Research Article, J Plant Physiol Pathol Vol: 6 Issue: 5

Technological System Construction on Artificial Embryos Synchronization in Artificial Seeds Production of Pinellia ternata (Thunb.) Breit

Ming-Sheng Zhang^*, Huan Li, Ye Hang, Gui-Xian Liu and Xiang Lv

School of Life Sciences, Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang, 550025 Guizhou, People’s Republic of China

*Corresponding Author : Ming-Sheng Zhang
School of Life Sciences, Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, 14, Xia-hui Rd., 550025, Guiyang, China
E-mail: mshzhang@163.com

Received: September 25, 2018 Accepted: October 11, 2018 Published: October 18, 2018

Citation: Zhang M, Li H, Hang Y, Liu G, Lv X (2018) Technological System Construction on Artificial Embryos Synchronization in Artificial Seeds Production of Pinellia ternata (Thunb.) Breit. J Plant Physiol Pathol 6:5. doi: 10.4172/2329-955X.1000190

Abstract

Keywords: Pinellia ternata; Loose callus; Cell micromass; Artificial embryo; Suspension culture; Synchronization

Abbreviations

2,4-D: 2,4-Dichlorophenoxyacetic Acid; 6-BA: 6-Benzylaminopurine; CH: Casein Hydrolysate; IBA: Indole-3-Butyric Acid; MS: Murashige and Skoog Medium; NAA: α-Naphthaleneacetic Acid

Introduction

Pinellia ternata (Thunb.) Breit. is a kind of medicinal plants (perennial herb) belonging to araceae, the medicinal parts are tubers which is an important major traditional Chinese medicine. It has many pharmacological effects such as dryness phlegm, downbear counterflow and check vomiting [1-3]. In recent years, the researchers found that P. ternata had the functions of antifertility, anticancer, antilipidemic and treating coronary heart disease [4-7]. According to statistics, there contain P. ternata in more than 200 kinds of proprietary Chinese medicine [8]. Because of the market demand increasing, together with the predatory excavation and changes of environment conditions, the medicinal materials of wild P. ternata appeared serious shortage. So artificial planting has become a major means to resolve the medicinal materials shortage of P. ternata. However, artificial planting easily led to lots of problems, such as the frequent damage of diseases [9-11], germplasm degeneration and yield decline [12-17] and it requires a large amount of reproduction materials and high cost. Although it provided a new approach that the tissue culture technology of P. ternate got success for to solve its reproduction and germplasm innovation, but the workload to transplant test-tube plantlets is very large, the survival rate is lower, and its management process is also complicated. Therefore, the technology has a slight practical significance in large-scale cultivation of P. ternata.

The emergence of artificial seed technology had brought expectation for the large-scale production of P. ternata, germplasm purification and rejuvenation [12,13,18-26]. The core of the technology is the synchronization culture of artificial embryos; it is also a key link relating to the mass production of artificial seeds can be fulfilled. So far, unfortunately, the technical issues of artificial embryos synchronization of P. ternata have failed to solve [18,20,26-34]. The reasons are that the intercellular junctions of P. ternata callus are very close and callus cells are difficult to disperse, so that the cells differentiation of callus different parts cannot be synchronized, and the small tubers (artificial embryos) by differentiation have no uniform size and they all connect tightly together, the operators have to adopt manual cutting to obtain independent small tubers using for artificial seeds embedding, it has seriously limited the artificial seeds industrialization of P. ternata. Therefore, it is a key and must first be solved technical problem in establishing artificial seeds industrialization system of P. ternata which how to culture the synchronization artificial embryos of dispersed to each other and suitable for artificial seeds production.

Our research team based on many years of trials [12,26,35], using the leaf blades or petioles as explants from aseptic seedlings of P. ternata to induce callus, then changing the intercellular bonding degree of callus cells by regulating carbon sources and plant growth regulators concentration and culture conditions to obtain the discrete and loose callus. Follow on to carry through the suspension culture to disperse callus cells into homogeneous cell masses (in this paper called “cell micromasses”) suspension system which is the important basis of subsequent synchronization differentiation. Finally, we successfully constructed and optimized the technology system of artificial embryos synchronization culture of P. ternata. This study laid a reliable technological foundation for solving required a large number of excellent-quality seed stocks and seedlings in large-scale cultivation of P. ternata.

Materials and Methods

Experiment materials

Pinellia ternata (Thunb.) Breit. was provided by Bijie Agricultural Sciences Institute of Guizhou in China.

Culture conditions

In this experiment, the pH value of all media was 5.8, temperature was (25 ± 1)°C, illumination intensity was 1500 lx to 2000 lx; illumination time was 12 h/d. For particular conditions were specified otherwise.

Explants preparation

The tubers of P. ternata were washed with running water for 2 h, then to sterilize the tubers for 45 s by 75% alcohol and to wash 3 to 5 times by sterile water, and to sterilize for 8 min with mercury bichloride and to wash 3 to 5 times by sterile water. Finally, the tubers using sterile filter paper to dry surface water were inoculated on the culture medium (MS+1.0 mg/L 6-BA+0.1 mg/L IBA+30 g/L sucrose+6 g/L agar) to induce the asepsis seedlings. Selecting the vigorous seedlings of 15 d age, cutting the leaf blade into 0.5 cm² small pieces or 0.5 cm petiole short segments to use as the explants.

Callus induction and propagation

The orthogonal table of L₉(3⁴) was selected for studying the effects of three factors including 2,4-D, 6-BA and explant sources in callus induction (Table 1). Each treatment contained 5 test bottles, each bottle inoculated into 3 explants, to repeat 3 times, and regularly checking the induction rate and biomass (fresh weight) of the callus, so as to screen the appropriate media to suitable for callus induction and proliferation. At the same time, the fresh and well glossy callus selected were inoculated on the appropriate media to subculture and proliferate for obtaining discrete and loose callus which will be used for suspension culture.

Table 1: Effects of different treatment combination for callus induction.

Establishment and optimization of suspension culture system of cell micromasses

Selecting the discrete and loose callus to inoculate into the suitable liquid medium (above appropriate medium for callus proliferation, but not contain agar) for suspension culture (rotating speed 90 rpm). After 7 to 10 days, the suspension cultures were filtered through 40 mesh screen to obtain homogenous cultures. The homogenous cultures was continued to culture in MS medium containing different concentrations of 2,4-D, 6-BA, sucrose and casein hydrolysate (CH) to set up suspension system composed of uniform cell micromasses (synchronization). The suspension system was optimized through L₉(3⁴) orthogonal experiment which factors and levels were shown in Table 2. The medium bottling volume was 30 mL per bottle, the inoculation quantity was 2 g loose callus per bottle, inoculating 5 bottles per treatment, and repeated 3 times. The rotating speed of shaking table was 90 rpm. After 25 days, sucking cell micromass to use for subculture and proliferation. Meanwhile, filtering suspension cells to weigh and calculate the biomass.

Table 2: Effects of different treatments combination for suspension culture of cell micromasses.

Synchronization differentiation of cell micromasses and formation of artificial embryos

Sucking the uniform cell masses (synchronization cell masses) from continuous subculture 3 weeks to inoculate in basal medium (MS+10 g/L sucrose+300 mg/L CH) and adding different concentrations of 6-BA and IBA (Table 3). The medium bottling volume was 30 mL per bottle, the inoculation quantity was 50 cell micromasses per bottle, inoculating 5 bottles per treatment, and repeated 3 times, the illumination intensity was 3000 lx to 4000 lx, the rotating speed of shaking table was 50 rpm. The differentiation rate of cell micromasses and the amount of artificial embryos were calculated when the process of artificial embryos formation was basic completed.

Table 3: Effects both 6-BA and IBA for cell micromasses to differentiate and form artificial embryos.

Statistical methods

Callus induction rate=(Explant numbers of formed callus/Explant numbers of inoculation) × 100%.

Differentiation rate of cell micromasses=(Amount of artificial embryos/Inoculated amount of cell micromasses) × 100%.

The SPSS 20.0 and DPS v 7.05 software were used for statistical analysis in this test.

Results

Effects of explants and plant growth regulators for callus induction

The effects of callus induction rate and its biomass (fresh weight) by orthogonal combining experiment both explant based sites and plant growth regulators were analyzed. The results showed that the combination both explant parts and plant growth regulators had different degree influence for the biomass, color and texture of callus, but the difference was not too big for the induction rate of callus (Figure 1 and Table 1).

Figure 1: The callus from different explants of Pinellia ternate (A) Callus from petiole (B) Callus from leaf blade.

The callus appeared early after inoculating 7 to 10 days when the petiole base of P. ternata was used explants. At the 15th day, most of the treatments formed callus. The rate of callus formation reached the maximum at the 25th day. The high concentration of 6-BA was advantageous to the callus induction and proliferation, and the callus was more loose while adding high concentrations of 2,4-D into the medium. Overall, for callus induction and proliferation, leaf blade was the most explants, petiole base was second, and petiole top was less.

Comprehensive considerating the fresh weight and texture of callus, we thought that the combination of treatment 3 or treatment 5 was the suitable medium for callus induction of P. ternata. ie. the medium of suitable to leaf blade callus induction was MS+2.0 mg/L 2,4-D+1.5 mg/L 6-BA+30 g/L sucrose+6 g/L agar, and the medium of suitable to petiole callus induction was MS+1.5 mg/L 2,4-D+1.5 mg/L 6-BA+30 g/L sucrose+6 g/L agar. The callus was subcultured and proliferated on this medium, to get the discrete and loose callus which well be used for suspension culture to establish cell micromasses suspension system.

Culture conditions of suitable for cell micromasses formation and proliferation

The above callus was subcultured and proliferated for 3 times to form the discrete and loose callus (Figure 2A). The discrete and loose callus was evenly dispersed into suspension liquid after 7 to 10 days by suspension medium MS+2.0 mg/L 2,4-D+1.5 mg/L 6-BA+30 g/L sucrose (Figure 2B). At the moment, the suspension liquid contained some large callus chunk, the cells showed kidney-shaped (Figure 2C). Filtering out large callus chunk to obtain uniform and milky white cell micromasses. The cell micromasses were subcultured 3 times to form the suspension system of homogeneous cell micromasses. In the suspension system, the cells became spherical shape and proliferated rapidly (Figure 2D).

Figure 2: (A) Loose callus and cell micromasses (B) Discrete and loose callus (solid culture) (C) Cell micromasses (suspension culture) and (D) Cell micromasses under a microscope by aceto-carmine dyeing (200X).

On the basis of preliminary experiment, screening medium compositions which had positive effect for suspension culture of cell micromasses carried out orthogonal combination experiment, the results were shown in Table 2 (basic culture medium was MS). By examining the fresh weight of cell micromasses, to find the fresh weight of cell micromasses in treatment 4 was the largest. Through extreme analysis, the contribution sorting of principal component for cell suspension culture was obtained, namely 6-BAï¼�?2,4- Dï¼�?CHï¼�?sucrose. The optimal combination of factors and levels as the medium for cell suspension culture was A₂B₂C₂D₃, in other words, the medium to suit for proliferation and growth of P. ternata cell micromasses was MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+40 g/L sucrose+300 mg/L CH. The formation of homogeneous cell micromasses laid a solid foundation for to realize artificial embryos synchronization in artificial seeds production of P. ternata.

The effects of plant growth regulators ratio for cell micromasses synchronization differentiation to form artificial embryos

The homogeneous cell micromasses of P. ternata were continuous subcultured for 3 weeks by MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+40 g/L sucrose+300 mg/L CH to form the larger cell masses (Figure 3A). Through the preliminary experiment based on different plant growth regulators combination, we found 6-BA and IBA combination showed a good effect for the cell micromasses synchronization differentiation to form artificial embryos. So 6-BA and IBA combination were selected in the suitable ratio test which the larger cell masses by successive transfer culture for three weeks were used for synchronization differentiation to form artificial embryos, the results were shown in Figure 3, Figure 4 and Table 3.

Figure 3: (A) The formation process on artificial embryos synchronization of Pinellia ternate (B) Cell masses by cell micromasses subculture (C) Artificial embryo of differentiating and developing (under the stereoscope) (D) Artificial embryos of completed differentiation and development.

Figure 4: Differentiation rate of cell micromasses by different treatments.

The process of cell micromasses synchronization differentiation was experienced several stages (A, B, C and D in Figure 3). The different concentration combinations of 6-BA and IBA showed much difference for the effect both cell micromasses synchronization differentiation and artificial embryos formation, the combination of 0.5 mg/L 6-BA with 0.05 mg/L IBA was well (Figure 4 and Table 3). From Figure 4, we know that the differentiation rate of cell micromasses was increased with time extension of cultivation, the different treatments almost always took the 9th day as the inflection point. All cell micromasses were differentiated to form artificial embryos after 15 day. From Table 3, we can determine that the suitable medium for cell micromasses synchronization differentiation to form artificial embryos is MS+0.5 mg/L 6-BA+0.05 mg/L IBA+10 g/L sucrose+300 mg/L CH, and no matter in which a group of medium, its artificial embryos after differentiation were all about 0.3 cm in diameter as dominant. The results show that the synchronization degree was high from cell micromasses differentiation of P. ternata to artificial embryos formation.

Discussion

The yield and germination rate of P. ternata natural seeds are all lower, and its medicinal materials yield is lower and the production cycle is longer when the natural seeds are used as breeding materials. So, people mainly adopt the tubers propagation in artificial planting of P. ternata. Because P. ternata is a weak herbaceous plant and the ability to compete against weeds is weaker. In order to reduce the heavy weeding workload in artificial planting, the planting pattern of high density planting to suppress weeds was widely used [12,36]. Therefore, the dosage of seed stock is larger and its production cost is higher in the present production conditions P. ternata. In view of this phenomenon, it has become the key problem to realize the industrialization of medicinal materials that how to construct the scale production technology of P. ternata seeds or seedlings.

In recent years, a number of scholars from home and abroad have carried out the research on the seedlings breeding of P. ternata, but most concentrate on the test tube seedlings through tissue culture to induce callus [37-45] and because of the culture process cumbersome of test tube seedlings, transplant workload larger, survival rate low, production cycle long of the medicinal material, and so on, so farmers are hard to accept the test tube seedlings breeding. In other words, the technique by tissue culture to produce seedlings of P. ternata has little practical significance.

For important medicinal plants like P. ternata which natural seeds do not suitable for medicinal materials production, tubers propagation need very high cost and test tube seedlings are inconvenient to field operation, its artificial seeds production is undoubtedly a promising technology choice, and this technology has also attracted many researchers. However, because artificial embryos of P. ternata must are the small tubers by callus differentiation and development, but the callus of P. ternata is quite tight and difficult to be dispersed, this is a fatal restriction factor for the embryo synchronization and it has not been broken through, causing the researchers almost still stay on these links such as artificial seed production process, artificial endosperm preparation and artificial seed coat screening, etc [13,20,39,46-48].

Conclusion

This study started with regulating carbon source and plant growth regulators concentration, cooperating with appropriate culture conditions, and solved effectively the problem which can reduce intercellular adhesion to form discrete and loose callus. Based on this, we established the dispersive and homogeneous cell micromasses suspension system; it realized the first breakthrough of P. ternata artificial embryos synchronization technology. This research work laid a good foundation for to build the industrialization technology of P. ternata artificial seeds.

Conflict of Interest

This research belongs to the research project of the tutor, which is accomplished by the unified guidance of the tutor in the same laboratory. There is no conflict of interest. All the data and materials in this paper are reliable and available from our lab.

Acknowledgements

This research was funded to MSZ by a grant from the National Key Research and Development Project in China (No. 2016YFC050260403), the Major Special Project of Science and Technology Plan in Guizhou of China (No. 2017-5411-06), the Science and Technology Support Plan Project in Guizhou of China. (No. 2018-2797), the Special Fund of Science and Technology Innovation Talent Team Construction in Guizhou of China (No. 2016-5624), the Project of High-level Innovative Talents in Guizhou of China (No. 2015-4031), the Major Projects of Innovation Group in Guizhou of China (No. 2016-023), the National key cultivation project of Guizhou University (No. 2017-5788), and the Modern Industrial Technical System Construction Project of Chinese Medicinal Materials in Guizhou of China (No. GZCYTX-02).

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