Effects of Plant Growth Regulators and Sugars on Ehretia asperula Zoll. et Mor. Cell Cultures

Plants are a crucial source of secondary metabolites with various applications. Research currently focuses on plant tissue and cell suspension cultures for the production of bioactive compounds when natural supply is limited or chemical synthesis is impossible (Gonçalves and Romano, 2018).
       
Xa den (Ehretia asperula Zoll. et Mor.) is widely distributed in some countries such as China, Vietnam, Myanmar and Thailand (Ly, 2016). In China, this species is usually planted at an altitude of 1000-1500 m. In Vietnam, Xa den grows in many provinces including Ha Nam, Quang Ninh, Ninh Binh, Hoa Binh, Cuc Phuong National Park, Ba Vi National Park, Thua Thien - Hue, Gia Lai and many others. The leaves have been used in traditional Vietnamese medicine to support the treatment of ulcers, tumors and inflammation (Ly, 2006). The EtOH extraction from Xa den stems showed potent cytotoxicity against hepatoma, nasopharynx carcinoma and anti-HIV replication activity in H9 lymphocyte cells (Kuo and Kuo, 1997).
       
To preserve these precious plants and produce raw materials for the medicinal industry, Xa den has been studied to multiply by micropropagation (Tien and Minh, 2015; Tuan et al., 2016) or cell cultures from leaf in vitro (Hong and Minh, 2019). However, the research on Xa den cell cultures is limited. This study investigated the effects of 2,4-D and BA concentrations along with sugars on callus induction from young stem explants of Ehretia asperula Zoll. et Mor. to create materials for further experiments.

Two year old Xa den (Ehretia asperula Zoll. et Mor.) plants blossomed in the garden, provided by the Northern Nursery, Dong Hoi village, Dai Dinh commune, Tam Dao district, Vinh Phuc province. Samples taken from young Xa den stems were less than two years old. Young stems with 2 to 6 leaves functioned as explants for callus induction. The stems were washed thoroughly under running tap water for 30 minutes followed by treatment with 70% alcohol for 1 minute and 20% bleach for 10 minutes. Then, they were rinsed in sterile distilled water about 4-5 times and cut into thin slices of approximently 1.0 mm. Explants placed on a B5 nutrient medium supplemented with 7.5 g/L of agar, 0.5 g/L of activated carbon, plant growth regulators and sugars were incubated at 25^oC, in the dark.
       
The study was carried out during the time period of June 2018 to December 2019, in the Department of Biotechnology, Ho Chi Minh University of Technology and Experimental Center of Thu Dau Mot University, Vietnam.
 
Effect of plant growth regulators on callus induction
 
Effects of 2,4-D: Explants were cultured on a B5 medium supplemented with 2,4-D in the amounts of 0, 0.4, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L; 0.1 mg/L of BA; and 30 g/L of sucrose.
       
Effects of BA: Explants were cultured on a B5 medium supplemented with BA in the amounts of 0.1, 0.2, 0.3, 0.4 and 0.5 mg/L; 2,4-D; 2,4-D with a suitable concentration in the above experiment; and 30 g/L of sucrose.
 
Effects of sugars
 
Explants were cultured on a B5 medium supplemented with BA and 2,4-D (suitable concentration in the previous experiment) and sugars: fructose, glucose and sucrose with three different concentrations of 20, 30 and 40 g/L.
 
The growth of callus of Ehretia asperula Zoll. et Mor.
 
Explants were cultured on a B5 medium supplemented with BA; 2,4-D; and sugar (suitable concentration in the previous experiment). The callus growth was determined by fresh weight after every week until the callus reached the death phase.
 
Cell suspension cultures
 
Two grams of callus was transferred into 250 mL flasks filled with 60 mL of B5 liquid medium. Suspension cultures were maintained at 25°C on a gyratory shaker at 150 rev/min in the dark.
 
Callus growth evaluation
 
Callus induction rate, morphology and fresh weight of callus was observed and recorded.
       
The determination ratio of friable callus induction after 4 weeks (Sahraroo et al., 2014) :
 
X: ratio of friable callus induction (%).
a: The number of explants for creating friable callus.
A: The number of explants.
 
Statistical analysis of the data
 
The experiments were randomly designed with 3 replicates. The data presented the average of the three replicates expressed as Mean ± SD. The statistical analysis of the data was evaluated by using Statgraphics Centurion XV at a level of 5% significance.

Effect of plant growth regulators on callus induction from the young stem explants of Ehretia asperula Zoll. et Mor.
 
Plant growth regulators were required to induce callus and to promote the growth of many cell lines. As an auxin, typically 2,4-D or naphthaleneacetic acid (NAA) is often used. Kinetin or BA as a cytokinin is occasionally required together with auxins for callus induction. Since each plant species requires different kinds and levels of phytohormones for callus formation, it is necessary to select the most suitable plant growth regulators to find out their optimal concentrations (Vanisree and Tsay, 2004).
 
Effects of 2,4-D
 
The investigation for the effect of 2,4-D concentrations with BA in a B5 medium was performed to find out the optimal combination of plant growth regulators for callus formation and making cell suspension material. After one week, the explants began to increase in size and form a small callus. After four weeks, callus was friable. The addition of 0.4 mg/L and 1.0 mg/L of 2,4-D resulted in statistically significant differences compared to the others (Table 1). Inside, a treated group with complex of 0.4 mg/L of 2,4-D and 0.1 mg/L of BA, friable callus was formed (85%) and the highest fresh weight was 0.048 g. When the 2,4-D concentration was increased, callus formation and fresh weight reduced (Fig 1). The exposure to high levels of auxins resulted in suppressed morphogenic activity and rapid proliferation of cells (Kumar and Jakhar, 2018).
 

 

       
Auxins normally encourage intensification of fragility and a decrease in cell differentiation. The friability tendency of the cells to round off and split immediately after division promoting a break from cell-cell contacts, which could contribute to differentiation mediated by nearby cells (Reis et al., 2018).  2,4-D is mostly used for callus induction (Michel et al., 2008), but it can also act as herbicide inhibiting the growth process by hindering detoxification of explants which results in preventing normal nucleic acid metabolism and protein synthesis (Behbahani et al., 2011).
       
The same result was also observed by Phua et al., (2016) where the percentage of callus formation in Clinacanthus nutans decreased because all concentrations of 2,4-D levels were above 1.0 mg/L. According to the authors, calluses successfully formed directly from leaf explants placed in MS media supplemented with 2,4-D concentrations below 1.0 mg/L were found to produce the highest callus formation. Similarly in 2019, Hong and Minh studied the effects of phytohormones on callus initiation from Ehretia asperula Zoll. et Mor. leaf explants in vitro. For 3.0 mg/L of 2,4-D, there was no callus induction but, in contradiction Guruprasad et al., (2016) found that 2,4-D (4.0 mg/L) alone reported to give the best result of callus formation at 90% from the mature and immature embryos of Zea mays L. In Arachis prostrate, the rate of callus formation from folded healthy immature leaves was higher in the Picloram supplemented treatments compared to NAA and 2,4-D. Picloram, from 0.5 - 5.0 mg/L, combined with 1.5 mg/L BA produced the highest callogenesis ranging from 99.7-100.0% (Bera and Gedia, 2014). Therefore, callus formation depends on the interaction of many factors like genotype, type of explants, environmental compositions, growth regulators, etc.
 
Effects of BA
 
The results of research on the effects of BA concentrations in combination with 2,4-D are presented in Table 2 and Fig 2. This study showed callus formed in a B5 medium with a 100% rate of callus induction. The treated group with 0.1 mg/L of BA and 0.4 mg/L of 2,4-D had the highest rate of friable callus induction at 84% weighing 0.055 g; the light yellow callus was friable. The treatment containing 0.5 mg/L of BA and 0.4 mg/L of 2,4-D had the lowest results with a rate of friable callus induction at 34% weighing 0.037 g.
 

 

       
In some cases, cytokinin is added to the culture medium to coordinate with auxin and stimulate the callus proliferation. Cytokinin is essential for cell division because without it, the metaphase during cell cycle elongates since protein biosynthesis gets interrupted (Luong and Tien, 2006). Auxin and cytokinin are often combined to form callus (Benítez-García et al., 2014). Similarly from Curcuma caesia leaves, the highest percentage of callus induction (66.70%) was obtained when 2, 4-D (0.5 mg/L) combined with BAP (0.1 mg/L) was used. No callus formed in the single concentration of 2, 4-D (Abubakar and Pudake, 2019).
 
Effects of sugars on callus induction from the young stem explants of Ehretia asperula Zoll. et Mor.
 
Carbon is one of the key factors as an energy source in the nutrient medium. Most plant tissue and cell cultures in vitro cannot synthesize organic substances effectively due to incomplete cellular and tissue development, lack of chlorophyll, limited gas exchange, etc. causing a lack in  auxotrophic ability which forces the  need  to add an external energy source (Vaezi Kakhki, 2008).
 
The variance analysis results related to the effects of different sugar types on callus formation were shown in Table 3. The callus fresh weights were high in all treatments obtaining glucose. The treatment   containing 30 mg/L of glucose had the highest weight of 0.165 g, in contrast to the treatment containing 20 g/L of fructose with the lowest weight of 0.015 g, at the induction rate of 26%. These results showed that the treated group with glucose would induce big light yellow friable callus compared to other sugars (Fig 3 and 4). The sterilized medium with fructose the explants to loosen and possibly sink into the agar. Since the autoclaved fructose was toxic, it could also inhibit callus formation (Vaezi Kakhki, 2008).
 

 

       
The benefits of glucose on callogenesis have also been studied in many plants. In 2006, Alina et al. investigated the effect of carbon sources like sucrose, fructose, glucose, mannose or sorbitol, which were in Pharbitis nil. This survey showed an autoclaved glucose addition instead of sucrose to the medium stimulating callus induction on flower buds and cotyledonary explants. According to the authors, the way of sterilizing the sugars was crucial to callus regeneration, particularly in the case of fructose. The stimulating effect of fructose was confirmed only when the filter-sterilization stock was used.
 
The growth of callus of Ehretia asperula Zoll. et Mor.
 
After one week of culturing, thin sections from the young stems of Ehretia asperula Zoll. et Mor. began to increase in size and small callus appeared around the explants. In the second week, the callus formed throughout the explants and continued to increase in size throughout the following weeks. However, at the beginning of week 6, callus began to turn brown. By the end of the sixth week, the callus fresh weight started to decrease. Based on the growth curve, callus should be subcultured at the fourth week or the first days during the fifth week (Fig 4 and 5).
 

 
Initially Ehretia asperula Zoll. et Mor. cell suspension cultures
 
Friable callus was placed in a B5 liquid medium supplemented with 30 g/L of glucose; 0.4 mg/L of 2, 4-D; and 0.1 mg/L of BA at a shake speed of 150 rpm. Cells slowly separated from callus into liquid medium. After culturing for 7 days, cell suspension formed, consisting of single cells and small cell clumps. After culturing for 14 days, cell suspension had turned from dark yellow to light brown (Fig 6). This may be due to the cells containing many phenolic compounds making the color gradually darken as the culture period is prolonged. Follow-up studies should focus on determining the suspension cell biomass after each week of culturing or at shorter intervals of 2 or 4 days to figure out the appropriate time to sub-culture cell suspension.
 

This study showed that the B5 medium supplemented with 0.4 mg/L of 2,4-D; 0.1 mg/L of BA; and 30 g/L of glucose is the most suitable option for callus induction from the young stems of Ehretia asperula Zoll. et Mor.
       
The friable callus was put into a liquid medium with the same composition as the callus culture medium to form cell suspension. After two culture weeks, the cell suspension cultures included single cells and small cell clumps with dark yellow to light brown color.

We are grateful to Thu Dau Mot University, Binh Duong and Ho Chi Minh City University of Technology, Viet Nam National University for providing the location and facilities to carry out the experiments. This research is funded by Thu Dau Mot University under grant number ĐT.20-006.