Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3627

Full Length Research Paper

Effect of hormones on the seed germination of Bupleurum species

Ma Yu
  • Ma Yu
  • Department of Agronomy, School of Life Science and Engineering, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, PR China.
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Guihua Lv
  • Guihua Lv
  • Dongyang Institute of Maize Research, Zhejiang Academy of Agricultural Sciences, Dongyang, Zhejiang, 322100, China.
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Hongzhang
  • Hongzhang
  • Department of Agronomy, School of Life Science and Engineering, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, PR China.
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Yuchan Li
  • Yuchan Li
  • Department of Agronomy, School of Life Science and Engineering, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, PR China.
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Cheng Yuan
  • Cheng Yuan
  • Department of Agronomy, School of Life Science and Engineering, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, PR China.
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Zhikang Tang
  • Zhikang Tang
  • Department of Agronomy, School of Life Science and Engineering, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, PR China.
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Xiaoyan Shu
  • Xiaoyan Shu
  • Department of Agronomy, School of Life Science and Engineering, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, PR China.
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Chun Sui
  • Chun Sui
  • Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193, China.
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Dabin Hou
  • Dabin Hou
  • Department of Agronomy, School of Life Science and Engineering, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, PR China.
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Jian-He Wei
  • Jian-He Wei
  • Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193, China.
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  •  Received: 18 November 2018
  •  Accepted: 21 December 2018
  •  Published: 10 February 2019

 ABSTRACT

In Asia, Radix Bupleuri (Bupleurum spp. root) is an important medicine used for treating many diseases over the past 2000 years. However, its cultivation is difficult due to poor germination rates and loss of seedling viability shortly after seeding. In the present study, the effect of 6-benzyl aminopurine (6-BA), daminozide, gibberellin (GA3), paclobutrazol, uniconazole, and mepiquat chloride (MC) on the germination of seeds of ‘Zhongchai No. 3’, which is the newest released commercial variety of Bupleurum chinense DC in China was analyzed. Only GA3 was found to promote seed germination significantly, and its optimum concentration was 223.84 to 238.78 mg∙L-1. In addition, incubation with GA3 on the first day of germination was extremely important for seedling survival. This protocol could also be applied to ‘Zhonghongchai No. 1’ (B. scorzonerifolium Willd.) and ‘Hubei chaihu’ (B. chinense DC.). The combination of GA3 treatment and centralized incubation could be an efficient way to industrialize the production of secondary metabolites from Bupleurum species.

Key words: Hormone, Bupleurum spp., germination, gibberellin (GA3).

 


 INTRODUCTION

Radix Bupleuri (root of Bupleurum spp.) is one of the most important medicinal herbs in Eurasia and North Africa for treating fever, chronic hepatitis, kidney syndrome, inflammatory diseases, menstrual disorder, and digestive system ulcers (Pistelli et al., 1996; Guo et al., 2000; Ikegami et al., 2006; Mabberleyl, 2008). Utilization of Bupleurum species in preparations was firstly recorded for more than 2000 years in Shen-Nong’s Herbal of China (Xie et al., 2009).

Nearly 250 bioactive compounds from this genus have   been   identified    through   phytochemical investigations; in particular, saikosaponin a and saikosaponin d are known for their pharmacological activities (Ashour and Wink, 2011). Bupleurum species are officially listed in the Chinese and Japanese Pharmacopoeias in addition to the WHO monographs of the commonly used medicinal plants of China and Korea (WHO, 1997, 1998). Due to its medicinal importance, the demand for R. Bupleuri has increased steadily in recent years. Nearly eight million kilograms of R. Bupleuri are required each year in China, and gross sales of manufactured prescription medicines containing R. Bupleuri in Japan amounted to 27 billion yen in 2002 (Pan, 2006; Zhu et al., 2009).

Due to the annual increased market requirement and considerable exploitation of Bupleurum species, natural wild resources have decreased sharply. Wild R. Bupleuri shows high variability in its pharmacological active components. Therefore, the cultivated Bupleurum has become economically important. In the last 10 years, three commercial varieties (Zhongchai No. 1, Zhongchai No. 2, and Zhongchai No. 3) of B. chinense DC and one commercial variety (Zhonghongchai No. 1) of B. scorzonerifolium Willd. were released to meet the market requirement in China (Yao et al., 2013). All these varieties showed uniform performance in their agronomic traits and high saikosaponin content. However, due to the short domestication history of Bupleurum species, there are still challenging traits to deal with, specifically poor seed germination and viability loss in a short period.

The aim of this work was to develop a protocol to promote Bupleurum seed germination, thus making the cultivation of Bupleurum easier. The study involved the initial screening of hormones to identify a candidate that could promote the germination of Zhongchai No. 3 seeds, which is the newest released commercial variety. The effect of this optimized protocol on other varieties of Bupleurum species was also validated.

 


 MATERIALS AND METHODS

Plant materials

Seeds of ’Zhongchai No. 3’ were used in all studies, except in validation of the optimal protocol. The seeds used in the validation of the optimal protocol were those of the commercial varieties ‘Zhongchai No. 1’, ‘Zhongchai No. 2’ and the landrace ‘Hubei chaihu’ of Bupleurum chinense DC; the commercial variety ‘Zhonghongchai No. 1’ and the landrace ‘Heilongjiang hongchaihu’ of B. scorzonerifolium Willd; the landrace ‘Rongxianzhuye’ of B. marginatum; and the landrace ‘B1’ of B. fruticosum. All materials were kindly provided by Professor Jianhe Wei from the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences & Peking Union Medical College. All varieties were planted in the Sichuan province in 2011–2013, and the seeding rate was 0.6–0.75 million plants per hectare. The planting area of each variety was over 0.1 hectares. All seeds were harvested in November 2013.

Incubation condition and germination evaluation

One hundred seeds were placed on moist filter paper at the bottom of covered culture dishes (diameter, 10 cm). The filter in every culture dish was initially moistened with 6 mL of hormonal solution at different concentrations. Distilled water was added to a few dishes to avoid drying during seed incubation. All culture dishes were placed in an incubator. Temperature was maintained at 20°C. Emergence of seeds greater than 0.5 cm was considered as successful germination, at which point the germinated seed was removed from the culture dish. The cumulative germination percent was calculated for each dish. Germination index was calculated as:

GI= ∑(Gt/t)

Where, Gt indicates the germinated seed at the tth day.

Experiment 1: Candidate hormone screening

The hormones in this screening study were 6-benzyl aminopurine (6-BA), daminozide, gibberellin (GA3), paclobutrazol, uniconazole, and mepiquat chloride (MC). For each hormone, six concentrations (i.e., 0, 20, 40, 60, 80 and 100 mg ∙ L-1) were utilized. Each treatment consisted of two replicates.

Experiment 2: Critical GA3 concentration and duration of treatment

The promotion of germination of Zhongchai No. 3 seeds was insignificant in a preliminary experiment when the GA3 concentration was above 500 mg∙L-1. Two sets of Zhongchai No. 3 seeds in 11 dishes were cultured in the presence of GA3 at concentrations of 0, 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500 mg∙L-1. The results from the experimental trials varied significantly and, consequently, different quadratic and cubic regression models were fit to each experimental trial. Critical GA3 concentrations were calculated from the different models.

The optimal concentration of 230 mg∙L-1 was used for the analysis of the duration of treatment for GA3. The seeds were incubated with GA3 for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 d. All seeds were incubated with distilled water following GA3 treatment for 10 d. The converse treatment sequence consisted of incubating the seeds with distilled water for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 d followed by GA3 treatment for 10 d. Then, all seeds were incubated with distilled water for 30 days.

Experiment 3: Validation of the optimal protocol

Seeds of ‘Zhongchai No. 1’, ‘Zhongchai No. 2’, ‘Zhonghongchai No. 1’, ‘Hubei chaihu’, ‘Rongxianzhuye’, ‘Fengshun landrace’, ‘Heilongjiang hong chaihu’ and ‘B1’ were incubated with 230 mg∙L-1 GA3. Distilled water was utilized as the control. Three replicate experiments were conducted.

Statistical analysis

All statistical analyses were performed using the SPSS 16.0 software (Norusis, 2008). Quadratic and cubic regression models were used to identify the critical incubation concentration of GA3. Duncan’s multiple range test (p < 0.05) was used to compare the differences.

 


 MATERIALS AND METHODS

To obtain the highest number of plants for the production of secondary metabolites, several studies based on micropropagation cultures of B. fruticosum, B. chinense DC, B. scorzonerifolium Willd, and B. smishii Wolff have been conducted (Fraternale et al., 2002; Li et al., 2008; Hao and Guan, 2012).

However, since the plants of Bupleurum species are small, the use of micropropagation cultures to industrialize the production of secondary metabolites is costly and has high labor requirements. Since plants of Bupleurum species can produce an abundance of seed, propagation from seed might be a more efficient method once the problem of poor germination is solved.

Seed germination represents the irreversible developmental-phase transitions from seed dormancy to germination  in  plants.  These transitions involved protein and hormonal alterations, especially the balance between abscisic acid and gibberellins (Graeber et al., 2010; Miransari and Smith, 2014).

A complex genetic network involved maturating genes, hormonal and epigenetic regulating genes may result this transitions (Bassel et al., 2011; Graeber et al., 2012). The germination of Bupleurum species seed is very difficult which considerably limits large-scale cultivation. This difficulty in germination might be attributed to seed maturity and germination inhibitors present in the seed coat (Wei et al., 2003). Dormancy of the harvested seeds also increases difficulty in germination (Hailu et al., 2008).

In the present study, the Zhongchai No. 3 seeds showed significantly different germination when treated with the different hormones (Table 1). Decreased germination was observed in the presence of daminozide, paclobutrazol, uniconazole, and MC. GA3 significantly promoted both cumulative germination percentage and germination index at all the concentrations used. In the case of 6-BA, the concentration of 20 mg∙L-1 increased the cumulative germination percentage and germination index, which decreased when the concentration exceeded 40 mg∙L-1. Since the root growth  of  ‘Zhongchai  No. 3’ seedlings was suppressed by 20 mg∙L-1 6-BA, we chose GA3 as the candidate hormone to further study germination promotion in Bupleurum species (Figure 1).

 

 

 

Gibberellins are diterpenoid, promoting germination and regulating plant growth. In previous studies, this plant hormone have been reported to stimulate the synthesis and accumulation of α-amylase, and regulate the expression of the Osem gene to control the production of one of the embryogenesis abundant proteins, resulting in the germination of seeds (Yamaguchi, 2008; Miransari and Smith, 2014).

Quadratic and cubic regression models were used to fit the GA3 concentration with cumulative germination percent and germination index (Table 2). Critical concentrations for promoting ‘Zhongchai No. 3’ seed germination were calculated from each model (Table 2, Figure 2). The predicted critical concentrations for increasing the germination index were in the range of 223.84–237.67 mg∙L-1, and the predicted critical concentrations for increasing the cumulative germination percentage were in the range of 230.21–238.78 mg∙L-1.

 

 

 

Therefore, the best GA3 concentration for promoting ‘Zhongchai No.  3’  seed  germination  might  be 223.84–238.78 mg∙L-1 When the ‘Zhongchai No. 3’ seeds were incubated with 230 mg∙L-1GA3, both the cumulative germination percentage and germination index were significantly higher than those of the control seeds when the incubation period was longer than 1 days (Table 3). However, a significant increase in cumulative germination percentage was observed when the converse treatment sequence was applied for more than 7 days, and a significantly increased germination index was observed when the duration of converse treatment was more than 9 days. These results indicated that incubation with an appropriate GA3 concentration on day 1 might be the most important factor in promoting ‘Zhongchai No. 3’ seed germination.

 

 

Significant germination variation was observed for other seed varieties. The commercial varieties showed a higher cumulative germination percentage and germination index than the landraces (Table 4). Only ‘Zhonghongchai No. 1’ and ‘Hubei chaihu’ seeds showed significantly increased cumulative germination percentages when the seeds were incubated with 230 mg∙L-1 GA3. However, the increase in the germination index of ‘Hubei chaihu’ was insignificant. This finding indicated that different germination mechanisms exist among different varieties of Bupleurum species. This could explain why several reported protocols for promoting Bupleurum seed germination were ineffective in other studies (Yao et al., 2011).

 

 

Therefore, in the case of commercial varieties other than ‘Zhonghongchai No. 1’ and ‘Hubei chaihu’, protocols using different hormones or varying incubation temperatures should be explored. However, in our study, a change in temperature for germinating Zhongchai No. 2 seeds was ineffective (data not shown).

Since Bupleurum species seeds are sensitive to water and temperature and have a long germination period, direct seeding in the field is difficult. In addition, the plants of Bupleurum species grow slowly at the early stage, leading to considerable competition in the field between small plants and weeds. Such competition can be potentially avoided by centralized incubation in a greenhouse  to increase seedling survival, followed by transplanting of these seedlings into the field. Hence, the combination of GA3 treatment and centralized incubation might be a more efficient strategy for large-scale cultivation rather than direct seeding.

 

 

 

 

 


 CONCLUSION

We found that treatment with 223.84–238.78 mg∙L-1 GA3 during the first day best promotes Bupleurum ‘Zhongchai No. 3’ seed germination. This hormone treatment is only useful in the case of ‘Zhonghongchai No. 1’ and ‘Hubei chaihu’ seeds. The combination of GA3 treatment and centralized incubation could be an efficient way to industrialize the production of secondary metabolites from Bupleurum species. The future work should focus on the contents variation of secondary metabolites for Bupleurum plants under transplanting and direct seeding.

 


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.

 



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