Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica

Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g−1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.

An Alternative to Increase Accumulation of Phenolic Compound in Grapevine Callus Cultures: Chemical Mutagen Applications

This research was carried out to investigate the effects of chemical mutagen applications on producing phenolic compounds in calli obtained from leaf petiole explants of the Royal grape variety.Petioles were cultured in B5 medium containing 0.5 mg/L benzyl amino purine and 0.5 mg/L indole acetic acid to obtain callus. Calli obtained during the period following planting were subcultured in the same culture conditions three times at one month intervals. In the fourth subculture, the calli were transferred to nutrient mediums with the same composition containing ethyl methanesulfonate, sodium azide, azacitidine and acridine orange in three different doses, 0.5 mM, 1.0 mM, and 2.0 mM, and cultured in these mediums for 60, 120 and 180 minutes. Then, they were cultured again for four weeks in mutagen-free nutrient mediums with the same content as their previous mediums. At the end of this period, calli were taken from their nutrient medium, and total phenolic compound, total flavanol, total flavonol and anthocyanin analyzes were performed.As a result of the research, it has been determined that all mutagens applied are effective in increasing the production of phenolic compounds depending on the dose and time.

Green Globular Body (GGB) Induction and Differentiation in the Medicinal fern Drynaria Roosii

Background: The green globular body (GGB) of ferns is a special propagule induced in plant in vitro culture systems. Owing to its high proliferation efficiency, GGB is widely used in the in vitro propagation of important ornamental and medicinal ferns. In addition, propagation using GGB shows great development prospects in the conservation of rare or endangered ferns and the breeding of new fern varieties. However, due to the lack of systematic studies on GGB ontogenesis, the morphogenetic aspects of GGB during induction and differentiation remain unclear.Results: We characterized the response of five types of explants of Drynaria roosii to GGB inductive medium and further investigate morphological and anatomical changes of explants that developed GGBs. We found that the rhizome explants directly produced GGBs through cell proliferation of the shoot apical meristem and lateral meristem. The leaf and petiole explants produced GGBs indirectly through the proliferation of meristematic cells of somatic embryos derived from the epidermal cells of the explants. The root and gametophyte explants failed to produce GGB under our induction conditions. We further investigated the differentiation process of GGB. During GGB differentiation, shoot primordia and leaf primordia differentiate from meristematic cells on the epidermis, and the root primordia develop from an inner meristematic tissue with developing vascular tissue connecting all these primordia, which indicates the involvement of multiple organogenesis processes.Conclusions: Our results suggested that preexisting or reestablished meristematic cells were the direct source of GGB in D. roosii. Somatic embryogenesis and organogenesis were involved in GGB induction and differentiation, respectively. The comparison with other common propagules revealed that GGB in D. roosii was largely different from somatic embryos, callus, and protocorm or protocorm-like bodies.

Micropropagation of Pelargonium odoratissimum (L.) L’Her. through petioles and leaves

Pelargonium odoratissimum (L.) L’Her is a hard rooting plant and the common methods of propagation via stem cuttings are not successful with this species. therefore, tissue culture methods have been experienced for the mass-propagation of this high-valued species. Intact leaves, leaf segments and petiole sections derived from nodal explants in vitro were employed for the optimization of P. odoratissimum micropropagation. The treatment combinations used were MS and 1/2 MS media supplemented with 6-benzylaminopurine, BAP (1, 1.5, 2 and 4.5 mg.L-1) and 1-naphthaleneacetic acid, NAA (0.1, 1 and 1.5 mg.L-1). With leaf segments, the lowest browning incidence, the greatest callogenesis and the highest number of shoots were obtained with the media containing 1.5 mg.L-1 BAP and 1 mg.L-1 NAA. Two mg.L-1 BAP + 0.1 mg.L-1 NAA kept the same results for petiole explants. Intact leaves showed the best results for the three mentioned treatments with 1 mg.L-1 BAP + 1 mg.L-1 NAA. 0.2 mg.L-1 NAA caused the highest rooting percentage and the greatest mean data for the number and length of the roots. Rooted plantlets were transferred to the pots containing 1:1 peat-moss and perlite. Acclimatization of the plantlets was followed by 90 % of survival rate in the greenhouse. The protocol employed would be a potent one to present for the extension section.

Regeneration and Genetic Transformation of Arabidopsis Pumila: An Ephemeral Plant Suitable for Investigating the Mechanisms for Adaptation to Desert Environments

Arabidopsis pumila is a type of cruciferous ephemeral plant, which in China mainly grows in the desert environments of northern Xinjiang. A. pumila not only has a short growth duration, but also has high photosynthetic efficiency, seed yield, salt tolerance, and drought resistance. It is an ideal species for the study of environmental adaptations in ephemeral plants. We induced callus tissue formation on the roots and hypocotyls of 8-day-old seedlings, and on the leaves and petioles of 4-week-old seedlings, and obtained multiple adventitious shoots on these tissues grown on Murashige and Skoog induction medium supplemented with 0.5 mg/L 6-Benzylaminopurine and 0.1 mg/L α-Naphthalene acetic acid. Young roots, hypocotyls, leaves, and petioles could all induce calluses, but the induction rate was highest on young roots. In addition, the leaves and petioles of 4-week-old seedlings were used as explants, the Δ1-pyrroline-5-carboxylic acid synthase gene 1 of A. pumila controlled by 35S promoter of cauliflower mosaic virus was used as target gene, and hygromycin B was used as screening antibiotic to explore Agrobacterium tumefaciens GV3101 mediated transformation. The results showed that the callus induction rate of petiole explants was the highest when they were treated with Agrobacterium suspension (OD600 = 0.6) for 10 min and thenco-cultured in dark for 2 d. The qRT-PCR results showed that the ApP5CS1.1 gene was overexpressed in the transgenic plants. These protocols provide working research methods for exploring the cellular level adaptative mechanisms of this species to desert environments.

Effect of Benzyl Adenine Concentration on Callus Induction of Geranium Plants (Pelargonium graveolens L'Her) from Petiole and Leaf Explants

Geranium plant (Pelargonium graveolens L'Her) is one of the geranium oil producing plants that has many benefits. Callus culture is a technique that can be used to plant multiplication and increase production of secondary metabolites. This study aims to determine the effect of the concentration of Benzyl Adenine on the formation of geranium callus from petiole and leaf explants. Callus induction was carried out by culturing petiole and leaf explants on MS medium + 0.1 mg.L-1 NAA + Benzyl Adenine (0; 0.5; 1; 1.5 and 2 mg.L-1). Callus morphological parameters, percentage of callus formation, and time of first callus formation were observed. The formation of geranium callus influenced by the explant type and the concentration of Benzyl Adenine. In the 2nd week, the geranium callus was initiated, light green colored with a compact callus texture. At 4th week, the percentage of callus formation containing NAA 0.1 mg.L-1 of petiole and leaf explants was 20% and 8%, whereas the percentage of callus formation on medium containing 0.1 mg.L-1 NAA combined with 0.5-2 mg.L-1 Benzyl Adenine of petiole and leaf explants was 52-80% and 24-52%. The best percentage of callus formation was found on the culture medium containing 1 mg.L-1 Benzyl Adenine, equaled 80% of petiole explants, and 52% of leaf explants.

Effect of Plant Growth Regulators on Coloured Callus Formation and Accumulation of Azadirachtin, an Essential Biopesticide in Azadirachta indica

For centuries, Azadirachta indica or neem has been utilized as a primary source of medicine due to its antimicrobial, larvacidal, antimalarial and antifungal properties. Recently, its potential as an effective biopesticide has garnered attention, especially towards efficient and continuous production of its bioactive compounds. The present study investigated the effect of the plant growth regulators (PGRs) thiadiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of colored callus formation and subsequent accumulation of azadirachtin (AZA) in A. indica. An efficient protocol was established for micropropagation and colored callus production of this species, followed by quantification of AZA (a mixture of azadirachtin A and B) and its safety assessment. For induction of the callus, leaf and petiole explants obtained from a young growing neem plant were excised and cultured on Murashige and Skoog (MS) medium supplemented with TDZ (0.2–0.6 mg L−1) and 2,4-D (0.2–0.6 mg L−1), either applied singly or in combination. Callus was successfully induced from both explant types at different rates, where media with 0.6 mg L−1 of TDZ resulted in the highest fresh weight (3.38 ± 0.08 g). In general, media with a single hormone (particularly TDZ) was more effective in producing a high mass of callus compared to combined PGRs. A culture duration of six weeks resulted in the production of green, brown and cream colored callus. The highest callus weight and accumulation of AZA was recorded in green callus (214.53 ± 33.63 mg g−1 dry weight (DW)) induced using TDZ. On the other hand, small amounts of AZA were detected in both brown and cream callus. Further experimentation indicated that the green callus with the highest AZA was found to be non-toxic (LC50 at 4606 µg mL−1) to the zebrafish animal model. These results suggested that the addition of different PGRs during in vitro culture could prominently affect callus and secondary metabolite production and can further be manipulated as a sustainable method for the production of a natural and environmentally friendly pesticide.

The effect of aseptic cultivation of Crambe mitridatis Juz. plants on its biochemical composition

The aim of the research was to establish efficient microclonal propagation conditions of endangered Crambe mitridatis plants in vitro and to study the possible effect of aseptic cultivation on biochemical composition (hydroxycinnamic acids, flavonoids, phenolic compounds) of plants. Methods. In vitro plant culture methods were applied. Seeds were used for aseptic culture initiation. Morphogenic potential of root, leaf and petiole explants was studied on Murashige-Skoog medium with addition of plant growth regulators. The content of biologically active compounds was measured using spectrometry in plants grown in aseptic conditions and in the greenhouse. Results. Morphogenic potential of root, leaf and petiole explants was studied and the highest regeneration frequency of plantlets was established for root explants (80 %), for petiole explants (50 %) and the lowest for leaf explants (20 %). It was found that plants cultivated in aseptic conditions have higher hydroxycinnamic acids, flavonoids and phenolic compounds compared to plants grown in vivo. Conclusions. It is advisable to multiply C. mitridatis plants in vitro via root and petiole explants. Aseptic cultivation contributes to synthesis of biologically active compounds (auxin synergists) in C. mitridatis plants.Keywords: in vitro culture, hydroxycinnamic acids, flavonoids, phenolic compounds, Crambe mіtridatis.