Nitrogen and three chemical mimics of saponin precursors enhance biomass and calculation saponin for Gynostemma pentaphyllum under hydroponic conditionsABSTRACT:
Gynostemma pentaphyllum (Thunb.) Makino (G. pentaphyllum) is used in traditional Chinese medicine to stimulate immune responses and reduce blood lipid concentrations, and has broad market growth prospects. In this study, a nutrient liquid culture method has been modified by adding nitrogen and or chemical mimic of saponin precursor to stimulate rooting from stem cuttings. Furthermore, total nitrogen concentration and ammonia/nitrate ratio in the nutrient solution has been optimized; And, the concentration of three ginsenosides precursors that applicated in nutrition solution together with total nitrogen and ammonium/nitrate ratio were studied. The results showed that, after 10 days of culture, adventitious roots developed from stem cuttings to form complete regenerated plantlets. The addition of 1 mg·L-1 indole acetic acid in hydroponic solution could effectively increase the length and number of adventitious roots. The optimal total nitrogen concentration in the nutrient solution for increase in fresh biomass of G. pentaphyllum was 7.0 mmol·L-1 and the optimal ammonium/nitrate ratio was 2:3 for increase in the plant fresh weight, and chlorophyll a, chlorophyll b, and total chlorophyll contents of G. pentaphyllum rooted cuttings. Addition to the nutrient solution of three chemical mimics of saponin precursors sodium acetate (0.5 mmol·L-1), geraniol (0.5 mmol·L-1), and DL-mevalonolactone (0.3 mmol·L-1) increased the content and yield of total saponins.
Key words:
hydroponics; Gynostemma pentaphyllum; stem cutting; nitrogen; chemical mimics of saponin precursors
RESUMO:
Gynostemma pentaphyllum (Thunb.) Makino (G. pentaphyllum) é usado na medicina tradicional chinesa para estimular respostas imunes e reduzir concentrações de lipídios no sangue, e tem amplas perspectivas de crescimento de mercado. Neste estudo, um método de cultura líquida de nutrientes foi modificado pela adição de nitrogênio e/ou imitação química do precursor de saponina para estimular o enraizamento de estacas de caule. Além disso, a concentração total de nitrogênio e a proporção amônia/nitrato na solução nutritiva foram otimizadas; e, a concentração de três precursores de ginsenosídeos que foram aplicados na solução nutricional juntamente com nitrogênio total e proporção amônia/nitrato foram estudados. Os resultados mostraram que, após 10 dias de cultura, raízes adventícias se desenvolveram a partir de estacas de caule para formar mudas completamente regeneradas. A adição de 1 mg·L-1 de ácido indol acético em solução hidropônica pode efetivamente aumentar o comprimento e o número de raízes adventícias. A concentração ótima de nitrogênio total na solução nutritiva para aumento da biomassa fresca de G. pentaphyllum foi de 7,0 mmol·L-1 e a proporção ótima de amônio/nitrato foi de 2:3 para aumento do peso fresco da planta e dos teores de clorofila a, clorofila b e clorofila total de estacas enraizadas de G. pentaphyllum. A adição à solução nutritiva de três imitadores químicos de precursores de saponina, acetato de sódio (0,5 mmol·L-1), geraniol (0,5 mmol·L-1) e DL-mevalonolactona (0,3 mmol·L-1), aumentou o conteúdo e o rendimento de saponinas totais.
Palavras-chave:
hidroponia; Gynostemma pentaphyllum; estaca de caule; nitrogênio; imitadores químicos de precursores de saponina
INTRODUCTION
Gynostemma pentaphyllum, also known as “Jiaogulan” in Chinese, is a member of the Cucurbitaceae family and is widely distributed in tropical and subtropical regions. Previous analyses of the chemical constituents of Jiaogulan have isolated saponins with the same molecular structure as ginsenosides, together with various major and trace elements (NORBERG et al., 2004; WANG & WANG, 2002). Phytochemical studies showed that Jiaogulan contains up to 90 types of pentaphylosides, which have a broad range of pharmacological effects (YIN et al., 2004; LI et al., 2019; WANG et al., 2022; WANG & LUO, 2007). Given that ginsenosides are well-known bioactive ingredients in ginseng, Jiaogulan has attracted extensive attention. Jiaogulan has been used to treat tumors, blood diseases, hyperlipidemia, digestive ulcers, insomnia, hormonal side effects, and other diseases (WANG & LUO, 2007; WANG et al., 2022; LI et al., 2016; LI et al., 2022). Jiaogulan stems and leaves can be used directly for medicinal purposes, or processed into a tea or health drinks. Because Jiaogulan is of extremely high medicinal value, wild Jiaogulan is widely exploited. However, most wild populations are scattered, and the resources are limited. Gynostemma pentaphyllum medicinal materials have long relied predominantly on wild resources and scattered cultivation by a small number of farmers, but wild populations are steadily decreasing. It is necessary to expand the cultivation and production of Jiaogulan to meet the market demand for traditional Chinese medicines.
Jiaogulan is dioecious and is commonly propagated from seeds. The ratio of male and female seed-raised individuals is approximately 1:20, hence natural seed production is very low, which is the main limiting factor for the development of new production areas (WANG et al., 2004). Seed propagation consumes much seed material, has a low propagation efficiency, and is strongly seasonal, which severely hinders expansion of Jiaogulan production. It has been reported that the 1000-seed weight is 7.7 g and, without pre-treatment, the average seed germination rate is 44%. Plant flowering and seed set directly affect the yield per unit area, and growth from seedling emergence to harvest requires more than 20 months. Therefore, no harvest is possible in the same year that Jiaogulan seeds are sown. An alternative method of propagation is stem cuttings, but previous studies have shown that many factors affect the survival rate of stem cuttings. The survival rate of cuttings taken from healthy middle stem segments is 86.9 %, whereas the survival of cuttings prepared from other parts of the stem is lower (GAO, 2018). Rapid propagation of Jiaogulan using biotechnological techniques has not yet achieved practical application (CHANG, 2005). Therefore, it is important to study its reproductive biology, and determine and optimize the most efficient propagation methods.
There are few reports on the preparation of stem cuttings of Jiaogulan and the factors affecting the survival of stem cuttings (ZHU, 2004; YAO et al., 2014). To date, no techniques for soilless cultivation and nutritional quality control of Jiaogulan have been reported. Therefore, the present experiment aimed to establish a soilless solution culture system for Jiaogulan. The saponins of Jiaogulan mainly accumulate in the stems and leaves, and nitrogen is the main factor affecting the growth of stems and leaves. Therefore, we investigated the effect of the total nitrogen concentration and ammonium/nitrate (NH4 +/NO3 -) ratio in the nutrient solution on plant growth. Given the strong interest in gypenosides, the effect of incorporating three chemical mimics of saponin synthesis precursors in the culture solution on the total saponin content and yield of Jiaogulan plants was also studied.
MATERIALS AND METHODS
Location
The present investigation was performed in a greenhouse at the crop science experimental station of the Henan Institute of Science and Technology, Xinxiang, Henan, China. The temperature in the greenhouse was maintained at 25 ± 0.5 °C with 75 % relative humidity, and a 16-h photoperiod with 45-55 µE m-2 s-1 photon flux density was provided. The study site lies in the Yellow River Basin, North China Plain, at 35°18’ N latitude, 113°55’ E longitude, and at an elevation of 79 m above mean sea level.
Plant material and sowing seeds
Jiaogulan seeds were purchased from the Jiaogulan Research Institute of Pingli County (Shanxi Province, China). The seeds were soaked in 50 ℃ warm water for 12 h, dried, and sown in pots with a sown volume of 7.7 g·m-2, covered with 1-2 cm of fine soil after sown, and sprayed with water to keep the soil surface moist. Seed emergence was observed for approximately 10 days. Seven days after emergence, seedlings were transferred to new pots for further cultivation.
Preparing stem cutting
When the seedling height exceeds 20 cm, the stem can be used to cut spikes. Stem cuttings were selected from robust fresh stems, according to the original growth direction of the stem. Stem segments were cut so as to include either one or two nodes. The lower cut was inclined (i.e. not perpendicular to the stem epidermis), and the upper cut was approximately 0.5 cm above the node, so as not to damage the axillary bud. One complete compound leaf was retained, and the leaf and tendril at the lower node were removed. Thus, two types of cuttings were prepared (Figure 1A): single-node cuttings (with one leaf at the upper node and the lower cut positioned in the internode) and two-node cuttings (with one leaf at the upper node and the lower cut positioned below the lower node).
Rooting and hydroponic process of G. gynostemma stem cuttings A: a:one node of stem; b:two nodes of stem; B: The rooting of cuttings was induced in nutrient solution; C: Single-node cuttings were rooted on day 10 in hydroponics; D: Stem cuttings were grown in nutrient solution.
Cultivating the stem cutting in water
The cuttings were placed in a culture bottle filled with pure water and the node with the leaf was kept above the water surface. The water was oxygenated with a super pump. In total, 30 cuttings of each type, with three replicates, were prepared. The experimental materials were placed in the greenhouse for observation and, after culture for 10 days, the number and length of adventitious roots and number of rooted cuttings were recorded.
Treatment of stem cuttings using hormone
The effects of naphthylacetic acid (NAA), indoleacetic acid (IAA), and indolebutyric acid (IBA) on rooting of the cuttings were assessed. Cuttings with two nodes were used in this experiment. The concentration range of 0 (pure water), 1, 10, 20, 40 and 80 mg·L-1 was evaluated for each hormone. The base of the stem cuttings was immersed in the treatment solution, which was oxygenated with a super pump, and the upper node with the leaf was kept above the fluid surface. In total, 30 cuttings were treated for each hormone concentration with three replications. After culture for 10 days, the number and length of roots were recorded.
Stem cutting growth under hydroponic condition addingnitrogen
Experiment 1
Well-grown rooted stem cuttings were selected, randomly divided into three groups each comprising nine plants with three plants per culture bottle, and placed in the greenhouse. Hoagland’s nutrient solution (HOAGLAND, 1920) was used in the experiment 1, nitrogen treatments (0, 1, 2, 5, 7, 10, and 12 mmol·L-1) was set (Table 1). The other components in the nutrient solution comprised 1.55 mmol·L-1 CaCl2, 0.45 mmol·L-1 CaSO4, 0.65 mmol·L-1 MgSO4, and 0.25 mmol·L-1 KH2PO4. The adjustment of four components of the nutrient solution for each treatment is shown in table 1. The concentrations of other trace elements and iron salts were identical to those of 100% Hoagland’s nutrient solution.
Experiment 2
Cultivated the stem cutting in Seven NH4 +/NO3 - ratio treatments were applied under a total nitrogen concentration of 7 mmol·L-1, namely, 5﹕0(T1), 4﹕1(T2), 3﹕2(T3), 1﹕1(T4), 2﹕3(T5), 1﹕4(T6), and 0﹕5(T7) (Table 2). The pH of the nutrient solution was adjusted to 6.5-7.5 at 3-day intervals and the culture solution was replaced every 6 days. Experiments 1 and 2 were completed after culture for 18 days, and the relative rate of change in biomass, and the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were measured.
Cultivated stem cutting in the nutrition solution adding chemical mimics of saponin precursors
Rooted stem cuttings with similar agronomic traits were selected, fixed in place in a Styrofoam board, and placed in nutrient solution for floating culture (Figure 1B). Based on the optimal total nitrogen concentration and NH4 +/NO3 - ratio determined in the preceding experiments, the total nitrogen concentration of 7 mmol·L-1 and NH4 +/NO3 - ratio of 2:3 were selected for the nutrient solution in this experiment. The effect of precursor compounds on the content of saponins was studied. Three chemical mimics of saponins precursors (sodium acetate, geranol, and mevalonic acid) were incorporated in the nutrient solution and the seedlings were cultured for 36 days. The culture solution was changed at 6-day intervals and the pH was adjusted to 6.5-7.5. Four concentrations of each precursor were tested according to an orthogonal experimental design (Table 3) with three replications. The mean values of the three replicates were analyzed. At the end of the culture period, the relative rate of change in biomass, total saponin content, and saponin production rate were measured.
Measurement parameters and methods
Determination of root number and length
Stem cuttings were induced to root in pure water, and the effect of hormones added to the culture medium on rooting was observed after culture for 10 days. Stem cuttings were gently removed from the culture medium, and water was drawn from the root surface using absorbent paper and placed on a glass plate to count the number of adventitious roots, followed by measuring the longest adventitious root length using a Vernier caliper.
Determination of plant fresh biomass
Changes in plant biomass were determined by weighing fresh samples. At the beginning of each experiment, plants with similar agronomic traits were sampled from each group and their fresh weights were measured; subsequently, after culture in the hydroponic solution with different treatments for 18 days, the plants were gently blotted dry with absorbent paper and the fresh weight of the plants was weighed. The relative rate of change in biomass was calculated using the formula: relative rate of change in biomass = [(biomass after treatment − biomass before treatment)/(biomass before treatment × number of days)].
Determination of photosynthesis pigment
To determine the contents of photosynthetic pigments, 0.2 g fresh leaf tissue was accurately weighed and placed in a 5 mL centrifuge tube, to which 5 mL of 95 % ethyl alcohol was added. The tube was sealed, incubated in a growth chamber at 25 ℃, and the content of photosynthetic pigments was determined after the leaves had become white. The absorbance of the supernatant was measured at 645 nm, 663 nm, and 470 nm to quantify the contents of chlorophyll a (Chl-a, Ca), chlorophyll b (Chl-b, Cb), and carotenoids (Cc), respectively, using the formulas Ca = 13.36A664 - 5.19A649, Cb = 27.43A649 - 8.12A664, and Cc = (1000A470 - 2.13Ca - 97.63Cb)/209 (SUMANTA et al., 2014).
Determination of total saponin content in leaves
The leaves were dried in liquid nitrogen and ground to powder, then 0.5 g powder was accurately weighed, placed in a 50 mL bottle, then dissolved by ultrasonication in an appropriate volume of absolute ethanol. The homogenate was filtered, the filtrate was dried in a water bath, resuspended in 10 mL water, then extracted four times with saturated n-butanol, the first extraction with 20 mL and the remaining three extractions with 10 mL, combined with n-butanol, then backwashed once with 10 mL distilled water, combined with n-butanol solution, evaporated, dissolved in methanol, transferred to a 10 mL measuring bottle, diluted to 10 mL volume, shaken well, and used as the test solution. The content of total saponins in each sample was determined by spectrophotometry, using ginsenoside Rb as the reference substance, at the wavelength of 550 nm (LU, 2007). The total saponin yield was calculated using the formula: Total saponin yield = net added fresh weight × saponin content. Saponin production rate = total saponin yield / days of culture.
Data analysis
All experiments were designed with three replications and the data were analyzed with the IBM SPSS Statistics (v25.0) software package. Significant differences between means were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) at the 5 % probability level (P < 0.05).
RESULTS
Relationship between number of nodes and rooting of cuttings
The single-node and two-node stem cuttings cultured in pure water differed in the rate that adventitious roots developed (Table 4). Roots developed more rapidly at the nodes than at the internode cut site. On the 5th day of the experiment, rooting was observed at the lower node of two-node stem cuttings, but no rooting was observed at the internode cut site. On the 10th day of the experiment, roots had begun to develop at the internode cut site (Figure 1C). Statistical analysis revealed that there was no significant difference in the number of roots nor in the average root length between single-node and two-node stem cuttings.
Effects of hormones on rooting of stem cuttings
The average number of roots on the stem cuttings was significantly different among the different hormone treatments (Table 5). For each hormone, the highest concentration (20 mg·L-1) inhibited root formation and the stimulation of rooting was greatest at 1 mg·L-1. The root-inducing effect of IAA was greatest among the hormone treatments at 1 mg·L-1. This finding was consistent with a previous report on the rooting effect of different hormones on the stem cuttings (WU, 2009). However, in that study, the concentration of IAA applied and the method of cutting treatment differed. In the present experiment, the hormone was added to the culture medium, whereas in Wu’s study the cutting base was immersed in the hormone solution for a finite period. The average number of roots per cutting in the 1 mg·L-1 IAA treatment was 8.3, which was significantly higher than that of the control. In addition, there was a significant increase in root length in the 1 mg·L-1 IAA treatment compared with that in the control.
Effects of nitrogen concentration on fresh biomass and photosynthetic pigment contents
Nitrogen is an essential element for maintaining a normal life cycle in plants. It has been reported that there is an optimal nitrogen concentration for Jiaogulan growth (ZHANG, 2005). The relative rate of change in biomass reflected the degree to which Jiaogulanwas affected by different nitrogen concentrations. The relative rate of change in biomass increased steadily with the increase in nitrogen concentration in the nutrient solution from N0 to N6 (Table 1). At the N0 concentration (0 mmol·L-1), the relative rate of change in biomass was the lowest; the biomass rapidly increased at concentrations from 1.0 mmol·L-1 to 7.0 mmol·L-1, and slowly increased from 7.0 mmol·L-1 to 12 mmol·L-1. Thus, the whole-plant biomass of Jiaogulan was greatest under the nitrogen concentration of 12 mmol·L-1.
Nitrogen is an important constituent of photosynthetic pigments and, accordingly, is strongly associated with photosynthesis (LIU et al., 2022). A deficiency of nitrogen will reduce the synthesis of chlorophyll and affect photosynthesis, resulting in lower yield. The contents of Chl-a, Chl-b, total Chl (Chl-a + Chl-b), and carotenoids showed the same trend under different nitrogen concentrations. For all treatments from 1 mmol·L-1 to 12 mmol·L-1, the Chl-a, Chl-b, total Chl, and carotenoids contents were higher than those of the control (0 mmol·L-1) (Table 1). This finding indicated that a lack of nitrogen directly led to difficulty in synthesizing photosynthetic pigments. When the nitrogen concentration increased from 1 mmol·L-1 to 7 mmol·L-1, the photosynthetic pigment contents rapidly increased. However, at nitrogen concentrations from 7 mmol·L-1 to 12 mmol·L-1, the photosynthetic pigment contents were only slightly increased. YODER & PETTIGREW-CROSBY, (1995) reported that there was a strong correlation between photosynthetic pigment content and nitrogen in plants. However, the present experiment showed that in the nitrogen concentration range of 7-12 mmol·L-1, the contents of photosynthetic pigments did not rapidly increase with the elevation in total nitrogen concentration, but maintained a consistent slight increase, which indicated that the nitrogen concentration of 7 mmol·L-1 represented an inflection point from rapid to slow increase in pigment content. Thus, an appropriate nitrogen concentration was required to enable full expression of the effect of nitrogen on photosynthetic organs. The photosynthetic pigment content of Jiaogulan in the 7.0-12 mmol·L-1 treatments group was higher than that in the 1-5 mmol·L-1 treatments group. These results indicated that nitrogen application promoted the synthesis of Chl-a, Chl-b, total Chl, and carotenoids in Jiaogulan.
Effects of NH 4 + /NO 3 - ratio on fresh biomass and photosynthetic pigment contents
Different NH4 +/NO3 - ratios significantly affected the increase in fresh weight per plant of JIAOGULAN (Table 2). The relative rate of change in biomass per day initially increased, but then decreased, with decrease in NH4 +/NO3 - ratio. The highest relative rate of change in biomass per day was observed in the T6 treatment. The relative rate of change in biomass among the treatments reached a significant level. The contents of Chl-a, Chl-b, total Chl, and carotenoids showed the same trend with different NH4 +/NO3 - ratios. The pigment contents in the T3 treatment were the lowest. The T6 and T7 treatments had the highest pigment contents. Compared with T7, the T6 treatment had a higher relative rate of change in biomass. However, there was no significant difference in all pigment contents between T6 and T7.
Effects of chemical momocs of saponin precursors on biomass and total saponin content
Intermediate compounds known to be precursors of saponins or chemical compounds that are helpful to increase the yield of saponins were added to the culture solution on the first day of culture. After culture for 36 days, the effects of these chemicals on the relative rate of change in biomass, and the content and yield of total saponins were determined. The experimental results are shown in table 6.
Visual analysis of the influence of chemical additives on biomass in the orthogonal experiment
An orthogonal experiment of L16(45) was designed. Among the four levels of the three chemical mimics of saponin precursors synthesis, the second treatment (A1B2C2) produced the optimal result, with an average relative rate of change in biomass of 23.4 %. The optimal levels of the three factors were A1, B2, and C2, namely, 0.5 mmol·L-1 sodium acetate, 0.5 mmol·L-1 geraniol, and 0.3 mmol·L-1 DL-mevalonolactone, respectively (Table 7). Visual analysis of the orthogonal experiment on the influence of Jiaogulan chemical additives on the relative rate of change in biomass showed that the primary and secondary factors order of influence on the relative rate of change in biomass of Jiaogulan was C > B > A (Table 7).
Visual analysis of the effect of G. pentaphyllum chemical additive on the growth rate of fresh weight by orthogonal experiment.
Visual analysis of the influence of chemical additives on the content of total saponins in the orthogonal experiment
An orthogonal experiment of L16(45) was designed. Among the four levels of the three chemical mimics of saponin precursors synthesis, the sixth treatment (A2B2C1) produced the optimal result, with an average total saponin content of 2.3132 %. The optimal levels of the three factors were A2, B2, and C1, namely 1.0 mmol·L-1 sodium acetate, 0.5 mmol·L-1 geraniol, and 0.1 mmol·L-1 DL-mevalonolactone (Table 8). Visual analysis and ANOVA for the orthogonal experiment on the influence of Jiaogulan chemical additives on total saponin content showed that the ANOVA results were consistent with the experimental results, and the primary and secondary factors order of influence on the total saponin content of Jiaogulan was B > C > A (Table 8).
Visual analysis of effects of three chemical additives on total saponins content in G. pentaphylosum by orthogonal test.
Visual analysis of the effect of chemical additives on the yield of total saponins in the orthogonal experiment
An orthogonal experiment of L16(45) was designed. Among the four levels of the three chemical mimics of saponin precursors synthesis, the sixth treatment (A2B2C1) produced the optimal result, with an average yield of total saponins of 0.5214 mg·d-1. The optimal levels of the three factors were A2, B2, and C1, namely, 1.0 mmol·L-1 sodium acetate, 0.5 mmol·L-1 geraniol, and 0.1 mmol·L-1 DL-mevalonolactone (Table 9). Visual analysis and ANOVA for the orthogonal experiment on the influence of Jiaogulan chemical additives on the yield of total saponins showed that the ANOVA results were consistent with the experimental results, and the primary and secondary factors order of influence on the yield of total saponins of Jiaogulan was A > C > B (Table 9).
Visual analysis of the effect of three chemical additives on the yield of total saponins in G.pentaphylosum by orthogonal test.
DISCUSSION
Jiaogulan has been used in both traditional and modern Chinese medicine over the past five centuries. More than 189 saponins have been isolated and identified from Jiaogulan (LI et al., 2016). Eight types of saponins are identical with saponins found in ginseng, namely, ginsenoside Rb1, Rb3, Rc, Rd, F2, Rg3, malonyl-RD, and Rf (XIANG, 2011). Ginsenoside Rb1 has a variety of pharmacological activities and is a chemical substance of significant medicinal value. This has led to the widespread use of Jiaogulan in place of the expensive ginseng. The rapid propagation of Jiaogulan is an important means to meet the production needs, which is currently achieved mainly through tissue culture regeneration and stem cuttings (KHAI & MINH, 2018; PHIP et al., 2021). Stem cuttings require loose soil generally and a suitable soil moisture content, which are beneficial to the survival of cuttings (GAO et al., 2018). The survival rate of stem cuttings is strongly associated with season, with 75.5 % survival in summer and 83.33 % survival in winter reported (PHIP et al., 2021). The present study showed that adventitious roots of Jiaogulan stem cuttings developed at the node within 5 days of culture in pure water. LIN et al. (2003) reported that adventitious roots of stem cuttings had developed at the stem node on the 10th day after stem cuttings were inserted in field soil. The present results showed that not only were the stem segments easier to root under a hydroponic environment, but the survival rate was 100 %.
As to whether rooting hormones influence the rooting of stem cuttings, WU et al. (2009) reported that the optimal concentration of NAA, IBA, and IAA was 400 mg·L-1 and the soaking time for stem cuttings was 45 min. The root number and root length were significantly increased in cuttings that survived in a soil matrix. ZHANG et al. (2017) reported that suitable concentrations of IAA and NAA can shorten the rooting time of cuttings and increase the percentage of rooted cuttings. In the present study, including a hormone in the nutrient solution had no effect on the rooting of cuttings, and its effect was mainly to increase the number of roots. However, the three hormones IAA, NAA, and IBA have different effects. All three hormones decreased the number of roots at a high concentration and the maximum number of roots developed at an appropriate moderate concentration. This finding indicated that the rooting of cuttings under hydroponic conditions requires a suitable concentration of a root-inducing hormone, and an excessively high concentration has an inhibitory effect on rooting. Among the three hormones, 1 mg·L-1 IAA was the optimal rooting treatment. The experimental results showed that addition of an appropriate hormone could increase the number of roots, which was consistent with the conclusion of ZHANG et al. (2017) that IAA was the most favorable hormone to promote rooting of Jiaogulan cuttings.
Regarding the relationship between the relative rate of change in biomass and nitrogen concentration, Jiaogulan plants were observed to grow rapidly in nutrient solution (Figure 1D), and the relative rate of change in biomass increased with the increase in nitrogen concentration. However, from 1 mmol·L-1 to 7 mmol·L-1 the increase in biomass was more rapid than that from 7 mmol·L-1 to 12 mmol·L-1. The present experiment showed that a nitrogen concentration of 7 mmol·L-1 represented an inflection point for the relative rate of change in biomass, so we suggest that the 7 mmol·L-1 concentration is suitable for the growth of Jiaogulan plants. ZHANG et al. (2005) studied the influence of nitrogen application mode on Jiaogulan growth in the field. These authors reported that the growth of the aboveground part of Jiaogulan was not promoted with increase in nitrogen concentration, but an optimal nitrogen application rate was observed. The present results suggested that the optimal nitrogen concentration for rapid growth of Jiaogulan was 7.0 mmol·L-1 under nutrient solution culture.
Under exposure to certain stress factors, plants will respond to adverse conditions through physiological and metabolic regulation, leading to changes in the content of specific substances. By studying the changes in these substances, we can clarify the response of plants to stress and adverse environmental factors (FANASCA et al., 2006). The Chl content is a measure of each plant’s photosynthesis and internal growth. WANI et al. (2016) reported that the application of nitrate nitrogen could significantly increase the Chl content during the development of lily. In the present experiment, the photosynthetic pigment contents showed a similar trend under different nitrogen concentrations, and increased with the increase in nitrogen concentration. These results indicated that nitrogen application promoted the synthesis of Chl-a, Chl-b, total Chl, and carotenoids in Jiaogulan. Under the present experimental system, the relative rate of change in biomass and photosynthetic pigment contents increased with the increase in nitrogen concentration, with the 7.0 mmol·L-1 concentration being an inflection point from rapid to slow increase in photosynthetic pigment contents. With continued increase in nitrogen concentration above the inflection point, the increases in relative rate of change in biomass and photosynthetic pigment contents slowed. Thus, Jiaogulan can be considered a high-nitrogen-tolerant plant and the optimum nitrogen concentration was 7.0 mmol·L-1. ADRIENN (2012) reported that the Chl content of maize showed the same trend of increasing with nitrogen concentration and there was an optimal concentration of nitrogen application.
Both ammonium and nitrate are important for the synthesis of photosynthetic pigments. Differences in the NH4 +/NO3 - ratio would lead to changes in the accumulation of certain intermediate components in the process of Chl synthesis, and the NH4 +/NO3- ratio that is optimal for growth differs among crops (GOU et al., 2020). In the present study, the relative rate of change in biomass, and Chl-a, Chl-b, total Chl, and carotenoid contents under different NH4 +/NO3 - ratios were highest when the NH4 +/NO3 - ratio was 2:3. A previous study has shown that the synthesis of 5-aminolevulinic acid is the rate-limiting step in the Chl biosynthesis pathway, and that 5-aminolevulinic acid is a direct precursor of Chl and tetrapyrrole ring formation, and thus is a crucial intermediate compound (LIANG et al., 2018). The content of 5-aminolevulinic acid is highest when the NH4 +/NO3 - ratio is 2:3 and lowest when the NH4 +/NO3 - ratio is 3:2 (WU et al., 2008). WANG et al. (2019) studied the growth status of Moso bamboo seedlings under different NH4 +/NO3 - ratios. The results showed that the root length and root surface area of Moso bamboo seedlings were optimal when the NH4 +/NO3 - ratio was 2:3, which is similar to the present results. We suggested that the optimal NH4 +/NO3 - ratio for relative rate of change in biomass and Chl synthesis of Jiaogulan is 2:3. 5-Aminolevulinic acid biosynthesis may be the rate-limiting step for chlorophyll synthesis of Jiaogulan.
Ginsenosides are an important bioactive constituent of Jiaogulan. Gynostemma pentaphylosides have a dammarane-type triterpenoidal skeleton, which is similar to the ginsenoside structure. Gynostemma pentaphyllum is the only plant species to date that has been found to contain ginsenoside compounds outside of the Araliaceae. Ginsenosides are mainly biosynthesized by the isoprenoid pathway through cyclization, hydroxylation, and glycosylation of 2,3-oxidized squalene (WANG et al., 2003; LIU & TANG, 2014). LU et al. (2001) reported that the addition of an elicitor to ginseng cell suspension cultures could stimulate saponin production. The basic precursor of saponins and sterols in Gynostemma pentaphyllum is acetyl-CoA, which is derived from glutaryl-CoA. In the process of plant secondary metabolite synthesis, an increase in the amount of precursors often promotes the combination of enzyme and precursor, thereby promoting product synthesis. The present production of secondary metabolites in many medicinal plants has been improved by precursor feeding (YU et al., 1999; WANG et al., 2003). Sodium acetate, geraniol, and mevalonic acid are important precursors for the synthesis of ginsenosides. The orthogonal experimental design method is used to establish a normalized table (or orthogonal table) to organize and simultaneously assess the effects of a number of influencing factors. It is a simple, convenient, time-efficient, and labor-saving method, and is widely used in agricultural research (SUN et al., 2021). The effects of the chemical mimics of saponin precursors on the content and yield of saponins under the optimum growth conditions were studied by means of an orthogonal experimental design. According to the orthogonal table of three factors and four levels, a total of 16 experimental groups were arranged (Table 5). Three optimal combinations were identified in relation to their influence on the relative rate of change in biomass, total saponin content, and saponin yield. The experimental group A2B2C1 had the greatest influence on the relative rate of change in biomass and total saponin content. The Chinese Pharmacopoeia stipulates that the total saponin content of medicinal Jiaogulan must not be less than 2.0 %. The total saponin content of the second experimental combination reached 2.3132 % in the present study (Table 8).
CONCLUSION
Our preliminary results indicated that Jiaogulan can be cultivated in a matrix-free nutrient solution. Plants for hydroponic culture can be generated from stem segments induced to form roots in pure water. It is feasible to induce rooting of two-node cuttings cultured in pure water for 10 days. For root induction from stem cuttings, 1 mg·L-1 IAA supplementation in pure water effectively increases the adventitious root length and number. The optimum nitrogen concentration in Hoagland’s nutrient solution is 7 mmol·L-1 and the optimum NH4 +/NO3 -ratio is 2:3 for hydroponic culture of rooted Jiaogulan stem cuttings. The content and yield of ginsenosides can be increased by supplementation with chemical mimics of saponin precursors in the nutrient solution. The optimal combination for saponin production is nutrient solution + 0.5 mmol·L-1 sodium acetate + 0.5 mmol L-1 geraniol + 0.3 mmol L-1 D-mevalonolactone.
ACKNOWLEDGEMENTS
The authors acknowledge the financial support of the Science and Technology Project of Henan Provincial Department of Science and Technology (No. 222102110282) . We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing a draft of this manuscript
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Edited by
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Editors
Leandro Souza da Silva (0000-0002-1636-6643)
