Effect of hormones on the seed germination of Bupleurum species

1 Department of Agronomy, School of Life Science and Engineering, Southwest University of Science and Technology, 59 Qinglong Road, Mianyang, Sichuan 621010, PR China. 2 Dongyang Institute of Maize Research, Zhejiang Academy of Agricultural Sciences, Dongyang, Zhejiang, 322100, China. 3 Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193, China.


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 andKorea (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 *Corresponding author.E-mail: dbhou@126.com;jhwei@implad.ac.cn.Tel: +86 816-6089523; Tel: +86 10-57833358.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 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.

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

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

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.

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.

RESULTS AND DISCUSSION
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 230.21-238.78mg•L -1 .Therefore, the best GA3 concentration for promoting 'Zhongchai No. 3' seed germination might be 223.84-238.78mg•L -1 When the 'Zhongchai No. 3' seeds were incubated with 230 mg•L -1 GA3, 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 223.84-238.78mg•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.

Figure 2 .
Figure 2. The quadratic and cubic correlation between gibberellin concentration with germination index and cumulative germination percentage of 'ZhongchaiNo.3' .

Table 1 .
Cumulative germination percentage and germination index of Bupleurum Zhongchai No. 3 seeds incubated after treatment with different hormones.

Table 3 .
Cumulative germination percent (GR) and germination index (GI) of Bupleurum seeds under direct treatment and converse treatment by GA3.

Table 4 .
The effect of gibberellin incubation on the germination percent (GR) and germination index (GI) Bupleurum varieties.