Micropropagation of Feverfew (Tanacetum parthenium) and Quantification of Parthenolide Content in Its Micropropagated and Conventionally Grown Plants

Feverfew (Tanacetum parthenium) is a well-known multi-functional plant with anti-inflammatory, cardiotonic, antiangiogenic, and anticancer effects. The therapeutic value of this plant is due to its phytochemical constitutes, especially parthenolide. Tissue culture techniques have been applied to improve the bioactive components of many herbal plants. Hence, this study, was carried out to establish a protocol for micropropagation of the feverfew plant and to quantify parthenolide content in its micropropagated and conventionally grown plants. To establish an aseptic culture, different concentrations of sodium hypochlorite (NaOCl) were investigated for seed surface sterilization. Besides, the effects of plant growth regulators (PGRs) on the callus induction, shoot organogenesis from callus and in vitro rooting were evaluated. Additionally, the parthenolide yield of the micropropagated and conventionally grown plants was determined by using high-performance liquid chromatography (HPLC). The results showed that surface sterilization of feverfew seeds with 6% NaOCl for 15 min obtained 65.00 ± 2.69% aseptic seeds. Murashige and Skoog (MS) medium supplemented with 0.4 mg/L thidiazuron (TDZ) and 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) resulted in 86.00 ± 1.72% callus induction. The highest number of shoots (5.00 ± 0.15) per explant was obtained in the treatment of MS medium supplemented with 5 mg/L zeatin. MS medium fortified with 3 mg/L indole-3-butyric acid (IBA) produced the maximum number of roots per plantlet (8.90 ± 0.35). A total of 90% of the micropropagated plantlets survived when planted in perlite + peat moss (1:1 v/v); the micropropagated plantlets were successfully established in the ex vitro conditions. According to parthenolide analysis, its level was significantly higher in the micropropagated plants than conventionally grown plants. Among different solvents, ethanolic extraction obtained the highest parthenolide content of the feverfew plant. Hence, it can be concluded that micropropagation of feverfew could be applied to produce disease-free planting materials and to improve the parthenolide content of the feverfew plant.

Sequential Sterilization of Banana (Musa Spp.) Sucker Tip Reducing Microbial Contamination With Highest Establishment Percentage

Sterilization procedure was standardized for grand naine cultivar of banana using various sterilants in combinations and alone. The observations were recorded regularly with respect to presence of fungal, bacteria as well as percentages of culture establishment. Results indicated that a treatment combination No. 3 [Lactic acid (0.15 %) + Tween-20 (0.1 %) + 0.8 % Commercial bleach (30 Min.) followed by Sodium chlorite (0.3 %) (20 min)] gave the highest percentage of aseptic culture establishment in vitro condition. The present study also showed that, single step aseptic inoculation was unable to control endophytic contaminants while sequential treatments were good enough to reduce microbial load as well as increase culture survival. Bangladesh J. Bot. 50(4): 1151-1158, 2021 (December)

The Efficient and Easy Micropropagation Protocol of Phyllanthus niruri

Phyllanthus niruri (P. niruri) or Dukung Anak is a herbal plant in the Phyllanthaceae family that has been used traditionally to treat various ailments such as diabetes, jaundice, flu and cough. P. niruri contains numerous medicinal benefits such as anti-tumor and anti-carcinogenic properties and a remedy for hepatitis B viral infection. Due to its beneficial properties, P. niruri is overharvested and wild plants become scarce. This study was conducted to develop an appropriate in vitro culture protocol for the mass production of P. niruri. An aseptic culture of P. niruri was established followed by multiplication of explants using different types of basal medium and its strength and plant growth regulators manipulation. This study also established the induction of in vitro rooting utilizing various types and concentrations of auxin. Treatment of Clorox® with 30% concentration showed the lowest percentage (%) of contamination, 4.44% in P. niruri culture. Nodal segments of P. niruri were successfully induced in full-strength of Murashige and Skoog (MS) basal media with 2.33 number of shoots, 3.11 cm length of shoot and 27.91 number of leaves. In addition, explants in full-strength MS media without any additional cytokinin were recorded as the optimum results for all parameters including the number of shoots (5.0 shoots), the length of shoots (3.68 cm) and the number of leaves (27.33 leaves). Treatment of 2.5 µM indole-3-butyric acid (IBA) showed the highest number of roots (17.92 roots) and root length (1.29 cm). Rooted explants were transferred for acclimatization, and the plantlet showed over 80% of survival rate. In conclusion, plantlets of P. niruri were successfully induced and multiplied via in vitro culture, which could be a step closer to its commercialization.

In vitro multiplication of wild Manihot species with different naphthaleneacetic acid and benzylaminopurine concentrations

In vitro multiplication is an important tissue culture technique that is capable of efficiently producing seedlings at any scale. It is a propagation method based on the aseptic culture of small propagules in a suitable culture medium to enable plant regeneration. Multiplication experiments conducted in vitro to set protocols adapted to wild Manihot species have used modified mineral salts and MS vitamins as basic culture medium. Here, 25 treatments based on combinations of the regulators benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) at 0, 0.025, 0.05, 0.075, and 0.1 mg L-1 were used for in vitro multiplication of three genotypes of wild Manihot species (M. violaceae Pohl Müll. Arg., M. pseudoglaziovii Pax & Hoff., and M. flabellifolia Pohl). Plant height and the number of 1 cm minicuttings, number of roots, shoots, green leaves and senescent leaves were recorded 120 days after explant inoculation. M. violaceae Pohl. Müll. Arg. and M. flabellifolia Pohl. presented favorable results with 0.05 and 0.025 mg L-1 NAA, respectively. Culture medium lacking NAA and BAP favored the in vitro growth of M. pseudoglaziovii Pax & Hoff.

Optimization Studies of Culture Media for In-Vitro Clonal Micropropagation of New Grape Varieties

This article describes the effect of Gautheret, White, Heller and Murashige & Skoog mineral salts during in-vitro clonal micropropagation of new grape varieties. The optimal mineral compositions of the culture medium that support the in-vitro regeneration of isolated grape explants were identified. The grapes that were studied were the Bart and Augustine varieties. Primary grape explants were cultivated for 30 days in a non-transplanted culture. Increased regenerative activity was observed in the Murashige & Skoog and White media. Increased haemogenesis occurred and shoots regenerated. The addition of cytokinin 6-BAP to the medium for obtaining aseptic culture led to an increase in the frequency of shoot-bud production by 5 to 6 times, depending on the type of medium. Combining 6-BAP with the auxin NAA provided an additional increase in the frequency of shoot-bud production, but to a lesser extent. Adding growth regulators to the culture medium also reduced the frequency of explant necrosis. Keywords: grapes, mineral salts, culture medium, microclonal propagation, in-vitro, cytokinins, auxins

Flowering and Seed Production across the Lemnaceae

Plants in the family Lemnaceae are aquatic monocots and the smallest, simplest, and fastest growing angiosperms. Their small size, the smallest family member is 0.5 mm and the largest is 2.0 cm, as well as their diverse morphologies make these plants ideal for laboratory studies. Their rapid growth rate is partially due to the family’s neotenous lifestyle, where instead of maturing and producing flowers, the plants remain in a juvenile state and continuously bud asexually. Maturation and flowering in the wild are rare in most family members. To promote further research on these unique plants, we have optimized laboratory flowering protocols for 3 of the 5 genera: Spirodela; Lemna; and Wolffia in the Lemnaceae. Duckweeds were widely used in the past for research on flowering, hormone and amino acid biosynthesis, the photosynthetic apparatus, and phytoremediation due to their aqueous lifestyle and ease of aseptic culture. There is a recent renaissance in interest in growing these plants as non-lignified biomass sources for fuel production, and as a resource-efficient complete protein source. The genome sequences of several Lemnaceae family members have become available, providing a foundation for genetic improvement of these plants as crops. The protocols for maximizing flowering described herein are based on screens testing daylength, a variety of media, supplementation with salicylic acid or ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA), as well as various culture vessels for effects on flowering of verified Lemnaceae strains available from the Rutgers Duckweed Stock Cooperative.

INDUCTION OF MICROTUBING OF NEW PROMISING POTATO VARIETIES IN ASEPTIC CULTURE

The studies were carried out with the aim of optimizing microtubing of promising potato varieties in aseptic culture. The experiments studied such factors as the use of vessels of various sizes (test tubes with a diameter of 25 mm and plastic containers 18x18 cm, into which 10 ml of agar and 400 ml of liquid nutrient medium, respectively, were poured, respectively), the density of planting plants in containers (40, 60 and 80 stem explants), the composition of the nutrient medium during ontogenesis, cultivation of mini-tubers from in vitro microplants (control) and microtubers (≥ 0.9 cm and 0.5 ... 0.9 cm in size). When studying the possibility of modifying the nutrient medium based on the Murashige-Skoog recipe to induce tuberization in one variant, the sucrose concentration during growth was changed from 2% before the formation of four internodes by 8% after this phase, the kinetin content during the entire observation period was 0.5 mg/l. In the second variant, microplants were kept on a medium with 6% sucrose and 0.25 mg/l kinetin throughout ontogenesis. The highest yield of microtubers of the standard fraction (27 ... 94%) with a multiplication factor of 0.8 ... 2.7 pcs/plant was noted in the variant with 60 cuttings placed in a container. When grown in test tubes with a change of medium, 1.0 ... 1.5 microtubers were collected per plant with a standard fraction yield of 64 ... 78%. The use of container technology with a similar alternation of nutrient media increased the yield of the standard fraction in most of the studied varieties to 75 ... 86%. In variants with a constant sucrose content in the nutrient medium (6%), a very low multiplication factor was noted, which did not compensate for a sufficiently high yield of the standard fraction, regardless of the laboratory vessel used. The multiplication factor of test tube microplants during planting in the ground was higher than when planting microtubers, with a high yield of the standard fraction

Plant secondary metabolites as bioactive substances for innovative biotechnologies

Plants are natural sources of bioactive compounds, and the intensive use of wild plants to obtain them, in particular secondary metabolites, depletes natural biocenoses. Instead, modern biotechnological methods, especially cell and tissue culture in vitro, make it possible to get environmentally friendly, highly productive plant raw materials that are able to synthesize and accumulate specialized substances, which are valuable for pharmacology, cosmetology, and medicine. Regenerating in vitro-plants of different plant species such as Acorus calamus L., Phalaenopsis sp. were obtained in our research. It was proved that by changing the cultivation conditions it is possible to change the content of substances of secondary metabolites in explants and in the nutrient medium under aseptic culture.