COMPARISON ON GROWTH DYNAMIC AND INDICATOR COMPONENTS OF DIFFERENT GERMPLASMS OF RUTHENICUM

YEHONG GUO1, QIAN LI1, MEI HUI1, MEIYING LI2 AND AIMIN WANG1

1 College of Agronomy, Institute of Traditional Chinese Medicine, Key Laboratory of Traditional Chinese Medicine Standardization Production Technology Innovation, Gansu Agricultural University, Lanzhou 730070, Gansu, People’s Republic of China 2 Gansu Endangered Animal Protection Center, State Forestry Administration, Wuwei 733000, Gansu, People’s Republic of China Corresponding author’s email:[email protected]; Cell number:+86 13519652912; Abstract This study report a comparison of growth dynamics, chlorophyll content of fresh leaves, main components content in -including polysaccharides, total flavonoids, anthocyanins and proantho cyanidins, moisture, total ash, and extract for three germplasm samples of Lycium ruthenicum. height, stem diameter, and branch number of the three germplasm samples all increased with time, but the growth rate was different in each case. Among the materials tested, Qinghai strains showed the maximum plant height, on August 31st, while the average height was 104.87 cm. Minqin Qingtuhu strains showed the largest stem diameter, on August 31st, while the mean value was 1.06 cm. However, the branch number of Wuwei, the native wild type Lycium ruthenicum, decreased after August 8th. Minqin had the highest chlorophyll content (39.92 mg/g). Qinghai had the highest chlorophyll b content (chlorophyll b and total chlorophyll were 13.05 mg/g and 51.96 mg/g, respectively). Polysaccharide, anthocyanin content, moisture content, and amount of extract from Qinghai were highest. Total flavonoid and ash contents were highest in Wuwei strains. Lycium ruthenicum introduced from Qinghai were the dominant strains and had better nutritional properties than the other two strain collections. Overall, results indicate that Qinghai Lycium ruthenicum is suitable for planting in the Wuwei area. Key words: Lycium ruthenicum; growth dynamic; index component; introduction cultivation

Introduction

Lycium ruthenicum Murray is a perennial flowering shrub that belongs to the . It is also called the Su wolfberry (Yuan et al., 2013; Han et al., 2014). Fruits of L. ruthenicum are rich in unique functional ingredients such as procyanidins and anthocyanins (Zheng et al., 2011; Wang et al., 2011), which are effective natural water-soluble free radical scavengers (Wei, 2014; Duan et al., 2015), with anti-cancer effects. Vitamin and fat content in these fruits are also much higher than in fruits of the close relative, Lycium barbarum L. The also contains polysaccharides, flavonoids, and other nutrients (Liu et al., 2014; Liu et al., 2016; Nzeuwa et al., 2017). L. ruthenicum is a valuable plant with antioxidant effects and useful for the prevention and treatment of cardiovascular and cerebrovascular diseases (Dong et al., 2008; Lin et al., 2012; Lv et al., 2014); It may also be helpful in preventing severe myopia and retinal detachment (Jin et al., 2015; Gong et al., 2016; Tang et al., 2017).

L. ruthenicum has long attracted considerable attention worldwide owing to its high nutritional value and remarkable effects on human health. However, no breakthrough with regard to development of cultivation technology for the species has been made despite years of research. Present limitations

owing to small scale production and germplasm scarcity are still key problems that limit industrial development of L. ruthenicum (Wang et al., 2011; Shen et al., 2012; Wang et al., 2014). In this work, the growth dynamics of the aboveground parts of L. ruthenicum, the anthocyanin content (Guo et al., 2016; Ma et al., 2017), and other indicators were studied to comprehensively evaluate the growth adaptability, fruit active ingredients, and quality advantage of L. ruthenicum (Lin et al., 2013; Li et al., 2017). This work aimed to provide a scientific sound basis for improving ecological adaptability, yield, quality, and standardized production and cultivation guidelines for L. ruthenicum. Our work can thus promote the cultivation of fine varieties and the expansion of the L. ruthenicum industry chain.

Materials and Methods

Experimental area: The experiment field is located northwest of Wuwei city (Gansu province) on the southwest margin of the Tengger Desert, which is characterized by a typical temperate continental climate. Geographic coordinates are longitude 102°43′-103°22′, latitude 38°05′-38°20′, and elevation 1632 m. The mean annual precipitation is 210 mm, evaporation volume amounts to 2600–3010 mm, and activity accumulated temperature is about 3003 ℃; The day/night temperature difference is 15 ℃ or more; The number of sunshine hours is usually around 3039, the frost-free period is 85–165 d. Light, heat, water, and soil conditions are suitable for L. ruthenicum cultivation.

Experimental plant materials: The experiment was conducted from April 2016 through April 2017, at the National Forestry Administration of Gansu Endangered Animal Protection Center and at the General Laboratory of Gansu Agricultural University. Test materials included introduced an L. ruthenicum Qinghai (QH) germplasm collection; Wuwei (WH), a native wild type; and Minqin Qingtuhu (MH), a native wild type. The experiment was laid out as a single-factor randomized block design with three treatments and three replications; the experimental plots were 3 m × 6 m = 18 m2. Ten strains of L. ruthenicum were randomly selected from each district and marked. Mature leaves were sampled at 23-d intervals, placed in preservation bags, and brought back to the laboratory for analysis.

Experimental methods:

Measurement of aboveground growth index of three different L. ruthenicum germplasm samples: Plant height, stem diameter and branch number were measured at 23-d intervals starting May 22nd. A total of 10 strains single were measured with a tape gauge and a Vernier caliper.

Determination of chlorophyll in leaves of different germplasm samples of L. ruthenicum: Chlorophyll content was determined and calculated as described by Mu et al. (2005).

Polysaccharide extraction and determination: Polysaccharide content was extracted and determined as described by Zhang et al. (2006) and Lin et al. (2013).

Preparation of standard curve: Anhydrous glucose (50.0 mg) was accurately weighed and transferred to a volumetric flask (50 mL), after which water was added to bring the solution to volume; 1.0, 2.0, 3.0, 4.0, 5.0 mL aliquots of this solution were drawn and transferred into 50 mL volumetric flasks, and again distilled water was added to bring the solution to volume. Finally, 1.0 mL aliquots of each standard solution were transferred into plug test tubes. Absorbance was determined according to the method described below (2.3.3.2). The standard curve was drawn with absorbance value as the abscissa and concentration (C) as the ordinate, and the regression equation was calculated. Precision experiment: Precise removal of the reference solution 1 mL and was set to 25 mL volumetric flask, respectively. One mL of each test solution was placed in a 10 mL plug test tube. The absorbance value was measured under 365nm and the RSD（Relative standard deviation） value was calculated (n = 5).

Reproducibility experiment: Five samples from the same batch of Qinghai L. ruthenicum were accurately weighed. The polysaccharide solution was prepared according to the ultrasonic method, and polysaccharide content was determined and RSD value calculated (n = 5).

Stability experiment: Test solution of Qinghai L. ruthenicum was sampled at different times (0.5, 1, 2, 4, and 8 h) to determine the absorbance; the RSD value (n = 5) was calculated in each case. The absorbance value was confirmed to remain constant for at least 8 h.

Recovery experiment: One milliliter of a glucose reference solution (1.0 mg/mL) was transferred to a 25 mL volumetric flask. The solution (0.5 mL) was then placed in a 10 mL stoppered tube, to which was added 0.5 mL of a test solution. Absorbance was measured, and average recovery rate and RSD value (n = 5) were calculated.

Determination of sample polysaccharide content: One milliliter of test solutions of Wuwei and Minqin Qingtuhu L. ruthenicum samples were placed in 10 mL plug test tubes, respectively. Absorbance was determined and polysaccharide content was calculated according to the following formula:

Polysaccharide content (%) = CDf/W × 100 (1)

Where, C is the glucose concentration (mg/mL) in the sample; D is the dilution factor for the sample solution; f = 3.17 (conversion factor (Chang et al., 2002); W is the powder quality (mg) of the sample.

Extraction and determination of total flavonoid content: Total flavonoids were extracted and reported as reported by Zhang et al. (2006).

Determination of procyanidins and anthocyanins: Anthocyanin content was calculated according to the following formula (Wang et al., 2016):

MF = (A × V)/(98.2 × M) (2)

Where, MF is the content of anthocyanins in the sample (mg/g); A is the absorbance at maximum wavelength; V is volume (mL) × dilution factor; M is the sample weight (g); and the value 98.2 is the average extinction coefficient of anthocyanin.

Determination of moisture, total ash, and extract content: Moisture, total ash, and amount of extract were determined according to "Chinese Pharmacopoeia" (2015 edition).

Statistical analysis:Excel 2013 software was used to generate images and the Duncan method was used for statistical analysis in SPSS 20.0 software.

Results and Discussion

Dynamic comparison of aboveground growth index of three germplasm samples of L. ruthenicum Dynamic comparison of plant height of three germplasm samples of L. ruthenicum: There were significant differences in plant height at different growth stages among the three different germplasm samples. There was no significant difference between L. ruthenicum from Qinghai (QH) and the wild type L. ruthenicum from Wuwei (WH), while significant difference between these two and Minqin Qingtuhu (MH) was evident (Table 1). In general, plant height increased initially, but then decreased towards the end of the growing season. L. ruthenicum grew significantly from May 8th to July 16th, while growth tended to be moderate after the latter date. Among the germplasm samples tested, the growth of WH was lower from July 16th to August 8th; Then from May 8th to August 31st, the growth of MH was lower and significantly different from that of the other two germplasm sample collections. Plant height of all L. ruthenicum samples reached a maximum on August 8th. QH was the tallest, with mean height of 104.87 cm, followed by WH, with a mean plant height of 102.12 cm; MH was the shortest, with a mean plant height of 65.58 cm. Overall, after August 8th plant height gradually reduced. In general, plant height of L. ruthenicum followed the slow-fast-slow growth law. Before July, plants mainly absorbed soil nutrients to supply vegetative growth. After July, flowering—that is, reproductive growth—was initiated. Blooming opposes vegetative growth, as a result of which plant height stopped increasing. The results showed that QH had the obvious advantage over MH or WH. This is consistent with the conclusion by Chang et al. (2002) about the effects of high temperature on growth dynamics in the case of Lycium barbarum growing in Qinling.

[Table 1 here]

Comparison of stem growth dynamics in different L. ruthenicum germplasm collections: The ranking of the germplasm studied with respect to stem diameter was WH > QH > MH. The mean stem diameter for these samples was 1.06, 0.97, and 0.75 cm, respectively. Although QH and WH were not significantly different from one another, MH differed significantly from QH and WH (Table 1). The stem diameter of the three germplasm collections studied increased as the growing season proceeded and the stem became obviously thick, especially from May 8th to July 16th, while diameter increase tended to be moderate after July 16th. Growth rate was much lower for MH. The nutritional growth period proceeded from May 8th to July 16th and the reproductive growth period occurred after July 16th; thus, stem growth rate was low. Results showed that stem diameter of WH was significantly greater than that of QH or MH, which is consistent with results attributed to the effects of high temperature on growth dynamics and photosynthetic rate of L. ruthenicum (Chen et al., 2007).These results also supported by Ma et al. (2016).

Comparison of branch-number increase dynamics among three different germplasm collections of L. ruthenicum: As shown in Table 1, there were significant differences in branch number at different growth stages of the various L. ruthenicum strains under study. On May 8th, May 31st, June 23rd and July 16th, the branch number of QH was significantly higher than that of WH or MH. On August 8th and August 31st there were no significant differences among the three strain collections tested. The branch number of QH and WH increased with time; indeed, branch number of QH increased more significantly than did that of WH. However, the branch number of MH decreased after August 8th. The root absorption capacity of QH is strong, presumably resulting in a larger number of branches. The results showed that the branch number of QH was significantly larger than that of WH and MH.

Comparison of chlorophyll content in leaves of different germplasm collections of L. ruthenicum Comparison of Chlorophyll a content in leaves of different germplasm collections of L. ruthenicum: Chlorophyll a content in the three germplasm collections tested first decreased and then increased towards the end of the sampling period. Germplasm ranking with regard to chlorophyll a content was MH > WH > QH and the corresponding value for mean content was 36.46, 34.29, and 32.78 mg/g, respectively. No significant differences in chlorophyll a content were found among any of the three L. ruthenicum strain collections under study. Because L. ruthenicum strains were in the active growing period, adequate light, moisture, oxygen, and nutrients for the synthesis of chlorophyll were available, the synthesis of chlorophyll a was assumed to be unaffected.

[Figure 1 here]

Chlorophyll a content decreased on July 16th, as shown in Fig. 1. Germplasm ranking based on chlorophyll a content was WH > QH > MH, with mean content of 31.86, 25.68, and 39.05 mg/g, respectively. However, the difference in Chlorophyll a content among strains was not significant. Because L. ruthenicum is a kind of light plants, but synthesis of chlorophyll a was hindered by plenty of rainfall from June to July.

Chlorophyll a content increased on August 8th. The germplasm ranking based on chlorophyll a content was WH > QH > MH, with mean content of 39.05, 38.92, and 35.05 mg/g, respectively. Chlorophyll a is a key element in photosynthesis, and photosynthesis also influences the synthesis of

chlorophyll a. CO2 content, light intensity, and absorbed nutrients altogether affected photosynthesis after July (Peng et al., 2014), thereby enhancing the synthesis of chlorophyll a. The results showed that the content of chlorophyll a in WH was relatively high, which is supported by previous findings (Wei et al., 2005).

Comparison of chlorophyll b content in leaves of different germplasms of L. ruthenicum: There were significant differences in chlorophyll b content among the three germplasm collections tested, as shown in Fig. 2. Chlorophyll b content in leaves of MH and WH was significantly higher than in those of QH on May 31st. No significant differences were observed among the three germplasm collections of L. ruthenicum on July 16th. On August 8th the ranking for chlorophyll a content was QH > MH > WH, with mean content of 13.04, 10.43, and 9.36 mg/g, respectively.

[Figure 2 here]

Content of chlorophyll b of QH increased, while that of WH first decreased and then increased again, while that of MH decreased as the growing season proceeded. Before July, L. ruthenicum was affected by rainfall, temperature, and other factors. QH had taller plants and leaves with large surface area, leading to an improved ability to capture sunlight, so the content of chlorophyll b in QH was relatively higher. After August, MH weakly adapted to the continued high temperature and dry summer weather, which resulted in diminished absorption of light, moisture and nutrients. This agrees well with previous findings(Guo et al., 2016).

Comparison of total chlorophyll content in leaves of different germplasm collections of L. ruthenicum: As shown in Fig. 3, there were no significant differences in total chlorophyll content among the three germplasm samples of L. ruthenicum at different growth stages. On May 31st, average chlorophyll content in MH, WH and QH was 48.13, 45.21, and 38.57 mg/g, respectively. On July 16th, chlorophyll content decreased significantly. Average chlorophyll content for QH, WH, and MH was then 41.99, 40.81, and 35.60 mg/g, respectively. On August 31st, the ranking with respect to total chlorophyll content was the same as on July 16th. Average chlorophyll content in QH, WH, and MH was 51.96, 49.76, and 44.41 mg/g, respectively. Moreover, total chlorophyll content in leaves of QH plants increased continuously, while in WH and MH leaves, it showed an initial decreasing trend which then subsequently reversed. Thus, chlorophyll content in QH was relatively high. The chlorophyll content increased with ambient temperature; external environment and soil organic matter may have inhibited the activity of chlorophyllase, thereby allowing for increased absorption of light energy by the chloroplast (Jin et al., 2015; Eva-Mari et al., 1993). The comprehensive analysis showed that chlorophyll content of QH was highest and the cultivation advantages were obvious.

[Figure 3 here]

Comparison of polysaccharides and total flavonoids in fruits of different germplasm samples of L. ruthenicum

Standard curve for polysaccharide: The standard curve for polysaccharide determination is shown in Fig. 4. The linear equation C = 0.1524A + 0.233, r2 = 0.9999 indicates a strong linear relationship.

[Figure 4 here]

Scientific examination results: The RSD for precision, stability, repeatability, and sample recovery rate was 1.66%, 1.96%, 1.46%, and 1.46%, respectively. These data confirm that the method is scientific and reliable.

Comparison of polysaccharides and total flavonoids in fruits of different germplasm samples of L. ruthenicum: There were significant differences in the growth index elements of different germplasm samples of L. ruthenicum. Polysaccharide content in fruits of L. ruthenicum was in the range 2.64%–5.97%; that of QH fruits was highest (5.97%); in fact, this was significantly higher than that in MH or WH. On the other hand, no obvious difference was noted in polysaccharide content between MH and WH (2.65% and 2.64%, respectively). This suggests that Qinghai L. ruthenicum has an obvious genetic advantage in this respect. Total flavonoid content in the fruits varied between 0.88% and 1.05%. Strain ranking for this parameter was WH > MH > QH, with values of 1.05%, 0.88%, and 0.88%, respectively. Analysis of variance showed no significant difference in flavonoid content between QH and MH fruits, but they both were significantly lower in fruit flavonoids than WH. Environmental factors, such as altitude, annual sunshine hours, light and heat resources, rainfall and soil nutrients resulted in the observed differences in polysaccharide and total flavonoid content in fruits of the strains analyzed (Liu et al., 2011), as has been previously reported (Zhang et al., 2006; Li et al., 2011).

[Figure 5 here]

Comparison of proantho-cyanidin and anthocyanin content in different germplasm samples of L. ruthenicum fruits: As shown in Fig. 6, there were significant differences in proantho-cyanidin and anthocyanin content in the germplasm samples of L. ruthenicum tested here. Strain ranking with regard to proantho-cyanidins content was QH > WH > MH, with values of 41.37, 24.95, and 24.84 mg/g, respectively. On the other hand, ranking for anthocyanin content was QH > MH > WH, with values of 7.37, 6.89, and 6.51 mg/g, respectively. QH plants might have gathered soil organic matter content more efficiently than their counterparts and this, together with better utilization of light and heat, would presumably have resulted in their being able to accumulate higher proantho-cyanidin and anthocyanin content (Hu et al., 2015). As a matter of fact, proantho-cyanidin content was much higher than anthocyanin content, a result consistent with that reported by Luo et al. (2015) and Chen et al. (2011).

[Figure 6 here]

Comparison of moisture, total ash, and extract content in fruit of different germplasms of L. ruthenicum: The moisture content of the different germplasm samples of L. ruthenicum varied between 7.71% and 9.45%, as shown in Table 2. The difference in moisture content among them was not obvious after drying at 105 ℃ (Peng et al., 2011) in compliance with the "Chinese Pharmacopoeia" 2015 edition. Percent extract ranged from 56.84% to 73.12%. Total ash and amount of extract was significantly different, as shown in Table 2.

There was a significant difference in total ash content among the three germplasm samples L. ruthenicum (Gong et al., 2015; Duan et al., 2015). The total ash content variation range was 2.42%–3.37% (Table 2). Percent extract was 73.12%, 62.09%, and 56.84% for QH, WH, and MH, respectively.

[Table 2 here]

Discussion and Conclusion

Dynamic growth index of aboveground biomass was significantly different among the various L ruthenicum strains under study. Plant height and branch number of QH plants were much higher than those of the other two strain collections considered; however, MH plants grew the thickest stems. As we know, the ability of plants to adapt to adverse conditions could be reflected by any of various growth indexes, including plant height, stem diameter, branch number, and so on. From the above results, we conclude that the introduced L. ruthenicum Qinghai was the best germplasm sample in terms of growth among those reported herein.

Chlorophyll content at each growth stage was significantly different. QH plants had the highest chlorophyll content among all strains considered. The chlorophyll content increased with increasing ambient temperature, indicating that appropriate high temperature is conducive to the accumulation of chlorophyll and photosynthesis enhancement, thus promoting plant growth and the ability to adapt to the environment.

Active ingredient content varied significantly among experimental materials. The QH strains had the highest procyanidins, anthocyanins, water, and extract content. Procyanidins and anthocyanins are index components of L. ruthenicum in the "China Pharmacopoeia". Our observations indicate that QH has the best quality among the germplasm samples under study. Therefore, high quality L. ruthenicum could be obtained by introducing these traits into high-performing cultivated Qinghai strains in Wuwei, Gansu, under conditions of non-limiting water and soil fertility conditions, as well as adequate light and heat.

This study aimed to provide a sound scientific basis for cultivation of L. ruthenicum in different areas and to provide a reference point for studying the relationship between nutritional value of L. ruthenicum and growth environment.

Growth dynamics, chlorophyll and active ingredients content in leaves of L. ruthenicum were affected by many factors including genotype and environmental conditions. Therefore, future research should focus on the difference in growth dynamics, yield, and quality of L. ruthenicum, cultivated in its native area and other areas where it might be introduced.

Acknowledgements

We gratefully acknowledge financial support from the Forestry departments of the Gansu Province. This work was also supported by other colleagues and the students in the laboratory.

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