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Effects of Mepiquat Chloride on Lateral Roots Initiation of Cotton Seedling and Its Mechanism

Liusheng Duan, Xiaoli Tian, Yi Zhang, Zuoshun Tang, Zhixi Zhai and Zhongpei He

China Agriculture University, #2 Yuanmingyuan Xilu, Beijing 100094, P. R. China, www.cau.edu.cn Email lshduan@hotmail.com

Abstract

This paper studied the effects of Mepiquat Chloride (DPC) on initiation and development of lateral roots of cotton seedlings, using the upland cotton (Gossypium hirsutum L.) cultivars Zhongmianshuo 16 and Zhongmianshuo 29 as experiment materials. Seedlings were cultured on glass boards after seeds were soaked with 400 mg/L DPC solution for 12 hours. DPC increased the number of lateral roots and root primordia, and the length of the segment where lateral roots and root primordia initiated. It was shown that DPC promoted both the initiation of primordia and their development to lateral roots in an experiment of lateral root removal. Under the stress of low temperature, the initiation of lateral roots was also significantly promoted by DPC. The levels of endogenous Auxin, Zeatin and Zeatin riboside in the middle segment of primary root were all increased by DPC, which might be the key reason for lateral root induction.

Media summary

The effects of Mepiquat Chloride on initiation of lateral roots and root primordia of cotton seedlings under normal and cold temperature were reported and its mechanism was illuminated based on hormone levels.

Key Words

Cotton, lateral root, Mepiquat Chloride (DPC), hormonal regulation.

Introduction

The root system is vital for plants to grow and develop well and the lateral roots are very important for the cotton root systems, so promoting the initiation and development of lateral roots will surely contribute to raising strong seedling and enhancing the resistance of plant to adversity like lower temperature, which is a frequent stress in seedling stage for cotton. It is reported that the initiation and growth of cotton roots were interrupted when the soil temperature decreased below 14.5C (Jackson 1986). It has been shown in numerous reports that the initiation and development of lateral roots is regulated by interior factors, of which plant hormones are a pivotal one, and also affected by environments (Tang 1994; Jackson 1986). Auxins (IAA) can induce the initiation of lateral root primordia and root development, while Cytokinins (CTKs) are necessary for a proper level of lateral root initiation, but its effects have been reported diversely, which might be related with cooperation or crosstalk among hormones (Li 1991; Tang 1994; Jackson 1986; Biddington 1982). Mepiquat Chloride (DPC) is a plant growth retardant that has been used world-wide on cotton fields to harmonize vegetative growth and reproductive growth to restrain overgrowth and to enhance lint yield. It is also reported to promote initiation and viability of lateral roots, but its mechanism is unclear yet. The objectives of this experiment were to explore the physiological mechanism of DPC on lateral roots, through inspection of lateral roots primordia, detection of endogenous hormones, and to supply theoretical support for application of DPC to promote roots and raise strong seedlings.

Methods

Seedling raising and treatment

The varieties used were Gossypium hirsutum L cv. Zhongmianshuo 16 (Zms16) and Zhongmianshuo 29 (Zms29). Cotton seeds were first delinted by sulfuric acid and then washed by abundant water until pH reached same as washing water. They were then treated by soaking in 400mg/l Mepiquat Chloride (DPC) solution at room temperature for 12 hours, while the control was soaked in the distilled water which was used to prepare DPC solution. The seeds were flow washed and their supernatant water was removed employing filter paper. Seedlings were then cultured between two layers of filter paper attached on vertical rectangular glass boards (20cm30cm) soaked in distilled water (described in detail by He ZP 1993). There were 20 seeds on each board and 10 boards for each treatment. The seedlings were grown in growth chamber at 25C with 12h dark/12h light. A low temperature treatment of 12C was imposed 2-5 days after germination. On the 7th day after germination, all the lateral roots of seedling on some boards were removed by scalpel, avoiding damage to the epidermis of the primary root. The lateral root primordia and roots on the primary root were inspected every day.

Inspection of lateral root primordia

The primary root was fixed in FAA (Ethanol 70%, Acetic acid 5% and Formaldehyde5%) for 24 hours, and then stored in 70% Ethanol. The lateral root primordia were dyed, hyalinized and inspected under stereoscope without slicing the roots, following He ZP (1993).

Detection of endogenous hormone levels

Primary root segments of 2cm, then another 3cm from the joint of primary root and hypocotyl (lateral roots initiating region) were sampled. Roots samples were first flash-frozen in liquid nitrogen and then homogenized and extracted by 80% Methanol under 0C-4C. Auxin (IAA), and Zeatin + Zeatin riboside (Z+ZR) were detected using ELISA followed He ZP (1993).

Results

Effects of DPC on lateral roots initiation

DPC treatment promoted lateral root initiation of cotton seedling remarkably, which could be owed to an increase of both the length of lateral root initiation region and the linear density of lateral roots. Zms29 was more sensitive to DPC than Zms16 (Table 1). At the 7th day after germination, for Zms16, the number of visible lateral roots per plant, the length of lateral root initiation region and its linear density was increased by 36.0%, 31.3% and 4.4% respectively, while these values increased by 43.0%, 55.0% and 22.0% for Zms29.

Effects of DPC on initiation of lateral root primordia

Lateral root primordia could be observed from the 4th day after seed germination and mainly happened in the segment within 2cm from base of the primary root. Lateral root primordia emerged in the middle region of primary root on 5th day after germination. After DPC treatment, the root primordia initiation was not noticeably changed in the base area of primary root while increased significantly in the middle area, which led to the elongation of the total initiation region of lateral root primordia (Table 2).

Table 1 Effects of DPC on initiation of lateral roots of cotton seedlings

Variety

Treatment

Lateral root number per plant

Length of lateral root initiation region (cm)

Linear density of lateral roots (roots/cm)

Zms 16

CK

25.0

2.68

9.3

 

DPC

34.3

3.52

9.7

 

Effect (%)

+36.0**

+31.3**

+ 4.4*

Zms 29

CK

21.0

2.20

9.0

 

DPC

30.0

3.40

11.0

 

Effect (%)

+43.0**

+55.0**

+22.0**

Measured on 7th day after treatment.* significant at 0.05 level. ** significant at 0.01 level. CK = Control.

Table 2 Effects of DPC on initiation of lateral root primordia of cotton seedling

Days after germination

Treatment

Number of lateral root primordia per plant

Segment length for lateral root primordia initiation (cm)

Linear density of lateral root primordia (primordia /cm)

4

CK

12.8

1.25

10.24

 

DPC

12.2

1.26

9.68

 

Effect (%)

- 4.7

+0.8

- 5.5

5

CK

20.9

2.06

10.15

 

DPC

27.3

2.81

9.72

 

Effect (%)

+ 30.6**

+36.4**

-4.3

6

CK

30.5

3.35

9.10

 

DPC

41.1

4.35

9.45

 

Effect (%)

+ 34.8**

+29.9**

+3.2

Variety was Zms 16. ** significant at 0.01 level. CK = Control.

Effects of DPC on initiation of lateral root primordia after lateral roots were removed

Visible lateral roots were removed in the experiment to test whether DPC promoted lateral roots through enhancing root primordia induction or accelerating the growth from primordia into lateral roots. As shown in Table 3, the lateral roots could still happen in the area where lateral were removed. This mainly owed to the development and growth from formed primordia, however new root primordia were also found in the region where lateral roots were removed. During 1-4 days after removing, both the new lateral roots and new root primordia were higher in DPC treatment than Control, which implied that both initiation of lateral root primordia and their growth into lateral roots were promoted by DPC.

Table 3 Effects of DPC on initiation of lateral roots and root primordia after lateral roots were removed

Days after lateral root removal

Number of lateral root primordia per plant
CK

Number of lateral root primordia per plant
DPC

Number of new lateral roots per plant
CK

Number of new lateral roots per plant
DPC

0

1.05

1.11

0.00

0.00

1

0.73

1.23*

1.00

1.23*

2

0.92

1.00*

1.69

1.83*

3

1.00

1.08*

1.70

2.00*

4

1.00

1.30*

2.09

2.50*

Variety was Zms 16. All lateral roots were removed on 7th day after germination. * significant at 0.05 level. CK = Control.

Effects of DPC on initiation of lateral roots and root primordia under low temperature stress

Under low temperature of 12℃, which was below the reported temperature that stopped root initiation and growth in cotton, the lateral roots and root primordia were found to be depressed remarkably too, while they were significantly promoted by DPC treatment (Table 4). On the 6th day after germination, the lateral root number and root primordia number were decreased by 53.75% and 9.3% under 12℃, while the lateral root number for DPC treatment only decreased by 28.4% and the root primordia number showed 16.3% higher than that under 25℃.

Table 4 Effects of DPC on initiation of lateral roots and root primordia under low temperature stress

Days after germination (d)

Treatment

Number of lateral root primordia per plant

Segment length of lateral root primordia initiation (cm)

Number of lateral roots per plant

Segment length of lateral root initiation
(cm)

3

CK1

0.0

-

0.0

-

 

CK2

0.0

-

0.0

-

 

DPC

0.0

-

0.0

-

4

CK1

8.0

0.95

0.0

-

 

CK2

3.7

0.58

0.0

-

 

DPC

6.9*

0.91*

0.0

-

5

CK1

10.4

1.47

5.6

-

 

CK2

10.9

1.51

2.2

-

 

DPC

14.2*

1.95*

2.2

-

6

CK1

12.9

2.02

13.0

1.25

 

CK2

11.7

1.75

6.8

0.77

 

DPC

15.0*

2.22*

9.3*

0.89*

Variety was Zms 16. CK1 = Control grown under 25℃, CK2 = Control grown under 12℃ from 2nd day after germination. * significant difference between CK2 and DPC at 0.05 level.

A. 2cm from base of primary root

B. 3cm in middle of primary root

Figure 1 Effects of DPC on IAA content in primary root of cotton seedling (Variety: Zms 16); CK = control

Effects of DPC on IAA and Z+ZR level in primary root

DPC treatment at seed soaking increased IAA level in the middle of the primary root and the IAA peak seemed to appear earlier than control (Figure 1).The Z+ZR in the base segment of primary root was little changed by DPC treatment and only was decreased in 3rd day after germination. The endogenous Z+ZR in the middle segment of the primary root was enhanced significantly (Figure 2).

A. 2cm from base of primary root

B. 3cm in middle of primary root

Figure 2 Effects of DPC on Z+ZR content in primary root of cotton seedling (Variety: Zms 16); CK = control

Conclusion

DPC seed treatment promoted lateral root initiation of cotton seedlings. In terms of the process of lateral root initiation, DPC not only enhanced the initiation of primordia of lateral roots, but also accelerated the growth and development from primordia into lateral roots. In terms of distribution of lateral roots and their primordia on the primary root, DPC not only elongated the segment of initiation of lateral roots and root primordia, but also increased the linear density of root primordia and lateral roots initiated on the primary root. Both the experiment results of removing visible lateral roots and cold stress that depressed roots supported this strongly. IAA and Z+ZR is the key regulating factors for lateral initiation. DPC increased significantly IAA and Z+ZR levels in the middle region of primary roots while it had little effect on the basal region, which could be a good illumination for its promotion of lateral roots and root primordia in the middle of primary root. DPC was shown to have potential to raise strong cotton seedling with better root systems and higher resistance to cold stress, which should benefit better plant growth and development, high yield and sustainable farming.

References

He ZP, Min XJ and Li PM (1988). Physiological effects of plant growth retardant DPC on roots activity of cotton. Journal of Beijing Agricultural University 14, 235-24.

Li ZH, He ZP and Li PM (1991). Studies on chemically induction of initiation of lateral roots of cotton seedling. Journal of Beijing Agricultural University Supplement 6-8

Tang ZS, He ZP and Li PM (1997). Development and chemical regulation of lateral roots of cotton seedling and its Mechanism. In ‘Crop hormonal physiological and regulation’ (Ed. He ZP). Beijing, China Agricultural University Press, 1-7.

Tang ZS, He ZP, Tian XL et al (1994). Studies on plant roots and hormones. In Proceedings on crop physiology for high yield and high efficiency (Ed. Zhou Q et al.), Beijing, Science Press, 178-186.

Jackson MB (1986). New root formation in plants and cuttings. Martinus Nijhoff Publishers, 1-200.

Biddington NL (1982). The involvement of the root apex and cytokinins in the control of lateral roots of emergence in lettuce seedlings. Plant Growth Regulation 1:183-193.

He ZP (1993). Experimental Guide for Crop Chemical Control. Beijing Agricultural University Press.

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