Zhong41

0.014 c

0.196 c

0.074 b

Zhong35

0.059 a

0.530 a

0.113 a

Zhong36

0.051 a

0.454 a

0.115 a

Values in each column followed by same letter were not different at P<0.05.

K+ absorption

After the seedlings were grown under different K+ levels for 21 days, K+ concentrations in the seedlings of cultivar DP99B and Zhong 35 were measured. The results indicated that transgenic DP99B showed a better K+ absorption ability over Zhong 35. K+ concentrations in roots and leaves of DP99B were all higher than those of Zhong 35 under both high and low K+ culture levels (Table 3). K+ concentrations of these two cultivars were not correlated with seedlings growth under different K+ levels. Under low K+ conditions, the growth of transgenic DP99B was reduced significantly more than Zhong 35. However, DP99B showed higher K+ concentration both in roots and leaves. The results also indicated that K+ absorption rates of these two cultivars could not account for the growth differences under low and high K+ levels (Table 4). After K+ starvation, seedlings from low K+ treatment showed higher K+ absorption rates than from high K+ treatment for both cultivars when the seedlings were provide sufficient K+. The absorption rate of DP99B was higher than that of Zhong 35 for the seedlings were treated with low K+ (Table 4).

Table 3. Effects of K+ levels on K+ contents (expressed as a % of dry weight) of roots (R) and leaves (L) of cultivars DP99B and Zhong 35 seedlings.

 

Cultivar

Root

Leaves

Low K+

High K+

Low K+

High K+

DP99B

1.04 aB

1.90 aA

0.58 aB

1.05 aA

Zhong35

0.90 bB

1.45 bA

0.47 bB

0.90 bA

Values in each column followed by the lower-case letters for comparison between cultivars and upper-case letters for comparison between K+ levels within cultivar and seedling part at P<0.05.

Nutrient efficiency is defined as the ability of plants to obtain higher yields at low nutrient supply, compared with other species (Steingrobe and Claassen 2000). Our results indicated that transgenic Bt cottons were more sensitive to low K+ conditions, with more growth reduction (Table 1 and 2). The K+ absorption results excluded the reasons of low K+ acquirement ability by the transgenic DP99B (Table 3 and 4). The growth differences between transgenic cultivars and conventional cultivars may be the result of the efficiency with which K+ is utilized in these cultivars. Zhong 35 could have a higher K+ use efficiency than transgenic DP99B. Low K+ efficiency could lead to premature senescence and obvious K+ disorder symptoms for transgenic cultivars. It was not clear that whether the K+ efficiency were affected by insert of the insect-resistant genes for the transgenic cottons. Further studies are needed to verify the mechanisms.

Conclusions

Transgenic cotton cultivars 99B and Zhong41 were more susceptible to potassium deficiency compare to conventional cultivars Zhong35 and Zhong36. This susceptibility was not the result of a difference in K+ absorption ability but from its lower K+ efficiency. The results indicated that transgenic cottons might need more K+ nutrient in order to achieve high yield and good fibre quality. However, this will be studied more in detail with ongoing research.

Table 4. Effects of K+ levels on K+ absorption rates (mg / g root fresh weight) of cultivars DP99B and Zhong 35 seedlings.

Cultivar

Low K+

High K+

DP99B

9.0 aA

2.0 aB

Zhong35

5.1 bA

2.7 aB

Values in each column followed by the lower-case letters for comparison between cultivars and upper-case letters for comparison between K+ levels within cultivar at P<0.05.

Acknowledgement

The research is supported by National Natural Science Foundation of China and the National High Technology Research and Development Program of China.

References

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Huang, JK, Hu RF, Pray C, Qiao FB and Rozelle S (2003). Biotechnology as an alternative to chemical pesticides: a case study of Bt cotton in China. Agricultural Economics. 29:55-67.

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