The Effects of 2,4-Dichlorophenoxyacetic Acid and α-Naphthaleneacetic Acid on Biomass Increment, Rhizogenesis and Somatic Embryogenesis of Suspension-cultured Dinh Lang Cells [Polyscias fruticosa (L.) Harms]

Dinh Lang [Polyscias fruticosa (L.) Harms, Araliaceae] is a dicot evergreen shrub and distributes in Southeast Asian countries, including Vietnam. The secondary metabolites in the leaves and roots of this plant (mainly polyacetylenes and saponins) have note-worthy health benefits such as reducing inflammation, neutralizing toxins, anti-bacterial effects, promoting diuresis, reducing fever and anti-dysentery effects. Thus, Dinh Lang has long been utilized as a tonic in Asian countries (Huan et al., 1998).
       
However, the amounts of triterpenoid saponins in Dinh Lang are not sufficient for meeting the demand of the pharmaceutical industry. Therefore, the cell culture of [P. fruticosa (L.) Harms] could be used as an effective method of increasing biomass to produce large amounts of secondary metabolites without depending on the traditionally cultivated plants (Ahn et al., 1996).
       
Despite studies on cell suspension culture and somatic embryogenesis of several species in the Araliaceae family, including Panax ginseng (Ahn et al., 1996; Arya et al., 1993; Chang and Hsing, 1980) and Acanthopanax koreanum Nakai (Choi et al., 1997), there has been no published paper about producing somatic embryos, rooted cells and large biomass of Dinh Lang by using cell suspension culture. 
       
Considering the lack of studies on Dinh Lang cell suspension culture, in this article, two auxins (2,4-D and NAA) were tested to initially determine the most suitable auxin and its optimal concentration for biomass raising, in vitro rhizogenesis and somatic embryogenesis of suspension-cultured Dinh Lang cells. By this means, a large source of Dinh Lang somatic embryos, roots and cell biomass could be created for bioactive compounds extraction and micropropagation.

This study (8/2019-4/2020) was conducted in the Department of Biology, Hue University of Sciences, 77 Nguyen Hue St., Vietnam.
       
The plant materials of the study were primary calli induced from the lower parts of Dinh Lang [P. fruticosa (L.) Harms] leaves. To obtain the calli, the leaves had beencultured on MS media (Murashige and Skoog, 1962) given 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) or 1.0 mg/L α-naphthaleneacetic acid (NAA) for 15 days before the beginning of  this study.
       
To find the most suitable amount of time for callus culture, firm, light-yellow primary calli were aseptically separated into 2-to-3-millimeter piles, sub-cultured on solid MS media supplemented with 1.0 mg/L NAA or 1.0 mg/L 2,4-D in different culture durations (2, 4, 6, 8 and 10 weeks). The optimal culture duration would then be used to create the source of cells for suspension culture.
       
To identify the optimal auxin concentrations for Dinh Lang in vitro biomass increment, rhizogenesis and somatic embryogenesis, two grams of 8-week-old calli were cultured in a 250 mL Erlenmeyer flask containing 50 mL of liquid MS medium given 30 g/L sucrose and different concentrations of NAA or 2,4-D (0.5, 1.0, 1.5 and 2.0 mg/L). All the flasks were shaken at 120 rpm. The best auxin and its optimal concentration for suspension culture of Dinh Lang cells were determined by the following factors:
· The release and clusterization of cultured cells and the numbers of somatic embryos and rooted cells.
· The fresh biomass showing the growth level of the cells.
       
The release and clusterization of cells in the flasks were observed after 4, 8 and 12 weeks of culture. After 12 weeks, all suspension cultures were poured out and vacuum filtered. The cultured cells were then washed on the vacuum filter by using double distilled water. Finally, the fresh weight of the cells and numbers of somatic embryos and rooted cells per flask were determined.  
       
All cultures were incubated at the temperature of 25 ± 2^oC and the photon flux density of 68 μmol/m²s. Liquid media (pH 5.8) were autoclaved at 121^oC, 1 atm in 30 minutes and poured into 250 mL Erlenmeyer flasks capped by two nylon sheets, with 50 mL each. Solid media (pH 5.8) were sterilized as mentioned above and distributed into 100 mL glass bottles covered by two nylon sheets. Each glass bottle has 20 mL of solid medium.
       
The means in each experiment were analyzed by ANOVA and compared by Duncan’s test  (Duncan, 1955) with an alpha of 0.05. All experiments were done in triplicates so that the sample size of each treatment was 30.

Optimizing callus culture duration
 
After being cultured for two weeks on MS medium supplemented with 1.0 mg/L 2,4-D, the calli did not show any sign of strong growth. Calli’s size commenced to increase fast in the fourth week of culture and continued to the eighth week. In this week, Dinh Lang calli (firm and light yellow) reached their maximum sizes. The brown color and death of calli were observed in the tenth week of culture (Table 1, Fig 1).
 

 

       
In contrast to calli cultured on MS medium containing 1.0 mg/L 2,4-D, the ones stimulated by 1.0 mg/L NAA began to grow fast from the sixth week. In the eighth week, calli with the largest sizes were observed. However, the growth level of calli cultured on the medium with 1.0 mg/L NAA was lower than on the medium with 1.0 mg/L 2,4-D. The brown areas indicating dead cells also appeared in the tenth week of culture (Table 2, Fig 2).
 

 

       
In our study, Ding Lang calli began to grow in the second week of culture. This result is in line with the work of Bhat et al., (2020), claiming that Solanum melongena calli were observed after 15 days. However, the time needed for callus to start growing could be shorter or longer than two weeks depending on the cultured species. Specifically, while Curcuma caesia calli were observed after five weeks of culture, it only took 7-8 days for Cajanus cajan’s calli to be induced from epicotyl explants (Abubakar and Pudake, 2019; Padmavathi et al., 2017).
       
The results in Table 1 and Table 2 prove that 2,4-D ismore suitable than NAA for Dinh Lang callus cell multiplication. 2,4-D was also considered the most efficient plant growth regulator for callus culture (Fonseka and Aluthgamage, 2020). This might explain why 2,4-D is often used for callus induction of species in the Araliaceae family. Specifically, Chang and Hsing (1980) used 1.0 mg/L 2,4-D to induce Panax ginseng C.A. Meyer calli. Moreover, Kim et al., (2019) reported that 2.0 mg/L is the optimal 2,4-D concentration for callus induction of interspecific ginseng hybrid between P. ginseng and P. quinquefolius, with a 100% induction rate.
       
There are also agreements among the results in Table 1, Table 2 and the ones in the study of other authors. Particularly, Thach et al., (2016) demonstrated that NAA could not stimulate the highest growth level of Dinh Lang calli (++++, profuse callus formation). Moreover, 2,4-D was also proven to be more efficient than NAA at enhancing callus induction and growth of other species, including Centella asiatica L. Urban, with the maximum induction rate of 83.33% (Tan et al., 2010).
 
Effect of 2,4-D on callus suspension culture
 
As is shown in Table 3, 1.5 mg/L is the optimal 2,4-D concentration for callus suspension culture. In particular, the released callus cells strongly grew in the 8 week before forming large and dense clusters containing a high number of somatic embryos and several rooted cells in the 12 week.
       
Table 4 and Fig 3 present more clearly the positive effect of 1.5 mg/L 2,4-D. Specifically, calli cultured in basal MS medium could not survive and then became a thin layer of dead cells in the 12 week of culture. There were insignificant increases of callus biomass cultured in MS media given 0.5 mg/L and 1.0 mg/L 2,4-D. Additionally, no somatic embryos and rooted cells were observed in those media. However, calli in MS medium supplemented with 1.5 mg/L 2,4-D formed 13 somatic embryos per flask, several rooted cells (1.2 roots/flask) and the heaviest biomass of 5.7 g in the 12 week of culture.
 

 

 

 
Effect of NAA on callus suspension culture
 
Of all the tested media, MS medium with 1.5 mg/L NAA could produce the highest quantities of somatic embryos and adventitious roots. Particularly, the number of somatic embryos began to surge in the 8 week. Moreover, long and thick adventitious roots, which formed large root clusters, were obtained in the 12 week of culture (Table 5).
 
 
 
The stimulatory impacts of 1.5 mg/L NAA on rhizogenesis and somatic embryogenesis are specifically clarified in Table 6 and Fig 4. In particular, there was noembryo and adventitious root in the medium given 0.5 mg/L NAA. Additionally, low numbers of 5.5 somatic embryos and 1.0 rooted cells were obtained from MS medium added with 1.0 mg/L NAA. However, these two values were rocketed by 1.5 mg/L NAA, with 489 embryos/flask and 259.5 roots/flask.
 

 

 
2,4-D is widely used for somatic embryogenesis and proven in studies on several species to possess a higher capacity of stimulating somatic embryo formation than NAA (Arya et al., 1993; Choi et al., 1997; Luo et al., 1999). However, in the case of Dinh Lang, the results in our study demonstrate that NAA is better than 2,4-D at stimulating somaticembryogenesis, with the number of NAA-induced embryos being approximately 38 times higher than that of 2,4-D-induced ones. This finding is similar to that observed by several authors such as Ozcan et al., (1993), Ozias et al., (1989) and Yang et al., (2018). Specifically, Ozcan et al., (1993) claimed that the number of Pisum sativum L. somatic embryos stimulated by NAA was higher than that in the medium added with 2,4-D. Ozias et al., (1989) concluded that the percentage of Arachis hypogaea L. nodular outgrowths in the medium given 20 mg/L NAA (72%) was larger than in the medium supplemented with 4 mg/L 2,4-D (24%). Yang et al., (2018) demonstrated that NAA at the concentration of 1.0 mg/L could stimulate a higher number of Koelreuteria paniculata Laxm. somatic embryos (54.75% induction rate) than all tested 2,4-D concentrations.
       
In addition to the great positive effect on somatic embryogenesis, Table 4 and Table 6 also clarified that NAA is more suitable than 2,4-D for inducing adventitious roots from suspension-cultured cells. Several studies also mentioned this observation. In particular, Ozcan et al., (1993) did not found Pisum sativum adventitious roots in the medium containing 2,4-D. Moreover, Sofiari et al., (1997) reported that there was a shift (created by NAA) from Manihot esculenta’s secondary somatic embryos to adventitious roots in several cultivars (“Gading”, “MCol1505”, “Line 11”, “TMS90853” and “Adira 1”), while 2,4-D was not able to induce rooted cells. Additionally, Geneve and Kester (1990) proved that 100 mM NAA could stimulate an optimal number of 30.5 Cercis canadensis’s rooted cells, which could not be obtained using 2,4-D. Finally, Lazzeri et al., (1987) claimed that Glycine max adventitious roots were only formed in media containing NAA.

The most strongly growing calli were formed in solid MS medium given 1 mg/L 2,4-D after eight weeks of culture. Therefore, these are the best culture duration and growth stimulant for callus multiplication of Dinh Lang.
               
While the heaviest biomass (5.7 g) was obtained in liquid MS medium added with 1.5 mg/L 2,4-D, 1.5 mg/L NAA is more suitable than 2,4-D for cell’s rhizogenesis and somatic embryogenesis, with 489 embryos/flask and 259.5 cells/flask, respectively.