Growth-Promoting Characteristics of Fungal and Bacterial Endophytes Isolated from a Drought-Tolerant Mint Species Endostemon obtusifolius (E. Mey. ex Benth.) N. E. Br.

Endophytes are primarily endosymbiotic bacteria and fungi that colonize the interior tissues of their host plant. They enhance the host plant's growth and attenuate adverse effects of biological stress. Endophytic species of many indigenous plants are an untapped resource of plant growth-promoting microorganisms that can mitigate abiotic stress effects. Thus, this study aimed to isolate endophytes from the roots and leaves of the medicinal plant Endostemon obtusifolius to evaluate their in vitro growth-promoting capacities and drought tolerance and to characterize the most promising species. Twenty-six endophytes (fourteen bacteria and twelve fungi) were isolated and cultured from the roots and leaves of E. obtusifolius. All 26 endophytes produced flavonoids, and 14 strains produced phenolic compounds. Of the 11 strains that displayed good free radical scavenging capability (low IC50) in the 1-1-diphenyl-1-picryhydrazyl radical scavenging assay, only three strains could not survive the highest drought stress treatment (40% polyethylene glycol). These 11 strains were all positive for ammonia and siderophore production and only one strain failed to produce hydrogen cyanide and solubilize phosphate. Seven isolates showed aminocyclopropane-1-carboxylate deaminase activity and differentially synthesized indole-3-acetic acid. Using molecular tools, two promising symbiotic, drought stress tolerant, and plant growth-enhancing endophytic species (EORB-2 and EOLF-5) were identified as Paenibacillus polymyxa and Fusarium oxysporum. The results of this study demonstrate that P. polymyxa and F. oxysporum should be further investigated for their drought stress mitigation and plant growth enhancement effects as they have the potential to be developed for use in sustainable agricultural practices.

Cytokinin-Facilitated Plant Regeneration of Three Brachystelma Species with Different Conservation Status.

In Africa and Asia, members of the genus Brachystelma are well-known for their diverse uses, especially their medicinal and nutritional values. However, the use of many Brachystelma species as a valuable resource is generally accompanied by the concern of over-exploitation attributed to their slow growth and general small size. The aim of the current study was to establish efficient micropropagation protocols for three Brachystelma species, namely Brachystelma ngomense (endangered), Brachystelma pulchellum (vulnerable) and Brachystelma pygmaeum (least concern), as a means of ensuring their conservation and survival. This was achieved using nodal segments (~10 mm in length) as the source of explants in the presence of different concentrations of three cytokinins (CK) namely N6-benzyladenine (BA), isopentenyladenine (iP) and meta-topolin riboside (mTR), over a period of 6 weeks. The highest (25 µM) concentration of cytokinin treatments typically resulted in significantly higher shoot proliferation. However, each species differed in its response to specific CK: the optimal concentrations were 25 µM mTR, 25 µM iP and 25 µM BA for Brachystelma ngomense, Brachystelma pulchellum and Brachystelma pygmaeum, respectively. During the in vitro propagation, both Brachystelma ngomense and Brachystelma pygmaeum rooted poorly while regenerated Brachystelma pulchellum generally lacked roots regardless of the CK treatments. Following pulsing (dipping) treatment of in vitro-regenerated shoots with indole-3-butyric acid (IBA), acclimatization of all three Brachystelma species remained extremely limited due to poor rooting ex vitro. To the best of our knowledge, the current protocols provide the first successful report for these Brachystelma species. However, further research remains essential to enhance the efficiency of the devised protocol.

Antibacterial, Mutagenic Properties and Chemical Characterisation of Sugar Bush (Protea caffra Meisn.): A South African Native Shrub Species.

Protea caffra is used as a diarrhoeal remedy in South African herbal medicine, however, its pharmacological properties remain largely unknown. In the present study, extracts from different Protea caffra organs were screened against drug-sensitive and -resistant diarrhoeagenic pathogens using the microdilution assay (minimum inhibitory concentration, MIC). Twig extracts (70% methanol, MeOH) of the plant were purified and the resultant fractions screened for antibacterial properties (MIC). The chemical profiles of the fractions were determined by Gas Chromatography-Mass Spectrometry (GC-MS), while ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to quantify the phenolic acids in the plant. The mutagenic properties of bioactive extracts were assessed using the Ames test. The extracts demonstrated weak-moderate antibacterial properties (MIC: 0.3-0.6 mg/mL). A cold ethyl acetate fraction of MeOH twig extract exhibited significant antibacterial properties (MIC = 0.078 mg/mL) against Enterococcus faecalis. The presence of antibacterial compounds (1-adamantane carboxylic acid, heptacosanol, levoglucosan, nonadecanol) in the plant was putatively confirmed based on GC-MS analysis. Furthermore, UHPLC-MS/MS analysis revealed varying concentrations of phenolic acids (0.08-374.55 µg/g DW). Based on the Ames test, the extracts were non-mutagenic thereby suggesting their safety. To a certain degree, the current study supports the traditional use of Protea caffra to manage diarrhoea among local communities in South Africa.

Synthetic strigolactone (rac-GR24) alleviates the adverse effects of heat stress on seed germination and photosystem II function in lupine seedlings.

There is increasing experimental evidence that strigolactones, a class of carotenoid-derived sesquiterpenoid hormones, and their downstream signal components play a role in plant resilience to abiotic stress. Strigolactones positively influence plant coping mechanisms in response to abiotic stressors like drought and high salinity. In this study, we examined the effects of rac-GR24 (a synthetic strigolactone analog) and strigolactone inhibitors on the physiological and molecular responses associated with thermotolerance during seed germination and seedling development in Lupinus angustifolius under heat stress. Photosystem I & II functions were also evaluated via Chl a fluorescence transient analysis in heat stressed lupine seedlings. Our results suggest a putative role for GR24 in mediating tolerance to heat stress during seed germination and seedling development albeit these responses appeared independent of D14-mediated signalling. Seeds primed with GR24 had the highest of all germination indices, enhanced proline content and reduced peroxidation of lipids. GR24 also enhanced the activities of enzymes of the antioxidant and glyoxalase systems in lupine seedlings. The JIP-test indicated that GR24 conferred resistance to heat stress-induced damage to the oxygen evolution complex while also preventing the inactivation of PSII reaction centres thus ensuring PSII thermotolerance.

Phytochemical Profiles and Antioxidant Activity of Grasses Used in South African Traditional Medicine.

Grasses are a valuable group of monocotyledonous plants, used as nourishing foods and as remedies against diseases for both humans and livestock. Phytochemical profiles of 13 medicinal grasses were quantified, using spectrophotometric methods and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS), while the antioxidant activity was done using DPPH and ferric-reducing-power assays. The phytochemical analysis included the total soluble phenolic content, flavonoids, proanthocyanidins, iridoids and phenolic acids. Among the 13 grasses, the root methanolic extracts of Cymbopogon spp., Cymbopogon nardus and Cenchrus ciliaris contained the highest concentrations of total soluble phenolics (27-31 mg GAE/g DW) and flavonoids (4-13 mg CE/g DW). Condensed tannins and total iridoid content were highest (2.3 mg CCE/g DW and 3.2 mg HE/g DW, respectively) in Cymbopogon nardus. The most common phenolic compounds in the grass species included ρ-coumaric, ferulic, salicylic and vanillic acids. In the DPPH radical scavenging assay, the EC50 values ranged from 0.02 to 0.11 mg/mL for the different grasses. The best EC50 activity (lowest) was exhibited by Cymbopogon nardus roots (0.02 mg/mL) and inflorescences (0.04 mg/mL), Cymbopogon spp. roots (0.04 mg/mL) and Vetiveria zizanioides leaves (0.06 mg/mL). The highest ferric-reducing power was detected in the whole plant extract of Cynodon dactylon (0.085 ± 0.45; r2 = 0.898). The observed antioxidant activity in the various parts of the grasses may be due to their rich pool of phytochemicals. Thus, some of these grasses provide a source of natural antioxidants and phytochemicals that can be explored for their therapeutic purposes.

Influence of biostimulants-seed-priming on Ceratotheca triloba germination and seedling growth under low temperatures, low osmotic potential and salinity stress.

Extreme temperatures, drought and salinity stress adversely affect seed germination and seedling growth in crop species. Seed priming has been recognized as an indispensable technique in the production of stress-tolerant plants. Seed priming increases seed water content, improves protein synthesis using mRNA and DNA and repair mitochondria in seeds prior to germination. The current study aimed to determine the role of biostimulants-seed-priming during germination and seedling growth of Ceratotheca triloba (Bernh.) Hook.f. (an indigenous African leafy vegetable) under low temperature, low osmotic potential and salinity stress conditions. Ceratotheca triloba seeds were primed with biostimulants [smoke-water (SW), synthesized smoke-compound karrikinolide (KAR1), Kelpak® (commercial seaweed extract), phloroglucinol (PG) and distilled water (control)] for 48h at 25°C. Thereafter, primed seeds were germinated at low temperatures, low osmotic potential and high NaCl concentrations. Low temperature (10°C) completely inhibited seed germination. However, temperature shift to 15°C improved germination. Smoke-water and KAR1 enhanced seed germination with SW improving seedling growth under different stress conditions. Furthermore, priming seeds with Kelpak® stimulated percentage germination, while PG and the control treatment improved seedling growth at different PEG and NaCl concentrations. Generally, high concentrations of PEG and NaCl brought about detrimental effects on seed germination and seedling growth. Findings from this study show the potential role of seed priming with biostimulants in the alleviation of abiotic stress conditions during seed germination and seedling growth in C. triloba plants.

Metabolite profiling and isolation of biologically active compounds from Scadoxus puniceus, a highly traded South African medicinal plant.

Scadoxus puniceus (Amaryllidaceae), a medicinal plant of high value in South Africa, is used as a component of a traditional herbal tonic prescribed to treat several ailments. Ultra-high performance liquid chromatography-tandem mass spectrometry quantified the phenolic compounds in different organs of S. puniceus. Gravity column chromatography was used to separate fractions and active compounds. The structure of these compounds was determined using 1D and 2D nuclear magnetic resonance and mass spectroscopic techniques. A microplate technique was used to determine the acetylcholinesterase inhibitory activity of the pure compounds. Metabolite profiling revealed a greater profusion of hydroxycinnamic acids (69.5%), as opposed to hydroxybenzoic acids (30.5%). Chlorogenic acid was the most abundant (49.6% of hydroxycinnamic acids) compound. In addition to chlorogenic acid, the study is the first to report the presence of sinapic, gallic, and m-hydroxybenzoic acids in the Amaryllidaceae. Chromatographic separation of S. puniceus led to the isolation of haemanthamine (1), haemanthidine (2), and a rare chlorinated amide, metolachlor (3), the natural occurrence of which is described for the first time. Haemanthamine, haemanthidine, and metolachlor displayed strong acetylcholinesterase inhibitory activity (IC50 ; 23.1, 23.7, and 11.5 µM, respectively). These results substantiate the frequent use of S. puniceus as a medicinal plant and hold much promise for further pharmaceutical development.

Antifungal and antioxidant activities of Coleonema album and C. pulchellum against skin diseases.

CONTEXT: Coleonema album (Thunb) Bart. & H. L. Wendl (Rutaceae) has been used in the formulation of skincare products, and the Khoisan people rub it on their skin to add luster. Coleonema pulchellum I. Williams has received less attention in the South African traditional medicine. OBJECTIVE: This study investigates the antifungal and antioxidant activities of C. album and C. pulchellum essential oil (EO) and leaf extracts; and analyzes the chemical components of their EOs. MATERIALS AND METHODS: Antifungal activity of leaf extracts was determined using the microdilution method with griseofulvin and ketoconazole as controls. Antifungal capacity of EO was investigated using the 'Volatile release plate method'. Trichophyton rubrum (ATCC 28188) and T. mentagrophytes (ATCC 9533) mycelia (0.3 cm diameter) were placed on fresh yeast malt agar in Petri dishes with filter paper (impregnated with 20 µL of EO) on the lid for direct exposure to EO volatiles while plates without EO were used as controls. The incubation time was seven days. Antioxidant activities of the leaf extracts were determined. RESULTS: Methanol leaf extract of C. pulchellum inhibited the growth of three fungi tested with MIC values of 195, 391 and 49 µg/mL for Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum gypseum, respectively. Terpenes formed the major components of the EO. The EO from both plants inhibited the growth of T. rubrum in vitro. DISCUSSION AND CONCLUSION: This study revealed the therapeutic value of C. pulchellum. Coleonema album and C. pulchellum should be considered as potential plants for skin ointment from natural origin.

Antimicrobial Activity, Phenolic Content, and Cytotoxicity of Medicinal Plant Extracts Used for Treating Dermatological Diseases and Wound Healing in KwaZulu-Natal, South Africa.

Medicinal plants used for wound healing and skin diseases are the key to unlocking the doors to combating problematic skin diseases as resistance of pathogens to pharmaceuticals and allopathic management continues to increase. The study aimed at investigating the antimicrobial efficacies, phenolic content, and cytotoxicity effects of 11 medicinal plant extracts commonly used for treating skin conditions and wound healing in traditional medicine within KwaZulu-Natal. Eleven plant species were separated into different plant parts (bulbs, roots, leaves) and extracted with different solvents. The extracts were assessed for antimicrobial activity against six Gram-positive and seven Gram-negative bacterial strains and four fungi commonly associated with skin conditions using disc diffusion and microdilution techniques. The aqueous methanolic extracts were screened for phenolic content while cytotoxicity tests were performed on all extracts using the brine shrimp lethality and tetrazolium-based colorimetric (MTT) assays. Extracts from Aloe ferox, A. arborescens, and Hypericum aethiopicum were the most active against almost all of the tested bacterial and fungal strains. All plant species exhibited some degree of antimicrobial activity. Total phenolic levels, flavonoids and tannins were also higher for A. ferox, followed by A. arborescens and H. aethiopicum, respectively. The cytotoxicity results of all plant extracts were in the range of 90-100% survival after 24 h in the brine shrimp assay. Extracts considered lethal would demonstrate >50% shrimp death. The MTT cytotoxicity test yielded LC50 values of >1 mg/mL on all extracts indicating that they are not cytotoxic. The observed antimicrobial efficacy demonstrated by some plant species and the general lack of cytotoxic effects on all the tested extracts presents some promising and beneficial aspects of these medicinal plant extracts in the treatment of skin diseases and wound healing. The two Aloe species and H. aethiopicum were among the best extracts that exhibited consistently good antimicrobial activity and warrants further investigations and possible isolation of bioactive principles.

Chromolaena odorata (L.) R.M. King & H. Rob. (Asteraceae) in sub-Saharan Africa: A synthesis and review of its medicinal potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Chromolaena odorata (L.) R.M. King & H. Rob. (Asteraceae) is a scrambling perennial shrub that originated in the Americas, but is now common in sub-Saharan Africa, Asia and Oceania, where it has become a serious weed. The species, particularly the biotype found in Asia and West Africa, has many ethnopharmacological uses, including treatment of malaria, wounds, diarrhoea, skin infection, toothache, dysentery, stomach ache, sore throat, convulsions, piles, coughs and colds. Furthermore, no attempt has been made to synthesise and review the available literature on the usefulness of the plant in the sub-Saharan African region, hence this paper examines the beneficial attributes of C. odorata in sub-Saharan Africa. MATERIAL AND METHODS: Published information on the species was gathered by the use of different database platforms, including Google Scholar, ScienceDirect, SciFinder and Scopus. RESULTS: Records indicate that two biotypes of C. odorata are present in sub-Saharan Africa viz. the more widespread Asian/West African C. odorata biotype (AWAB) and the southern African biotype (SAB). While the usefulness of the former is well elucidated in the literature, such information on the latter is still scarce. Although the importance of AWAB C. odorata as a fallow species and as a soil fertility improvement plant in the slash and burn rotation system of agriculture in West Africa is increasingly being recognised, its usage in traditional medicinal practice is far more appreciated. The species has a wide range of ethnopharmacological uses, possibly because of the presence of flavonoids, essential oils, phenolics, tannins and saponins. The plant is reported to have antibacterial, anti-inflammatory, antioxidant, anthelminthic, antifungal, cytotoxic, anticonvulsant, antiprotozoal, antispasmodic, antipyretic and analgesic properties. CONCLUSION: While the results of this review suggest that the AWAB plant can be exploited as an alternative to other threatened plant species known to possess similar medicinal potential, the medicinal and pharmacological potential of the SAB plant remains to be established. Further studies on the phytochemistry and pharmacological properties of the SAB plants will not only advance our knowledge of ethnobotany and ethnomedicine, but may also improve the health and knowledge of the local people.

Nutritional and pharmacological potential of the genus Ceratotheca--An underutilized leafy vegetable of Africa.

ETHNOPHARMACOLOGICAL RELEVANCE: Ceratotheca (Pedaliaceae) is an endemic African genus comprising of five species. The genus is commonly used as a leafy vegetable with medicinal properties. AIM OF THE REVIEW: The review aims to highlight the unexplored nutritional and pharmacological potential of African indigenous leafy vegetables Ceratotheca sesamoides and triloba, in order to conserve and domesticate these species. METHODS: The information was obtained from various search engines such as Scopus, Google Scholar and Web of Science as well as Ethnobotanical books. RESULTS: Ceratotheca sesamoides and triloba have good nutritional potential. The species are high in energy levels, fat content, proteins and carbohydrate values. The species have also been reported to have good antibacterial, antidiarrhoeal, antidiabetic, antiplasmodial and antiviral properties. These species have slight toxicity and cytotoxic activity when extracted at high concentrations but no mutagenic activity was detected. CONCLUSIONS: To date, few studies have documented the usage (nutrition and pharmacology) of Ceratotheca sesamoides and triloba. More studies investigating the nutritional content as well as methods of its improvement are necessary if the plant is to be included as a domesticated vegetable crop. Safety and toxicity analysis of this leafy vegetable need to be extensively studied as the plants are consumed in high quantities.

Effects of Cd and Al stress on secondary metabolites, antioxidant and antibacterial activity of Hypoxis hemerocallidea Fisch. & C.A. Mey.

This study investigated the effects of cadmium (Cd) and aluminium (Al) on the accumulation of phenolics, flavonoids and the bioactive compound hypoxoside in Hypoxis hemerocallidea. In addition, antioxidant scavenging and antibacterial activity were assessed to evaluate if Cd and Al stress affect the accumulation of bioactive compounds in H. hemerocallidea. In vitro grown plantlets of H. hemerocallidea were acclimatized for seven months in a greenhouse. Thereafter plants were exposed to various concentrations of Cd and Al both singularly and in combination in the form of Cd(NO3)2 (2, 5, 10 mg Cd/L); Al3(NO3)3 (500, 1000, 1500 mg Al/L) and combinations of Cd and Al (Cd 2:Al 500, Cd 5:Al 1000 and Cd 10:Al 1500 mg/L) for a further six weeks. The highest amounts of Cd and Al translocated to the shoot were 34 and 1608 mg/L respectively. Phytochemical screening showed significantly high amounts of total phenolics and flavonoids at the moderate Cd treatment (5 mg/L) compared to the controls. Exposure to Cd and Al significantly decreased the accumulation of hypoxoside. There was a significant increase in diphenylpicrylhydrazyl (DPPH) antioxidant scavenging activity in most of the metal-treated plants compared to the positive control ascorbic acid. Extracts from Cd 2 mg/L treatment exhibited moderate antibacterial activity against Staphylococcus aureus compared to the control. The results of the present study revealed that cultivating H. hemerocallidea on metal contaminated soils affects the accumulation of the bioactive compound, hypoxoside.

Dissecting the role of two cytokinin analogues (INCYDE and PI-55) on in vitro organogenesis, phytohormone accumulation, phytochemical content and antioxidant activity.

There is a continuous search for new chemical entities to expand the collection of suitable compounds to increase the efficiency of micropropagation protocols. Two cytokinin (CK) analogues, 2-chloro-6-(3-methoxyphenyl)aminopurine (INCYDE) and CK antagonist 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55) were used as a tool to elucidate the auxin-CK crosstalk under in vitro conditions in the medicinally important plant, Eucomis autumnalis subspecies autumnalis. These compounds were tested at 0.01, 0.1 and 10 µM alone as well as in combination with benzyladenine (BA) and naphthaleneacetic acid (NAA). The organogenesis, phytohormone content, phytochemical and antioxidant response in 10 week-old-in vitro regenerated E. autumnalis subspecies autumnalis was evaluated. INCYDE generally favoured shoot regeneration while the effect of PI-55 was more evident in root proliferation. Overall, INCYDE promoted the accumulation of higher concentrations and varieties of endogenous CK relative to the PI-55 treatments. In contrast, higher concentration of indole-3-acetic acid and 2-oxindole-3-acetic acid were generally observed in PI-55-supplemented cultures when compared to plantlets derived from INCYDE. Both CK analogues (individually and in-conjunction with exogenously applied PGRs) significantly influenced the phytochemicals and consequently the antioxidant potential of the in vitro regenerants. These results provided insight on how to alleviate root inhibition, a problem which causes considerable loss of several elite species during micropropagation.

Screening for adulticidal activity against Anopheles arabiensis in ten plants used as mosquito repellent in South Africa.

BACKGROUND: Due to the development of resistance to synthetic insecticides, adverse effects to human health, non-target organisms and the environment, there is an urgent need to develop new insecticides, which are effective, safe, biodegrable and target-specific. This study was undertaken to evaluate the adulticidal activity of 10 plants used traditionally as mosquito repellents in South Africa. METHODS: The dried plant materials were extracted with dichloromethane (DCM) and ethanol (EtOH). The extracts were evaluated for adulticidal activity against Anopheles arabiensis mosquitoes, a potent malaria vector in South Africa. Adult mortality was observed after 24 hours of exposure. RESULTS: All the extracts showed adulticidal activity. The highest activity was observed in both DCM and EtOH extracts of Aloe ferox leaves with 98 and 86% mosquito mortality, respectively. The DCM extract of A. ferox leaves was then subjected to a dose-dependent bioassay to determine the EC50 value. The extract exhibited an EC50 value of 4.92 mg/ml. CONCLUSION: The results of the present study showed that the DCM extract of A. ferox leaves may have the potential to be used as an insecticide against An. arabiensis. Further studies to isolate and identify active compounds are in progress.

Pharmacological potential and conservation prospect of the genus Eucomis (Hyacinthaceae) endemic to southern Africa.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Eucomis (Hyacinthaceae) consists of 10 species that are extensively used in African traditional medicine. This review is an appraisal of current information on the distribution and morphology, traditional uses, pharmacology, toxicology and approaches devised to enhance the conservation of the genus. METHODS: A systematic and comprehensive literature search using electronic searches such as Scopus, Google Scholar, Web of Science and ethnobotanical books was conducted. RESULTS: Evidence from traditional medicine usage shows wide utilization of this genus for ailments such as respiratory, venereal diseases, rheumatism as well as kidney and bladder infections. Pharmacological screening reported antimicrobial, antiplasmodial, antitumor, cytotoxic, phytotoxic and anti-inflammatory properties. CONCLUSIONS: The potential of the genus Eucomis especially in terms of pharmacology cannot be overemphasized. Apart from the anti-inflammatory properties, the antifungal activity of Eucomis remains a valuable reservoir with potential application in the agriculture sector as a source of an affordable biocontrol agent. Based on the speculated toxic constituents in the genus Eucomis, it will be valuable to conduct detailed toxicological studies. Extensive utilization of members of the genus Eucomis is causing severe strain on wild populations. Although conventional propagation has been relatively effective in the alleviation of the declining status, micropropagation of members may be vital to guarantee the conservation of wild populations.

Carbon-nitrogen ratio and in vitro assimilate partitioning patterns in Cyrtanthus guthrieae L.

In response to variations in nutritional composition of the growth medium, plants often adjust their metabolism and progressively alter their growth patterns. Carbon (C) and nitrogen (N) constitute the major plant nutritional components influencing plant growth and development patterns. This study examined the growth dynamics and patterns of assimilate partitioning to primary and secondary metabolites in response to varying levels and combinations of C and N in the culture media of Cyrtanthus guthrieae. In vitro callus-derived C. guthrieae plantlets were cultured on solid Murashige and Skoog (MS) media with different concentrations and combinations of C and N. Relative growth rate (RGR) increased proportionally with an increase in C concentrations up to 88 mM sucrose (0.58 d(-1)) beyond which it was hardly influenced by further increases in C. Growth was also significantly favoured in media with high concentrations of N at all C concentrations tested. In C-limited media regimes with growth saturating N conditions, alkaloid accumulation became favoured while polyphenol content increased with an increase in C levels in the medium, a characteristic pattern that appeared to be less influenced by the amount of N. Of the primary metabolites, only proteins showed small significant variations across different media treatments, with starch and soluble sugars increasing proportionately with C levels. In the medium with a high sucrose concentration (175 mM), soluble sugars, amino acids and polyphenols increased markedly, possibly as an adaptive response to the reduced osmotic potential in the media and/or a storage mechanism for excess C and N reserves in the media. From a medicinal perspective, with regard to polyphenolic compounds in C. guthrieae, growth medium conditions that allow for high levels of C pools in the tissue would thus be favourable for the enhanced synthesis of this group of compounds. The medium conditions with 175 mM sucrose and 10.3 mM NH4NO3 gave the highest total polyphenols, flavonoids and proanthocyanidins with a moderate growth rate.

Endogenous cytokinin profiles of tissue-cultured and acclimatized 'Williams' bananas subjected to different aromatic cytokinin treatments.

Endogenous cytokinin (CK) levels of in vitro-cultured and greenhouse-acclimatized 'Williams' bananas treated with six aromatic CKs were quantified using UPLC-MS/MS. The underground parts had higher endogenous CK levels than the aerial parts. Control plantlets had more isoprenoid CKs while the aromatic-type CKs were predominant in all other regenerants. Following acclimatization of the control and 10 µM CK regenerants, there was a rapid decline in both isoprenoid and aromatic CK in the greenhouse-grown plants. Apart from the control and 6-(3-Methoxybenzylamino)-9-tetrahydropyran-2-ylpurine (MemTTHP) treatment with higher level of isoprenoid CK, aromatic CK remain the predominant CK-type across all CK treatments. The most abundant CK forms were meta-topolin (mT) and benzyladenine (BA) in the micropropagated and acclimatized plants, respectively. Micropropagated plantlets had cis-Zeatin (cZ) as the major isoprenoid CK-type which was in turn replaced by isopentenyladenine (iP) upon acclimatization. On a structural and functional basis, 9-glucoside, a deactivation/detoxicification product was the most abundant and mainly located in the underground parts (micropropagation and acclimatization). The results establish the wide variation in metabolic products of the tested aromatic CKs during micropropagation and acclimatization. The findings are discussed with the possible physiological roles of the various CK constituents on the growth and development of banana plants.

Antioxidant and phenolic acid profiles of tissue cultured and acclimatized Merwilla plumbea plantlets in relation to the applied cytokinins.

Merwilla plumbea (Lindl.) Speta is an important medicinal plant widely used in traditional medicine. We evaluated the effect of five cytokinins [benzyladenine (BA), 2-isopentenyladenine (2iP), meta-topolin (mT), meta-topolin riboside (mTR), and meta-methoxy-9-tetrahydropyran-2-yl-topolin (MemTTHP)] on the level of phenolic acids and antioxidant activity of M. plumbea during the tissue culture and acclimatization stages. Two cytokinins (mT and mTR) significantly improved the antioxidant activity of tissue culture plantlets while the control plantlets were better after acclimatization. Using UPLC-MS/MS, the levels of hydroxybenzoic and hydroxycinnamic acid derivatives (phenolic acids) varied significantly during tissue culture and acclimatization, depending on the cytokinin and plant part analyzed. Vanillic acid (24.9 µg g⁻¹) detected in underground parts of tissue culture plants supplemented with BA was the most abundant phenolic acid detected. The current findings indicate that the phytochemicals together with the bioactivity during in vitro propagation of M. plumbea is influenced by the cytokinin type used and the stage of plant material collection.

Dissecting the stress metabolic alterations in in vitro Cyrtanthus regenerants.

Metabolic plasticity in plants allows for continuous adjustments of defence strategies in suboptimal environments. Proline and other metabolites figure prominently in most stress-mediated responses. This study examined the expression of salinity and osmotic adjustments in the enzymatic activity and accumulation of solutes and metabolites in response to imposed water and salt stress in Cyrtanthus contractus N.E. Br. and Cyrtanthus guthrieae L. Bolus regenerants. In vitro-derived plantlets were cultured on solid Murashige and Skoog (MS) media with three different polyethylene glycol (PEG)-induced osmotic levels and four NaCl stress-induced levels at 25 °C. The levels of proline and phenolic compounds measured at intervals of three, four and five weeks from initial plantlet culture increased in a stress-dependent pattern. The levels of these metabolites also showed a significant increase with an increase in the duration of plantlets under stress conditions. The highest proline concentration (9.98 µmol g(-1) FW) was recorded in C. contractus at 300 µM NaCl after five weeks. A corresponding high level of total phenolic compounds (147 mg GAE g(-1) DW) was also recorded in the same treatment for the same species. The activity of proline dehydrogenase (PDH) (EC 1.5.99.8) was shown to decrease with an increase in proline levels from week three to week five in almost all the stress conditions. The high levels, particularly of phenolic compounds obtained under osmotic and salinity stress conditions in this study present a promising potential of manipulating culture and/or growing conditions for improved secondary compound production and hence medicinal benefits.

Physiological responses and endogenous cytokinin profiles of tissue-cultured 'Williams' bananas in relation to roscovitine and an inhibitor of cytokinin oxidase/dehydrogenase (INCYDE) treatments.

The effect of supplementing either meta-topolin (mT) or N(6)-benzyladenine (BA) requiring cultures with roscovitine (6-benzylamino-2-[1(R)-(hydroxymethyl)propyl]amino-9-isopropylpurine), a cyclin-dependent kinase (CDK) and N-glucosylation inhibitor, and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine), an inhibitor of cytokinin (CK) degradation, on the endogenous CK profiles and physiology of banana in vitro was investigated. Growth parameters including multiplication rate and biomass were recorded after 42 days. Endogenous CK levels were quantified using UPLC-MS/MS while the photosynthetic pigment and phenolic contents were evaluated spectrophotometrically. The highest regeneration rate (93 %) was observed in BA + roscovitine while mT + INCYDE plantlets produced most shoots. Treatment with BA + roscovitine had the highest shoot length and biomass. Although not significant, there was a higher proanthocyanidin level in BA + roscovitine treatments compared to the control (BA). The levels of total phenolics and flavonoids were significantly higher in mT + roscovitine treatment than in the mT-treated regenerants. The presence of roscovitine and/or INCYDE had no significant effect on the photosynthetic pigments of the banana plantlets. Forty-seven aromatic and isoprenoid CKs categorized into nine CK-types were detected at varying concentrations. The presence of mT + roscovitine and/or INCYDE increased the levels of O-glucosides while 9-glucosides were higher in the presence of BA. Generally, the underground parts had higher CK levels than the aerial parts; however, the presence of INCYDE increased the level of CK quantified in the aerial parts. From a practical perspective, the use of roscovitine and INCYDE in micropropagation could be crucial in the alleviation of commonly observed in vitro-induced physiological abnormalities.