Caralluma tuberculata is used to accumulate biomass and secondary metabolites. Because of their ability to produce uniformly industrially relevant secondary metabolites, elicited plant in vitro cultures are becoming more popular.
The present study examined the effects of different amounts of silver nanoparticles and plant growth regulators (PGRs), on in vitro culture for sustainable production of biomass and antioxidant secondary metabolisms using callus cultures of Caralluma tuberculata. When combined with PGRs from the MS (Murashige or Skoog), results showed that different concentrations of AgNPs had a significant effect on callus proliferation and significantly increased callus biomass.
Cultures raised in vitro at 60 Ug/l AgNPs and 0.5 mg/l 2,4D plus 3.0 Mg/l BA showed the highest callus biomass accumulation (fresh (0.78 g/l), and dry (0.01 g/l). The callus cultures were examined phytochemically and showed higher levels of phenolics (TPC 3.0 mg), flavonoids (1.81 mg), phenylalanine ammonia lyase activity (5.8 U/mg), and antioxidant activity (90%), when they were grown on MS media with 90 ug/l AGNPs. The enhanced activities of antioxidant enzymes like superoxide dismutase, peroxidase, peroxidase and catalase (POD) were also found at higher levels (90 ug/l AgNPs). The AgNPs could be used to enhance bioactive antioxidants in C. tuberculata callus cultures. There are many applications for nanomaterials, including in medicine, biology, agriculture, and medicine.
Silver nanoparticles (AgNPs), among other types of nanomaterials have attracted a lot of attention due to their remarkable physiological properties and higher anti-microbial potency. AgNPs are also known for their ability to influence plant cell growth, biomass production, and the production of noteworthy secondary metabolites in cell cultures. The use of AgNPs in growth media alone or in combination with PGRs, such as a-naphthalene Acetic Acid (NAA), profoundly affected the callus growth in P.vulgaris. It also increased the antioxidants potential. Caralluma tuberculata is part of the Asclepediaceae family. It is a leafless, fleshy and important medicinal and edible plant. It has a angular stem that can grow up to 15cm.
The branches end with dark purple flowers. C. tuberculata can be called chung, boteri and marmut in different parts of the world. Caralluma tuberculata has a wide range of bioactive metabolites that have higher antioxidant potential. This plant is used to treat diabetes, inflammation, asthma and paralysis. It also helps with joint pains, fever, arthritis, obesity, paralysis, weight loss, and other conditions. However, in vitro tests against human carcinoma cells have shown positive results. C. tuberculata species have been drastically reduced in their natural habitats in different parts of the globe due to overexploitation and ruthless collection. C. tuberculata has been classified as a critically endangered species according to the red list categories of the International Union for the Conservation of Nature (IUCN). Two species of Caralluma in Pakistan, C. tuberculata (and C. edulis) are found wild in the hills of Sulaiman in the Khyber Pakhtunkhwa region. C. tuberculata seed viability is low, so the chances of germinating are very low. Stem cutting is the most time-consuming way to propagate the plant. Alternative methods are needed for the plant’s propagation, conservation and production of biomass. Plant cell cultures are a promising technology that allows for rapid growth and production of secondary metabolites in plants within a short time. Plant cell culture technology can also be used to preserve plants that are at risk of extinction or threatened in their natural habitats.
Callus culture, among the many methods of plant cell culture, is the most suitable for the production of plant material and bioactive phytochemicals. Further manipulation of callus can be done to in vitro regenerate whole plants. There is no literature that has shown the effects of AgNPs in callus biomass production and antioxidant production in C. tuberculata callus cultures. The current study was designed to assess the potential effects of AgNPs on the formation of callus biomass, induction of callus growth, and production of desired levels of metabolites in C. tuberculata, including phenolics, flavonoids, and flavonoids. In the callus cultures, the free radical scavenging and antioxidant enzyme activities were also measured.
Establishment of callus culture
tuberculata shoot explants were harvested from C. tuberculata growth room potted plantlets. Practically, 0.5 cm explants were inoculated (fresh apical tips) on Murashige  media. Initial supplementation with 0.5 mg/l 2,4–D + 3.0 mg/lBA was added. The MS media was also supplemented with 30 g/l sucrose (Oxoid) as a carbon source and 8 grams/l agar from Oxoid, England. The pH of the MS media containing PGRs was then adjusted to 5.7 (pH510, Eutech Instruments Karnataka Culture Singapore). The media-containing flasks were then placed in an autoclave, and sterilized at 120°C for 20 minutes (Systec Germany). The cultures were then placed in the growth chamber under 40-50 umol/m-2 s-1 light at 25°C. For callus culture development, the chamber photoperiod was 16/8 h.
AgNPs can be used to control callus proliferation and biomass production
After 49 days of culture, calli were taken from cultures that had been grown on MS media supplemented with 2,4-D (0.5%/l) + BA (3.0mg/l). This was done to evaluate the impact of AgNPs (and PGRs) on callus proliferation. The calli were cut using sterile forceps, surgical blade and sterile filter papers. They were then cultured in MS media containing AgNPs alone or mixed with PGRs. Sigma (Aldrich), catalogue number 730807, purchased AgNPs. AgNPs were characterized to reveal a size of 40 nanoparticles.
Varying concentrations (30-60, 90 and 90 mg/l respectively) of AgNPs were tested in combination with 2,4D (0.5 mg/l), + BA (3.0mg/l) in MS media. As a control, the MS media was supplemented with 2,4-D (0.5mg/l) + BA (3.0mg/l). In the culture of the MS media, sucrose (30g/l) + BA (3.0 mg/l) were used as a control treatment. The media was then solidified with agar (80.0 g/l). After setting the pH to 5.7, all media were autoclaved at 120 degrees C for 20 minutes. Micro filter was used to sterilize AgNPs solution. After filtration, AgNPs in different concentrations were added to autoclaved MS media. This was done under aseptic conditions. The autoclaved test tubes were then filled with approximately 9 +- 1ml medium and sealed with a cotton plug. All cultures were kept in the growth chamber at 25 +-1 degC with a 16/8 hour photoperiod. After 49 days of culture incubation, data were collected to assess the effects of different concentrations of AgNPs and PGRs on fresh (FW), dry (DW), callus moist content, and callus morphological characteristics. The calli from each treatment were washed in sterilized distilled waters to determine FW. Each callus was then placed on filter paper and pressed with forceps to extract any extra water. Finally, it was weighed. Each callus for DW was placed in an oven at 50 degrees Celsius for 24 hours before being finally weighed. The data for FW and DW were converted to g/l.