0967-B1

Improved Germination of Pine Seeds by Electrostatic Field Treatment

Zhi-bin Gui[1], Li-min Qiao and Jun-jun Zhao


Abstract

Tree seed of pine, Pinus tabulaeformis Carr., as well as other species, such as fast-germination tree seeds that are widely used in aerial sowing for reforestation in China, were treated by electrostatic fields to enhance germination, improving germination percentage, and to explore theoretical relationships between electrostatic field treatment and changes in seed vigour. Results indicated that the effects of treatment depended on treatment dosage, process and index of early stage of seeds. The optimal dosage was 500 kV/m for 10 minutes for treatment of dry or wet seeds for improvement of both germination and root development during initial germination and middle and later stages of seedling development.

Materials and methods

Seeds of pine, Pinus tabulaeformis Carr., from north China, with a thousand-seed weight of 40.2 g, moisture content of 7 to 9% and 80% germination when tested for 16 days at 20/25 °C, were selected and divided into two equal lots. The first lot, designated as the dry lot, was mixed and divided into four sublots. One was the control. The other three were treated at an electric field intensity of 300 kV/m, 500 kV/m, and 700 kV/m, respectively, for a time of 10 min.

The second lot, designated as the wet lot, was placed into cold water for 24h, then removed from the water and drained for 5 minutes, and then divided into four sublots in the same way as the dry lot. One sublot was retained as a control and three sublots were treated at the same electric field intensities as the dry sublots, 300 kV/m, 500 kV/m, 700 kV/m, respectively. Treatment times were selected for these sublots as 10, 20, and 30 min., respectively.

The electric field gradient, or electric field intensity, E, was calculated as E = v/d, where v is the electrode voltage and d is the electrode separation distance, as used by Murr (1965), but the circuit for high voltage production was not the same. Treatment electrodes were two horizontal, circular metal plates 300 mm in diameter 4 mm thick, separated and maintained in parallel orientation by three insulating posts, 3 cm in length and 2 cm in diameter. The top one was the (+) positive electrode; and the bottom one was the (-) negative electrode as shown in Fig. 1. Seeds were placed on the top surface of the bottom plate. Treatment electrodes of the high- voltage circuit were similar to those of Murr (1965). Positive and negative electrodes were connected to the positive and negative output terminals, respectively, of a 1 to 30 kV high-voltage generator with adjustable output voltage. The positive electrode was connected by means of an insulated high-voltage line, and the negative electrode was also connected to a deep ground. The distance between the plates was fixed at 3 cm by the insulating posts, providing a uniform electric field between the parallel plate electrodes. A kilovolt meter and a micro-ammeter were used to measure the voltage between the electrodes and the electric current delivered by the high-voltage generator. Based on the very small electric current measured, its effect was considered negligible.

For stimulation treatment, the seeds were placed in a single layer (or more than a single layer) on top of the negative plate with 50 to 100g containing about 1000 to 2000 pine seeds at a time, (In outdoor test, 50 to 100kg pine seeds were treated in the box at a time). Seeds were placed on the negative electrode and in contact with each other, the distance between surface of seeds and positive electrode is decided by electrical electric field intensity. Then the power was turned on, and after the selected time, it was turned off. The plates were then discharged with an insulated conducting rod for safety before removing the treated seeds. Both wet and dry control seed samples were not treated and were kept a distance of 3 to 4 meters away from the treatment equipment.

Fig. 1 Circuit diagram of equipment for electrostatic seed treatment

Four replicates of 100 seeds each of treated and control sub-lots were randomly selected and placed into glass culture containers with sandy medium. They were watered in timely fashion and germinated at 24°C constant temperature with full photoperiod. Seeds were considered to have germinated when the length of the radicle was equal to the length of the seed. The number of germinated seeds was recorded daily. Germination percentage was calculated according to Rules for Seed Testing (ISTA, 1993) and Zhu et al. (1986). For data analysis, we used standard deviation to express degree of variation. The F-test was used as to examine differences among tested samples. Values of F>F0.05 or F>F0.01 and least significant differences (LSD) were used for mean comparisons (p < 0.05, 0.01), Zhu et al. (1990).

Results

Germination results at 10 days for the control and treated samples of the wet lot of pine seed are summarized in Table 1.

Table 1 Comparison of 10-day germination of treated and control samples of Pinus tabulaeformis Carr. seeds

Test samples

Average germination (%)

Difference of treatment and control

300 kV/m, 10min

55.3

6.3*

300 kV/m, 20min

52.3

3.3

500 kV/m, 10min

55.0

6.0*

500 kV/m, 20min

47.0

-2.0

700 kV/m, 10min

38.0

-11.0

700 kV/m, 20min

37.7

-11.3

control sample

49.0

---

The treatments at 300 kV/m for 10 min and 500 kV/m for 10 min improved germination from 49% to 55.3% and 55.0%, respectively, compared to the control sample. Because both LSD0.05=6.3 for the 300 kV/m, 10 min treatment and LSD0.05=6.0 for the 500 kV/m, 10min treatment surpassed LSD0.05=5.69, but were less than LSD0.01=7.90, these tests demonstrated that germination improvements for both treatments were significant (p < 0.05) at 10 days. Increasing the electric field intensity or the exposure time tended to reduced germination because stronger electric fields and long exposures restrain and damage seeds and slow down the germination. Another test results of germination tests on the 500 kv/m wet sublot samples at 7 days are summarized in Table 2.

Table 2 Comparison of 7-day germination of treated and control samples of pine seed soaked

Test samples

Average germination (%)

Difference of treatment and control

500kv/m 10min

42.0

7.0*

500kv/m 20min

39.7

4.7

500kv/m 30min

39.7

4.7

control

35.0

-

The 10 min treatment improved 7-day germination from 35% to 42%. This demonstrated that treatment at 500 kV/m for 10 min was a suitable dosage. Even when exposure time was extended to 30 min, no reduction in germination was observed. Longer exposures would restrain germination at 500 kV/m, especially showing inhibition of root development of young seedlings. Effects of the electrostatic treatment wet lot seeds on radicle or root length of germinating seeds at 7 days are shown in Table 3.

Table 3. Comparison of 7-day root growth of treated and control samples of pine seed soaked

Test groups

Average young seedling root length (cm)

Difference of treatment and control

500kv.m 10 min

5.9

1.2**

500kv/m 20 min

5.5

0.8*

500kv/m 30 min

5.4

0.6

control

4.7

-

Root lengths of young seedling from seeds treated at 500 kv/m for 10 min, and 20 min were improved from 4.7 cm to 5.9 cm (p < 0.01) and 5.5cm (p < 0.05), respectively, at 7 days. These tests thus demonstrated that treatments gave highly significant increases in root length (1.2 cm) and significant increases (0.8 cm) for 10 min and 20 min exposures, respectively, when soaked pine seeds were treated at 500 kV/m. Treatment for 10 min was the best (p < 0.01). Results of electrostatic electric field treatments of dry pine seeds are summarized in Table 4, where the germination at 6, 8 and 10 days, and the germination index are shown. The germination index, GI, is an another indicator of seed vigour and is defined as , where n is the germination percentage on each day counted and d is the number of that day after the germination test is started. Thus early germination seeds had a larger germination index.

Table 4. Germination effects of electrostatic field treatment on dry pine seeds

Samples

Germination
6 days

Germination
8 days

Germination
10 days

GI

MGT

300kV/m 10’

8.0±2.0

24.7±4.9

29.3±5.2

3.3±0.6

8.9±0.5

500kV/m 10’

13.0±3.9

29.0±4.0

35.0±4.2

4.1±1.0

8.9±0.2

700kV/m 10’

13.3±1.2

27.3±3.2

34.0±3.5

3.9±0.3

8.9±0.3

Control

9.7±1.2

21.7±1.5

26.3±2.9

2.9±0.4

9.1±0.2

The mean germination time (MGT), an international common statistic for speed of germination of seeds, is also shown in Table 4. The data for MGT in Table 4 show that the germination time for dry pine seeds treated by electrostatic fields for 10 min at three different field intensities was reduced. Treatments at 500 and 700 kV/m accelerated germination percentage and shortened the germination time a little. Treatment of the dry pine seeds at field intensities greater than 700 kV/m decreased germination because of damage to the seed are not shown in Table 40, An effective improvement of germination index was close to that of treatment of presoaked seeds, but germination was later for the dry pine seed.

As experimental examples, earlier aerial seeding reforestation tests were successful, we conducted reforestation tests during 1989 to 1994 by aerial sowing of seeds in the southern mountains of Shaanxi Province of China at 700 to 1700 meters above sea level. Firstly, 20 percentage of water comparing to seeds was added to dry seeds, and mixed them uniformly, and then, the seeds were loaded into a plastic box that had top and bottom plate electrodes, and treated at 1 kV/cm for 20 minutes. After treatment, 800 kg of pine seeds were loaded in an airplane and the seeds were sewn in an area of mountains at 0.25-0.5 kg seeds for 666 square meters (depending on species size, germination index, raining and soil or vegetation conditions).

Table 5. Results of reforestation tests by aerial sowing seeds in mountains in 1992


Area
(hektare)

Sowing rate
(kg/ hektare)

No. of seedings
(hektare)

Seedling
Height (cm)

Seedling
Diameter (cm)

Species

Electrical treated area

426

3.7

9705

5.8

0.1

Pinus mass.

Untreated area

214

3.7

5700

5.9

0.1

Pinus mass.

Electrical treated area

90

4.6

11160

5.0

0.2

Pinus tabu.

Untreated area

114

5.3

9000

4.5

0.2

Pinus tabu.

Table 5 shows results for a reforestation test using P. massoniana seeds in an area of Yuan-Shan Mountain of Chenggu County of Shaanxi Province in 1992. The test results showed that there were 9705 young pine young seedlings per hektare in the electrostatic sowing area, and 5700 young seedlings in the untreated sowing area when sowing the same weight pine seeds per hectare. The results were shown clearly that the electrostatic treatment improved germination of seeds and enhanced quantities of young seedlings. In another reforestation sowing area of Ningqiang County, Pinus tabulaeformis seeds were used as test materials. The results showed that there were 11160 young seedlings per hekeare in the electrostatic-treatment area, and 9000 young seedlings per hektare in the untreated sowing area. Thus, the treatment enhanced germination of seeds. The height of young seedlings in the treated sowing area was 5.0 cm, and 4.5 cm in the untreated sowing area. Thus, the electrostatic treatment accelervated growth of young seedlings.

Seed vigour is defined as the potential capacity of seed germination and seedling growing speed. The quick germination of seeds, uniform germination of seeds, and robust growth of seedlings indicate high vigor of seeds. In using electrostatic fields as a form of seed treatment, no temperature increase was produced in the seeds. Increases in vigor index were achieved by some contribution of the electric field force. The electric field used was more uniform than that produced by metal wire electrodes used by Yan et al. (1987). We expect, after determining a treatment method, that positive or adverse effects depend on electric field intensity, exposure time, treatment process, shape of electrodes, and germination characteristics of seeds. The results demonstrate that if a treatment at 400 to 600 kV/m for 10 min is selected, it can increase germination of seeds and young seedling development, and the optimal parameter is about 500 kV/m for 10 min for dry and wet pine seeds. It not only can improve germination percentage but also root development of young seedlings during initial, middle and latter stages of germination. If greater treatment dosages (more than 700 kV/m and longer exposures) are used, germination of seeds and development of seedlings will be reduced. If small dosages are selected, such as 100 to 300 kV/m for 10 min, they can improve germination but results are not ideal.

Discussion

Seed treatment with electricity includes other methods, such as low and high frequency electric fields. Electrostatic field is a special case arising from d-c potential differences between conducting electrodes, and the direction and magnitude of the electric field does not change during the exposure. In the treatment method used here, seeds being treated were in contact with only one electrode. Seed vigour was apparently changed by electrostatic field force, because there was no temperature change in the seeds before, during, or after seed treatment.

The Germination percentage of seeds was accelerated by electrostatic field because each seed has some electrical nature with electric potential differences existing in all tissue cells. Thus, polarization phenomenon occurs in the seed tissue when there is an external electric field. Thus, positive and negative ions in the seed tissue would move and be concentrated on top and bottom surfaces of each seed during electrostatic field exposure. Positive ions would concentrate from original positions toward the negatively polarized electrode, and negative ions in the seed tissue would concentrate from original positions toward the positively polarized electrode. Therefore, an inner electric field induced by the external electric field would form in the seed, and its direction is the reverse of the external electric field. Therefore the magnitude of the inner electric field in the seed tissue will depend on the magnitude of the external electric field according to theory of conducting electricity and electrical interactions of matter.

Exposure of pine, Pinus tabulaeformis Carr., seeds to electrostatic fields between horizontal, parallel-plate metal electrodes, spaced 3 cm apart, with the seeds lying in a single-seed layer on top of the lower electrode, produced significant improvements in germination and root development during the germination period. The best results were obtained, on both dry seed and seed presoaked in water, with exposures of 10 minutes to an electric field intensity of 500 kV/m. This same exposure of dry pine seeds accelerated germination and produced significant improvement in the germination index. Results are attributed to effects of the electric field forces in seed tissue, but mechanisms of the action are mainly unknown. The pre-sowing treatment offers potential for improving stands for reforestation by aerial seeding in mountainous regions.

Acknowledgements

We thank Professor Hu Jing-jiang, Mr. Liu Jian-chao and Mr. Wen Jian-lei, physiological and Biochemical Laboratory of Northwest Forestry College for their assistance in the seed testing, and part of the analyses. The work is supported by the National Natural Science Foundation of China.

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[1] Microelectronics Institute, Xidian University, Xi an, 710071, China. Email: zhbgui@xidian.edu.cn